SEMINAR ON
               MAY  4-5, 1972
                 EDITED  BY
            DISTRIBUTED BY
                JULY 1972

                       SEMINAR ON
                  IN INDUSTRIAL WASTES
                      MAY 4-5,  1972
                        Edited by

 Robert H. Bordner, Chief, Microbiological  Methods,  AQCL
                    Cincinnati, Ohio


Bobby J. Carroll, Chief, Microbiological Services  Branch
               Southeast Water Laboratory
                     Athens, Geo rg ia
                      U.S. EPA Region 8
                      Technical Library 8OC-L
                      999 18th Street, Suite 300
                      Denver, CO 80202-2466

                     Distributed by
                  Office of Enforcement
          National Field Investigations Center
                    Denver, Colorado

                        July 1972



Introduction and Summary	     vii

Statement of Problem
   Thomas P. Gallagher 	      1

History and Background on Occurrences of Fecal Coliforms in
   Industrial Wastes
   R. Richard Bauer 	      4

Experiences with Coliform and Enteric Organism Isolation from
   Industrial Wastes
   Donald L. Herman	      27

Observations of Selected Waste Discharges from Region IX
   Kathleen Shimmin 	      42

Review of Research Regarding Coliforms in Pulp and Paper Mill Wastes
   Dr. Martin Knittel 	,      54

Brief Literature Review of Klebeiella as Pathogens
   Dr. Leonard Guarraia 	      94

The Detection and Significance of Klebsiella in Water
   G. J. Vasconcelos	      98


Appendix A; Considerations for Permit Preparation Pulp and
   Paper Industry	     A-l

Appendix B; Tables 	     B-l

Appendix C; Experiences With Coliform and Enteric Organism
   Isolations From Industrial Wastes 	     C-l

Appendix D; Identification of Coliform Colonies 	     D-l

Appendix E; Figures 	     E-l


Discussion                                                            Pages

Coliform colonies on EMB and Endo agar 	    12-13

Treatment Methods - pulp and paper processing wastes 	    14-17

Nutrient Reduction 	    18-22

Regrowth of coliform organisms 	       23

Effluent quality criteria  	    25-26

Klebsiella Resistance to Antibiotics  	    39-40

Fecal coliform test results from environmental samples
   and areas remote from civilization 	    41-43

Fecal coliform MF test for industrial wastes  	       46

Elevated temperature test  	    48-49

Total and fecal coliform values from  industrial wastes -
   reconciliation of results  	    50-52

Bacteriological standards  for industrial wastes  	       52

Fecal coliform MF test - background growth  	    60-62

Pathogens - paper and pulp mill wastes	       63

Fecal coliform test - incubation temperature  	    64-65

Coliform MF tests - time and  temperature limits  	       66

Fecal coliform test - incubation temperature  	    67-68

Klebsiella  	       69

                             (Contents - cont'd.)


Indicators versus pathogens	      70-73

Correlation of bacteriological and chemical parameters 	         76

Statement of proposal for monitoring permit program 	         78

Total-versus-fecal coliform tests - shellfish-harvesting waters ...         80

Research Needs - microbiology of industrial wastes 	         89

Standardization of Methods 	      92-93

Suppression of indicator organisms 	         97

Need for improved total and fecal coliform tests 	    103-104

Klebslella - Pathogenicity and public health hazard 	    124-125

Standardization of Methods 	    106-116

Standard Methods 	    117-119

Quality Control 	        113

Microbiology Meetings 	    116-117

Laboratory evaluation 	        119


                                    EPA Seminar
                        The. Significance of Fecal Coliform

                               in Industrial Wastes

Mr. Richard R. Bauer
Acting Chief, Technical Studies Section
Surveillance Branch
Surveillance and Analysis Division
Region X
1200 6th Avenue, Seattle, Washington

Dr. Gerald Berg
Chief, Virology, AWTRL, NERC
4676 Columbia Parkway
Cincinnati, Ohio 45268

Mr. Robert H. Bordner
Chief, Microbiological Methods
Analytical Quality Control Laboratory
Cincinnati, Ohio 45202

Mr. Francis Brezenski
Chief, Technical Support Branch
Edison Water Quality Laboratory
Edison, New Jersey 08817

Mr. Bobby J. Carroll
Chief, Microbiological Services Branch
Southeast Water Laboratory
College Station Road
Athens, Georgia 30601

Mr. Ralph Christensen
Microbiologist, Region V
1 North Wacker Drive
Chicago, Illinois 60606

Mr. Howard S. Davis
Microbiologist, Region I
John F. Kennedy Bldg, Room 2303
Boston, Massachusetts 02203

Mr. Thomas P. Gallagher
Director, National Field Investigations
  Center, Bldg. 53, Denver Federal Center
Denver, Colorado 80225
Mr. Edwin E. Geldreich
Chief Bacteriologist
Water Supply Research Laboratory, NERC
4676 Columbia Parkway
Cincinnati, Ohio 45268

Dr. Leonard J. Guarraia
Microbiologist, Office of Water Programs
Water Quality Protection Branch
Applied Technology Division
1921 Jefferson Davis Highway, Rm. 902
Arlington, Virginia 22202

Mr. Donald L. Herman
Microbiologist, National Water Quality
6201 Congdon Blvd.
Duluth, Minnesota 55804

Dr. Martin D. Knitted
Research Microbiologist
Pacific Northwest Water Laboratory
200 South 35th Street
Corvallis, Oregon 97330

Mr. David N. Lyons
Sanitary Engineer
Office of Refuse Acts Program
Crystal Mall #2, Rm. 716
Washington, D. C. 20460

Mr. John P. Manhart
Microbiologist, EPA, Region VIII
Suite 900, 1860 Lincoln Street
Denver, Colorado 80203

Mr. Louis A. Resi
Chief Microbiologist
National Field Investigations Center
5555 Ridge Avenue
Cincinnati, Ohio 45268

Mr. Courtney Riordan
Office of Technical Analyses
Crystal Mall, Building #2
Washington, D. C. 20460

Miss Kathleen Shimmin
Assistant Chief
Surveillance and Analysis Division
Technical Support Branch
620 Central Avenue
Alameda, California 94501

Mr. William J. Stang
National Field Investigations Center
Bldg. 53
Denver Federal Center
Denver, Colorado 80225
Mr. Murray Stein
Director, Enforcement Proceedings
Office of Enforcement and General Counsel
Crystal Mall No. 2, Room 1116
Washington, D. C. 20460

Mr. G. J. Vasconcelos
Northwest Water Supply
Research Laboratory
Gig Harbor, Washington 98335

Mr. William H. Winders
Microbiologist, Region VI
Suite 1100, 1600 Patterson Street
Dallas, Texas 75201

                       Introduction and Summary

   On May 4 and 5, 1972,  at the invitation of the National Field

Investigations Center-Denver,  a group of EPA microbiologists, sanitary

engineers, and other concerned participants held a seminar to discuss

the significance of fecal coliforms in certain industrial wastes and the

use of bacteriological parameters as effluent standards in the permit pro-

gram.  Additional topics  that  were discussed at the meeting included the

relevance of Klebsiella as an  indicator of sanitary quality, the need for

standardization of methods within EPA, and improved communication among

agency microbiologists.

   The seminar included six formal presentations pertinent to the subject

of the meeting.  Open discussion followed each presentation and a general

discussion period was held at  the end of the seminar session.  The par-

ticipants made specific recommendations for monitoring nutritive indus-

trial wastes and reached the following general conclusions:

   1.  The fecal coliform test is the most valid microbiological

       parameter for industrial wastes presently available.

   2.  The excessive Klebsiella densities in fecal-coliform test results

       from certain industrial wastes such as the pulp and paper, sugar

       beet, and food-processor effluents are significant because they

       reflect the high nutrient levels in these wastes.  They can suppress

       E. ooli detection, they can be pathogenic, and they are coliforms by

       definition.  In addition, Klebsiella pnewnoniae is found in the in-

       testinal tract of approximately 30 percent of humans and 40 percent

       of animals.

3.    Nutrient-rich wastewaters provide the capability for bacterial

     re-growth in the receiving streams.

4.    Salmonella are recoverable from a number of high nutrient

     wastes.   They are also capable of re-growth and of producing a

     potential public health hazard.  However, pathogen detection

     alone is not adequate for monitoring because no one test will

     detect all possible pathogens that might be present, and

     negative results do not ensure the absence of all pathogenic


5.    Effluent standards for highly nutritive, industrial wastes

     shall be below 30 mg/1 and preferably in the range of 14-30 mg/1,

     as measured by BOD or the equivalent TOG.  A TOG level was

     not recommended, pending the establishment of a value that

     will correlate with the recommended BOD limits.

6.    The fecal-coliform bacteria test shall be used to monitor the

     nutrient reduction, by measuring the bacterial levels in the

     treated wastes.  A geometic mean density of fecal coliforms,

     not to exceed 1,000/100 ml, was recommended as an index of

     adequate treatment.  Bacteriological analyses will be performed

     on final effluents prior to disinfection, if disinfection is


 7.   When the receiving waters are classified for contact sports

     or shellfish harvesting,  the bacteriological and chemical

     limits shall be reduced in order to comply with the estab-

     lished water quality standards.

 8.   The discharger has the primary responsibility for conducting

     the required monitoring.   EPA or other agencies may need to

     provide analytical support for small industries.  Preferably

     EPA will conduct the initial monitoring in order to provide

     basic data and to evaluate the performance of the proposed

     system.  At a later date the industries or other agencies may

     assume the monitoring responsibility, with periodic checks

     by EPA.

 9.   Sampling should be conducted initially at a higher frequency,

     such as weekly or biweekly covering maximum flow periods. After

     adequate baseline data have been attained, less frequent samp-

     lings may be adequate.

10.   High priority should be given to research devoted to the im-

     provement of bacteriological and chemical parameters for in-

     dustrial wastes.  Improved tests for the indicator organisms

     and methods for pathogen isolation are needed.  Further research

     should be conducted in order to define the public health risk

     associated with Salmonella, Klebsiella, and other pathogens

     in industrial waste discharges.

11.   The importance of standardization of methods within the

     agency was emphasized.

12.   Better communication among agency microbiologists was whole-

     heartedly endorsed.  The seminar was in favor of periodic

     meetings to deal with microbiological problems.

13.   Suggestions were made for needed additions and supplementation

     to Standard Methods.

                           Statement of Problem

                            Thomas P. Gallagher

     The National Field Investigations Centers (NFIC)  have been

assigned the responsibility for preparing effluent guidelines that can

be used by the Permit Program in developing permits under either the

Refuse Act or, in the event of its passage, the new water pollution

legislation.  We have had meetings with technical representatives of

some of the industries, namely the pulp and paper industry and the

canning industry.  Many of the effluent guidelines that we are pre-

paring for the industries contain a fecal coliform or a coliform bac-

teriological constraint.  The pulp and paper representatives as well

as the sugar beet people have questioned the sanitary significance of

fecal coliform and whether or not it can be required in developing a

permit.  To them it has some very practical significance in terms of

the economics involved.  Generally the industries have not been re-

qired to put in disinfection equipment and to hold down their coliform

levels with the kinds of rather expensive installations that would be

necessary if some of the standards in the effluent guidelines are


     About two months ago in Corvallis the pulp and paper people pre-

sented at a seminar what has been reported to me as a rather strong

case against coliform standards for this industry.  We felt that, be-

cause of the casethat had been made in Corvallis, it was necessary to

assemble the best people in EPA who had familiarity or experience with

this problem and aee whether there is sufficient evidence and sufficient


technical rationale for requiring a. coliform or some kind of bacteriological

standard in the effluent guidelines that will be written into the permit

program.  Now let me tell you this: it must be a technically defensible

standard and it must* be backed up by evidence.  We've got to stay away from

things that are "desirable" or that "we would like to see."  It must have

strong technical substantiation.

     Now, on that basis, we have Murray Stein, who is the Chief Enforcement

Officer for Water Control and in charge of the Enforcement Proceedings

Division in Washington.  He will have a lot to say about what happens with

this problem.

     (Mr. Stein is recognized.)

     I want to welcome you here, and if you need anything, just let either

Robert Schneider or William Stang know and they will accomodate you.  We

want to have a real free-wheeling discussion.  We have a tape recorder, but

it's not being used to inhibit anybody.  As a matter of fact, we plan to

assemble the results of these discussions in a report and send it to you,

the active participants, for comment, and forward it to Headquarters for

recommendation on this whole problem, at least from the technical people

within EPA.

     I'm going to get out of the way and Richard Bauer, from Region X,

who has had some experience in this particular problem, will moderate

this discussion.  We'd like to hear presentations from the participants

and then we would like to have a rigid question-answer session and dis-

cussion back-and-forth on the particular information, and a decision on

whether or not you think what's been presented is defensible.  Thank you.


     Bauer:   Thank you.  It's a real pleasure to be here today.   I would

like to start by all of us introducing ourselves.

     (Attendees proceed to introduce themselves.  See preceding list of

participants and attendees.)

     I hope personally that this is the first of a number of meetings of

this sort.  I think that we can profit a great deal by, to use an old

cliche, " that cross-fertilization of having people who are working in the

field, discuss the kind of problems that they are facing," and, if this

turns out to be a profitable experience, I hope that perhaps we can make

this an annual affair.

     In any event, we have a policy that was tentatively formulated by

the NEIC, and I believe copies of it have been passed out.   [See Appendix A:

Considerations for Permit Preparation, Paper and Pulp Industry.]   I feel

that the most important purpose of this meeting is to try to come to some

agreement  as to whether this policy is relevant.  If there are any diffi-

culties with it, will you point them out to Mr. Stein?  Again, I agree

completely with Tom Gallagher that the policy can't be based upon personal

feelings; we should be  able to back up what we  say with numbers and some

hard data, because the  industry is vitally concerned with this problem  and

is willing to go  to the wall concerning it.

     If we can dispose  of this issue in the next two days, or day and a

half,  I think it would  also be relevant while all of us are here, to enter

into some  discussions on the adequacy of Standard Methods.  There have  been

some complaints made about  it from various people.  I  think this would  be  a


good opportunity to discuss that, and then perhaps a general discussion on

ideas that people in the field have, to help us in our enforcement.  I know

a number of people are using new techniques in bacteriological analysis for

enforcement purposes like fluorescent antibody techniques, enteropathogenic

E ooli identification and that type of thing.

     With that in mind, I would like to kick the meeting off with a little

historical background on the subject, "The Significance of Fecal Coliforms

in Industrial Wastes."

History & Background on Occurrences of Fecal Coliforms in Industrial Wastes

                          R. Richard Bauer

     I first became involved with the questioning of the relevance of

coliform tests to pulp mills in about 1968 when I was assigned to NERC,

Corvallis.  The State of Oregon had adopted a rather ambitious program for

waste treatment on the Willamette River.  This included both municipal and

industrial wastes.  It required an implementation plan that all the in-

dustry and municipalities be on-line with secondary treatment and with

adequate disinfection for the municipalities by 1972.  The municipalities

had to have their treatment by 1968.  All the municipalities complied, and

all the bacteriological standards were still fractured rather badly.  We

were experiencing total coliform counts, in numerous places throughout the

river, on the order of 100,000/100 ml.  Fecal coliform values were con-

siderably lower, by and large, and on the order of magnitude of approxi-

mately 50.

     Coincidentally, about this time the National Council for Air and


Stream Improvement (hereafter referred to as the National Council) had funded

some research.  A gentleman by the name of Ching Chang, in a master's thesis

at John Hopkins, came to the conclusion that total coliform tests were not

relevant.  I think that most of us agree that certainly there is some ques-

tion on its applicability.  Mr. Chang recommended that we go to a fecal coliform


     Since then the National Council has also challenged the fecal coliform stan-

dard, and the basis for their challenge is a series of studies they did on

these pulp and paper mills on the Willamette River.  They selected mills in

which, they claimed, they completely separated all their domestic sewage.

They dumped dye down all the toilets, and checked all the plumbing, and

I am convinced that they tried quite hard; in all probability, domestic

sewage is being excluded from these plants.

     Nevertheless, the coliform levels being discharged in the effluent

are extremely high.  In one particular case, Crown-Zellarbach's sulfite

mill (Lebanon, Oregon), total coliforms were consistently 100,000,000/100 ml

and fecal coliform values are on the order of 100,000.  They went further

to start IMViC-typing all these total and fecal coliforms.  From  the fecal

coliform plates, in all cases except Crown-Zellarbach, they claimed they

found absolutely no E coli.  They will admit to having isolated some Type I

E aoli out of Crown-Zellarbach (Lebanon).  They have done some additional

testing to try to isolate Salmonella out of the wastes.  They were unsuccess-

ful, and came to the position at that time that the fecal coliform test  is

not relevant.


     This was argued between the State of Oregon, the National Council, and

the EPA Water Laboratory (Corvallis).  It was decided at that time to form

an ad hoc committee to see if we could shed some light on this problem.  We

started getting together in October 1969 to try to coordinate our research.

Although not a researcher, I am capable of applying logic to some basic

bacteriological questions and I, with the concurrence of the National Council,

the states of Oregon, Washington, and Idaho (we included them in this at this

time), and some of the food processors, put together a proposal to study five

mills on the Willamette River to see whether we could isolate any potentially

pathogenic organisms out of the wastes, to study the effect on the receiving

water, and to ascertain if we could define the areas that need further research.

     I now refer to the data sheets which were passed out (See Appendix B)-

Please refer to Table I.  We picked five mills, both sulfite and kraft process,

with various types of pulping techniques and various types of waste treatment.

The American Can kraft mill (Halsey, Oregon) is what I think we could call

the state-of-the-art in pulp mill waste treatment.  There is a series of

area lagoons for secondary settling of kraft process mill wastes, and the

best equipment and engineering went into the plant.  Likely, it is con-

siderably overdesigned.  We looked at the mill effluent and then came up

with counts upstream and downstream.

     I conducted a whole series of filtrations, picked individual colonies,

and confirmed their ability to ferment lactose with production of gas,

yielding confirmed total and fecal coliforms.  We inoculated EC broth at

44.5°C, and fecal streptococcus KF agar.  This number of Klebs-iella was cal-

culated based on a percentage of colonies identified as Klebs-iella from


a percentage of the colonies that were picked.  If they were Klebsiella, I

referred to the original count on the filter and got what I would consider

a semi-quantitative value for Klebsiella pnewnoniae,  Criteria for iden-

tifying Klebsiella were —-H- IMViC test, lack of motility, and the ability

to decarboxylate ornithine.  These results were confirmed by the National

Communicable Disease Center (NCDC), so I am confident that the numbers

are reasonably accurate; if anything, they are low.

     Referring to the Crown-Zellarbach mill and the verified tests, note

the extremely high coliform counts.  The fecal coliform counts are also

high.  One thing that is worth noting on that page is the first value of

4^DOPOO/100ml  I discovered that the waterbath was at 44.1°C, which was at

that time within the limits of the test (that was 44.5 plus or minus 0.5°C)

I then raised the temperature to 44.5; there was an apparent, significant

drop.  I suspect that the drop has to do with an increase in temperature

of 0.4°C.

     Note the high coliform, fecal streptococci, and Klebsiella counts  for

Crown-Zellarbach mill effluents and  for the S. Santiam River.  Upstream,

there are a number of municipalities that are discharging treated wastes

into the river.  Coliform counts increased significantly, and there was

a 9,000-fold increase in the concentration of Klebsiella pnewnoniae in

the area downstream receiving the effluents.  The kraft mill at American

Can Company consistently had low concentrations of  all indicators.  With  a

few exceptions, I could not isolate  any Klebsiella  there.

     In attempting to isolate Salmonella out of these wastes, I took  a

liter of sample and filtered it through diatomaceous earth.  I  inoculated


the "plug" into an enrichment medium and tried to get a qualitive Salmonella

isolation.  In all cases, including those of the effluent and receiving

water, I failed.  This doesn't mean Salmonella weren't there; it just means I

couldn't get Salmonella as far as Im concerned.

     Brezenski:  Are the Klebaiella counts based upon total coliform plates?

     Bauer:  Correct.

     Brezenski:  What was your rule of thumb in terms of how many relative

percentages of your columns checked out?

     Bauer:  I assumed that the colonies were layed down on a filter in

a random manner.  I would start at the upper, left-hand corner of the

plate, pick everything that looked like a typical coliform until I hit

20  or  30 colonies, calculate the percentage on that figure, and apply it

to  the original number of colonies on the plate, then multiply that by the

correct figure.

     Gallagher:  You're only measuring process wastes?

     Bauer:  Absolutely.

     Vasconcelos:  You said you had a portion of these checked?  They were

all confirmed by NCDC?

     Bauer:  I  took the first 60 isolates and sent them to CDC.  They came

back 60 positives (Kleba-iella).  I'm convinced that the method used  is, if

anything, conservative.  I understand, for instance, that M-Endo Broth is

slightly inhibitory to KlebB^ella^ so some strains probably would not grow.

If  anything, those results are low.  I also excluded any culture that was

indole-positive, and approximately five percent of Klebsiella are;  so again,

these  results are low.


     Vasconcelos:  Did they refer to this Klebeiella as Type I or a mixture?

     Bauer:  There are 77 serotypes of Klebs'iella.   They gave me a specific

serotype where there was sufficient capsule to type.  I can tell you right

now that you'll get every kind of colony morphology in the world:  smooth,

rough, mucoid; on EMB agar you just cannot tell.  There was no way I could

look at the plate and say, "That is Klebsielld*  If there was a great big

mucoid colony, I was very suspicious, but I tried,  at first, to differentiate

smooth from rough; it was completely hopeless.  The biochemicals were the

only way I could tell.

     Indicated in Table II are data that were prepared by Dr. Tom Aspertarde

at Crown-Zellarbach.  The total-coliform bacteria numbers indicate that

either the MPN or/ the MF will give reasonably comparable data.  Moving to

Table III, I then attempted to do frequency distribution of the IMViC

types from different isolates.  Approximately 600 coliform and 300 fecal

coliform colonies are represented by these statistics.  In most cases, the

—H- IMVic type is certainly predominant.  With the exception of Crown-

Zellarbach, and I didn't calculate a percentage of -H—IMViC types, they

were negligible.  I recall something on the order of three percent.  I was

not satisfied that I really had enough colonies in order to put down a

number.  That is what is represented in the column entitled, "all mills."

Those numbers represent approximately 500 isolates.  I just lumped them

all together, 3.3 percent Type I E ooli.  Bear this in mind:  those all

came from  Crown-Zellarbach.  That was my particular experience.

     From fecal-coliform bacteria plates [Table IV] the percentage producing

these IMViC types, which are Enterobaater types, is even higher — 89 percent

at Publishers, 100 percent at American Can, 87 percent at Weyerhaeuser


(a biological treatment process); Crown-Zellerbach somewhat lower.  In this

case for the fecal coliform, I came up with a healthy percentage of Type I

E ooli.

     Then the next question arises:  How many of these "aerogenes" IMViC

types were Klebaieltal   It appears that most of them are.  Combined data,

from all the mills (Table V), of all the "aerogenes" IMViC types, approxi-

mately 50 percent of everything, indicate 84 percent of these are Klebsiella

pneumoniae; from the fecal-coliform bacteria tests, 92.5 percent.

     Then, I compared frequency distribution of IMViC types from my data

with some of the numbers that Ed Geldreich has in his publication "Signi-

ficance of Fecal Coliforms in the Environment."  Certainly, there is a

significant difference in the IMViC type distribution (Table VI).  The

majority of them are Enterobaate? IMViC types.

     In Table VII is indicated, in the first column, a description of the

colonies from a very minimal effort I made.  These colonies are the ones

that could be called fecal coliforms.  The colonies were blue from edge

to edge, cream color with a blue center, or cream color with a light purple

center.  The next columns (Table VII) indicate, respectively, the number

of colonies picked, the percentage of those that were lactose-positive,

the percentage of those that were subsequently positive at the elevated

temperature, and the percentage of Type I E ool-i.  It does appear that

one can discriminate, at least to some extent, based on colony morphology,

on the MF membrane.  Although there is a small number of colonies, you will

note the ones with the blue centers that are Type I E ooli.  I should like


to show you a few pictures of the plates which I photographed.   (I am sure

you have all seen them a million times).

    (Projection of color slides demonstrating total-and fecal-coliform

bacteria colonies on the membrane filter).

     That is the kind of problem that we frequently run into in industrial

wastes.  This was from Publishers Paper Company (Oregon City, Oregon).

which is a magnafite-process sulfite mill.   At that time primary settling

was its type of treatment.  Magnafite process involves a degree of chemical

recovery.  The BOD of this waste would not be as high as that of an

ammonia-base sulfite mill.  When JDU see something like that, you wonder

what you count.  They are not all that bad.  Standard Methods says:  "Do

not count the gray colonies," and sometimes I am not sure I am seeing gray

or blue.

     Geldreich:  You have an overloaded membrane to start with.

     Bauer:  Yes, let us move on to the next one, a little cleaner plate

to count.  This was Crown-Zellarbach (Lebanon) with 0.005 ml of inoculum.

These colonies were blue.  They photographed as black, but this M-FC medium

with an agar base, a personal preference of mine, tends to make the colonies

a little darker.  This (pointing to slide)  is the type of thing that I

was calling blue center, somewhat mucoid in appearance.  This is what I

was calling blue, edge-to-edge.  Let us try another slide.  This is again

Crown-ZeHarbach (Lebanon).  The total-coliform bacteria membrane shows a

reasonably good sheen production.  I should say that you would normally

identify those as being coliforms.  Number 37 was the mill waste itself, and


this is the downstream sample, 38: the filtration volume was 50 ml.

     Next, these cultures were streaked on EMB agar.  This is a fairly

typical plate (referring to slide).  They are not all that way.  Some will

give a nice green sheen.  There is every kind of colony morphology you

can imagine.  As I recall, Harold Jeter, National Training Center  (Cincinnati,

Ohio), counsels you on the completed tests to streak them out on EMB agar^

turn the plates over, and look for a nucleated colony.  Is that correct?

     Geldreich:  That is correct.  There is one thing about EMB agar that

is not very well understood by most microbiologists.  The textbooks have

never clarified EMB reactions.  The differential characteristics of EMB

agar are pretty poor.  Sheen colonies may or may not be E aoli.  The fish-

eye colony may or may not be "aerogenes."  The literature or textbooks give

you the impression that there is a sharp differentiation with this medium

between E ool'l being a metallic sheen organism and Enterobaotev looking

like this.  In this case it is probably Klebs'iella..

     Bauer:  That is Klebsiella, by the way.

     Geldreich:  There is not that sharp a differentiation on EMB  agar.

     Bauer:  I found every kind of IMViC type in the world, just by

streaking out "aerogenes" types on the EMB medium.  After a while  you

get a tremendous variety of colony morphology.  One characteristic in

common is they are all nucleated.  I just streaked them out to purify the

culture, by the way.

     Geldreich:  This EMB agar procedure is really nothing more than a

way to isolate colonies, a process you are going to carry through  to a

completed test.  There have been options made in Standard Methods  to use


EBM or Endo agar.  In our country the textbooks, Standard Methods, up until

recently, (as well as Difco's or BBL's label), state that a colony is E ooli

if it produces a sheen colony on Endo agar.  This is not true.  Red colonies

are something else again.  Endo agar will produce a sheen colony, if it is

properly made, but this is a misconception.  When you sterilize Endo medium,

you destroy its capability for giving the sheen production with all coliforms.

What you are seeing is just a few of the coliforms with sheen colonies on

Endo agar.  If colonies are picked from the membrane filter and streaked on

properly made Endo agar, all coliforms will have a sheen colony.

     Riordan:  You have percent "aerogenes" IMViC types, and then "aerogenes"

identified as Klebaiella pnewnan-iae', where would E ooli, be represented in

the data?

     Bauer:  In the frequency distribution tables that I referred to

earlier, the -H-— types.

     Riordan:  My basic question then is, can you make any inference?  With

respect to the E <30Zij..can you say what percentage of the fecal coliform

are E oolil

     Bauer:  I think itds not in that table you are looking at, but it is

in this other data (Tables III and IV) under "all mills," indicating that

3 percent from a total coliform plate, and 15 percent from a fecal coliform

plate are E aoli.  You can compare those with the different types of processes.

The percentages will vary depending on the pulping process and  the treatment


     Riordan:  Referring to earlier tables, could you break down  the data

from Crown-Zellerbach to get E oolil


      Bauer:  Yes, you could do that.  This Is one of the Issues that we

have to come to grips with.  Can we use straight fecal-coliform bacteria

tests, or is it necessary to go on to determine whether or not they are

E Qoli, and can we base the standard on E oo'iit  This is going to be a

very controversial subject.

      Stein:  In addition to determining the kinds of organisms, did you

attempt to correlate the information with other chemical data and the type

of treatment?

      Bauer:  Yes.  We measured the BOD, COD, and every kind of nutrient

Imaginable in the attempt! to correlate them with the count, without success,

      Stein:  The notion Is that no matter what type of treatment you are

thinking of for the pulp and paper business—and the assumption is that

the American Can plant is the pptlmum example of treatment today—we are

not going to obtain a significant reduction.

      Bauer:  That is correct.  American Can has a beautiful effluent.

      Stein:  No, I mean on the coliforms.

      Bauer:  The coliforms were uniformly very low, less than 1,000.

In the final effluent, fecal coliform counts are, consistently, in the

range of 20.

      Stein:  But there are some high numbers here.

      Bauer:  The percentage may be high, but the number is low.

      Stein:  Let us assume that they are going to ask all the paper mills

to match the American Can standards.  Will we, by that process, lower

the coliforms to such a low level that we may not have to think in terms

of disinfection?


      Bauer:  That is precisely what should be recommended.  May I expound

more as long as you have asked this question?  Crown-Zellerhach had a micro-

fermenter in that particular biological treatment system which, as you know,

EPA subsidized, a situation that allows us to get in to sample it at any

time.  The system has approximately eight days of detention time in it.

The counts in the intake waters are about 103 per 100 ml total coliforms,

                                  7      9
and they come out anywhere from 10  to 10 .  There is a lot of regrowth

going on in the plant process.  American Can puts this material in a

microfennentor and lengthened the detention time to 18 days.  The result:

the total coliform count is 9,000.  The effluent looks beautiful.

      In contrast, the effluent at Crown-Zellerbach is so viscous and

rancid that it might congeal momentarily.  No more than 10 ml could pass

through a membrane filter.  One can biologically treat these wastes to

low bacterial levels and, while doing that, one gets rid of the BOD.

The material finally starts to settle.  One of the biggest problems in

biological treatment is the inability of the floe to settle, and the

tremendous wastage of solids.  Increase that detention time to 18 days

and, naturally reduce the coliform levels to some reasonable level. The

result is stabilized waste; so stabilized that it is not going to cause

an added persistance of bacteria downstream from domestic wastes.  If one

increases the nutrient level in the stream, then the persistence of

enteric bacteria in the receiving water will be increased.  If one

stabilizes that waste, the metabolizable carbohydrate is removed.

There will not be any large re-growths of undesirable organisms  that are

potentially deleterious to life.


      Stein:  One can get these numbers down with a reasonable detention

time, whatever the treatment is (one has the American Can or its equivalent,

or one has your 18-day retention time, or physical-chemical treatment).

Obviously, when one uses physical and chemical treatment, there will be

a lot of resistance from the mill.  Then, there is the other problem. Mills

are either going to own the land and be able to give us the retention time,

or with other mills, there will be a determined amount of resistance because

there is no space.  We can talk about 18 days, but in large companies, we

are not going to get it.  The question is:  If one can get this treatment

from American Can, below 1,000 fecal colifortns, should we require these

companies to go below 1,000?  Should we require them to do it any way they

want to do it—retention, physical and chemical, or disinfection—or would

it really make any difference?  The point is this:  If they are above 1,000,

what over-all significance will that have?

      Bauer:  I would prefer to see treatment to the level of 1,000 used

rather than to see disinfection employed.  If the nutrient is still in

the waste, those organisms that survive, oftentimes re-grow, returning to

the level they were.  One still has, in the waste, the unstabilized car-

bohydrate that is going to initiate re-growth in the stream, increase the

persistance of enteric bacteria, etc.  If it is at all possible I would

recommend to biologically treat to a "reasonable" coliform level.

      Stein:  You're not saying biological?

      Bauer:  Well, no, physical chemical treatment.  Polyelectrolytes

work beautifully on these wastes.

      Stein:  But if the industries do not have the space to give us


biologlcal treatment, we should at least offer a counter proposal if we

are interested in coliform reduction.  It is not clear whether we should

put a restriction on them.  We have to answer the basic question:  Is

there any pathogen problem from pulp and paper wastes, however high the

fecal or the general coliform count?  If we answer that question, the in-

dustry may be faced with the problem of not enough land and with either putting

in physical and chemical treatment or disinfection.

     Unless I'm wrong, there may be some other problem here.  The price

difference may be so great that the companies may say, "If we're going to

be forced to, we want to disinfect and we are not going to put in physical

and chemical."  If so, we should determine from a scientific point of

view just what we shbuld require.  If we have massive resistance from

industries in getting them to put in something directed specifically

toward reducing the coliforms, we should have some very specific evidence

of the dangers before we can ask them.

     Bauer;  I hope people subsequent to me can shed some light on that


     Lyons:  From the engineering standpoint, when one is formulating

the rules, one is considering several trade-offs in retention time, in

microbiological concentrations, and in how they all affect the reaction

rate.  With 18-day retention time there is quite a decrease in rate of

return as far as substrate removal is concerned because of less organisms.

The whole thing is the cost trade-off between doing biological treatment

or some form of physical and chemical treatment or disinfection.


No one is going to look into it right now, but have these considerations

been investigated?

     Bauer:  Along the engineering line, I am convinced that it is tech-

nically feasible to treat them physically, chemically, and biologically—

some way to reduce those numbers without having to resort to disinfection.

     Stein:  It is our view that disinfection is not satisfactory, because

of the re-growth.  That has nice implications, for disinfection is what

municipalities are doing along the Willamette River.

     Geldreich:  I have been giving Mr. Stein testimony for years that we

have a problem in this same area:  nutrient removal.  A case in point if

Wynopscot Bay.  If the nutrients were lowered, this would then solve the

problem.  It is necessary to recognize, somewhere, what is the value of

the downstream water use.  Is that recognition going to be so important

that it is going to demand that industry treat its waste in order to

meet a certain limit?  It may be shellfish we're talking about and that's....

     Stein:  That is another case.

     Geldreich:  Yes, but it is a case we have to recognize.

     Stein:  We shall come back to that specifically.  I think that you

get the point with all these economic questions.  The industry would be

much happier to spend money on nutrient removal or on removing the solids,

than to be tagged with bacterial removal, which  of course, may mean that

they (the companies) are creating a public health hazard.  If one can

give the rationale in order to put this another way, where we are going

to achieve this removal, and not really hit the industry head-on, then

it will be a great advantage, because the companies are going to  fight

to the death to disprove that there are health hazards.  If we can achieve


these reductions with other benefits to the stream, this method is going

to be the more acceptable one.

     Geldreich:  We recognize this in the sugar beet industry.  In the

reports, we are saying that if the industry achieved nutrient reduction,

say 85-90 percent  BOD removal, in that area, we would very well accomplish

acceptable treatment.

     Bauer:  The State of Oregon requires coliform data from all the

municipalities, and the municipalities have considerable amounts of

correlative data with suspended solids, BOD, etc.  If one measures the one

parameter, the coliform, as a monitoring tool, then a lot of inferences

from that can be made.  Oregon has found, with municipal waste, that if

the total coliform value in the effluent is less than 1,000, and if

municipalities use a very high level of chlorination (1 mg residual

after 60 minutes detention time) the result is excellent correlation

with good treatment efficiency.  Suspended solids are uniformly below

20 mg/1; BOD is uniformly below 20 mg/1.  "Since I am in the surveillance

business now I am looking for tools like this.  If I can tell our Permit

Compliance Division that if we look at coliforms and they are at a

certain level, I can give you reasonable assurance that the waste is

well stabilized—BOD is within this range, etc., we have a tool."

     Stein:  On the Missouri River it was unsatisfactory if they treated

wastes or if they didn't treat the wastes.  When regrowth occurred it

has to be recognized that if we chlorinate and disinfect the wastes,

and if we are satisfied with what is being discharged from the outfall

coming out of that pipe, the effluent may not be enough to protect the

stream downstream if regrowth occurs and something else happens.

     I am trying to indicate one of the significant problems:  Just

giving us a number, say X number of coli forms, after the waste comes

out of the effluent pipe, may or may not solve our problems.  You have

to indicate to us a method and you have to specify a way of reducing

the nutrients, whether the preferred way is a retention time or a

physical-chemical method rather than disinfection.  This is something

we should know about because we may require a company to spend several

million dollars and then we have to come back in a year or two because

that company has not cleaned up the stream.  This should be the phil-

osophy we have to follow.

     Again, let me make this last point because it is a big one.  Suppose

we ask companies to put in a complete system, ie, similar to that of

American Can, and suppose their fecal count is higher than the EPA limit

so that the company is in violation.  The firm says:  "OK, now you

(EPA) tell us what to do."  EPA is obligated to tell them something, and

something with some meaning.  Maybe it is to be disinfection.  Now, if

the policy suggested does not have any meaning after EPA has asked for

the treatment equivalent to that of American Can, there are going to

be more problems.  Over the next few years as these better mills go into

operation, those are the alternatives.

     Very soon it will be necessary to make a determination as to

whether we are going to ask for any kind of restriction in terms of


bacteriological parameters, particularly In canning and sugar beet plants

and in several other industries.  We have to assume that we will have to

require a certain amount of BOD and solids removal.  The question is: If

we are go-ing to ask for removal, can we forget the coliforms?  Can we ask

for a complete treatment system, including nutrient removal and disin-

fection?  Uhat is the BOD of the American Can Comp'any, for example?

     Riordan:  With reference to American Can, what are they doing in

terms of BOD and suspended solids removal?  What have we got, 10 pounds

per ton production-could it be less than that?  I think the company wants

6 to 8 pounds per ton production at 30 mgl BOD in terms of effluent.

     Knittel:  In contrast—something which may come up later for dis-

cussion.  We are trying to set American Can up as being the ultimate

in removal, and, especially, we are addressing ourselves to the aspect

of coliform removal.  The firm is a relatively new mill (approximately

two years) whereas the other mills that we have been talking about,

during the date presentation, are older, well-established mills.  These

older, established mills, over a period of time, have probably become

colonized with coliform organisms and they do not represent merely

domestic sewage contribution.  What we need to do is to keep looking

at American Can from a certain standpoint:  Do the processes become

colonized?  The waters that they are using are fairly high in coliform

organisms.  Over a period of time the system is going to look and act

somewhat like a continuous culture.

     Lyons:  To get back to Mr. Stein's point, what do we do initially?


We have about two weeks to come up with some reasonable starting point.

Now, let us understand that  this is not going to be hard and fast; in-

stead, it is going to be a reasonable working point—as were the other

limitations that were pointed out.

     Bauer:  Could we hold the discussions, please, until we get through

all the presentations and then we can start arguing about it.

     The second handout (Appendix B) is a collection of analyses on a

variety of waste sources.  The National Council has become convinced

that the only bacteriological parameter of sanitary significance is

E aoli, so we screened all these different places for the presence

of EC positive cultures  (Table VIII).

     Resi:  Yes> but these are natural "elevated temperature bacteria."

     Bauer:  Right.  In  fact, the Council will not call them fecal

coliforms.  The National Council refers to them as "elevated temperature

coliforms."  Tables IX and X show some data that were recently obtained

out of the Snake River.  Included are both effluent data, and data from

the receiving water, by  the total coliform, fecal coliform, and fecal

streptococci tests.  These are not verified counts.  (The normal Standard

Methods membrane filter  technique was used).

     A form of bacteriological mass balance was performed on all these

numbers.  Known quantities were the flow in the river, and the contri-

bution of all the municipalities and industries; we computed the expected

concentration of coliforms.  If one assumes complete mixing from all

these various sources, the predicted coliform concentration in the Snake


River is about 11,000.  Obviously,  from some of these numbers in the

receiving waters, the counts are considerably higher than that.   Note,

for example, the 260,000 average below the Idaho Falls sewage treatment

plant.  You will notice, on these receiving waters, that the arithmetic

average for total coliforms in the upper Snake River is well within a

recreational water quality standard.

     Downstream  further, around Burley, Idaho, the regrowth of  total

coliforms is very apparent.  The averages were several hundred thousand.

There is no way to account for the presence of all these coliforms except

as a regrowth phenomenon.

     In Table XI, I played the game with the frequency distribution of

IMViC types again, grouping together the various types of treatment

plants with food processing, meat processing, etc.  With the exception

of fruit-packing plants, E ooli were present in all of them.

     Brezenski:  Would you summarize your presentation, Mr. Bauer?

     Bauer:  In summary, I think that some of the contentions of

the National Council that no E ooli are present are not completely

accurate.  The percentages are low, but nevertheless, in most cases the

bacteria are there.   (I think that Dr. Martin Knittel may be able to

corroborate that later).  Klebsiella pneumoniae is present in sulfite

mills, consistently, in very large numbers.  Whether or not that rep-

resents a potential health hazard I shall leave up to the medical

doctors, the epidemiologists, and so forth.  These organisms will regrow

quite readily if you attempt to disinfect them, unless the BOD is re-

duced significantly.  I am convinced that methods are available  to

either biologically or physical-chemically treat all of these various


types of industrial wastes to achieve a low bacterial content.  In my

own opinion this is desirable.  I would prefer treatment without having

to go to disinfection.  I have not found Salmonella in any of these

wastes.  Some of the other people here may or may rot have found these

organisms.  The National Council says no bacteriological criteria should

be applied to their effluents.  Are there any questions?

     Brezenski:  In general, you are telling us that on the whole,

your beliefs are compatible with what the NCASI states except that the

Center is saying it is not passing E. aoli in their effluents.

     Bauer:  My opinion is not compatible with theirs, to the extent

that I think that no bacteriological criteria should be applied to

their wastes.

     Breaenski:  No, looking at the data, not looking at the Council's

conclus ions.

     Bauer:  But that is the conclusion they come to.  The inference,

if you say I agree, it is that I agree with their conclusions, and

positively, I do not.

     Riordan:  Why is there so much dependence on E soli?  Are not

Entevobacter also found in the human intestine?

     Bauer:  Yes.  Klebsiella pneimoniae are also present in the human

intestine; 20 co 30 percent of the people are carrying it all the time.

     Riordan:  Is E ooli more specific?  If that  is a much better in-

dicator, is that why?

     Bauer:  Uniformly, it can be found in the gut of warm-blooded

animals.  There is a percentage of humans who do  not carry Klebsiella.


Kleb8i,el'Lat Enterobacter, Servatia, etc. are found in the environment,

in decaying vegetation, and in many sources, although the concentrations

are not particularly high.

     Lyons:  You said significant BOD reduction; what level are you

talking about?

     Bauer:  The waste at Crown-Zellerbach goes in at 800 and comes

out 300; that is not adequate.  What is adequate, is as yet unknown.

     Lyons:  Are we going for percent removal?

    Bauer:  I would prefer not using percent removal.

     Lyons:  I would just like some rationale on which to set effluent

quality, because if we chose a quality of 30 mg/1 BOD, is this a level

that will greatly inhibit this regrowth?

     Bauer:  I can live with that; I do not know about other people.

That figure is certainly a vast improvement over the proposed policy

passed around today.

     Lyons:  I would like to comment on that.  I wrote that permit

proposal based upon only my sanitary engineering background and about

three hours of microbiology.  It was not intended as a final document,

but to be a basis for discussion on what can be written into permit

standards.  The concentration is about 30 mg/1, using statistical

flows and unit operations.  This sounds good except that I am wondering

if sulfite mills will be considerably higher because of the status  of

treatment technology as applied to this particular waste.  You said,  too,


that you restricted the presence of Klebs'ie'Lla to sulfite wastes.

     Bauer:  No, we are looking at kraft also.  It is present there

but in low concentrations.  The basic difference is related to the type

of sugars that are left.  One has chemical recovery in the kraft, and

the sulfite process contains up to 15 percent pentosans, pentose-type

sugars.  Likely they are responsible for the preponderance of that

particular type of organism.  Kleba-iella can use pentoses, quite readily

with a large variety of enzymes.  In the kraft process wastes there is

not nearly that level of sugar available.

     Geldreich:  With high nutrient waste of this type, 30-60 percent

removal is relatively poor.

     Speaker from the floor:  The elevated temperatures used in these

processes and the high bacterial populations certainly emphasize the

need for monitoring these wastes.

     Geldreich:  I think the figure of 30 mg/1 is reasonable, looking

at it from the research point of view.  Some of the experiments that

we or others have done with fecal coliforms in highly treated sewage

effluents indicate that 14 to 30 mg/1 could very well control this

problem of nutrients, if we have to measure it by something as crude

as the BOD.  Temperature certainly is important, but if you can throw

a block in one of these things—and these would be nutrients—the

temperature in the summertime would not be too much of a problem to

overrun what one is trying to do with nutrient removal.  This makes

more sense than our older approach to the problem i-e. asking for 85

to 90 percent BOD removal.

     Lyons*  This approach makes more sense,  if it is technically attain-

able with the type of treatment available today,  than to call for percent


     Geldreich:  Yes.  What we are trying to  remove here, in terms of

the nutrients—so those of the engineering group can understand— are

the nitrogen and carbon sources.  Throughout  this discussion we are not

talking about phosphates and nitrates, but about carbon and nitrogen

sources.  These nutrients and temperatures, as well as the bacteriological

population, are very critical items to control,

     Lyons:  Actually, we are trying to control nitrogen and the com-

bination of materials that are in our treatment plants, and we would

just as soon have a bacterial monitor to do the work.

     Geldreich:  For a monitor, one needs a bacteriological measure-

ment to back up what your chemical parameters indicate.  Two systems

are available to support a contention that this would do it.

     Bauer:  One of the issues Mr. Stein raised was:  Are there other

things in these wastes that might be pathogenic?  Therefore, I asked

Don Herman from National Water Quality Laboratory (Duluth) to go through

the data that has been working up during the last five or six years

and try to give us some insight into that.

           Experiences with Coliform and Enteric Organism
                  Isolation from Industrial Wastes

                           Donald L. Herman

     A lot of the industrial material which 1 shall be discussing was

collected prior to my affiliation with EPA except for the data from  the


paper and pulp mills and domestic secondary effluents.  These few

slides from an area in Minnesota will demonstrate the problem.

     (A series of slides depicted obviously high quality stream water

becoming progressively more polluted downstream.  Areas below the entry

of pulp and paper mill wastes were shown.  Recreation use was illustrated)

     This stream headwaters in the Superior Forests.  During an entire

siimmer we found a positive coliform count only from five to a dozen

times.  One may find one fecal coliform count during the summer.  This

water is very low in nutrients.  As we go on downstream we observe the

effects of pollution.  Some of this nutrient addition is due to the pulp

and paper industry.  There is some water where we have complete dissolved

oxygen loss.  Then there are some fish kills.  Then down close to the

communities involved in various discharges, the river is merely devoid

of any form of life.  On downstream we find floating material and sludge

development.  We are involved, in most cases, with some recreational

uses at one time or another—fishing, swimming, and bathing.

     (Slides show total and fecal coliform colonies on the membrane


     These slides show the coliform colonies on the membrane and the

type of colonies we find.  The fecal coliform membrane illustrates the

type of colony morphology.  I will discuss this later.  We picked any

colony that had any blue to it at all.  Very often if we incubated these

plates a little longer, colonies picked up the blue color regardless of

morphology.  As far as Klebaie'ila goes, I think we have to consider


reduced metabolism of the lactose.   The color addition results from

metabolism of that type of sugar.  Here we made an isolation from EMB agar

and this one is Klebsiella.  We found various types of morphology.  It

shows some of the encapsulation you get around the cell.

     I now refer to the handout sheets, page 7 in Appendix C.  These

data were collected—before I became associated with EPA--when I was

working with the canning and food processing industry.  Most of the work

was with treatment lagoons.  During the initial treatment stages of

aerated lagoons in this industry, the BOD was approximately 1400 mg/1;

our range was 160-2400 mg/1.  These data represent three different plants.

The survey extended over a two-year period.  Flow rate averaged about

125,000 gpd.  The total coliform bacteria level was 320,000/100 ml,

ranging from 2,000 to 1.5 million/100 ml.  The fecal coliform was

15,000/100 ml and the range was 100 to 60,00/100 ml.

     The fecal coliform work was performed using the MF and the

Salmonella work was with the MF on XLD medium.  We tested -E ooli Type I

because most of the states we worked with did not care about E ooli but

wanted to know what we had in E ooli Type I.  These effluents had 35

percent Type I.  Klebsiella pneumoniae in any work we do is a nonmotile

organism and we key it on down to the pneumoniae organism because other

species of Klebsiella are involved in animal infections.

     The Pectobaoteriwn was 6 percent.  Salmonella species were running

0.7 percent.  Now after post treatment, and this is after some twenty-one

days, the total coliform density was 3,000, the fecal coliform density

140/100 ml.  E ooli was running. 32.1 percent, Klebsiella pneumoniae  42.9  percent,


Enterobaater species 5 percent, Peotobacteriwn 9.3 percent, and in this case

we had an increase in the percent that now is going out, Salmonella 10.7 percent.

    Bauer:  Don, that's percent of what, samples analyzed?

    Herman:  No, in this case, I refer back to the fecal coliform factor there, 140.

    Bauer:  How about the Salmonella species?  .1 percent of all the samples?

    Herman:  Yes.

    Bauer:  That's a qualitative result?

    Herman:  Yes.

    Resi:  One point here - I do not think this is the best way to recover

Salmonella; therefore, I think this point should be made.

    Herman:  I was just going to get to that.

    Bauer:  But, you can conclude that they were there in a certain percentage.

    Herman:  In the first few samples we could not even find a pair, then we

got into some bottom samples.  In these cases you can go to the stream bottom

and find if the organisms are there.  We ran XLD, SS, Tetrathionate and

various combinations of media to find them.  On page eight are carbonated and

non-carbonated beverage waste data.  We got very high reducing sugar levels here.

The initial BOD was 1600 mg/1 and we had a range of 800 to 18,000 mg/1.  The

flow was not too high, about 36,000 gpd.  Total coliforms were 7,500,000/100 ml;

fecal coliform, 50,000/100 ml.  The types of isolation were:  E ooli Type I,

fairly low, 5.6 percent; Klebsiella pneumonias 68 percent; Entevobaoter 15

percent; Peotobaoteriim 7.0 percent, and Salmonella 4.4 percent.  On post

treatment, the persistence seemed fairly high on some of them.

     I will proceed to  discuss  potato  wastes.   On the initial  stage,  BOD was

 approximately  14,000  mg/1  and  the range of 1500  to 25,000 mg/1.   Flow rate was


75,000 gpd.  The total colifonn bacteria was 160,000,000/100 ml; the fecal

collforms, 265,000/100 ml, and in a range of 18,000 to 850,000/100 ml.   In

this area, E ooli type I was only 0.9 percent; Klebeiella pneumoniae 81.1 per-

cent; Entevobaoteria 9.4 percent; Peatobaoterium 6.9 percent; and Salmonella

species were 1.6 percent.

    Bauer:  How many samples did you process to achieve this Salmonella

species percentage?

    Herman:  I would have to go back over my figures on that topic.

Put it this way:  There is not any waste reported here that involves less

than 200 samples.

    Bauer:  O.K.

    Herman:  Regarding isolations, we have about 25,000—all types of isolations.

Most of them were recovered in a period of about two years.  On post treatment

the total  coliform was 1700/100 ml; fecal coliforms 45/100 ml.  Isolations gave

E ooli Type I of 15.5; Klebsiella pneumoniae 60.1 percent; Enterobaater 8.9 per-

cent; and  Salmonella at 15.5 percent.   Note the shift of what is represented

within these ratios.  We had confirmations hack from NCDC on the Salmonella

typhimurium and the 5". St. Paul.

    Meat and slaughter wastes were just loaded (page ten).  Initial BOD was 26,000

mg/1; flow rate, 28,000 gpd; and total and fecal coliform 670 and 6.5 million/100 ml

respectively.  Results of the isolations were: E ooli type I of 56.9 percent;

Klebsiella pneumoniae 21.5 percent; Enterobaoter 13.8 percent; Peotobacterium

0.5 percent; and Salmonella was at 7.3 percent.


After treatment, the BOD was reduced to 400 mg/1, a 98.5 percent

reduction.  That is quite a high BOD for a discharge to receiving

waters.  The retention time in the lagoon was thirty six days.  Total

coliforms were 250,000/100 ml and fecal coliform 18,500/100 ml.

Isolations gave:  E aoli- 65.8 percent; Klebsiella pnewnoniae 24.1

percent; Enterobaetev 6.9 percent; Peatobaoterium  0.9 percent; and

Salmonella at 2.3 percent.  Initial random isolates confirmed as

Salmonella typhimurium and 5. St. Paul.  Resampling—because there

was a question on these results—confirmed these findings, and added

the variety S. Copenhagen as well as Shigella sonnei and S. flexneri.

    There were firm orders, from that time on, that one really did not

need any more sampling of this type.  All that was required for both

Federal and State tests were total coliform counts; that was all that

the regulatory agencies were going to get.

    Now, I refer to the paper and pulp wastes.  This phase of

work resulted in some real challenges and the utmost use of tact

and diplomacy in order to obtain valid samples.  It is still a

question of whether the samples represented full production stress

of the plants that were involved.  The initial state was aerated

lagoons.  They never would tell me the flow rate.

    Question  from the floor:  What kind of mill was>:that,  kraft or



    Herman:  Two of these are sulfite and one is kraft.

    The initial BOD was about 2200 mg/1; total and fecal coliforms

1.5 million and 8,000/100ml respectively.  We made initial checks;

the correlation work was carried out by the State, and indicated

that this waste was extremely devoid of nitrogen content; in other

words we were looking at nitrogen.  E ooli Type I was 4.4 percent;

Klebeiella pneumoniae 85; Entevobaoter species 9.5; Peatobaoterium

0.8, and Salmonella 0.3 percent, and we could only get Salmonella

when we got to the interface sample.  If we took a sample out at

any other part of the lagoon we did not find Salmonella.  For

post treatment discharge without chlorination, BOD was about 3,600 mg/1,

Retention time was two days in the lagoon.

    The total coliform bacteria density was 57 million/100 ml and

fecal coliform ran 130,000/100 ml; E ooli. Type I was 0.4 percent;

Klebslella pneumoniae 92.3; Enterobaoter species 6.7; Pectobaaterium

0.6; and Salmonella species at 0.0008 percent.  There were more than

200 samples.

    For a lot of these wastes, one is talking about nitrogen de-

ficiency.  A number of these organisms, especially Klebs-Lella employ

the nitrogen from air—a selective medium is created.  There is no

reason why one should not have some selection of the microflora

response to these various kinds of wastes.

     We did conduct some additional field survey work on the re-

ceiving rivers.  One river demonstrated fish kills due to lower

DO in 1969 and 1970.  The CODs in the river ranged from 12 to 260.

That picture of the dead fish on all that high quality water was

taken during that period when we were out in the boat getting samp-

les.  Total coliforms ranged from 10 to 171,000/100 ml and fecal

coliforms from 3 to 75,000/100 ml.  Fecal streptococci /100 ml

ranged from 6 to 1350 and, in the course of events, eleven types

of pathogenic bacteria were found and confirmed by CDC including:

Klebsiella pneumon-iae, Shigella sonneij Salmonella typhimurium,

Staphyloaoaaus aureua, E. aoli, and Pseudomenas aeruginosa.

    We found one isolate of Klebsi-ella pneumoniae among these

areas of major discharge while recovering types 14, 58, 21, 22,

31, 33, 35, 65, 7, 56, and 67, up to and including twenty seven

river miles below the outfalls.

   Within the river we found active colonized zones of Klebsi-ella

pnewnoniae, types 14, 15, 21, 22, and 31.  These types were tested

for mouse pathogenicity and were shown to be positive.  Furthermore,

in this particular area, these types and some similar ones that we

obtained from clinical patients were analyzed biochemically, and were  also

sent to CDC for typing.  We exchanged cultures with Dr. John Matson, University

of Minnesota School of Medicine.  Clinically, we could not tell the

difference between the ones recovered from fatalities in patients versus

the ones we isolated but of the river.  We got mouse fatality titers of

10  and 105 on patient strains; on the river strains it took titers of

10° to 10' to achieve the same effect on mice.  Again, in the river, in

these zones of colonization we picked up bottom rocks where you could

see the cultures just growing right on them.  These were taken to the

Minnesota State Board of Health.  Quite a few of them turned out to be

pure cultures of Klebe'leVla pneumoniae growing on these surface edges.

     The chemistry factors of interest were, of course, the decreased

dissolved oxygen.  We had none for some seven miles.  Also, we found

that as the pH decreased inthe receiving water the more the enteric

organisms there were present.  There was a presence of reducing and

hexose sugars; this was found for the entire twenty-seven-mile stretch.

We found Salmonella once when fecal coliforms were 3/100 ml and con-

tinuously when the fecal coliform was more than 40/100 ml.  The fecal

coliform-to-fecal streptococcus ratio was averaging 1:14.6.  Then, on

downstream the ratio started to be 325:1.  Bottom samples downstream

from that area of discharge ranged from 50,000 to 5,600,000 total coli-

forms /100 ml.  Fecal coliform bacteria were from 300  to 23,000 /100ml.

In all bottom samples E ooli. Type I, Kleb'Siella pneumonlae, and

Salmonella typhimurium were demonstrated.

     For comparison, we ask, "What does this mean for  some other areas?"

In connection with some disinfection work we have some similar data  on


secondary effluents.  We have disinfected with ozone, chlorinated, and

dechlorinated, and then we followed through on the raw effluent.  (These

data are recorded over 2 1/2 years and 14,000 isolations).  Total coli-

forms per 100 ml were 581,000; fecal coliforms 32,500.  The E ooli Type

I was'62 percent, Klebaiella pnewnoni-ae was 18 percent.  Dr. Matson and

co-workers report that, in cases of urinary tract infections over a ten-

year study, that Klebsiella pnewnoniae represent 18.6 and 18.7 percent

of the total urinary tract infection from humans.  Are we talking about a

population factor being shown through here?  It's curious.  Peotobacter-ium

was 3.6 percent and Salmonella species 2.1 percent.

     Tests were conducted  for Pseudomonas aerugi-nosa.  We found these

extremely difficult as compared to the other tests.  The findings ranged

from3/100 ml to 6,700/100 ml.  The bacteria were highest in the meat and

slaughter wastes.  This area became a major workload to isolation and to

confirmation with a limited staff.  We tried Hektoen agar, which is

suggested by King in a 1968 publication.  Many times the results were

open to question—especially when we got into higher quality water, as to

what the colonies really indicated.  Finally, we had to pick every colony

on the plate.  We found Pseudomonas aeruginosa present in low numbers

where fecal coliforms were not detected.  We also found fecal coliforms

and Salmonella when we could not find Peeudomanas aevuginosa.  I agree

with the concern related to the organism, but prefer to think that it is

part of the total picture of the microflora and not just the only indicator.


     I have the results covering two pages from antibiotic sensitivity

analyses on randomly selected Klebsiella pneimoniae  from the secondary

effluent, raw, chlorinated, ozone,  dechlorinated, and back to the second-

ary.  Then we have one from the Lake Superior and the Duluth water intake;

the remaining were isolates made by Dick Bauer.  We were informed by

certain individuals, and especially the National Council, that all

Klebe-iefia isolated from their wastes were not resistant to any anti-

biotics.  There were no factors involved.  So I decided to take a look

at this.  In summary, we will find that almost all of the Klebs-Lella

are resistant to ampicillin.  Some articles state that all Klebsiella

should be sensitive to a Cephalothin.  Clinically we have found this to

be the case.  These result from the environmental samples.  From the

clinical viewpoint angle we find that about 39 percent of the Kleba-Letla

are resistant to that antibiotic.  We have the strongest antibiotic

resistance from the Isolates from the paper and pulp wastes.  We have

one isolation of Ktebe-iella Type 33 that is resistant to every antibiotic

available.  In checking with the local physicians, we find there are a

number of reported intestinal tract problems in the summer.  Again, these

are secondary infections and often not reported.  When we reviewed the

CDC reports, they confirmed the same thing.

     To summarize quickly, my own experience indicates there may be a

relationship between the bacterial flora of receiving waters and the flora

of the intestinal tract, and to their behavior toward nutrients.  In

addition, too much nutrient in the substrate can totally disrupt the normal


human, natural flora balance.  The high quality of water we sampled was

found to be extremely low in total coliforms and devoid, in many areas,

of fecal coliforms—except downstream from a beaver pond or shore birds,

and so forth.  It just depends upon what other substrate is there.  The

solution has been pointed out in expert investigations by Gallagher,

Anderson, and our FWPCA reports, the reports from the Red River of the

North—they are all talking about nutrients and waste addition.

     From working with industry, I know that the technology is available

for better reducing of their wastes.  If industry has the land available

and makes use of the more modern waste reduction techniques, this can be


     Review of the E coli isolations revealed that 97.2 percent of these

are typical fecal coliforms, E aoli Type I.  In all cases where the fecal

coliform counts were greater than 100/100 ml, Salmonella was recovered.

In 1972, Eickhoff, in his report to the Council, emphasizes the critical

need for epidemiological work on the Klebsiella pnewnoniae in relation

to recreational waters.  A proposal of this type was recommended in 1971,

by Dr. John Matson.  At last report, this proposal was completely lost in

Washington.  For developing legal actions, we certainly will need additional

bacterial identification within these coliform counts.  They are no longer

going to accept what we say  ("We have found so many coliforms"), instead

they want to know which, specific organisms are found.

     In conclusion, as microbiologists, we must become well organized,

work closely with the chemists and biologists, engineers, administration,

and fellow microbiologists.  I hope more meetings of this type are forthcoming.


     Geldreich:  May I ask what your explanation of the question:  Why

did we have more resistance of Klebs-Lellas to antibiotic sensitivities

in paper mill wastes?  I can see that doctors up in your area must be using

the shotgun technique, with gantricin, to treat all their patients, i.e.

a broad spectrum approach, but why it is we have so much resistance in

paper mill wastes?  Is there a logical explanation?

     Herman:  I hope some data on the nutrients and trace elements in

this waste can come to light.  On antibiotic resistance, in Anderson's

doctoral thesis, it was strongly emphasized that, in the presence of

sodium maganese and magnesium, there was a direct relationship to the

ability of the organism to develop stronger resistance to antibiotics.

My question is, do we have these trace elements in this type waste?

     Bauer;  That is a magnifite process where those isolates come from.

Magnesium oxide is involved.  Another possibility is that that particular

mill is located downstream from a number of municipal discharges.

     Herman:  Some work by Buck shows strongly that in the laboratory

recovery of KlebB'lel'ia the encapsulation factor is important.  If the

right carbohydrate nutrients are available, the capsulation becomes very

thick and mucoid.  So, we are talking about nutrient conditions again.

     Berg:  Was not the question one of difference between antibiotic

sensitivity of free-living forms with that from those coming from patients?

     Brezenski:  I think there is another factor here that you are for-

getting about completely; it is the R transfer factor.

     Herman:  This is another factor, and please do not forget this  area.


We have strains of Klebeiella Type 33, from hospital patients who are

resistant to every antibiotic.  We also have isolates of Klebsiella Type

33 from recreational areas that are resistant to every antibiotic that

we have.  Review the literature on the R transfer.  There are a number of

articles on the transfer of antibiotic resistance from Klebsiella  to

E coli to Salmonella.  In work by the Council, Klebsiella is found every-

where.  The Council  claims it found it on lettuce up to ICr/lOO ml.  We

analyzed a number of samples of cabbage  (from the grocery store) and,

out  of some 200 samples, we got one Klebsiella isolation.  Although it

is found in animal infections, it is  usually not Klebsiella pneumoniae.

It would not be a surprise to find it there because Klebsiella pneumoniae

is considered  to be  a part of the normal flora of the intestinal tract.

     Bauer:  Some of  the participants  at  this meeting have asked what

was  discussed  at the Corvallis Seminar.  I will try to summarize.  The

National Council has been spending large quantities of money for admittedly

defensive research,  if they call it that, in order to try to discredit

the  fecal coliform test.  They had some  data that if they are accurate,

taken on face  value, are quite damaging.  They would indicate that perhaps

the  fecal coliform test is not applicable to industrial wastes.  You have

heard very briefly,  reference to some comments made by Dr. Razzel.  He

used to be with the  pulp and paper industry in British Columbia and is

a member of the ad-hoc committee.  He and a technician went up into the

headwaters of  a watershed in Vancouver,  British Columbia, and took samples

of moss and various  pieces of vegetation and then conducted fecal coliform


tests on them.  He spoke in generalities and did not give us any hard

data, but he alluded to the fact that they found EC positive Klebsiella

pneumonias in the order of 10  per gram.  These are rather surprising

figures in light of the data that have been published by Bob Bordner,

Ed Geldreich, and others.

    They did  this same thing with some of the wastes from the wood products

industry in Canada.  Essentially, the findings were the same.  The one

statement that was his real psychological cruncher was that they went

down to the market in Vancouver and randomly picked a series of leafy

vegetables and root crops.  They carried out fecal coliforms to the

Klebaiella identification, including EC.  His statement that at any time

you  eat a head of lettuce, and at any time you have a salad in Vancouver,

British Columbia, you're eating at least 40,000 EC positive Klebsiella.

     All of the industry representatives  said, "Hurray, hurray, we've

got  'em whipped."  They went off with big smiles on their faces and we

did not have  any data to refute it, at least not at that particular

moment; that was a frustrating experience.

    Geldreich:  You do not need to refute it, Dick.  Data are in the

literature not only from our group.  Bob Bordner and I have put out a

paper summarizing much of the work of the South Platte irrigation water

study.  The data are not only on the South Platte but also cover areas

where I had been involved within other areas in Utah and Wyoming, and

Lou Resi also contributed data from certain areas, such as Snake River

and a few other places in the lower Colorado River.  Plenty of data, not

only from our own organization, are available.  There is plenty of data


In the literature from the English, and also from Greek research that is

being done.

    Bauer:  Unfortunately, I wasn't sharp enough nor was anyone else

at that meeting to really hit them with it.  I was very skeptical, but I

could not quote anything off the top of my head.  It made us look pretty


    Geldreich:  I was going to ask you Dick; the vegetables they were

talking about, were they actually grown in Vancouver?

    Bauer:  I don't know.  It was never stated.

    Geldreich:  You know I was wondering.  If they came from Mexico4

they would be loaded.  Was the study done in the winter?  Obviously, the

vegetables were not grown in Vancouver, but it would have been interesting

to know more.

    Bauer:  I mentioned to you that these people were going to deliver

this coup de grace at the last American Society for Microbiology meeting.

I was hoping that someone here would have been sitting in on that par-

ticular presentation.

    Vasconcelos:  It was the worst session I had ever been to.  It was

just before lunch and it was packed, right next to the lunch counter.

It was an open room and everybody was eating and I could not hear a word

they were saying.  No one else could either.  I don't think there were any

questions for that reason.

    Bauer:  I think that's the worst thing that could have possibly

happened.  That will get into the record without being contested.

    Geldreich:  Another problem Dick, is that apparently they failed

to recognize, when they were discussing their results with you in that


other meeting, that we do not live in an environment which is sterile in

terms of not discovering some fecal contamination.  In the remote areas

we have a very high animal population about which people quite often

forget.  We have rodents in remote sections.  In some areas deer popula-

tions are high.  These people who are reporting this material are failing

to recognize, intentially or otherwise, that animal populations could very

well contribute something to the particular site.  They may have actually

gone to an area and sampled where they know these animials are going to

be coming to a water hole, or in an area where bacteria will colonize.

There certainly are reasons why you can occasionally find them.

    Bauer:  I think that we probably could have refuted every one of

Dr. Razzel's findings, but at that moment the rebuttal was not at hand.

He and his associates left a very definite psychological impression which

I hope that perhaps we can reverse today.

    Our next speaker is Kathleen Shimmin from Region IX.

        Observations of Selected Waste Discharges from Region IX.

                          Kathleen Shimmin

    I would like to talk to you about some of the recent work that

we are doing.  When Bill Stang called and told me that this presentation

was going to be forthcoming, we were in the process of verifying some

permits of discharges in the San Francisco region.  There are data being

passed around right now that I am going to be talking from  (Appendix D).

You will see that it is of a very limited nature  compared with Mr. Bauer's

data or Mr. Herman's three-or-four-year observations.  But, still it speaks

to the topic that we're talking about today, and  so I would like to present

it to you.  There are two main sections.  One of  them covers  the data  that


were collected from sugar refinery and pulp mill discharges in the San

Francisco bay area.  The second is data that were collected in 1968 from

a kelp processing plant in San Diego.

    The data that you have here are gathered from total and fecal coliform

determinations by the membrane filter technique.  The samples were processed

within three hours of collection.  Colonies were then picked from the

membrane filters, streaked on EMB agar to isolate them, and transferred

to nutrient agar slants.  They were then inoculated into Enterotubes,

EC broth, and Brilliant Green Bile broth.

    The first page (Table I, Appendix D) is identification of selected

total-coliform bacteria colonies that appeared to be metallic green on

the M-Endo plates.  We have indicated the numbers of colonies and the

numbers of types that were found by Enterotube identification.  You will

see for the sugar wastes, that primarily the total coliforms were of the

KlebB'ie'lla.-Enterobactev group.  These were very low in positives, as

revealed by the EC tests.  All of them were confirmed in Brilliant Green.

    In the pulp mill process water, the same outline was observed.  The

total counts were as indicated:  8,000-11,000/100 ml for the sugar wastes,

23,000 total coliforms/100 ml, and greater than 60,000/100 ml fecal

coliforms by appearance on M-FC plates.  For the fecal coliform colonies,

needless to say, that was the surprising result.

    We looked then at selected colonies from the fecal coliform plates.

There we found that for the sugar processing plant wastes, the colony

appearance was primarily gray-white colonies and of these, the majority

were the Klebs-iella type.  We looked at just a very small number of blue

colonies.  As you can see in the sugar plant these were primarily of the


Klebeieila Enterobaoter type also.  In the pulp processing water, pink colonies

were distributed across the board as far as the Enterotube identification went.

A large proporation were of the rum-Enterobaoteraceae type.  The blue

colonies in the process water from the pulp mill were, also, a large

majority of non-Entepobaateraaeae type,  although they did  turn out positive

so  far as  the brilliant  green went.  I think  the difference between those

two findings  is  that  the Enterotube is incubated for twenty-four hours

and the brilliant  green,  forty-eight hours.   We picked up  some slow

lactose fermenters.   (If you want to compare  the counts  they are listed

on  Table  II.)

     The conclusions  that we made  after viewing that data were that

the pulp mill blue colonies were, primarily,  not fecal coliforms, as

would be  identified by Standard Methods.   Eighty-five percent of the

blue colonies were not fecal  coliforms.   In the sugar wastes 90 percent

of  the gray-white  colonies on  the M-FC filters were the  Klebsiella group;

only 3 percent of  these  were  EC positive.  In the  same samples, again

very limited  samples, 85 percent  of the  blue  colonies were typical coliform

 type, but  were not positive in EC broth.   Ninety percent of  the total

 coliforms  found both  in  sugar  and pulp wastes were from  the  Klebsietla-

Entevobaatev  group.   We  have merely summarized the 1968  data.  The plant

that was  investigated was one  that processed  kelp  for  the purpose of making

alginate  salts.  The  kelp process is one that would encourage  the growth

of  anything in the kelp.  It was  steeped in warm water,  at about  35°C,

 for up to  about  five  days in  this process; then the water was  discharged

into the bay.  There  were some studies performed in which the  investigators

found a very  high  coliform count, 32,000,000/100 ml.   The count  was  also


high for fecal coliform, an average of 580,000,/100 ml.  Fecal strepto-

cocci were very low.  Of the EC positive isolates, many were not typical

E ooli.  We found 19 percent of the EC positive isolates were E. soli

Type I; 17 percent was E aoli Type II, and 74 percent not E ooli, based

on thirty-six different isolations.  Many of the M-FC positive colonies

were also EC negative.  Seventeen percent of the M-FC positive colonies

were EC positive and 83 percent, negative.

    The conclusions that we came to after reviewing these data are that

if we  continue to use fecal coliform by MF as a parameter, we are either

going  to have to put in an extra step in order to verify the colonies

or we  shall have to make the medium more specific.  Secondly, we certainly

should consider choosing different parameters.

    Geldreich:  What do you propose?

    Shimmin:  Klebeiella might be a good possibility.

    Herman:  What did you give as your definition of E ooli Type II,

Miss Shimmin?  Many people are confused when we speak of E ooli type.

    Shimmin:  Yes.  Type I is -H— and Type II is either -\— or III!.

    Herman:  What about —H- and other IMViC types?

    Shimmin:  These are not data that I did myself.  These are data that

were collected in our region in 1968.  IMViC Type —H- was 0.  This is kelp

process waste that was not mixed with domestic waste, but had the addition

of sea-gull droppings.  When the processor brings the kelp 'out of the

bay, there are all kinds of fish and other materials caught in it.  So,

when it is being chopped up and transferred	

    Herman:  Did you say Type —H- was negative?

    Shimmin:  Yes.

    Herman:  What Is represented then when you say Klebsiella-EnterobacteT?

Are you essentially taling about Enterabaoter and not Klebsiella?

    Shimmin:  No.  I am talking about two different things.   The first

section of data that I talked about from the San Francisco Bay is not

IMViC data; they are Enterotube data.  That is when I was talking about the

Klebeie'lla-Enterdbaoter group.  The second set of data were IMViC data

from the San Diego kelp processing.

    Vasconcelos:  Which Enterotube are you talking about?  Was it the

new improved Enterotube with the ornithine?

    Shimmin:  It does not have ornithine.

    Vasconcelos:  They now have included ornithine.  They solved the

Klebs'Letla problem; because, I understand the system was not working too

well for Ktebs-lella identification.

    Shimmin:  Right, that is why I called it Klebs-iella-EntevobaoteT.

    Resi:  Do you ever perform the biochemicals to check out what you got

with these Enterotubes?

    Shimmin:  We did not do it on this particular study because we only

gathered the data last week.  But we have checked the Enterotube results

we have gotten with Salmonella isolations and they have always confirmed

exactly what we were getting in the data in the individual media.

    Knittel:  That Enterotube is designed for Salmonella and related work;

it is 100 percent in the isolation.  When one starts digging into some of

the other Enterobaoteraceae3 one is going to be in trouble because the

percentage is not that high; I think it is something like 85 percent positive.

    Bauer:  Excuse me, but I hope we do not get too involved right at
the moment in the details of what we are identifying.   We are still
trying to come to grips with the problem:  Should we apply a bacteriological
standard to the pulp mill wastes?
    Herman:  We are not talking about pulp mill wastes.
    Bauer:  Industrial wastes, more specifically, one that has domestic
sewage excluded.  Are there any questions for Miss Shimmin?
    Dr. Leonard Guarraia:  Are all these out of sea water, Miss Shimmin?
    Shimmin:  These are effluents.  There is sea water in the kelp effluent,
but the cooling water and all the source water in industries along San
Francisco Bay are primarily fresh water, I would imagine, because of the
location of the plants.
    Riordan:  Could you relate this to the elevated temperature fecal
coliform test?
    Knittel:  That is a misnomer.  We had better put that to rest right
now.  That elevated temperature coliform idea is the biggest bunch of
garbage they have come up with.
    Riordan:  But this is what they are saying.
    Knittel:  Just recently, I asked a representative of the Council,
"What is your definition of an elevated temperature coliform?"  He said
that it is anything that grows on the FC membrane at 44.5°C.  Well, there
is a lot of different bacteria that grows on there.
    Questioner:  Whether it is blue or not?
    Knittel:  Yes, whether it is blue or not.  I said, "Now, what about
E aoUl"  He said, "That is a different ball park."  He began to back
off on this question.  The only thing the National Council is really

going to believe at this point in time is E ooli.
    Bauer:  May I ask Miss Shlmmin a couple of questions?  On these
particular wastes we are dealing with, you mentioned that with the
kelp processors, there was evidence of rapid multiplication of
bacteria in the processing of kelp.
    Shimmin:  That was a hypothesis, because of the kelp processing
    Bauer:  Yes.  Is there an indication of that type of thing
occurring in sugar processing, i.e. considerable regrowth through the
process water?
    Shimmin:  We have not checked it that far.  There is a great reluc-
tance on the part of the industry to let us come in and sample.  Therefore,
we were only able to sample at selected effluents.
    Bauer:  May I press the point a little bit further then? You said
that, with  the kelp processing wastes, there was a good chance that
fecal contamination from sea gulls was mixed with the raw product as
it entered  the plant.  What would be your judgment with reference to
sugar cane?
    Shimmin:  Well, sugar cane, as it comes from Hawaii, is rather
dirty; it has been rinsed but it certainly is not sterile, so  I would
imagine since in Hawaii they do use some sewage effluents to  irrigate
the cane fields, that there is a possibility of contamination.
    Bauer:  They also have a large rat population.  Isn't that somewhat
of a problem?
    Shimmin:  Yes.
    Geldreich:  I have a question, when trying to interpret  this  last

page.  Are these individual numbers, where you have "total and fecal
coliforms per 100 ml?"
    Shimmin:  These are averages.
    Geldreich:  What is bothering me is that you show total coliforms
less than fecal coliforms, after talking about lactose fermenters.
    Shimmin:  Yes, but this is according to the appearance on M-FC
plates when the value is greater than 60,000; these are blue colonies
that appear.
    Geldreich:  You mean to tell me then, that you found lactose fer-
menters, which are blue colonies on the M-FC plates, that were greater
in number than 60,000 and then, only 23,000 which will ferment lactose
at 35°C?  This is very difficult to reconcile because we are talking
about lactose fermenters.  Your lactose fermenters at 35°C are more
numerous than they are at 44.5°C.
    Shimmin:  That is true and that is why I made the remark when I
was reporting the data, i.e. that this was highly unusual.  I believe
that those were all fecal coliform colonies that are appearing as
blue colonies.
    Geldreich:  What were the densities of organisms on these total
coliforms you used on the MF?  Are we talking about numbers like 23
on the MF or 80?
    Shimmin:  We are talking about statistically significant values.
    Geldreich:  What was that?
    Shimmin:  On totals, that would be twenty-three colonies per
    Geldreich:  What was your background count on these membranes?

Did you have a problem?  I wonder if we have an overloaded membrane
    Shimmin:  No.
    Geldreich:  You say that you do not have one.  That is why I cannot
reconcile something that will not ferment in the same magnitude of pop-
lation at 35°C but it does at 44.5°C.
    Guarraia:  Did you try this with different lots of media?
    Shimmin:  No.
    Vasconcelos:  You say this was an average.  Is that correct?  One
could get some distortion if one has a high point, a mean in other
words.  How much data....
    Shimmin:  I  lifted the data myself and this is what I would consider
statistically significant.
    Vasconcelos:  Is it not skewed?
    Shimmin:  No.
    Another question:  What you are asking is, "Is it an arithmetic
or  geometric mean?"
    Vasconcelos:  Right.  When you say average, right away	
    Shimmin:  It is an arithmetic average, but the range of  counts was
not a great enough range that one would need  to use the geometric mean.
    Vasconcelos:  It looks, you know, a little	
    Shimmin:  It looks very strange, but  that  is my point.   These things
are appearing on the M-FC plate.
    Bauer:  Have you experienced this frequently?  Is this  the  only  time?
    Shimmin:  This is when we first started running industrial  effluents.
We  did  not  experience it in any of the sea water  or sewage types.


    Vasconcelos:  You said the San Joaquin River.

    Shimmin:  This is the San Joaquin River, close to the intake, and

I have a feeling that there are other discharges from the pulp mill

coming down.  I don't think you are looking at anything too much different

than from the pulp mill itself.

    Bauer:  I would like to cut this particular point off.  Perhaps we

can bring it up again.  Tomorrow I would like to talk about methodology

a little more.  I am hoping now that we can continue to focus our atten-

tion on the question whether or not bacteriological criteria or effluent-

limiting criteria should be applied to industrial wastes.

    Vasconcelos:  Will the pulp mill companies agree on a bacteriological


    Bauer:  No, they will not.  That is the whole problem.  They say that

we are way out of line to even consider suggesting one.

    Vasconcelos:  I am talking about an Esoher-ichia eoli standard.

    Bauer:  They would agree to an Esoheriahia eoli but not fecal coliform.

    Geldreich:  If the difference is two million versus twenty

million; so what, we still have a problem.  Taking your data, Dick,

where you might have EC as you partition it off, maybe two million on

some of them and your total fecal coliform or your Klebs-letla counts

were even higher, the magnitude is tremendous.  We have to achieve re-

duction.  This is a smoke screen the companies are throwing out.

    They tried three different ways.  They tried to get EPA to get around

using the word "fecal" because that is a nasty word.  They won't look

for Salmonella which I have asked them repeatedly to do.  They do not

want to carry out this reduction, which is the problem, and they have

attacked the methodology in terms of an MF and MPN problem.  Now they are

going on the idea of trying to throw a smoke screen on what we find in

our methods, like Klebs-iella versus other coliforms, not realizing of

course, that Klebs'iella, as we find, are found in some human intestinal

tracts and other warm blooded animals.  It is a series of probes that

they are trying to put out to cast confusion over the real crux of the

problem:  Have they got the nutrients down?

    Riordan:  I think that is the thing we have got to do.  Sit down and

decide whether we have a method.  I don't think one can obtain the de-

sired result on the basis of 30 mg/1.  There will be some nutrient re-

duction, but we do not have the same experience with pulp and paper wastes

that one has with municipal wastes.  Five-day BOD with paper wastes has a

ratio, I believe, of about 20 percent ultimately, rather than the 67 percent

that we have in municipal wastes.  This means that even with 30 mg/1 one

is going to have the nutrient there.

    Geldreich:  To start with there are more sugars there.

    Riordan:  One may not be able to think even of 30 mg/1 as being

adequate nutrient reduction.

    Geldreich:  I think that number is the workable thing that we have

to look at really; it is between 14 and 30 mg, from our own experiences.

I would like to check it out more thoroughly.

    I would like to let Dr. Martin Knittel present his data.  Then per-

haps we can get down to some serious discussion about the points raised by

Mr. Stein this morning.


           Review of Research Regarding Colifortns in Pulp
                        and Paper Mill Wastes

                          Dr. Martin Knittel

    I would like to give a progress report on research we have conducted

during the last eight months, on the presence of Klebsiella pneumoniae in

pulp and paper mill wastes.

    We are interested in the ranges of Klebsiella pneumoniae in various

pulp mill and fiberboard wastes and in the local streams.  From this data

we would develop a research^plan to meet the objectives of the program.

Our isolation technique is very easy because of the large numbers of

Klebsiella pneumoniae in pulp mill effluents.  We have found that close

to 100 percent of the total coliforms appearing on the M-Endo agar can be

identified as Klebsiella pneumoniae.  We picked these colonies and in-

noculated them into lactose broth and those that were positive for gas

in forty-eight hours were transferred into the IMVic media and ornithine

decarboxylation medium, checked for motility, and typed using pooled

sera.  A representative number of these was sent to NCDC for confirmation.

    Some brief observations on the Klebsiella pneumoniae that we have

isolated:  They are usually late lactose fermenters, delayed forty-eight

hours.  For some Klebsiella there is a temperature dependence on gas

production in lactose.  Some are not positive at 35°, but will produce

gas in lactose broth when incubated for an additionalfour or five hours

at room temperature.  Not all of them are encapsulated so not all of them

are typeable.   They make up between 90 and 100 percent of the total

coliform colonies found in pulp mill effluents.  We have been able to


identify Klebsiella pneumoniae following any of the criteria for identi-

fication.  Because there are a number of classification techniques, one

of the problems was to determine if the isolates were either Klebsiella

pneumoniae or some other Klebsiella.

    Let us run through these slides.  (A series of slides demonstrated

Klebsiella colonies and growth curves).

    These are cultures that we received from the American Type Culture

Collection that we wanted to use as comparative organisms.  We plated them

out to get some idea of colony morphology.

    Geldreich:  Notice that on overloading the membrane bigger colonies

occur toward the edges while the center ones are sometimes quite tiny.

    Knittel:  Right, we made no attempt to count these colonies.  We

merely wanted to see what kind of reaction would result on M-Endo and

LES agars.  The next slide is more  typical of a pulp mill effluent.  The

mucoid colonies show a variation in metalli  sheen production, a rather

soupy type of colony.  This is more typical of pure cultures than have
been carried in the laboratory for  some time.

    Geldreich:  Is the alcohol in that particular LES media?  It would

keep down the confluency.

    Knittel:  Yes.  This is a highly mucoid organism, difficult to count

on the plates because it spreads very rapidly.  Let us go to some of the

data we have collected (Appendix E).  On  four successive days we sampled

the mill for total-and fecal-coliform bacteria determination  (Figure I).

I do not have the fecal coliform data prepared, but they would present


a similar profile with lower numbers.   The intake water had low densities.

As we got into the primary settling ponds, the numbers came up.  There

were day-to-day fluctuations in concentrations.  When we looked at the

secondary pond effluents, the numbers  of coliforms were higher showing

that regrowth was occurring during treatment.

    The next slide (Figure 2) shows the comparison between the number of

total coliforms and percentages of Klebsisella in primary and secondary

influent-effluents.  There is a large number of coliform organisms coming

into this primary, indicating that they are coming out of the mill some

place.  Somewhere within the mill is the source of inoculum, in this waste,

which increases in numbers during treatment.  There is a slight but re-

producible decrease owing to settling going through the primary settling


    As the waste enters secondary treatment, which is merely an aerated

lagoon, there is an increase.  There are more total coliform bacteria

than there are Ktebsi-ella.  The higher numbers may represent the effect

of pH adjustment as the effluent goes in the secondary treatment system.

There is a long travel period from the primary to secondary.  By the

time the effluent comes out of the secondary aeration, there is approxi-

mately a two-orders-of-magnitude increase in numbers, most of which are


    Speaker from the floor:  Is this the sulfite mill?

    Knittel:  Yes, this is the Crown-Zellerbach (Lebanon), one of the

easier mills to sample because we have money invested there.  We have


trouble getting Into the other places.  I think if we could sample other

mills we could show some kind of relationship.  We found it in kraft mills

and one other mill that we have sampled.  There is a presentation, in fig-

ure 3 of the data on a grab sample of secondary influent, treated in

various ways to determine whether regrowth of these Klebsiella and fecal

coliforms could occur at the treatment temperatures the mill was using.

We took a portion of the sample, incubated it, and called these total

coliforms the indigenous cold form population.  We compared this level

to the coliform increase in the waste itself; we did have an increase.

We sterilized the sample either by autoclaving it or by filtering it

through 0.45u membranes.  We inoculated the autoclaved or filter-sterilized

wastes with an EC positive, Type I, E ooli or a Klebsiella pnewnoniae

which we had isolated from their system.  We incubated it at 20°C, on a

shaker in a waterbath.  We got good growth as one would with any standard

nutrient medium.  We had a discrepancy with the filter-sterilized waste:

we did not have much growth or die-off.  This result may be explained

on the basis that some of the nutrients that are available for growth

have been removed by filter sterilization.  The particular material may

act to adsorb the nutrients onto the surface so that essentially  they

have been removed.

    Speaker from the floor:  You can always break them down in the

autoclave, too.

    Knittel:  This is true.  We may have solubilized the nutrients.   These

results did convince us that we».when sampling the various waste  streams


in and out of the various treatment facilities, were seeing an overall in-

crease in coliforms and Klebsiella.

    Geldreich:  Did you try this with the Salmonella?

    Knittel:  No, we have not.

    Geldreich:  We have done similar experiments, and the Salmonella take

off just like that.

    Herman:  We compared maximum growth rates of E aoli and CDC-confirmed

Type 33 Klebsiella pneumoniae at 15°C and they followed the same pattern.

The Klebsiella grew faster than E ooli,

    Knittel:  These observations probably come back to what we have been

saying:  Growth is really a function of nutrient availability.  If these

coliforms can grow in the plant, and if the plant itself is contributing

nutrients, Klebsiella pneumoniae can grow.  Then Salmonella^ Shigella, or

some of the other Klebsiella can also grow.  This problem is not so much

that the wastewater represents immediate fecal contamination, but that growth

of the coliforms does occur.  If these wastes can grow coliform, Salmonella

or other pathogens may also grow.  This increases the chances of water-borne

infections.  On the other hand, these large numbers of coliforms may mask

coliforms from other sources that are more of a health hazard.

    As a result of the Corvallis meeting, and of the statements that were

made to the effect that Klebsiella could be found anywhere in the un-

civilized environment, we went to an experimental forest, on the campus,

which has limited access.  We took samples of vegetation, pine needles,

leaves from trees and bushes, leaves on the ground, soil, and water from

a small stream.  We found no total coliforms, fecal coliforms, or fecal

streptococci (Figure 4).   We did find some total coliforms in the water

samples after forty-eight hours incubation, none of which were lactose

positive.  We did not carry them further but we hope to follow this up

at a later time.

    The representatives of the pulp and paper industry at the Corvallis

meeting contended that they were willing to go along with E ooli and they

kept bringing up the "elevated temperature coliform" theory.  They denoted

"elevated temperature coliforms" as those bacteria that are found on the

fecal coliform membranes, including typical and atypical colonies.  I

find this term very confusing and suggest that EPA microbiologists de-

velop a statement to rid the future literature of this term and to de-

fine more precicely the term fecal coliform.

    We investigated the effect of temperature on the growth of fecal

coliforms at elevated incubation temperatures.  We shall proceed very

rapidly through these slides.  This is an E ooli (National Center for

Communicable Disease, Atlanta, Georgia) plated at 44.5°C.  It is a

typical blue colony, easily identifiable as a fecal coliform.  Next is

the same organism incubated at 45°C.  There is not much difference.  The

counts  go  down a little, but they will survive at 45°C.  Next at 45.5°C

the colonies are larger.

    The next slide shows a Klebsiella pneumoniae culture from NCDC plated

on M-FC medium at 44.5°C.  This is the reason the pulp and paper people

have been after this coliform problem.  These organisms are the ones

they are calling "elevated temperature coliforms."  They are lactose-

fermenting, thermal-stable Klebsiella pneumoniae that will  look just

like E ooli at 44.5°C.   They do grow at 45°C.  At 45.5°C they do not grow.

This colony appearance is not true of all Klebsiella.   The culture from

the American Type Culture Collection that I showed before were plated

in a similar manner and incubated at those same three temperatures.

They do not survive.  We had no colonies appearing on the medium.

    Next, we see the fecal coliforms from a mill effluent.  This is the

appearance of the colonies when the plates are incubated at 44.5°C.

There are some dark blue colonies typical of fecal coliforms intermixed

with some atypical colonies of various colors from white to pink and

those with blue-centers.  The next slide is a close-up one to emphasize

the high numbers of atypical coliforms that crowd the membrane and obscure

the actual numbers of fecal coliforms.

    The next MF was incubated at 45°C; the background growth has been

decreased and the colonies are countable.  The problem is to demonstrate

the presence of fecal coliform colonies intermixed with the atypical

smaller colonies.  A close-up of the previous plate shows that the

suppression of the atypical colonies by a 0.5°C temperature increase

makes the dark blue typical colonies more distinct.  On the next plate

incubated at 45.5°C, the number of atypical fecal coliform colonies has

been suppressed further.  When light blue or blue-centered atypical

colonies are examined in culture they are found to be slow lactose-

fermenting, thermal-resistant Klebaiella pnewmoniae.   The large, dark

blue colonies isolated are about 100 percent E aoli Type I and follow

all the criteria set forth for typical fecal coliforms.  At 45°C and


and 45.5°C one notices that the number of atypical colonies has been

suppressed, and it is easier to define the fecal coliforms.  The next

close-up of the previous membrane shows atypical white, blue, blue-

centered, and cream-colored colonies that are not E aoli3 when we com-

pare them with typical dark blue, somewhat irregular E ooli Type I

colonies which gave the typical -H— IMViC reaction.

    The next membrane is more typical of the mill effluent, a very

crowded membrane.  We can pick atypical colonies without contamination.

If we use a higher dilution we do not have a countable plate, just one

or two blue colonies.

    Geldreich:  That is an overloaded membrane, though.

    Knittel:  If we go to the next highest dilution, we lose the E coli.

    Geldreich:  What is the next highest, five-or ten-fold difference?

    Knittel:  One-tenth, a decimal dilution.  For example, this would

     -1                     9
be 10   and the next one 10" .  The next membrane was incubated at

45°C.  We immediately have eliminated many of the interferring atypical

background organisms in the same sample dilution.  The next slide is

a close-up of the previous plate.  We suppressed the growth of the

slow lactose-fermenting Klebsiella, and exposed the typical type I E coli..

    The size of the blue colonies, the fecal coliforms, is larger

when the membranes are incubated at 45.5°C. Temperatures above 45.0°C

inhibit cell division of E aoli\ however, cell growth continues and

the cells become filamentous.

    This is an interesting organism represented on the next slide.

Of course, we are not going to count this one as a coliform at all


because of its green color on this media.  We have pLcked several of

these colonies and performed several biochemical tests on them.  They

turn out to be Enterobaeter hafnia.  A close-up will reveal colonies

with a blue center.  They are slow lactose fermenters and will turn

blue after longer incubation.  At 45°C this organism still survives

very well.  At 45.5°C we have definitely lost some of those atypical

colonies but the larger E hafnia colonies survive.

    We took an average of several runs at these incubation temperatures

on three different effluents.  We plotted the numbers of fecal coliforms

that appear versus the temperature of incubation (Figure 5).  There

really is not a lot of difference between the numbers that survive at

44.5°C and 45°C.  However, with the  increase of the incubation tem-

perature to 45.5°C, only about 50 percent of the fecal coliform popu-

lation survives.

    The first 2 columns in Figure 6  represent a comparison of the two

types of colonies appearing on membranes incubated at 44.5°C.  We picked

representative numbers of colonies and recorded the results from a set of

biochemical and cultural tests.  From the wastewater examined 89 percent

of the dark blue colonies tested were EC positive.  Further in this table

we reported the typical and atypical colonies that appeared at the three

incubation temperatures and compared those two types of colonies on a

variety of media.  The dark blue colonies gave the classical reactions

for E ooli.  The light blue colonies were a mixture of Klebs-iella and

other Enterobaotev species, usually  hafnia, once or twice l-iquifaciens.

The same picture remains at 45°C.  At 45.5°C the fecal coliform count

went down, but those that did appear were definitely E coli.  The back-


ground colonies were Peeudomonae of various kinds.

    We have also attempted on a limited basis to use the enrichment

technique for Salmonella but have not been successful.  On every occasion

that we have tried to enrich for Salmonella we have been very successful

in isolating Pseudomonas aeruginosa from the same wastes.

    Guarraria:  Have you ever tried to use internal control for seeding?

    Knittel:  No, we have not.  i would like to conclude my remarks with

what I consider  to be two important recommedations:

    1.   In  any survey a confirmation procedure should be followed for the

coliform bacteria that appear on either the M-Endo or M-FC media.  For

example, since pulp and paper industries personnel have stated that

E aoli* type I is their main  concern, confirmation of  colonies from these

industrial  wastes is particularly  important.  This information will pro-

vide a firmer foundation  for legal action.

    EPA  needs a  central bacteriology laboratory devoted to classification

and confirmation not only of members of the coliform  group but also of

other problem-causing microorganisms.  We have been depending upon the

good nature of NCCD to confirm  enteric pathogens.  We must develop our

own expertise.

    2.   EPA needs to develop a  document that clearly  states the  correct

classification of the members of the family Entevobaateviaceae.   (Some

microbiolegists  are still using the outdated taxonomic keys of Bergey's

Manual).  EPA must refute the term "elevated temperature coliform" that

is being proposed by the  industries.  This term is confusing and does not

have a sound foundation.

    Gallagher:   Let me ask you  this one question.  You indicated that at


the symposium in Corvallis about two months ago, the industry said it

would be amenable to accepting E aoli.  Did the representatives say  that?

    Knittel:  I do not think that they said that directly, but that  was the

way they were pushing it; they put  great emphasis on the presence of

E  ooli  type  I as being significant.

    Bauert   There were certain members of  industry, such as Matthew  Gould

of Georgia-Pacific, who indicated that, in trying to clarify the sig-

nificance of the elevated temperature tests.  They believe E aoli is a lot

more acceptable than fecal  coliforms.

    Bauer:   I think the representatives would accept the presence of

E  aoli  as evidence of fecal contamination, with all that that implies.

    Geldreich:  I am very much  impressed with what you're doing here on

temperature. I should like to  give you a  little background on elevated

temperature  tests.  When we began to work  on the elevated temperature

concept and  looked through  the  literature, we found elevated temperature

tests recorded back as far  as when  Eichmann was playing with the prin-

ciple using  glucose broth.  Through the history of elevated  temperature

tests there  have been many  choices  made as to what temperature to use.

We finally settled on lactose broth and we started incubating at  45.5°C.

We were much concerned that we  were very close  to the  upper  limit where

we would get a sudden and very  sharp kill-off if the water-bath deviated

very much.   We were also  aware  of the World Health Standards which were

set at  44.5°C.  We began  a  series of experiments with  the environmental

waters  in our area at 44.5°C, 45.0°C, and  45.5°C.  We  found  that when

we reached 45.7°C, we had just  about wiped out  everything.   If we found

a  colony growing at 46°C, it was one of these oddballs, a huge colony.

    Therefore, we had a real problem to face in the laboratories that

were going to do this test.  We know that many of the laboratories did

not have a reliable waterbath (a temperature deviation +0.5 tenths or

less).  We felt that if we put out this procedure at 45.5°C we would run

a very good risk of having people come up with negative answers because

waterbaths would not be performing correctly.  We set the incubation

temperature of the test at 44.5°C to be sure that waterbaths, at that

time, would be able to cope with this test (it is really very sensitive

to temperature).

    The World Health Organization is using, at this time, 44.5°C in its

standards.  I had the opportunity to talk with the water laboratory

directors of twelve European countries last month in Belgium on another

assignment and discovered they are all using 44.5°C.  They may call it an

E ooli. test; it is actually the MPN version with a confirmation very

much like we perform.  They are using a different medium, but I am sure

if we made measurements under similar conditions, we would get identical

answers.  We have done some parallel tests with MacConkey broth and

other media.  We are talking about the same numbers and the same pop-

ulation worldwide.

    In the light of your findings, it might be desirable to elevate

the temperature of the test to 45.0°C now that we have more labora-

tories using better waterbaths.  I do not want to go to 45.5°C because

I am afraid of the results.  We have to recognize a trade-off here.

     Knittel:  You can see from that curve that there is a plateau

between 44.5°C and 45.0'C.  The results are going to depend a lot upon

the kind of population present other than E ooli.

     Geldreich:  You made one interesting observation, that if one would

incubate the membrane filter for the total plate count at 35°C for the

normal time, but then set the plates out at room temperature for a few

hours, one would see more sheen colonies which one would then pick.  Am

I reading this right?

     Knittel:  I was not referring to the colonies that appear on the total

coliform plate, but to the isolates from the colonies growing in lactose broth.

     Geldreich:  This is a very critical thing when we are looking at this

whole problem.  For instance, on an elevated temperature test we must

place the membranes in the incubator within thirty minutes and then we

must take them out and get them counted within thirty minutes.  On this

particular test I have had many people tell me they leave the plates

setting on a work bench for several hours—while perhaps they are having

lunch, etc.—and then they count them.

     Knittel:  They might as well throw them away.  They do not have

typical blue-centered colonies.  They immediately are released from the

inhibition of temperature and they're going to grow very rapidly.  Within

an hour and a half to two hours they will change.

     Geldreich:  This also occurs on a total coliform test.  The counts

become excessively high because you suddenly get the slow lactose fer-

menters occurring in such populations.  Instead of having 20 or 30


coliform colonies when you count, you might have 100.  I do not think

this is well understood.

    Knittel:  I do not believe people are following the procedures that

strictly.  I believe that there has to be precise quality control of

media use, sterilization, and techniques to reproduce results within

defined limits.

    Geldreich:  Both tests involve a transient reaction.  With the total

coliform test actually the Enterobaoter aerogenes and Klebsiefla will

grow very fast.  I was wondering whether those plates that you made had

been out on the work bench for a while.

    Knittel:  Yes, by the time they were photographed, they had been out

a while.

    Geldreich:  I have had this problem.  I was going to photograph some

colonies like this, and they had lost their sheen.  They have completely

consumed all the lactose.  They have now started to attack the aldehyde,

and then the color turns gray.  They were originally sheen colonies and


    Brezenski:  On the question of 45.0°C as an incubation temperature,

this is also being perpetuated by FDA right now.  At the last shellfish

meeting in Washington, this was one proposal which came up regarding

44.5°C and 45°C incubation.

    Geldreich:   I do not think that you are going to find much difference

in coliform IMViC patterns between 44.5°C and 45°C.  If the higher in-

cubation temperature will clear up the background of confusing organisms,

I am all for it.  I have looked at this very carefully, and I do not

th ink that changing 0.5°C is going to upset anything we have done in the

past or will do in the future.  If it will help in paper mill wastes, I

would be all for just making a standard across-the-board change to 45.0°C,

in the next edition of Standard Methods.  We had originally set the tem-

perature to be compatible with World Health Standards and to recognize

the problem with incubators at the time.  We have reliable equipment now,

and perhaps we ought to raise the temperature to 45.0°C.

    Brezenski:  I think that is a good point.

    Knittel:  We use Blue M Magniwhirl waterbaths, cover the tops, keep

an extended scale thermometer on them, and check them two or three times

a day.  As well as I could judge, these plates were within 0.2°C variation.

The temperature ranges from 44.9 to 45.1°C, but it averages out that the

length of time at 45°C is greater than at the two extremes provided one

keeps the waterbaths covered.

    Geldreich:  The important point to remember also is that this is

still a fecal coliform test and not and E ool-L test.  We are going to

find Klebsiella pnewnoniae even at 45°C and it is important.  Also, the

members of the fecal coliform populations may differ even within one

individual.  For a period of time a fecal coliform with one IMViC

pattern may predominate, and then, three or six months later the popula-

tion may shift into another pattern.  That those organisms are all

elevated temperature positives, is the important thing.

    Bauer:  I would like to bring this discussion of methodology to

a close here if I may.  If you are in agreement, let us start the

discussion of the question at hand:  "Is a bacteriological limitation


necessary in industrial wastes where municipal waste is excluded?"

    Brezenski:  This is not a Separate question.  This question is tied

very closely to the one we are talking about.  This is methodology, also

definition:  what organisms we are talking about.  Apparently, there is

not complete agreement on the significance of some of these intermediate

types or of Ktebs-Lefia.  I do not think we can make a decision on one

problem without considering the other.  I think this interpretation is


    Bauer:  Let me be more specific.  Do you feel that fecal coliforms

at 44.5°C are a relevant parameter to apply to industrial wastes?

    Brezenski:  1 think that is the more pertinent question.

    Howard Davis:  What do you mean by fecal coliform?

    A speaker from the floor:  Any coliform that will produce gas at


    Bauer:  We are talking about how one definition fits on top of

another.  Coliforms that will grow and ferment gas at 44.5°C regard-

less of IMViC type, are good fecal coliforms.

    Geldreich:  Klebs-iella is a coliform, a lactose fermenter.  Sometimes,

some of us forget this.

    Brezenski:  I agree.

    Gallagher:  Maybe you can be a little more specific.  Some of the

material that has been prepared by the NFIC, to go to the regions as

guidelines for the permit program, has "organisms isolated in the fecal

coliform test and associated with pathogens shall not exceed  1000/100."

Is that a defensible statement?  Do the processed wastes have sanitary

significance?  Secondly, if so, are the fecal coliforms or the organisms

isolated in fecal coliform tests valid indicators of health hazards in

these wastes?

    Geldreich:  This is a loaded statement.

    Gallagher:  I know that it is.

    Bauer:  Can we take them one at a time then?  Are we to be concerned

about the high bacteria counts in industrial wastes, per se; do they have

potential pathogenicity associated with them?  If we can agree to that,

then what kind of a test do we apply to it to determine that point?

Alright, anyone willing to give his opinion?

    Geldreich:  First of all, if we are talking about fecal coliforms,

regardless of whether there are 3,000 or 20,000, we are talking about

fecal pollution.  This is what we are trying to measure.  We are trying

to measure fecal hazard where there is no pathogen test that will

guarantee that one can recover all possible pathogens that will be


    Now as far as Salmonella is concerned, it is great when you get an

answer.  However, when one does not get an answer, first of all, it does

not mean Salmonella are not there.  I had a project, in the Northwest

Laboratory, in which we had been looking at serotype recoveries in many

different kinds of media and determinations for Salmonella.   It is

most frustrating because you cannot be sure they are not there when you

get a negative result.  Secondly, Salmonella is only one pathogen we

ought to be concerned about.  We have a man here interested in virology,


and we have enteroviruses.   I am also Interested in enteropathogenic

E oot'i, which is a fecal coliform.  There are also Shigellae and Leptospira,

if we were to talk about feedlots.  The one thing that we can correlate

best with this concern for pathogen hazards is the use of some procedure

that will measure fecal pollution.  The only thing we have in bacteriology

is a fecal coliform test that correlates between 90 and 95 percent of the

time with this type of pollution.

    Brezenski:  Let me stop you right here.  The question that was put to

me by  the Council at that point was, "Yes, you can show data.  Mr. Geldreich

has shown us in the literature that one arrives at 95 percent correlation

when one is working with sewage and human excreta.  However, this fecal

coliform test was not developed for use with industrial waste, and one will

not come up to a 95 percent value in such wastes."

    Berg:  The problem here is that one is talking about two different

things.  One is talking about organisms as indicators on the one hand,

but that is not what the issue really is here.  The question here is:

What kind of organisms....(interrupted, mixed talking).

    Stein:  As many of you know, I am kind of an amateur scientist and

I am very sympathetic to what you are doing here.  Let me tell you how

difficult this is.  We are going to have to determine whether there is

a pathogenic significance (and we might want to stretch pathogens if you

do not think we cover viruses) in the pulp and paper mills and sugar beet

wastes.  If there is a pathogenic significance, then there has to be a

measurable limit, and this is probably going to be a coliform test.

The point is:  Are we going to say a high fecal coliform count in these


wastes is a hazard to health?  This is the hurdle we have to get over.

I think we can back you all with the kind of test you set.

    Geldreich:  What you really want is definite, positive evidence

of the pathogens present in an effluent as some of the states do....

    Stein:  No sir, we do not.

    Gallagher:  We want to have some evidence that there is a hazard

associated with process wastes from the industries.

    Lyons:  If you cannot establish that, everything else is a lot of

good scientific significance, but in the enforcement field we are

trying to build a house without a foundation, and we have to have that


    Geldreich:  Now, you have fecal coliforms present, indicating a

fecal contamination.  What you want is to correlate it with potential

pathogen occurrences if possible.

    Gallagher:  We would like to have what evidence is available.

    Stein:  Let me read this.  (It is a very good and accurate expla-

nation of the industry's position).  "Pulp and paper industry research

has shown that Klebs-iella is often found in process waste waters that

are completely isolated from the domestic wastes; it has no sanitary

(fecal) significance."  The statement is credited to Mr. Blosser.  To

me it is unacceptable.  A waste does not necessarily have to be asso-

ciated with sewage or packing house waste to have significance.  Let

us assume that we have an industrial waste like sugar beet or pulp

and paper effluents where the process wastewater is completely isolated

from domestic wastes.  Do we have to control these waters in any way


to keep the fecal count down?  What significance do they have for man?

    Geldreich:  It is to tell them something they do not want to realize:

There is so much nutrient present that those organisms are multiplying out

of proportion.  There are two things we are getting out of this indicator

concept:  One, there is fecal pollution there, and two, industries are

magnifying the hazard because the nutrients are there.

    Stein:  Right.  You made that point very clear.  If we can convince

the industries to keep the nutrients down, the industry will buy that

and spend a lot more money than to keep pathogens down.

    Geldreich:  There is a method, the fecal coliform, to measure whether

or not they are reducing the nutrients, because coliform counts are going

to go down after adequate treatment.

    Stein:  If the right policy is to keep the nutrients down and we

have some measure for nutrient reduction, we can put in a tight control.

If, by controlling the nutrients, we are automatically going to get the

bacterial and virus count down, I think we can push for a much harder re-

striction.  If we put the control on the nutrients, and if we can get the

industry to accept it, we are on firmer ground, particularly if we get

into the area of arguing about the effects of the microorganisms.

    Geldreich:  There is one thing, one cannot rely on the chemistry

test alone to be sure that nutrients are down.  One has to have a back-up

test in order to show that the coliforms are there in smaller numbers.

We cannot be sure that the BOD or some other chemical measure is that good.

    Stein:  We are talking in terms of nutrients and we figure that the

nutrients provide a medium for bacteria to grow.  We just want the nutrient

level down.  Let me try to oversimplify:  I think a company might get ex-

cited if I were to say they are pouring a test tube of bacteria in the

water and contaminating the stream.  But if I tell them they are dumping

into the water some molasses which is relatively benign although it will

provide a good medium for the bacteria to grow on, I can tell them to cut

down on the molasses.  If we say we want to keep these nutrients down and

we are using this coliform level as a check, this may be a way of getting

around first base.

    Berg:  May I raise a question here?  I still think there is something

very wrong with these arguments.  Unless we can show that the Klebsi,e11a

are pathogenic and should be kept out for that reason, the question that

is going to be asked in the situation where one is obviously getting am-

plification is:  "How many organisms of fecal origin did you have originally

since you now consider them such a problem?"  Are the nutrients also

bringing about the multiplication of pathogenic forms?  If they are not,

you are dead.

    Geldreich and others:  They are.  They are.

    Berg:  I mean pathogenic forms.

    Geldreich:  Ralph Christienson has found Salmonella in paper mill wastes,

    Berg:  Then it is based upon the pathogens; what are you worrying about?

    Geldreich:  We are trying to establish, if we can, a policy from the

standard procedures that we use.  If we spend our time searching for


pathogens all the time, we are really going to have some great problems.

    Lyons:  Are the nutrients you are talking about not soluble substrate

levels or are you talking about this total group?

    Geldreich:  I am talking about a nitrogen and carbon source.  I am

vague because I do not know how else to put a handle on it.

    Lyons:  Because you could have it in either a solid or soluble form.

    Knittel:  Well, the nutrient has to be soluble and degradable, in

usable form.  A. W. Anderson, at Oregon State University, carried out some

research related to slim growth control in pulp and paper mill wastes.

This was a very sophisticated gas chromatographic analysis and it was

beautifully done.  He was able to point out that the six carbon sugars

were very rapidly removed during the first portion of the treatment

process.  The five carbon sugars were left.  They are a very good substrate

for most of the Entevobacteriaeeae including E aoli and Klebsiella, so that

we have really developed a selective medium for them.  There is a tre-

mendous amount of nutrients in these wastes.  He calculated that five

pounds of carbon sugars were going over the weir daily, based upon the

flow.  The number in tons was fantastic.  A seven-day retention time is

apparently not long enough to reduce them.

    Geldreich:  Other people have tried to measure nutrient levels

with a chemical test.  Lou Resi, Lois and Carl Shaddix, and others have

tried to find some chemical test that will relate to this nutrient prob-

lem.  We have not yet found one that is completely reliable.  That is

why I am cautious.  Use the best chemical measurement whether it  is

CCE, BOD, total carbon or another.   However,  we still need a bacterial

test as a back-up in case that one  system is  not measuring all the nu-

trients that might be degradable.   That is my only concern.

    Stein:  Assuming the complete  separation  of these industrial wastes

from the domestic ones, we have always had doubts about whether we could

relate the microorganisms present  to a pathogenic hazard.   We want to

stress practical treatment in the  permit program.  As far  as I can see,

likely this is going to be the treatment that the industry is going to

use between the years 1976 and 1981.  At this stage,  we may be able to

get a big start on this program if  we talk about keeping the nutrients

to the minimum, with the exception that the five-day  BOD test is, at best,

a very rough operation.  We can get more definitive results by having a

bacteriological test.  This may very well be  the fecal coliform test.

In one sense the fecal coliform test may be in a research  stage for the

job of relating it to the presence of a health hazard in these wastes.

Still, we have to use this test as the only certain back-up that we have

to be sure that we have controlled the nutrients coming from the plant.

We may have to adjust the numbers  if we use the test for this purpose.

If the statements made here today  are correct, treatment technology is

available to reduce the nutrients  in these wastes and also lower the

coliforms.  The correlation is pretty good and we will not have these

coliforms in the effluents.

    Geldreich:  I think we have a two-pronged problem.  One is to keep

the nutrients and the bacteriological counts  down, and the other is

the problem of the downstream user.  If the industry started out with

poor source water, let us say 1,000 fecal coliforms, and they knock them

down and still come out with 1,000 fecal coliforms/100 ml, we have some


    Stein:  The point is, what we are doing here is not going to solve

every problem.  In the permit system we cannot hold the industry resp-

onsible for what they take in from a poor source water if that water comes

from the same stream.  We probably have a better line-up on holding them

responsible if they take it from an underground water supply.  For example,

we could never hold an industry responsible for mercury in the water when

they picked up the mercury in the intake water.

    Geldreich:  You are hoping that what goes into the system at least

comes out no worse.

    Stein:  Yes, no worse than they take in.  This regulatory permit pro-

gram is what we are going to have to live with for the next decade unless

there is a tremendous change in thinking.

    Geldreich:  If there are polluters upstream of an industry we can get

a hook on them.

    Stein:  That is different.  But if an industry takes in water with

1,000 coliforms/100 ml, they are going to be able to put out 1,000.  We

just hope the industry runs the mill right.

    Bauer:  In pulp mills some of the sources of large inputs of coliform

organisms into the treatment system are the barker wastes, the white water

from the paper mill, and so forth.

    Knittel:  You experience this tremendous proliferation of the organisms

once they get into the biological treatment systems or in the primary


    Bauer:  Industry has rather expensive water treatment systems for

intake water at most of these mills.

    Herman:  They do not consider these costs?

    Bauer:  Yes, industry does, but it is forced to live with them.

    Stein:  It's a 'production' cost.

    Herman:  Why is not nutrient removal in waste a 'production' cost?

    Stein:  The answer is obvious.  The industry went into that plant

knowing what they had to pay for the raw materials.  They consider water

as a raw material.  They consider cost of getting that raw material into

shape a 'production' cost.  However, once the industry gets the water in,

they have a blind spot for spending a nickel to clean it up before they

dump it into the receiving stream.

    Gallagher:  A proposal has been put forward to form the rationale

for the permit system on the basis of reducing the nutrients and making

the industries measure this reduction by some test, presently undefined,

and to use the fecal coliform test as a monitoring device to be sure

that fecal coliform values are being reduced to prevent the regrowth.

That is the proposal.  Is there any problem in using that kind of rationale

in the permit program?

    Davis:  We seem to be talking about two things.  One part of the prob-

lem is using the tests as a monitor.  We are trying to institute a civil

suit against a paper company and show damages.  The other part of the

problem we have been discussing is the sanitary significance.  We are going

for an injunction and maybe treatment later.

    Gallagher:  I think we all feel that there is sanitary significance

involved here.  The problem is a real logistical one in having to prove

that in every case if we are going to get a permit.  If one is going after

a civil suit, one should have to prove that.   But we are talking about

hundreds of permits that have to be written,  and about trying to keep the

major logistical problem away from the Surveillance and Analysis Division,

not having to prove it in every case.  We are looking for a rationale to

get the results and to put to the industry in a rational way.

    Shimmin:  I think concomitantly, if we are going to talk about nu-

trient removal and using fecal and total coliforms as indicators, we

should have a statement in there about not allowing disinfection.  If

one is using total coliforms and fecal coliforms to indicate whether or

not the nutrients have been removed, and the industries have added these

dumps of chlorine, the indicators just might be temporarily wiped out.

    Another speaker:  Who is chlorinating?  I do not think most of these

industries are disinfecting.

    Shimmin:  My point is, if we do not have some other way of testing

the amount of nutrients in there besides growing these organisms....

    Stein:  I know of no pulp and paper industry that chlorinates their

waste, do you?  Now, if you are going to open some shellfish beds, this

is a specialized problem as I indicated to you before.  If you get over

70 coliforms, you have the shellfish beds closed.  If the pulp and paper

plant or anyone else is putting in something that  goes in the shellfish,

you have a damage case, and you are going to have  to work that out in  special

terms.  I think your point is well taken.  It might be possible  to get your


sample before you put in the chlorine for monitoring purposes.

    Riordan:  If you have a control on BOD,  and you have a control on

organisms, how is the industry going to escape?  If they chlorinate,  it

is not going to bring the BOD down.

    Speaker from the floor:  You chemically  oxidize the wastes  with the


    Geldreich:  I should like to forget about total coliforms.   I do  not

think we are getting any additional information out of that test.  Let

us stick with the fecal coliform test.  I think we are moving out of  that

area in the development of drinking water standards and in the  develop-

ment of the new water quality criteria.  We  are going to the fecal con-

cept that we are phasing out of total so let us not continue to carry

it on the books.

    Gallagher:  Do you think you can sell it to the shellfish people?

    Geldreich:  We are working on it.  It is hard.

    Stein:  I agree with you, but you see what you are up against with

shellfish.  I have not been able to get the  shellfish monitoring people

to change.  Here is the problem.  We are going to be able to approach

every paper mill with this concept except the paper mills upstream of

shellfish areas.

    Geldreich:  In relation to the shellfish, let me give you something

you may not have seen.  This letter is dated April 24 to Mr. Kelly in

our Office of Water Programs from Robert Schneider, Chief Biology Branch

NFIC-D, who is chairman of a committee on shellfish testing.  The point


is that the committee is not going to total coliforms.   That is rather


    Gallagher:  Yes, but the Food and Drug Administration is our problem.

    Geldreich:  My problem with them is that they call their test a test

for E aolij rather than fecal coliforms.  For shellfish we probably need

another paragraph that zeros in on this specific problem in the permit


    Stein:  We have that paragraph.  With the shellfish we are going

along with the total coliform parameter.  The real test will be to see

whether they are representing the shellfish concept.  I do not think there

are too many paper mills upstream of shellfish areas.

    Speaker from the floor:  What kind of tests, other than the fecal

coliform test, would you suggest could be used to control nutrient levels?

Total organic carbon?

    Bauer:  That was the chemical parameter that showed some correlation

with bacteriological levels in the mills that I investigated.

    Gallagher:  The objection that the industries have to total organic

carbon (TOC) has been "who owns the stock in Beckman and why do they

want us to buy these $9,000 instruments?"  That is the problem; they are

not equipped for TOC work and they do not want to make that investment in

instrumentation.  They suggest they will live with the COD.  There are

a lot of problems there, too.  Why do you not discuss what kinds of tests

would be the best technically?  We need a rationally defensible test for

accomplishing this.

    Geldreich:  Looking at the data that we have obtained from field surveys

over the years where the chemistry tests were run in parallel, we  find


difflculty in zeroing in on any one test like TOC or BOD.  I cannot give

you that answer right now.

    Stein:  One of the problems here is cost.  Some of the larger com-

panies will do these tests, but if you expect that of a canning plant, for-

get it.  That plant does not even want to spend the money for labor costs

to send an employee out to the end of the lagoon to take a sample and return.

The possibility of really securing s sophisticated test into all these per-

mits is practically nil.  If you include as a requirement for this permit

that these sophisticated tests be carried out, you are going to get a

tremendous complaint, and my bosses and yours are going to sympathize

with them.

    Geldreich:  The only thing that we can do then is to emphasize that

the fecal coliform level input and output be identical and base it on

that arrangement.

    Stein:  And have them check it?

    Geldreich:  No, I would not have them check it.

    Stein:  No, I would not give that up.  This is perhaps one of the

more useful things that we can do.

    Lyons:  May I invite comment from the S & A Division people as to

what that would mean if they had to check all of these parameters, i.e.

the fecal coliform and the TOC?

    Bauer:  I do not see the TOC as a big problem because it takes perhaps

a 10 ml sample, which can be acid stabilized, placed in-a bacteriological

mailing tube, and tossed into the mail.  It is a very quick analysis.  I

would not be at all adverse to all S & A Divisions being required to run


    Lyons:  Is It a part of their regular monitoring?

    Bauer:  Yes.

    Geldreich:  That is the answer then.  It would be the kind of infor-

mation we would need.  It would provide a ball park number, and we would

back it up with a bacteriological test.

    Lyons:  Now,  of course, the next question is that there is not suffi-

cient data base on TOG.  What number do you choose for the effluent?  It

will not be a monitoring tool until there is a number.

    Stein:  We agreed at least in the pulp and paper industry on that.

The paper industry would be ready to use TOG instead of BOD or any other

test if we could find the correlation.

    Bauer:  I do not think it would be too difficult to do it.  Again, I

relate back to the microfermeter work that was done at Crown-Zellerbach.

You could pei'form TOG analysis using instrumentation, together with bac-

teriological tests on various kinds of mills, and see whether you can get

a decent  correlation.

    Stein:  The industries will not buy the principle that they have to

put out the same water quality that they take in.  Do they not add something

in the plant?

    Knittel:  Can we go with a net increase type of statement?

    Geldreich:  That is the best way.

    Knittel:  A net increase that would be equal to the water quality

standard  for the water that they discharge into, or something like that.

    Geldreich:  The quality should not be any worse than what they took in.

    Knittel:  What I meant was a net increase through the plant.   Is it

entirely possible not to get any increase of fecal coliform through the

plant?  With adequate in-plant controls and best practicable technology?

    Geldreich:  I would certainly think it is possible.

    Question from the floor:  How is it possible to control within the mill?

    Bauer:  You can establish the kind of BOD level, TOG level,  or whatever,

that you consider acceptable and, which is indicative of best practicable


    Remark from the floor:  That has been done.

    Question from the floor:  Yes.  What is the bacteriological level that

would be associated with that?

    Remark from the floor:  That has not been done.

    Lyons:  Let us say that 30 mg/1 might be the upper limit for substrate


    Geldreich:  It is somewhere between 14 and 30 mg/1.

    Herman:  I would consider 30 mg/1 the maximum.

    Lyons:  This fits nicely except for the canning industry.  The rationale

in both the pulp and paper and the sugar industries is based generally

upon 30 mg/1 in the processes which are most used.  We can ask them to go

to bat with the TOG as a monitoring tool to develop a data base and with

the fecal coliform as a back-up for monitoring.   Do both of these tests

have to be run by EPA?

    Stein:  For industries other than the pulp and paper industry,  you


are going to have to do the testa.  I think the reason is obvioun.   The

pulp and paper industry is a big business and they have a scientific

staff.  I do not think you are going to find that in the canning or in

the sugar beet industry.

    Herman:  I have had seven years in canning, International Gartners'

Association.  I know that when the Qanners wanted to treat, they did.

From waste effluents between 1,600 and 2,000 BOD they produced a final

effluent which routinely for over three years held at eight.  They can

do it.

    Stein:  Well, let us try them.

    Speaker from the floor:  That can be backed up.  I do not think there

is any problem.  They will admit they just have not designed facilities.

    Herman:  They will recognize that they can do it.

    Bauer:  I am hesitant to give my complete blessing to having EPA

perform all these fecal coliform tests, recognizing what it will take

to get them done. The TOC tests can be punched out quickly on the in-

strument, but fecal coliform tests will require time.

    Knittel:  Fecal coliform samples cannot be preserved for later


    Geldreich:  How about state agencies?

    Stein:  That is what we may be able to do.  The question is, who

is responsible?  This is the basis of the problem.  I have been out  in

the field and looked at these canneries, big and small.  These are seasonal

operations, small plants.  Some companies may cooperate with us and  do

the fecal coliform tests.  The canning associations and some of the  pulp


and paper mills will.  With other plants, we should probably indicate

that we are going to monitor on a spot-check basis.  As a regulatory de-

vice we do not have to do this with every plant.

    Geldreich:  I think once you have established a baseline to deter-

mine the TOG number, you will not have to do very much bacteriological

work; it will diminish.

    Stein:  If we had the TOG we could go ahead.   We do not have the


    Gallagher:  This would be set up as a monitoring device.  When they

feel the data base is sufficient, maybe two years from now, the permit

program would amend the permits to incorporate the baseline which we are

talking about.  This is a way of getting at this whole problem of bacteri-

ological distress.

    Herman:  We should have the authority to make this check when we


    Gallagher:  That is taken for granted as part of the monitoring


    Herman:  That was not the case when I was with industry.

    Stein:  I would not give any industry a permit unless they would

agree that any authorized representative can come in at any appropriate

time and take a sample.  Otherwise the firm does  not receive the permit.

    Berg:  Are you talking about one TOG level for all industrial wastes?

    Gallagher:  No, we are establishing TOG as an indicator for all wastes


but different levels for different kinds of wastes.

    Herman:  The National Canners Association was bucking some of these

coliform tests; so was the National Council and the potato and sugar

industries.  You will have a united effort from all of these industries.

    Gallagher:  That is right.  That is why we are having this meeting.

    Stein:  Do not feel bad about the Corvallis meeting.  In my opinion

it would have happened anyway.  Every time we finish a meeting with an

industry, their associations are going to get together.  It was inevitable.

It is Very easy to talk with these industry representatives when you are

discussing research.  When you are talking about regulations, the situ-

ation is different.

    We have this fecal coliform testing problem with all these industries.

It appears to me that we are going to have a very hard time selling this

permit policy on the basis of pathogen effect, viral effect, or health

hazard, where we have a segregated waste.  If we push the pathogen approach,

the natural reaction would be to disinfect, and I am not sb sure we want

that because of the regrowth problem.  I think the two alternatives may

dovetail if we can push these guidelines to remove the nutrients.  We may

get the same results and be five to ten years ahead in cleaning up the

water.  It may be a blessing in disguise, to approach it the other way.

    Gallagher:  We are going to ask the regional programs, with their

monitoring programs, to examine for pathogens and see if they can get  a

data base on them.

    Geldreich:  If you get positives, it is great, but when you get ne-

gative answers, you are just spinning wheels.


    Another speaker:  That is right - look at the Snake River Report.

    Herman:  In the 1966 meeting of the National Canners* Association

many of these points were discussed.  At least some people within the

industry felt then that the Federal Water Pollution Control measures

within three years would be what you are suggesting today.  This would

not be any shock to them.

    Stein:  I think that is right.  When representatives of these in-

dustries go to a scientific meeting with you, they are constructing and

testing a case that they throw at us when we meet with them.  They have

all our arguments  before we put them out.  The way they get those ar-

guments is by going around with their scientific contacts.  After all,

where do we get our arguments, but from fellows like you.  If they have

tapped you fellows at the source, then when we have all the arguments,

they have the rebuttal all lined up.  I am not against free scientific

information exchange with anyone, but please remember what they do with

this information.

    Bauer:  Are we satisfied with the proposition that Mr. Gallagher

has summarized?  Are there any violent disagreements with that?

    Knittel:  Since we do not have a baseline, I am looking for areas

of research to support this.  Perhaps we need microbiological research

to support these tests with regrowth studies and correlations of coli-

form content with nutrient level.  Will this kind of information help

the permit program?

    Geldreich:  You need authorization, and that has been on the books

for a long time.

    Stein:  If you can pass up some of the bureacracy to get such re-

search through, we shall back you.

    Lyons:  You will have the support, at least from our programs to

highlight points with which we are involved, and problem areas that

need more exploration and definition.

    Knittel:  We do not need a lot of money or a million dollars worth

of equipment to do it.  We do need people, but if we cannot find the

people, we shall do it anyway,  If we can get the support from the top

level, we shall do as much as we can.

    Stein:  We can break through this impasse.  We need your help.  If

you have a project that you think will go, just keep pushing it.  When

we have problems which are partly biological or microbiological that

an engineer cannot solve, we will need an aquatic biologist or a micro-

biologist.  When it comes down to the real expertise in your areas, you

are going to have to make the judgments yourselves, because the engineers

and ourselves are something like educated amateurs.  By the time we had

the second enforcement case years ago, we dragged Harold Clark (deceased)

from the Taft Center because I recognized that we could not move without

this information.  I think that essentially the next law suits are going

to be in the area where the people, in this room, who have the expertise

are going to lead us.  This is one of the areas where we are going to need

some monitoring and sensitive tests.  We have a pretty good record with

with the engineers and with the fish biologists.  Now we are going to need

some of the more sophisticated testing.  We have to depend more and more

upon the kind of information we have discussed here today.  We shall have

to work together in identifying the problems.

    Let me say one last thing on this subject.  No lawyer or engineer

or aquatic biologist can tell you what you should do.  You have to de-

termine it yourself.  I picked up more ideas by watching your presen-

tations here today than I ever did from meeting with people who are not

working in your particular field.  You have to identify these research

needs yourself and put them forward.

    Gallagher:  Let me say specifically, that if any of you people in

Office of Research and Monitoring (ORM) has a particular project that

could be of benefit to the permit program or in the enforcement program

generally, let us know about that project.  If you need the external

support, and it is compatible with the enforcement program, we shall

try to get you that support.

    Shimmin:  Could we hear again the statement that you are going to

make about the coliforms?

    Gallagher:  We are not going to make any statement.  We are pro-

posing to summarize what has been said to you, and write up a statement

based upon our understanding of the discussion.  We will very quickly

put it back out to you to see whether or not you think that is what you

said.  Then we shall make a recommendation to Headquarters.

    Speaker from the floor:  In other words, we shall all see that state-



    Gallagher:  Well, I do not know whether you will all see the state-

ment, but the summary will be sent out.  We want to do this very quickly.

    Stein:  There will be no surprises in the statement.

    Shimmin:  Is this statement under the "Considerations for Permit

Preparation" on coliforms going to be deleted?

    Gallagher:  Yes.  This is one that was set up with the pulp and paper

industry for the permit proposal to which they strenuously objected at

the Corvallis meeting.  I would think that this would be replaced, except

for the last paragraph on receiving waters which are classified for shell-

fish harvesting.

    Lyons:  That was a sanitary engineer's poor attempt to set a number

on bacteriological significance.  We are looking for recommendations.

    Stein:  The real problem we have to face is that although we are

going to have lawyers and engineers drafting permit and enforcement policy,

we will have to work with these other professional groups, aquatic biolo-

gists, bacteriologists, and specialists in other fields.  You, the micro-

biologists, are going to have to give us the formulation that we are

going to sell.  We are not going to have any lawyer or engineer or any

specialist in Washington who is going to sell this policy and then face

the rebuttal.  We are dealing with your field, bacteriology, and the

rebuttal is going to come from bacteriologists.  We are never going to

make it unless we get the formulation from you people.

    Bauer:  There are a few things we ought to discuss tomorrow —

additional research needs, problems with Standard Methods, and this type

of thing.  Another question we have talked around all day  today is whether

or not we should be concerned about the presence of Klebs'iella. in large

numbers.  Are they potentially pathogenic?

    Stein:  I can find another problem for you when you need it.  I sensed

it today, and it was proven by several other people in the room.  We are

going to have a lot of problems unless someone can get the methodology

lined up so that it is uniform.  I do not suppose you would want such

tests as you are coming up with now in Standard Methods.  I do not know

whether you can split samples, or how you do it.  I do not want to pre-

sume to tell you how to do your work.  However, from what we have cut

out for ourselves here today, the sooner you get at this problem of

uniformity the better off you are going to be.  Perhaps you can do this

while you are all here, or perhaps you are going to set up committees.

Even if you are all agreed now on what you are doing, when you go back

to your various laboratories, carry out these tests, and start to get

different results, you will wonder why the results are so disparate.

Unless the microbiology checks out very closely, we are going to have

another meeting like this six months from now to discuss these differ-

ences.  Then we are going to have to get Mr. Gelreich and a committee

of four others to go around from laboratory to laboratory to pull you

together, and this is a wasteful activity.  This is approximately six

months off and unless you have coliform methods, you have problems.
    Geldreich:  I have some encouragement for you, Mr. Stein.  Several

of us in this room are on Standard Methods committees.  I was on the

committee for the last edition and we can handle getting it into the

next edition of Standard Methods which we are now preparing.

    Stein:  In the interim this is the problem we are dealing with.

    Geldreich:  Right.

    Stein:  The point is, if you go away today and do not. have your

methods standardized, you will have problems.  For example,  one man

may work for the next four months and come in with all those beautiful

counts, but his results are a little different from those of another

laboratory.  So you decide he is not doing the tests exactly right.

He is going to complain loudly because he has that tremendous invest-

ment in this data.

    Geldreich:  I do not think you will have that problem.

    Stein:  I hope not, but if not, this will be the first professional

group not to have that problem.

    Geldreich:  We will discuss this more tomorrow, but I do not think

we shall have any problem.  We are closer to solutions than you think.

    Gallagher:  I should like to thank all of you for coming today because

we are over a tough problem.  I think that we have something that we

can defend, and call upon you for, in our confrontations with industry —

our search is after truth with the industries.

    Bauer:  One of the questions we have been touching on at this

session is whether or not Klebai-el'ia is considered potentially pathogenic.

Bill Stang asked Leonard Guarria to do a brief literature research for us

in preparation for this meeting.  To kick off the discussion of possible

pathogenicity of Klebsiella, I should like him to review this literature

for us.


         Brief Literature Review of Klebsiella as Pathogens

                       Dr. Leonard Guarraia

    Briefly, I think it is important to distinguish bacterial infection

from disease.  Infection merely seems that an organism is capable of

living within a host.  We are infected by many organisms - E ooli,

streptococci, staphlococci, and so forth.  When the organism causes a

disease, that is a pathogenic condition; it becomes important in terms

of human health.  Klebeiella pnewnoniae infects approximately 30 percent

of the human population.  It has also been isolated as a saprophyte from

soil.  It probably is more significant as a human-related organism; at

least this is my feeling from my search of the literature.

    Klebsiella is a gram-negative asporogenous, non-motile rod which

ferments lactose.  That is the definition in Sergey's manual and in all

the textbooks.  As a disease organism it was first recognized as

Friedlander's bacillus, and it was associated with upper respiratory

distress, pneumonia.  It causes approximately 2 percent of the total

cases of bacterial pneumonia, and about 60 or 70 percent of the mortalities

of all bacterial pneumonia are related to Klebsiella pneumoniae.  This

organism is resistant to many antibiotics and is so encapsulated, so

slimy,that normal defense mechanisms of the host break down.

    The Klebsiella have been associated with liver absesses, infant

diarrhea and urinary tract infections.  Many healthy people (10 to 20

percent) have been found to shed Klebsiella pnewnoniae in their urine.

The organisms are not in the kidneys, but occur on the way out.  They

have been associated with urinary tract infections and with oculitis,


eye Infection.  Klebaiella have been cultured with increasing frequency

in certain hospitals in human blood cultures.  This is a rather serious

finding by medical microbiologistH.

     KLebs-Lella have also been implicated in bovine infections, rnnrtltis.

Many healthy cows have antibodies to Klebsiella antigens in their blood.

Many healthy animals (about 30 to 40 percent) have Ktehsiella pneumonias

in their gut.

     An a medical problem, Klebsiella penwmoniae certainly does present

some concern.  As an indicator of pollution, it is a legitimate one.  One

cannot say, a priori, that because we have Klebsietla pneumonias occurring

as a high temperature organism, that it invalidates a test.  That is an

erroneous assumption.  The elevated temperature test is still a very

valid test in my opinion.  Klebsiella pneumonias certainly is associated

with the gastrointestinal tract.  As to whether or not Klehniella comes

as a normal habitat of the stream, or the soil, is really not relevant.

The data are not strong enough to suggest that Klebaiella would lie  there

in quantity as it would be from a sanitary source.  That sums up xvhat

I have found on Klebaiella.

     Herman:  According to some medical and  veterinary texts,  there is

some question as to whether many of these organisms would be  classified

as Klebaiella pneumonias.

     Vasconcelos:  At present there are nine binomials (genus  and species

names) and the rest are referred to as Klebsiella  serotypes  up  to  72.

     Herman:  Out of the 72 serotypes, we now have 68, in  the culture

bank at Duluth, that come from environmental sources.  I  am  convinced

that we shall find them everywhere, depending upon concentrations  present.


     Knittel:  In reviewing the literature, what is your opinion of the

validity of the mouse test in proving or disproving pathogenicity?  This

is one point that has been rather salient with the industry people.

     Guarraia:  I think we have to look at the reason for using this test.

They have used it for Diploaoaaus pneumonia; if it will kill them it is

a pathogen.  This is then related to encapsulation.  1 guess in lieu of

another test, it is all right.  It is very dangerous to extrapolate from

a mouse to any other animal because they are exquisitely sensitive to

this type of organism.

     Bauer:  I think from talking to people who do it that we are on

particularly shakey ground with mouse pathogenicity test.

     Geldreich:  As a further insight into this problem, a closely

related study was carried out in Argentina in 1956 (Revista de Obras

Sanitarios de la Nacion, Vol. 20, p 169-172).  It was on bacteria of

the,Klebsiella genus in water, particularly of the La Plata River, which

empties near Buenos Aires.  They found 61 serotypes of Klebsiella.  The

author cites 14 serotypes from urinary tract infections, 17 from sputum,

2  from fecal material, 9 from abscesses, and 7 from blood infections.

He  also found them in the La Plata River.  This organism is found in

feces.  It is potentially pathogenic from some people, not everyone,

but we certainly have to be concerned about it.

     We are  concerned about some of these organisms such as Klebsiella,

PseudomonaSf and Flavobacteviian in drinking water; all three  are  secondary

invaders.  They do cause problems in hospitals.  We have had  a  lot  of  re-

ports around the country that the potable water in hospitals  from the


organisms get in the whirl baths, the aspirators, the respiratory materials,

and patients' wounds.  Some very nasty infections result.

     Our concern is that as we get higher and higher on the standard plate

count, our ability to detect coliforms starts to tail off.  It relates to

competition from other organisms.  I have asked two of our laboratories,

in addition to our own, to look at this problem not only in problems of

potable water but also in those of paper mill wastes.  I want you to be

aware that we have some limitations.  If you get enough Klebsiella in the

water, your chances of finding total and fecal coliforms may be suppressed,

may be inhibited, or may be reduced significantly so that some of these

counts that we get, for instance in paper mill wastes of fecal coliforms,

may only represent part of the total number present.  I do not want to

discuss drinking water here, but this will give you an idea of how we got

interested in the problem.  We have found in experiments and some few

papers in the literature that Paeudomonas aemginosa in certain densities,

between 1,000 and 10,000, and upward, actually does suppress the detection

of total coliforms in water.  Klebeiella is apparently another organism

that can cause this suppression.

     Guarraia:  I should like to add one more thing about Klebsiella.

The reason that these organisms have had a decided growth advantage in

pulp and paper wastes is because Klebeiella has  a very extensive complement

of extracellular enzymes.  They are able to break down complex carbo-

hydrates that are found in these types of wastes.  This  is why Klebsiella

will overgrow so rapidly and other organisms probably  can not  compete.

Also, the data are not available to make the correlation  on  how  many

Klebeiella you need  to cause infection.


     Geldreich:  Mr. Vasconcelos do you want to pick up the discussion

on suppression of indicator organism and describe the work at the

Northwest Laboratory?

        The Detection and Significance of Klebsifilla in Water

                          G. J. Vasconcelos

     We got interested in Klebsiella from the standpoint of potable

waters.  Our next step was to go into the influence of the waste liquors

on this balance between Klebsiella and E. ooli.  I tried first  to expose

a wild strain of E. ooli isolated from a river source to a Klehr.iella

strain.  I had to have a label.

     I needed a rapid way of differentiating F,. aoli from Klvbsiella.

I ran across esculin hydrolysis that is unique for members of the

Klebsiella-Entevobaatev-Servatia group.  They apparently possess the

ability that 77. ooli does not, to hydrolyze esculin, producing  dehydroxy-

coumarin which reacts with ferric ions to form a black compound.  With

this medium I could differentiate Klebsiella from F,. coli. rapidly.  Also,

E. ooli is motile and Klebsiella is nonmotile.  I had pure cultures of

E. coli, a wild strain which I had isolated, and .Klebsiella Type 22, that

was isolated from the stream off the membrane filter.  It would have been

counted as E. ooli or fecal coliform by that method.  I lypholized the

cells so that, in my experiments, I used the same cultures, and they

would not have any variation.  Before the experiments, I grew the cells

in a carbohydrate medium, washed them, and re-suspended them in phosphate

buffer.  I then determined the cell concentration photometrically.  In

three flasks, in a shaker bath maintained at 18°C, I had two controls,

one containing a concentration of E. ooli, another a concentration of


KLebeiella, and then the two of them together in the experimental  flasks.

     I tried these at three different Klebeiella levels, maintaining

my E. ooli level at approximately 100 organisms/ml.  I had to have a

fairly high concentration of E. ooli because I was working with quite  a

differential in counts.  1 was working with 10  E. ooli, as opposed

sometimes  to 10  Klebaiella,  I had to have a way of picking them  out

in the same petri dish.  In the first experiment I tried them at compar-

able levels, 100/ml of E. ooli and 100/ml of Klebsiella.  With these

levels I obtained good results; I could differentiate E. coli from

Klebeiella without any trouble.  The same thing was true on M-Endo.

When I used bile esculin there was no question which was E. coli. and

which was  Klebeiella.

     In the second experiment I increased the level of Klebaiella  to

  5                 2
10  as opposed  to 10  /ml of E. coli.  I could recover E. coli fairly

well by the M-FC, but on the M-Endo the E. ooli- was completely overrun

by the Klebeiella.  By increasing the concentration of  10  Klebsiella

     2                                '
or 10  E.  ooli, I could in no way detect E. ooli with  the M-Endo;

however, with the M-FC I could.  In fact the  levels remained  almost

equal to the control, E. ooli.

     In the last experiment I went on and compared  the MF with  the MPN

method in  recovering E. ooli in the presence  of  10  Klebsiella.   I did

a correlation based on a ratio of the number  of E.  ooli recovered  by

the MF method,  as opposed  to the number of E.  ooli  recovered by the

MPN method.  In this  first  experiment with  the Klebsiella Type 22  the

MPN method appeared to be  a little better  in  recovering E.  ooli.    If

unity is one, the ratio with the MF method  gave a mean of 0.3;  with the


MPN method the mean was 0.9.  I did not draw any definite conclusions,

because I wanted to include other serotypes.  It is a good thing I did,

because it did not work out that way.  However, the other two experiments

showed that the MF method used with the fecal collform medium was better

with the MPN method of recovery of Klebsiella Type 38.  I intended to

get the results for the base difference between the two.  I started

getting higher counts with EC in the tubes containing Klebs-iella as

opposed to the control with the MPN method.  I did not get this result

as much on the MF method, using the M-FC medium.

     Bauer:  The Klebsiella you had were EC-negative?

     Vasconcelos:  There are three types of Klebsiella:  non-aerogenic

at 48 hours with good growth, aerogenic strains in EC, and this particular

Type 22 which was not aerogenic after 48 hours.  Klebe-iella Type 2 and

Type 38 were aerogenic.  I differentiated by motility-  I inoculated

each tube into motility agar; if the stab was all motile, I counted it

as E. ooli.  This is how I arrived at my MPN.  Basically, it seems that

the MF method, with the use of the fecal coliform medium under  these

experimental conditions, tends to inhibit the Klebeiella more than the

MPN or the M-Endo medium.  The reason for this is not quite clear.  I

have not explored it, but I presume  that it is because the cells were

under stress and at this period perhaps E. aoli, could compete more

successfully than Klebsiella in this medium.

     Mr. Bauer, I noted on  the data  that you presented yesterday  that

downstream, the Klebaiella  tend to die off, or one did not recover as

many Klebsiella as E. ooli.  Now, this may be  a consideration.   Under

stress maybe the cell does not proliferate.  I have not carried out any
survival studies, but I plan to include some.
     As far as differentiating E. eoli from Klebeiella, I think it can
be performed easily if this is what the pulp-mill people are interested
in.  This bile esculine medium works very well with every strain 1 have
ever worked with.  They are all esculin-positive.  You can almost dif-
ferentiate EnterdbaQter from Klebsiella by motility:  one is motile and
the other is not.
     Geldreich:  What is the length of incubation that you use with the
bile esculin agar?
     Vasconcelos:  You can get esculin hydrolysis within one hour.  I
incubated these  for 18 to 24 hours.
     Geldreich:  Do you think a medium could be developed for a membrane
filter procedure?
     Vasconcelos:  I was thinking about that.  I tried taking the  filter
containing both  E. aoli- and Klebsiella and placing  it on an agar plate
containing bile  esculin.  Within an hour,  there was a blackening of the
medium; that indicated KlebBiella were predominant.  With a pure culture
of Klebsiella placed on the medium, within four hours the colonies had
grown out.  Another consideration with esculin is that it absorbs  in
the UV range.  If one eliminates the  ferric  ion  from the medium and puts
it under a black light, the black zones showing  esculin hydrolysis appear
distinctly.  There is no question about it;  whereas, the rest  of  the
medium is fluorescent.  There is an article  — in the January  1971 issue
of Applied Microbiology, indicating that  99  to 100  percent  of  the  E.  ooli
were found to be esculin-negative, whereas the Klebaiella-Enterbacter-


Serratia were all positive.  I have not subjected this to  a comprehensive

examination, but to differentiate E. coli. from Kle'osiel'la.  shows promise.

     Guarraia:  Have you tried the urease test for this purpose?

     Vasconcelos:  I have tried that and found too much variation  in  the

strains I tried.  Motility and ornithine decarboxylase are the other  two

tests that  are fairly consistent in addition  to  this  esculin hydrolysis.

     Knittel:  Where do you obtain your esculin?

     Vasconcelos:  It is available from Difco; I think it  is a nutritional

biochemical.  I found the prepared bile esculin  medium that is used  for

streptococci  to be slightly inhibitory to Klebsi'-clla, so I used a  modified

formula with  onepercent lactose.

     Geldreich:  There are two points of interest in  what  you have presented.

One  is that we may have a tool to use in work with K'tebsie'Lla.  It may be

very helpful  for field investigation work because it  is not to complicated

and  could be  used in mass sampling field surveys.  Secondly, your  are

beginning to  demonstrate that Klebsie'lla in the  excessive  numbers  that we

arc  worried about Is actually suppressing our ability to detect •indicator

systems which in thnm«elvns may reprfiBont some hazardous situation,

whether it  is in stream pollution or potable?  waters,  which are

not  chlorinated.

     We need  a better indicator system  for  these tests.  We arc  running

into problems with laboratory results around  the country,  in  stream

pollution work, potable waters, and source  waters.  We  are running into

problems in the southwestern  and  southeastern parts  of  our country where

there  are excessive backgrounds of  organisms  on  the  M-Endo MF that are

interferring  with coliform detection.   Frankly xje  need  a better  ff-Flndo

MF medium or a better indicator system than the Schiff reaction of l>asir
fuchsin and sodium sulfite.  The problem is occurring In these -irons
because they have warm water temperatures all the time.  I have also
known of this problem in the tropics, Puerto Rico, and in the Virgin
Islands.  In San Paulo, Brazil, some water supplies and surface waters
are producing an excessively high background of organisms.  We need a
better way to suppress these background organisms in order to let the
coliforms grow and be differentiated.  They are there, but we are
losing them.
     The problem is not just with the MF methods.  The. Florida Health
Department,  for example, has been getting some wildly erratic MPN data
in estuarine areas and in  some of their fresh water streams in northern
Florida.  These results relate to the problem that excessive numbers of
some of these organisms are actually suppressing  coliforra detection.
Skips in the MPN data and  unusual results occur.  The most critical
problems are with 10 ml and 1 ml inoculations where more of the original
sample is present, and oddball results occur like 2, 5, 3, 1 positive
tube combinations.  Sometimes, they have actually streaked out some of
these unusual combinations of negative tubes, at  the highest sample
amount used, and found a coliform, but it is difficult  to isolate.  T
wanted you to be aware that this problem is occurring not only in  the.
MPN methods but in the MF  method because we need  some better media.
Actually we need chemically defined media, which  are more uniform.
     Bauer:  I should like to direct the conversation back  to  this
pathogenicity aspect and to suggest  a person to  contact who has  been


very helpful to us, Dr. John Matsen, (School of Medicine, University of

Minnesota, Minneapolis, Minn.) who is extremely knowledgeable about the

clinical aspects of infections due to Klebsielta as an etiological agent.

Dr. Matsen is very cooperative and helpful.  He gave a presentation at

the meeting in Corvallis and made a couple of important points with

respect to whether or not we should consider Itf-ebaifilla environmental

isolates to be potentially harmful.  The laboratory at the University

of Minnesota Medical School periodically experiences what amounts  to

localized epidemics of Klebsiella.  He documented one case recently in

the nursery in which 12 infants  acquired infections where Klebsiella

Type  33 was isolated as the causative agent.  The sites of infection

were  quite varied, but eight of  those children died.  The investigators

performed stool isolations from  all of the children in the nursery.

Upon  entering, none of them had  Klebsietla and shortly thereafter,

approximately  89 percent of all  these infants were carriers  of  the

organism.  They were also able to  isolate the organism off the  hands  of

the attending  nurses.  So, although the  rate of infection ir> low,

Kleb3'ie11ai can be a very virulant  organism.

      Dr.  Matsen described another  interesting investigation,  lie and

his co-worker,  routinely screen  the stools of a number of incoming

patients  and they  find approximately  20  percent are carriers of the

organism.  Then they compare  the number  of people who  develop a Klebs-iella

infection while they are in the  hospital.  The people  who  are pre-

colonized prior to entrance run  four  times the risk of  acquiring a

hospital-acquired  Ktebs-iella  infection  as  those people who  are not

colonized.  Let me emphasize  that  if  these organisms  are allowed to


proliferate in the environment, we may see an increased rate of coloni-

zation among the public, and persons under conditions of stress in the

hospital, e.g., antibiotic chemotherapy, catherization, etc., run an

elevated risk of acquiring infection.

     Guarraia:  Another point he brings out is that when you have an

anitbiotic regimen even at home, the Klebsiella can supplant the normal

bacterial flora, because they are not as susceptable.

     Geldreich:  May I read into the record here a comment Dr. Matsen

wrote on this subject, dated June 16, 1974:  "Klebsiella is a normal

flora bacterium in humans although certainly not in the same numbers or

to the same extent found with E. ooli.  In spite of being found in the

normal flora of fewer individuals than  are E. ooli, Klebsiella is more

often a cause of septicemia, pneumonia, and serious post-operative

infections.  Klebsiella is the second most common organism, next to

E. cotif as a  causative organism in urinary tract infections.  One of

the  features of Klebsiella which makes  it a particularly troublesome

organism is its propensity to become  resistant to anitbiotics.  In

this regard it is probably our most troublesome hospital bacterium

and  indeed we have strains at  the present time that  are  resistent  to

every antibiotic but  gentomycin.  This  worries me as  we  know  that

resistance to this particular  antibiotic  can  also occur.   I would  not

recommend that anyone swim, drink, or bathe  in water as  heavily  con-

taminated as you described.   (This is in  reference  to a  problem  of

paper mill wastes).   That Klebsiella  can  cause  a primary pneumonia and

the  aspect of  creating  a human population with  an increased incidence of

Ktebs'ie'Lla colonization are both worrisome to me."  These are the

critical points which were made and they back up your comments.

     Herman:  I am curious why that proposal which was requested by

Region X and others and which Dr. Matsen submitted to Project Coordination

in Washington was not acted upon.

     Geldreich:  Dr. Johnstone, with the State of Washington, has been

very interested in a grant in this particular area also.  He has not

been able to get funded either.

     Knittel:  Dr. Johnstone has submitted a grant proposal to study

the pathogenicity of Klebsiella, cultural comparison of environmental

isolation and disinfection techniques.  The disposition of the proposal

was not known for a long  time; it was not funded.  I think this is

ridiculous.  When we got  out into the field and meet capable people

like this we are continually in the posture of apologizing for the

inadequacies of this agency-

     Geldreich:  "Credibility gap" is the word.

     Knittel:  Really,  it's  embarassing.

     Herman:  It has been more than a year ago that Dr. Matsen submitted

his proposal.  It is unfortunate that these people with recognized

reputations submit proposals to EPA and they wind up lost.  Dr. llatsen

wants  a straight answer.  This makes it very embarrassing.

     Vasconcelos:  In Streptococcus work we use  the blood agar as  an

indication  of pathogenicity  and  for staphylococci we use  the  coagulase

reaction.  How about Klebsiella pneumoniae  on DNase medium?

     Knittel:  Negative


     Vaflconcelos:  IB it negative?  Huve you trir"l nil  tlui.'!i- environ-

mental strains?

     Knittel:  Yes, this is one of our cultural screens.  We ran eleven

of our strains through the DNase and we have had all negatives up  to

this point.  We did some pathogenicity work x^ith some of our environmental

isolates on a very limited basis and we thought we should process  them

through this capsule medium.  If one increases the capsule, one should

get increased pathogenicity.  Unfortunately they were all negative. •

However, in a very close observation of the mice during the time they

were post  inoculated, one could t«ll they were not feeling too prc.-it.

     bauer:  1 should like to discuss the memoradum, on standardization

of methods, that  originated with Sid Verner, Office of  Monitoring.  Are

we as microbiologists satisfied with Standard Methods,  or what are  the

areas of dissatisfaction?. I should like to poll the group individually

as a means of discussing this request and ask whether or not you respond

to the memorandum and if you did, what areas you were dissatisfied  with.

     Geldreich: I will give you the background.  A meeting was called

by the Office of  Monitoring in Washington, on February  10, 1972, to

explore the need  to develop guidelines in microbiological methods.

Kathleen Shimmin  had written, under Mr. DeFalco's signature, a strong

request to take a look at the needs that we might have  for some micro-

biological methods book within EPA.   (Mr. Balllnger might like to  prefer

to call it an EPA .Standard Methods.)  The outcome of  this meeting  war;

that it was decided to circulate  through the Office of  Monitoring  a

memorandum asking each of the Regional Laboratory Directors,  each  of

the Regional Offices, all of the NERC's and anybody we knew within EPA
at the supervisory top level to designate one person who would be their
leading microbiologist, and ask him/her to respond.  Some questions were:
What do you see as weaknesses in Standard Methods, are you satisfied with
it, and if there are weaknesses, what are they?
     We were hopefully going to get this information.  I have not received
any as yet.  It would all be sent to Sid Verner in the Office of Monitoring
and he, in turn, would send it out to me.  I would separate the comments
covering different areas.  I would give it to other scientists in the
community who are very closely associated with certain aspects in order
to collate this material.  We would form some sort of a report and then,
from there, we would decide what we are going to do.  For example, we
decided we would ask Dr. Chang to review and collate any comments on
protozoans, Gerald Berg on viruses, Francis Brezenski and Bernard Kenner
on fecal streptococci, Donald Spino on Salmonella, Rocco Russamanno on
Salmonella, Victor Cabelli on PseudomonoB3 Richard R. Bauer on Klebsiella,
and I see, I am also on coliform.  Some of you don't even know you have
been put on the committee because I did not have any material to send
you yet and I did not want to generate something that has not happened.
     The idea was to put this in a collated form and then call this some-
what of a  "blue ribbon" committee if you want, to make following decisions:
first, is  there a need for a microbiological-guidelines manual to be
circulated among EPA,  and secondly, should we try to incorporate the
necessary  material where it differs from the potable water needs into
Standard Methods?


     Standard Methods is supposed to cover the needs both for potable

water and stream pollution analyses.  In the last edition we began to

spell out the differences in some of the paragraphs wherever there were

divergencies of needs.  I hope we continue this in the 14th edition,

where there is a definite need to have some separate procedure included,

or to perform a test differently because there are problems with bacterial

floras, etc.  The problems of stream pollution and marine waters are not

characteristic of potable waters.  Since I am on Standard Methods com-

mittee — as well as Bob Bordner, in this particular group, we should see

whethere there are some weak spots  (there are always areas for improve-

ment in Standard Methods), and we should certainly try to eliminate them

in the development of the 14th edition.  I believe you are also on the

bathing water committee, Mr. Brezenski?

     So, this in the background material.  I personally would like to

find out which of you have received this assignment, and briefly, what

your comments were.

     Guarraia:  I never  received the memorandum.

     Comment from the floor:   I think it went to  the Regional Adminis-

trators .

     Another comment:  It took mine a full month  to get  to me.   It went

to the Regional Administrator.

     Herman:  All I can  say is I never saw it.

     Bauer:  Mr. Davis,  can we go on with you?

     Davis:  First of all, I received the memorandum.  My  comments were

that we have to stick to the routine total and  fecal  coliform,  etc.

We don't have any trouble as far as Standard Methods  go.   I  hardly ever


read the book.  Sometimes things come up, like sample handling procedures.

There is a statement in the introduction of Standard Methods, 13th Edi-

tion, which says something to the effect that we are not recommending a

time condition, but more specific information is given on page 659 of

Standard Methods.

     Geldreich:  I do not recall seeing that.  I think that we are going

to have to search through this 13th edition.  If any of you find areas

with which you are dissatisfied, please send them to us.  We shall submit

proposed changes to the committee for consideration and review.  We are

asking for your input.  Believe me, you are doing as, as well as your-

selves, a favor.  If possible, we shall try to make Standard Methods more

suitable for your needs, as well as more accurate, and have it give us

the kind of data we want.

     Davis:  We are concerned, basically, with sample-handling procedures

and quality control.  We want to get as valid results as possible.  Other

than that we have to stick basically to  'tried and true1 methods.  We

have no problems there.

     Brezenski:  I received the letter and we responded.  We have diverse

opinions, among the microbiologists in the region, concerning a need for

an additional manual besides Standard Methods.  Some people  felt that

Standard Methods was adequate,  for the routine parameters, the way it was.

We have another faction that felt EPA should have a manual just for the

sake of having a manual.  My opinion, with respect to this point, was

that we really do not need another manual if we are going to just dupli-

cate Standard Methods and put the EPA name on it.  I think this is a waste

of money and  time.


     On the other hand, I feel that we need standardization in some

critical areas, and whether we do it in EPA or accomplish this in

Standard Methods is something to be worked out.  I think we need it.

This need affects the following areas:  (1) bacteriological sampling

of sediments, (2) sediment analysis — what types of samples, sample

volumes, and methods should be used, and (3) the dearth of material

in Standard Methods concerning the marine environment.  These are main

areas where we feel that we need some standardization.

     The final point here was that if an EPA manual is written, in

addition to standard Methods, it should be a representative of new

people coming in who have been absorbed from industry, from food, from

beverages, etc., and not only the people who have been set in the order

for a number of years.  We are saying that we need standardized; we do

not care whether it is done through Standard Methods or a separate EPA

Manual.  If we are going to go to an EPA Manual, we should like to have

representation from a  greater number of people.  If we are going to stay

with Standard Methods, then I feel Standard Methods is Inadequate,  as it

is, for sediment sampling from the bacteriological point of view and also

for the marine environment.

     Shimmin:  I responded to the effect that  I  felt  that Standard

Methods was inadequate as it stands because  (1)  it certainly  lacks  in

methodology, which I shall go into, and  (2)  it has to  appeal  to  a wider

range of laboratories  than we in EPA should have to address  ourselves  to.

For example, it has to include considerations  from states that  receive

their samples in the mail.  So, I think Standard Methods needs  to  be  a

11ttie less precise than perhaps EPA procedures should be.  As far as
the methodology goes, I agree with Mr. Brezeuskl In the marine area of
sediments; there is nothing in there for air; there Is certainly a lack
in pathogen methodology, e.g., Vibrio, Shigella* and Leptospira; proto-
zoan methodology is not addressed.  In the Southwest (particularly Indian
reservations) we have need for such special procedures.  Also in
Micronesia and in American Samoa, within Region IX, we have great prob-
lems with amoebic dysentary.  Standard Methods does not concern itself
with techniques like fluorescent antibody tests.  We need some type of
round-robin procedures, something on the order of those used for testing
chemical methods.  Analytical quality control, including sample handling,
is not adequately addressed to in Standard Methods either.  I do not
think Standard Methods should be discarded; I think it should be
separate reference.
     Christensen:  I have not received the memorandum.
     Carroll:  We received it two weeks after it was due.  We fully
intend to  respond to it.  I think all the points that have already been
covered, mainly the  one of sample handling time, were an issue among
the people in our region.  Also  they would like to see an expansion of
the area of pathogen identification and a section on the fluorescent
antibody application.  We felt no real critical need for a new manual
if it simply would be  a duplication of Standard Methods.  There  are some
feeling that Standard  Methods was not updated  frequently enough  and
could be supplemented  with round-robin tests,  as suggested a while ago.


     Manhart:  Primarily, I have gotten the impression that we should

just as well leave Standard Methods pretty much as it is because pro-

cedures are covered in a broad spectrum.  There were a few places, for

example, in the time element for sampling handling where we would stipu-

late a firmer statement.  If a sample is too old, say so, and throw it

out.  We should leave it like it is, because if we start putting all

this information in we have another bacteriological journal.  We feel

you can go to other texts or literature sources for such information.

     Resi:  I responded.  I feel that Standard Methods is adequate for

routine use.  It is accepted as having precedence for enforcement.  No

manual that can be put out would totally meet our needs as  far as

enforcement goes because we have to play it off the cuff and many times

we have to improvise.  No methods  that could be published could ever

fulfill our needs totally.

     There is a real need for analytical quality control in EPA.  We

should have  a group that surveys laboratories, as Mr. Geldreich does

the state laboratories.  The purpose is to help our people  in our own

organization to make sure that we  all  comply with Standard  Methods

especially in the enforcement area.

     Winders:  Our region received the request and we did  reply to

the memorandum.  I think we agree  that the  credulance supplied by

Standard Methods is something we would not  like  to see  changed by

another EPA manual.  In  our opinion, however,  it  does not  address  itself

to  the  areas we work in.  We do not work with  water  supplies.  We work

with industrial effluents.  Invariably, we  are trying to locate coliform

and fecal coliform bacteria in  industrial  effluents  of  very complex


mlxtures and compounds.  Neither the literature nor Standard Methods

address themselves in any way to the interpretation of the data we

obtained.  We realize that there is an influence of these compounds

on our data, but we do not know how to interpret the data.  We need

some help in this respect.  I am not sure that there is an answer.

     Also, we are very much opposed to the lengthening of the time of

sample storage.  We would prefer to see testing performed in the field

or at small local laboratories.  If we do this, of course, we need

some sort of quality control techniques.  I think our biggest point is

that we felt that Standard Methods did not address itself to the types

of waters we were working with.

     Geldreich:  I agree with you we have to work on this area and expand

it more in wastewater, which is the second part of Standard Methods.

You have  got a point.

     Brezenski:  For example, when you get samples from a meat-packing

house they are loaded with grease,  fat globules, etc.  You treat these

samples differently  from  a normal water sample.

     Stang:  I did respond in  four  or five categories, but not in much

detail.   One specific problem was that there is no recognized standard

procedure for sea water  (FDA does not even recognize the MF procedure),

while there is a standard for  the MPN.  Another point was that the MF

procedure for chlorinated wastewater is specifically denied in Standard

Methods.  I think everybody uses this.  I agree that we do need  a more

detailed  procedure for pathogens, specifically for the people in EPA.

My other  point was the industrial wastewater guidelines — we are

working on these now.


     Bordner:  We feel quite strongly about the need for some type of a

manual, but I would like to make it clear that we at the Analytical

Quality Control Laboratory are not talking about another Standard Methods

and I would like to dispel this idea.  Any manual of methods and quality

control should recognize and closely reference Standard Methods, but I

think we could supplement it and really help ourselves by going beyond

what Standard Methods offers.  The primary objective would be to create

a greater uniformity of methodology within our agency.  For example,

there are certain specific instances where we would like to direct

microbiologists to the MF procedure as opposed to MPN; Standard Methods

offers both alternatives but does not show this direction or guidance.

We would consider this type of manual as a guideline for bench methodology

rather than a repetition of Standard Methods.

     For pathogens, I think that it might be possible to indicate common

procedures that we In the agency could follow but not to the extent that

we would restrict ourselves from recognizing natural variation in samples.

Some of us have very good information on which of these media work best

in water and other samples.  How else are we going to get this information

than through some type of written communication and more frequent meeting

of microbiologists.  Perhaps an interim manual could accomplish much

along this line.  Another point, which we picked up from field personnel,

is that microbiologists who are in rather remote areas feel  isolated  and

need a laboratory bench type of commentary that would go beyond Standard

Methods and indicate common practice in testing routine and  special

types of samples.  Treatment of industrial effluent samples  is  a  good

example of this type of problem.

     It seems to me that the reason we really came here today, to discuss
the special problems with fecal coliforms and KlebsieHa. in industrial
wastes, is a good example of a subject that requires such guidelines.
There will be a need for uniformity in methods, for the identification
and differentiation of Klebsiellat that are certainly not going to be in
Standard Methods for some time.  Quality control is a big area where we
could get together and build such control into an additional or supple-
mentary guideline manual for EPA.  Lastly, because EPA's authority goes
way beyond water, there may be microbiological involvements in solid
waste or air; perhaps we should share information with these areas.  We
are thinking about a broader scope of manual of guidelines — call it
what you will, that would complement and not replace Standard Methods.
     Can the microbiologists who have not had a chance to reply to the
memorandum still do so?  Do they have to have an official invitation or
can they just go ahead and address a reply to Mr. Verner?
     Geldreich:  I was in hope that they would volunteer.  The bench
people are working with these problems and they are certainly having
experiences about which we would  like to hear.
     Brezenski:  That is the point I make before.  We should have a
greater representation.
     Resi:  We have a basic meed  for a list of microbiologists; I am
talking about all of EPA — I do not care who they are or where they
are.  I think that this is one of the ways we can have better communication.
     Winders:  For instance, we have some microbiologists at Research
Triangle, but there is not one of them here.

     Brezenski:  A directory could be circulated listing several people
with their areas of special knowledge or experience.  I received such a
listing of specialists in solid wastes.  They assembled a list of names
and* if you have problems in solid wastes, or a particular type of oil,
organic compound, or some process, for example, you can contact these
people, who are very helpful.
     Bordner:  That is a great idea.  That information could be included
in a manual and updated from time to time to help them people who need
such contacts.
     Bauer:  I received the letter and have not responded to it.  I will.
In general, 1 agree that if the manual would do nothing other than to
reproduce Standard Methods, it would be a waste of time.  I must confess
that,  up until now, I have not given it too much thought, but in coming
up last in this discussion and listening to the other points that have
been brought up, I shall have to agree with what appears to be a need
for a  supplemental manual that addresses itself specifically to the
problems that are peculiar to this agency, our responsibilities, and our
abilities to meet those responsibilities.  I would  favor something on
that order as a supplement to Standard Methods.  We certainly need a
vehicle in order to get some of this new technology into the hands of
the people who need to use it.
     Mr. Geldreich, would you like to  respond  to the questions  that have
been raised?
     Geldreich:  There are things that appeared in  the  13th Edition
which  I did not like at all.  What bothered me the  most were  the  comments

on the MF being used on chlorinated effluents.  We wanted to spell out
a limitation on using the membrane filter in chlorinated primary
effluents.  You just cannot use the MF on primary chlorinated effluents.
     The membrane filter method works very well on chlorinated secondary
effluents or other combinations of treatment, but it is not worded this
way in Standard Methods.  I have had a series of phone calls from state
Health Departments and from private industries selling sewage treatment
package plants to the Navy, the Air Force, and the Army and to various
cities.  All had the same comment:  They did not buy this idea that
the MP does not work on secondary effluents; I do not eithe*.  The
method works, and I am sure all of you who have worked with the MF in
this better quality of wastewater will agree.
     The problem can be demonstrated another way.  If one blends the
primary effluents sample one will find that one can actually get an
Increase in coliform count.  Part of the reason we are getting erratic
results is a gelantlnous mass  of material plugging the pores.  When one
blends secondary effluents one does not have these micrope1lets; one
does not have these erratic counts.  The MF gives one reproducible
results because the secondary  is a better quality effluent.  We are
hoping that we can really show where the limitations on the MF are and
make it clear that the MF is applicable to secondary effluents.
     The section on swimming waters has a series of Pseudomonas tests
in it that absolutely do not work.  I  repreatedly wrote to Archibald
Greenburg, even while Standard Methods was in manuscript, and told him
about the data collected by our Laboratory and  others revealing that  the

methodology on Pseudomonas in the existing Standard Methods is inadequate.
(I was not on that subcommittee.)
     The Standard Methods committee is divided into several subgroups.
Some of the members are from the academic world, some from Federal groups,
some from state groups, and some are from city and county health depart-
ments.  Microbiologists are divided into areas where they have particular
experience.  For example, Mr. Brezenski may be working on the swimming
waters and marine waters and I may be working in another section — on
standard plate count that has to be expanded, or perhaps in this MF
section.  Mr. Bordner may be working on another part, too.  We have
specific responsibilities, but we do not have the final decision all the
way around on everything that goes into that microbiology section.  We
are going to try hard to incorporate much of these comments that you arc
giving us today and those of others who, we hope in the future, will
reply to a second invitation.
     There are areas on pathogens that need more work.  The 13th Edition
was the first that had anything on pathogens In that book, and we are
hoping to expand that area, certainly for enteropathogenic E, coli.  We
need something on Leptospira because we have problems in  feed lots.  As
we have all been saying, we have a need for more expanded methods in
wastewater treatment.
     A meeting was held April 7, in our office  in Cincinnati, with  people
from the Office of Research and Monitoring.  The group  included Tom
Stanley, Gordon Roebeck, Norm Clark, Earl McFarren,  and Robert Clark  and
took up discussion of the water quality control program.  There  is  a
need for a laboratory evaluation program of the EPA  laboratories  that


are involved with field investigations.  Many of the data collections

that you are making are going to be involved in court actions, in hear-

ings, and in determinations that will affect particular pollution

problems.  We want to be sure that the data are reliable and that the

quality of the laboratory work is beyond reproach.  We do not want any-

body casting any comments that EPA laboratories are turning out inferior

data.  We have to protect and update ourselves so that we are all using

what we think are the best procedures in order to obtain the most

accurate and the most reliable data.

     The Analytical Quality Control Laboratory, where you are working,

Mr. Bordner, should become involved with the evaluations of the media.

For example, we have problems with the commercial medias that are

becoming very noticeable around the country.  The commercial manufacturers

do not  turn out a uniform quality media at  all times.  All of you have

seen  this problem from  time to time.  Years ago we  tried to set up some

kind  of a certification program on medium,  but we could not do this

because, in the Federal Government, it would in a sense be endorsing a

product.  However, NCCD has  found some mechanism whereby they have some

control on  the serological materials  that some of us  are using.  The

center  has been able  to at least have some  quality  control worked out.

I think this  is something  that  should be  included in  your program if

you have enough man  power  to  do  these things.

      Bordner:  Yes,  how do you  get  a  standard  madiura by which you can

test  other  media?  Does any  one  have  any  suggestions?


     Geldreich:  This is one of the problems that we are going to have

to solve.  In the final analysis if we cannot handle them this way, we

are going to have to come up with chemically defined media that will

meet our needs in coliform and other tests.  For example, we use bile

salts; they are variable.  Difco makes them one way; BBL makes them

another; Oxoid in England makes them a different way.  Peptones involve

trade secrets.  One company makes a peptone one way and goes to a certain

point, stops, and calls it Peptone X, while another company boils a

little longer, adds a little more of something, and calls it Peptone Y.

They are not quite the same, but they use them in our media.  As a

result,  the quality of some of  the medias we have developed fluctuates,

depending upon which product was used.

     We  certainly need evaluation of the procedures and equipment.  There

is  going to be more and more instrumentation forthcoming.  We need back-

ground information which  could  very well be developed out at your shop,

Mr. Bordner,  that would help all of us.  If we are  going to buy some  of

this equipment, we should like  to know what are  the desirable specifi-

cations; you have been doing it on membrane filters for  us since  I  got

out of that area some years ago.  You have performed analysis on  the

waterbath recently and supplied this Information.   This  could be  expanded.

     Brezenski:  You brought up the question about  what  is a  standard

medium.  Is there a possibility of setting up  specifications  based  on

what we  think  the specifications should be?  For example,  total coliform

media should have a  recovery of a  given percent  using  certain organisms.

Suppose  we have  a list  of specifications  that, we say,  have  to be met by

a medium.  If the medium meets these specifications, this is the quality

control provided.  For example, NCDC has a problem with dye concentrations

used in the fluorescent antibody test for beta hemolytic strep.  This is

creating a big problem in the different diagnostic laboratories.  NCDC

issued a publication when they evaluated a number of different dyes.

They found out that some of the dyes have 10 percent dye and 90 percent

inert material — in others, 30 percent dye.  When you make up a 20

percent solution of this dye, it really may not be the same as Allied

or some other product.  The Center is specifying that the dye must meet

this percent concentration, and then, the quality control program is

more effective.

     Geldreich:  This is one of the troubles we have in getting a decent

sheen on M-Endo medium.  The dye content of the many different lots of

basic fuchsin range anywhere from 80 to 99 percent.  The inert material

is a great unknown; it could be toxic.  The concentration of the active

ingredients varies.  Since we are doing a sulfite-basic fuchsin titration

to arrive at the proper proportion to put in the medium for sheen develop-

ment, it is very important to know the concentration.  I do not believe

that the manufacturers, once they establish the proportion, continue

the quality control.  They cut corners because of costs.  The quality of

these materials  fluctuates.  We need some way to correct this situation.

     Bordner:  We have written to BBL and Difco on two occasions about

their quality control procedures.  BBL provided a detailed description

of the Company's quality control.  It stated that the firm uses a series

of organisms in  order to test  the water quality media.  Difco carries

out quality control procedures on all lots of their products, but the

Company did not send prepared descriptions of them.  We are suggesting,
here, that if we determine the cultures that should be used, it would
be a more directly controlled program for which we have some input.
     Geldrelch:  We need to develop specifications or guidelines that
we could circulate among EPA laboratories for review and comment.
     Resi:  They still certify dyes for staining, but certification for
our needs is rather fuzzy.
     Shlmmin:  Something else that we need to do is to establish some
stronger contacts with Dlfco and BBL than we have at present.  We dis-
covered that the manufacturers slightly changed the formulation of
brilliant green agar and did not bother to inform anyone.
     Herman:  Dr. Gordon with the EPA Laboratory. College Station, Alaska
and  I  found a marked difference among lots of Difco and BBL M-FC media.
We wrote both BBL and Difco.  We got quick, straight-forward answers
back from BBL, but none from Difco.
     Resi:  We find that we do not achieve the same responses  from the
organisms, owing to differences in the water characteristics in different
geographical areas.  There are differences within  the medium,  but  there
are  also differences in the samples that we are testing.
     Herman:  1 am talking about the same sample in our  area versus  the
medium; not the medium versus various samples.
     Brezenski:  I feel that we are not really talking  about any water
that is different in a major way.  For example,  I  would say that when
we are testing different  types of water,  be  it fresh  or salt water,  we
have to look at the flora.  This is probably  the biggest variable  that


is going to affect our testing for indicator or background organisms and

it is going to continue to vary.

     We have been receiving reports that a lot of organisms grow, on the

M-FC medium, that are not fecal coliforms, when one checks the blue

colonies.  This is particularly true in warm marine waters and in shell-

fish areas.  We do not see this in temperate waters, for example, off

the New Jersey coast in the Atlantic Ocean, where we investigated this

problem with the shellfish people.  We did some work with the people at

the Needham Laboratory to confirm pink and blue colonies on M-FC medium.

We obtained a high recovery of fecal coliforms in terms of the blue

colony characteristics.  The shellfish people say that they get a high

percentage of pink colonies that turn out to be fecal colifortn and yet,

a high percentage of the blue colonies are not fecal coliforms.  I think

the problem here is the microflora, the indigenous flora in one area.

     Geldreich:  Part of this relates to the rosalic acid.  If the

rosalic acid solution is made up and put into that medium within a

few minutes, that indicator does cause some color additions to the

colonies.  It is not an indicator system in a sense, but it produces

some non-fecal coliform colonies that yield other colors, such as yellow

and red.  Rosalic acid should be evaluated.

     Periodically the manufacturers come up with a new gadget.  One of

these gadgets is called the Coli Counter.  Of course, I immediately was

flooded with requests from our  regional offices:  "What do you think of

this product?"  Pan American Health called me and commented:  "Say, this

looks great.  We can send it down with our engineers and they can monitor

the (potable) water."  The Coli Counter only takes one mlllillter, and
this la not enough volume to look at potable water; so they recognize
immeidately that they cannot use it for that purpose.  It does have
some crude application as a quick-and-dirty test to determine the
density in a stream.  Do not use this method when you are trying to
establish reliable data.  It would give you a rough answer on some
surface waters.
     I think that you have evaluated these, Mr. Bordner, and I have too.
Your data and mine indicate that the Counter has considerable limitations.
The service you render in taking a look at some of these products would
be of help to the rest of us for use as background material in order to
answer questions on  this type  of subject.
     Bordner:  The brochures are very appealing, and a lot of people
have asked about it.
     Geldreich:  There are some comments made in the brochures that
imply  that it  is a  standard method.
     Bordner:  They  say  it is  a "standard  material," but  the  implication
to some  readers is  that  it is  a standard method; it  is not!
     Winders:  We use  the Coli Counters when the engineers go into  the
plants for pre-plant surveys before we do  industrial waste surveys.   The
results  tell which  outfall stream has  sanitary  wastes  in it.   We do not
use them as  a  quantitative method but  only to  show the presence  of  that
type of  bacteria.
     Geldreich:   If one  had to use  those  results  as support  data in some
court  hearing,  one  certainly is going  to  have  a problem.


     Winders:  We do not record it as data at all.  When we go back in,

we sample these streams to run a quantitative analysis,

     Bordner:  So, we are saying, "Use it, but do not use the data."

     Winders:  Well, let us say one plant has 15 outfalls; it is better

to use this method than to swamp our laboratory with 15 different micro-

biological analysis samples every day.  We find that coliforms are in

only two outfalls; then, we concentrate on those.

     Bordner:  I think that is the type of rough testing that was

originally proposed for the Coli Counters.

     Geldreich:  As a final comment, some of you may have seen a manual

called Evaluation of Water Laboratories, which we produced for the

evaluation of state and other laboratories that test potable waters.

The manual has tied in with the Standard Methods book and with what we

considered to be the best operating practices.  We are now in the process

of rewritting this manual to include not only the bacteriological exami-

nation but also the chemical examinations of potable water.  Earl McFarren

has  finished his part.  I am expanding the bacteriology to cover not

only the needs for potable water testing but also to give the rationale

of water quality testing for stream pollution or recreational waters.

The manual is about 60 percent finished; it will be about 150 to 200

pages.  I hope to finish it by the end of the year.  We shall circulate

it and we hope that this manual will be of some value, to give you some

additional information at the bench level and to explain material that

is only vaguely referred to in Standard Methods.

     Actually. Standard Methods  is a  cookbook approach; it does not

explain the background for the procedures.  We are hoping between this

interest in a guidelines' approach that many of us have mentioned here
and this new second edition of an Evaluation of Water Laboratories, that
we shall be able to give you the supplemental reference material to go
along with Standard Methods and that it will be a greater help to you
than is Standard Methods alone.
     When we do laboratory evaluations, we have a form which we follow,
based not only on Standard Methods, but also on our best laboratory
practices.  We analyze the procedures the laboratories are doing, their
equipment needs, the kinds of equipment they have, and the media.  We
even get into areas involving stream pollution and recreational work
now because I have sections in the evaluation form that cover methods
for pathogen detection,  etc.  This type of material is very helpful
when we do a laboratory  evaluation.  The check list is a starting point
that we use to make a lot of notes.  We then come up with a report
covering the capabilities of that laboratory in order to examine not
only potable waters but  also recreational water.  I wanted you to be
aware of the manual; it  may fit into your concerns about Standard Methods.
We hope that we can come out with some material in the future that will
be more helpful.
     1 understand there  are 125 EPA facilities scattered around  the
United States —• not only in the  continental area but also in Alaska
and other  areas of our control.  We certainly have to know where  these
mlcroblologists are.  We would like to identify them, offer our
assistance to them, and  ask them  for ideas  from their experiences.
I think we are making a  start with this meeting and  I hope somehow we


can find a way to have this kind of meeting once a year.  Mr. Bauer, do

you have any thoughts on how we can do this?

     Bauer:  I talked to Mr. Gallagher before the meeting started, and

he indicated a certain willingness on the part of the NFIC to sponsor

this type of meeting on a regular basis.  I certainly think that a meet-

ing of this sort is in order if Field Investigations is willing to assume

the responsibility.  I intend to talk to Mr. Gallagher about this again

if you are in agreement with that approach.

     Brezenski:  There is only one problem.  I attended a meeting on

methods in Washington with Mr. Verner's and Mr. Stanley's groups.  They

may have some ideas along these lines; at least it was made evident at

that meeting.  I think someone will have to touch base with them.  I

think we should make it definitely known that if they do not identify

people and sponsor such meetings, we will.

     Guarraia:  I think this purpose is a little different from theirs.

You were talking here of addressing problems of microbiologists


     Brezenski:  They would cover the same  thing.

     Guarraia:  Yes, but would all  the microbiologists be attending

this meeting?

     Brezenski:  They would make it open to all the microbiologists

whom the regions will let go.  For  example, the region will not let go

of every microbiologist they have,  but I think it is up to the region

or the microbiologist to express his/her desire, to make it  important

enough so  that  the administrators are willing  to let them go.


     Geldreich:  We need some sort of a strong recommendation from who-

ever Is going to sponsor the meeting to the people in charge of the

mlcrobiologists so that we get the change to go.  Some of us may find

It rather difficult to convince our supervisors, but if we get the

letter from somebody requesting our specific services and explaining

the program needs and its value, I think many of us will get the


     Bordner:  This approach from a Washington office would help along

that line, would it not?

     Geldreich:  It worked in this particular instance.

     Bordner:  Would this invitation be coming from the Office of

Monitoring and include microbiologists who are specifically research

people or would it include all microbiologists?

     Brezenski:  It includes both th NERC groups and regional people.

     Bordner:  If this is going to be a periodic meeting would it be

advantageous to meet in different geographical areas of the country,

as the large organizations do, to give a better chance for attendance

in local areas?  Should this be a consideration?

     Brezenski:  Yes, as they do for chemistry.

     Bauer:  The permit people do that same thing.  They hold meetings

in different regions each time.  I would like to wrap  up the meeting at

this time; there are a number of people who have planes to catch  and I

should like you all to have the opportunity to meet  any individuals whom

you have not met thus far.  I should like to  thank you for attending and

thank the National Field Investigations Center-Denver  for  organizing and

sponsoring this meeting.


             APPENDIX A



                                   PULP AND PAPER INDUSTRY

          Effluent  Limits

               1.   Production Basis.  The average permitted effluent level, in
          pounds per  day, shall be computed based on maximum daily production,
          in  air-dry  tons,  as determined at the time of application.

               2.   Schedule Selection.  A permittee with existing abatement
          program should be at  least  achieving Attachment B levels and should be
          put on a  schedule to  achieve Attachment A levels prior to January 1,
          1976.  A  permittee that is  presently without an approved pollution abate-
          ment program should be required to expeditously achieve Attachment A

               3.   Coliform.  This is a significant parameter for admixtures of
          industrial  wastewater and sewage.  Because of the complex sewering of
          most mills  the absence of sewage must be established by sampling and
          analysis  for fecal coliform organisms.  If sewage is present the follow-
          ing effluent limit shall be imposed:

               "Organisms isolated in the fecal coliform test and associated
               with pathogenes, shall not exceed 1000 organisms per 100 ml (1).

               (1)  Where receiving waters are classified for shellfish
               harvesting or contact  recreational sports, the effluent limits
               shall  be reduced to comply with the established water quality

               4.   Toxic Materials, Oil and Grease.  These parameters should be
          considered  to determine their significance on an individual basis.  If
          they are  determined to be significant then the appropriate "Special
          Conditions" should be applied.

               5.   pH. The pH  shall  be maintained between 6.0 and 8.5 unless un-
          usual receiving water considerations necessitate a variance (i.e., the
          natural pH  is outside this  range).

               6.   Other Limits.  The following may be significant parameters
          depending on production and receiving water characteristics:




                                           TABLE I
                                   PULP AND PAPER MILLS STUDIED


     Boise Cascade


     American Can


Oregon City.
Salem, Ore.

Lebanon, Ore.

Halsey.  Ore.

unbleached sulfite
magnesium base

bleached sulfite
ammonia base

unbleached sulfite
ammonia base

bleached kraft
Springfield,  Ore.      unbleached kraft



primary settling

primary settling

aerated lagoons
aerated lagoons
secondary settling

aerated lagoons
Mill effluent
Willamette River
upstream of
Publishers Co.
Willamette River
downstream of
Publishers Co.

Mill effluent
Willamette River
upstream of
Boise Cascade
Willamette River
downstream of
Boise Cascade

Verified Verified
TC/100 ml FC/100 ml FS/100 ml K. pneumoniae/100 ml
i 2



       TABLE I (Cont'd.)
Mill effluent

McKenzie River
upstream of
Weyerhaeuser Co.
McKenzie River
downstream of
Weyerhaeuser Co.

Mill effluent

Willamette River
upstream of
American Can Co.
Willamette River
downstream of
American Can Co.

TC/100 ml
FC/100 ml
FS/100 ml

K. pneumoniae/100 ml

Mill effluent

S. Santiam River
upstream of
Crown Zellerbach
S. Santiam River
downstream of
Crown Zellerbach

                                    TABLE II

                      COLIFORM DATA - EFFLUENT FR.OM POND I

                 Date          MPN*/100 mis.       MF**/100 mis.

                6/11/69                              10,300,000
                6/12/69                               3,900,000
                6/13/69                               8,400,000
                6/16/69           24,000,000          19,000,000
                6/18/69                              24,000,000
                6/23/69                               8,300,OpO
                6/24/69            2,200,000           3,600,000
                7/10/69            9,200,000          11,000,000
                7/14/69            2,100,000           1,400,000
                7/16/69            1,400,000          16,000,000
                7/21/69           24,000,1000           4,800,000
                7/22/69            1,400,000           1,500,000
                •J/^/Ao              350,000             400,000
                 *MPN  - Most  Probable  Number  of  organisms/100 mis.
                       by  the  multiple  tube  method.

                **MF   - Actual  count ojE  organisms/100  ml.  as-deter-
                       mined by  the membrane filter  (MF)  techniques.

                                   TABLE  III
                               TOTAL COLIFORM
                               MILL EFFLUENTS

                                      TABLE IV
                                  FECAL COLIFQRM
                           IMVIC TYPE DISTRIBUTION IN PULP
                             AND PAPER MILL EFFLUENTS
iMViC Types Publishers
— + 0
— +- 0
-+-- 0
+— 0
++-- 0
--H-- 0
---H- 89.4
-+-+ 0
+--+ 0
+-+- 0
I [ i n
1 i r ™ \j
-H--+ 0
+--H- 8.5
_-i t 1 ^
Mil 0
Crown Z.
American Can
All Mills
   *  Insufficient data.

                     TABLE V
Oregon City, Ore.
Salem, Ore.
Crown Zellerbach
Lebanon, Ore.
American Can
Halsey, Ore.
Springfield, Ore,
Combined Data
* Insufficient culti
MF Total
IMViC Types




jres examined
Coliform Test
K. pneumonia




MF Fecal Coliform Test
% Identified
Aerogenes as
IMViC Types K. pneumoniae

89.4 90.5

* *

37.8 100

100 75.0
87.5 100
68.4 92.5

                                               TABLE VI
                                COMPARISON OF TOTAL COLIFORM iMViC TYPE
                                    OCCURRENCE FROM VARIOUS SOURCES
7., From
Pulp Mill *
IlMViC Types Effluents
-H— 3.3
— H- 52.9
-+-+ 12.4
-+++ 9.9
++++ 5.8
+-++ 4.1
-H--+ 5 . 0
-- H—
% From
Animal £./



7. From
Foliage £/

7. From
Flowers —'


7. From
Insects £/



% From
Undisturbed —'

70 From
Polluted — '



* Combined data from five mills.

a/ Geldreich, E.E., Sanitary Significance of Fecal Coliforms in the Environment.,
   U. S. Department of the Interior - Publication WP-20-3.

             TABLE VII

CZ Lebanon, Ore. Fecal  Coliforra  Confirmations
Colony Description
All Blue
Blue Centers
Light Purple Center
Lactose +
B.C. +
Type I E. Coli.

B"10                                       TABLE  VIII
Jefferson STP Influent
Lebanon STP Influent
Sweethome STP Influent
STP Effluent & Rec. Waters
Wapato STP Effluent
U.S. Plywood Log Pond
Idaho Frozen Foods(Potato)
IDA Frozen Foods
IDA-Frozen Foods
Independent Meats
Independent Meats
H & H Packing Inf.toLagoon
H & H Pacing Effluent
Northwest Packers
Kummer Meats
TC/100 ml
                                                                    Colonies   %       %
                                                                    Picked   Lactose  E.C.-t
                                                                    20        75      27
                                                                    20        95      37
                                                                    20        90      67
                                                                    19        50      .0
                                                                    14        79       0

                                                                    19        84      31
                                                                    19        74       0
                                                                    20        70      27
                                                                    20        45       0
                                                                    20        40       0
                                                                    12       100      42
                                                                    15        87      15
                                                                    20        95       0
                                                                     8        88      14
                                                                    20        95       5

                                                                     7        57       0
                                                                    20        75      33
                                                                    20        55      55
                                                                    20        80      63
                                                                    19       100       0
                                                                    11       100      91

Fruit Packing Effluent
Fruit Packing Effluent
Fruit Packing Effluent
Fruit Packing Effluent
U & I Sugar
Alpenrose Dairy
Tillamook Dairy
Ridgefield Wildlife Refuge
Ridgefield Wildlife Refuge
Ridgefield Wildlife Refuge
Ridgefield Wildlife Refuge
Ridgefield Wildlife Refuge
Shadybrook Dump
Columbia Slough
TC/100 ml
TABLE VIII  (cont'd)                              1*11
                             Colonies   %        X
                             Picked   Lactose  E.C.+
                             14       64        0
                             20      100        0
                             20       90        0
                             14       79        0
                             20      100        5

                             20      100        0
                             12       92       67

                             20       65       23
                             20       85        6
                             20       90       40
                             20       80       18
                             20       10        0
                             13       62        8
                             12      100      100

October It Survey
Total Co]lform
Sea. No.
+ Sampling Location
River Mile
Avg/100 ml
Range/100 ml
Fecal Cnliform
Avg/100 ml
Range/100 ml
Fecal Streptococci
Avg/100 ml
Ranr,p/100 ml
Snake River Above; American Falls
Above Idaho Falls Upper
Power Plant
At Grandview Drive,
Idaho Falls
At Broadway Bridge,
Idaho Falls
At 17th Street Bridge
p .L-|r| <:. .-U
Below Idaho i-^l-ts-S^P „,.
Pull* iff "£-
Above Shelley at
Bcimut Bridge
2 Miles West of Shelley
Near Firth
2 Miles North of Blackfoot
Near Blackfoot
At Blackfoot
5 Miles Above Tilden Bridge
At Tilden Bridge
6 Miles Below Tilden Bridge
40-300 (4)
190-500 (4)
100-800 (4)
100-1000 (4)
13,000-58,000 (4)
16,000-42,000 (4)
7,000-20,000 (3)
10,000-38,000 (4)
16,000-35,000 (4)
21,000-45,000 (4)
5,400-34,000 (4)
20,000-60,000 (4)
5,400-34,000 (4)
(4) 42
< 4-20
4. 10-20
10 (3)
< 4-90 (3)
< 10-140
20-200 (3)
5-45 (3)
25-130 (3)
40-500 (3)
340-480 (2)
150-3,6(10 (3)
120-1000 f4)
200-5000 (A)
150-2000- (3)
100-7,000 (4)
60-210 (4)
100-4,100 (4)
60-210 (4)

                                                                          TABLE X
                                                      BACTERIOLOGICAL ANALYSES OF WASTE DISCHARGES

                                                                   October  1971 Survey

Total Collform
ta. Ho8.000,000
> 31 ,000 ,000->flO ,000 .000
No data
Coll Corn
Range/100, ml
One sample
One sample
One sample
Avs/100 nl
Ho data
1,000.000(2) 1
RniEf/100 r>l
One maple
• One sample
,000,000- 1,000,000
53080     U 4 I Sugar Co.
          + Storet Station Number

          * Estimated flow

                                                     TABLE X  (cont'd)
                                                     October 1971  Survey

. No.+ Sampling Location
067 A & P
RM 648.4
065 Amalgamated Sugar
RM 646.9/8.7
1201 Paul STP
RM 646.9/7.2
X>57 Main Drain
RM 646.9/1.8
Snake R.
Main Drain
Main Drain
Snake R.
Total Coliform
Avg/100 ml
15",000, 000(2) 5,500
19,000,000(4} 7,000
Focal Coliforiu
Rango/100 nl
One sample
One sample
Avg/100 ml
No data
Range/100 ml
* One saziple
One sample
One sample
AvR/100 ml
. 8
R*nge/100 nl
One sample
One •aaple
* Estimated flow

                                           TABLE XI
                            COMPARISON OF TOTAL COL IFORM INViC TYPE
                                OCCURRENCE FROM VARIOUS SOURCES

              % from                   %  from                 %  from                  %  from
              Sewage Treatment  Plants  Potato Waste            Meat  Packing  Effluent   Fruit Packing Processes
 IMViC TYPES   (data from 5  plants)     (data  from  9  sources)   (data from  4  plants)    (data from 4 studies)
— +
+-H--                  -                         -                       1.5                     -            w

                APPENDIX C



          INDUSTRIAL WASTES,  Donald L.  Herman, Research  Microbiolegist

          National  Water Quality Laboratory,  Duluth,  Minnesota.

          For:   Environmental Protection Agency  Seminar,  THE  SIGNIFICANCE  OF

                 FECAL  COLIFORM IN INDUSTRIAL  WASTES,  Denver,  Colorado,  May 4-5,


          A.   Introduction  and Review of the  Coliforms:

                Aquatic microbiologists are presently  faced with  pertinent, direct,

           and challenging questions regarding the use of "coliforms" in water quality

           surveillance relating to standards.  Messrs. Geldreich,  Butterfield, Wattle,

           Clark, Kabler,, Bauer, and many others have  faced these questions

           in the past, resulting in numerous  publications.  The  microbiologist in

           EPA is now faced with .taking a critical review of oneself, one's work,

           results, and questions if the "coliforms" can indeed survive the enduring

           test of  time.  New microbiologists  in our agency must  aquire a working

           knowledge of the literature review (past, present and  future needs).  They

           must have a fundamental understanding of chemistry, metabolism, engineering,

           biology  (including fish and other aquatic organisms),  and a practical

           understanding  of bacterial survlvial.   All microbiologists should have

           field experience and  openly exhange information with other EPA micro-

           biologists in  order to gain self-confidence.  All other parts of  the EPA

           team  can be of considerable aid to the microbiologist who is not  scared

           to ask questions.  He must be  able to define "coliforms"  and certainly

           what  bacterial types  generally make up the  group called "coliforms."  The

           days  of  "just  a  coliform  count" are not  enough  when one may be  faced with

           a  possible court action.

     Brecher and Nestle. (1970) in their book entitled ".Environmental Lav;

Handbook" state that contamination of water by disease-producing organisms

is always a distinct possibility.  Bacteriolectistr. look for organisms

that inhabit the human  intestine, since these arc the type that breed

the most dangerous diseases.  Then, these bacteriologists make the state-

ment "Coliform bacteria are harmless organisms that also inhabit the

intestine."  This is a  comment we hear many times from the parties not

wanting to  face the possibility  of having a high coliform count resulting

from their  discharge.   It  is  for this reason that a quick review of the

"Coliform Group" should be made  to refresh our minds.  Smith  (1969),

Morgan  (1965), Burrows, and Moulder, Lewert and Rippon (1969), and Merchant

(1950)  all  define the  coliform group as Escherichia coli, Enterobacter

aerogenes (Aerobacter), Kelbsiella pneumoniae (Friedlander's  bacillus),

Paracolobactrum, Knterobacter freundii, Enterobacter cloacae  and Merchant

(1950)  adds Proteus.

     A  more prer.Lne .literature review  results In the following facts  about

each of these "harmless organisms."

E.  coli - is unabashed as  an  opportunist when out Its natural settling.

It  is one of the most  common  causes of pyelonephritis and  urinary  tract

infections  and is an important cause of epidemic diarrhea  in  nurseries

for newborn infants.   It  is in the intestinal tract of practically all

vertebrates and some invertebrates (Geldreich,  et al, and  Snoddy  (1971).

Causes  "White scours"  in  calf dysentery  (Merchant 1950)  and  infantile

diarrhea  (0rskov  1951).   Rail (1970) reports E.  coli in  swine;  in  house-

hold pets,  as reported by  Meyer, et al  (1971),  Mian  (1959)  and  Hackel,

et  al (1960).  Pfuhl  (1902) reported survival in  contaminated soil for

101 days  and Kokolios  et  al (1971) reported  survival  of  E.  coli  in soft

agar for  41 years.

        Enterobacter  aerogenes  (aerobacter)-  Found  frequently in  the  same

        environment as IS.  coli  and  is also  found  on grain, plants and soil

        (Smith 1969 and Geldreich,  Simonds (1915) used the term

        "foamy organs" when involved in some  types  of  human  infections.

        KLebsiella pneumoniae (Friedlander's  bacillus)-  This organism is not

        new to the water quality field since  it was adopted  as a  fecal indicator

        organism in  water surveillance in England in the late iSOO's  (Heukelekian,

 196^).  It is considered normal flora of  the  intestinal tract

        (Kendall 1916, Morgan 1965  and Hentges 1967).   Klebsiella pneumoniae

        has become known as serious infection when involved  in pneumonia with

        mortality rates reported in humans  from 12^ to as high as 90% in untreated

        cases resulting in concern  for the  young  and the aged patients (Baehr 1937>

        Morgan 19&5i  an^ Branson 1968).  Blazevic, (1972)  ranks Klebsiella

        as number two in urinary infections (18.8%) with E_.  coli  being number one

        (33.995).  Matsen (1970) states that Klebsiella comprise  18.7% of all

        urinary tract bacterial isolates and  the  disturbing  "propensity"  to

        become resistant to antibiotics which is  also confirmed  by  Burrows,

        (1969).  Montogomerie, (1970)  review Klebsiella  in fecal flora of

        renal-transplant patients and from  other  environment sources.  He  cites

        cases of infection related to milk shakes having Klebsiella present at

        the level of 10  and finding the organism in potato  salad,  luncheon

        sausage, brawn, curry, bacon, porridge, cheese and vegetable mixture,

        egg custard and in milk shakes thirteen times.  Montogomerie states

        "These results differ from other studies of hospital infections in which

        predominant Klebsiella serotypes were isolated within an institution and

        which implicated predominant strains of Klebsiella in the environment as

a source of these infections".  Burrows, (1969) cites Klebsiella

as paralytic disease of moose and metritis of marcs; however, Merchant

(1950) states the same cases but clarifies that _K. genitalium is found

in mares, _K. paralytica is related to the moose transfered by ticks and

also pathogenic for the bull, sheep and chicken.

Proteus-  Proteus morganii is thought to cause infectious diarrhea in

infants, Proteus mirabilis is ranked second in urinary tract infections

by Smith (1969) and third by Blazevic, (1972).

Paracolobactrum- Smith (1969) includes the Bethesda-Ballerup group,

Arizone group, Providence group, and the Hafnia group which may be

associated  with the intestinal disease area.

       This  review then would seem to make a different picture than the

one of coliforms being referred to as "harmless organisms", however, one

should also remind  themselves that almost any bacterial agent in the

wrong  place at the  right time could lead to a infection within the human

or animal body.

       Mr. Bauer asked that I include the organism Pseudomonas aeruginosa

in my  discussion, therefore, I have also made a short literature review

of this organism.

             Ringen,  (1952)  found PsGudomonas  aoruginosa present  in

        of healthy humans  and considered the  normal  habitat  of  this  organism

        to be the human intestine.   He  also found  aeruginosa in 90$  of  sewage

        samples,  2$>  of stable manure samples, ~5% in  soil  samples and none in

        natural waters. Reitler (1957) concluded  that this  species  should be

        taken into account,  as well  as  E.  coli in  assessing  the suitability of

        water for drinking and also  cited  finding  10^  present in normal health

        persons.  Hoadley  (1968) states concern of aeruginosa in recreational

        use  water related  to outer ear  infections  and  suggest that numbers

        in excess to 100/lOOmls in  swimming waters should be in suspicion.

        Hoadley further indicates no correlation between  populations of E_. coli

        and P.  aeruginosa  in drinking  waters.  Hunter,  (19^+7) cites a

        epidemic  of  diarrhea in a new-borne nursey caused by P_. aeruginosa resulting

        in 9 deaths  related to contaminated milk supply.

        B.  Experiences with Coliform and  Enteric  Organism Isolations from

            Industrial Wastes and Secondary Effluent:

              The majority of the information reported in this  section was data

        collected prior to the time  I joined  EPA,  except  the data on domestic

        secondary effluent and part of  the data on paper  and pulp.   Since I

        become  involved in water pollution in 1962 my interest  developed around

        the enteric  survival  and isolation both from industry and clinical cases.

        Since I joined the National Water Quality Lab we  have also had a chance

        to obtain results from  some of the nations highest quality natural water.

I must admit it was a rude awakening when one roalizeu how little

"mother nature" haa paid attention to both the clinical and engineering

text books regarding survival of the enterics in natural receiving waters.

The engineering courses I had covered the coliforms very quickly and from

that point on we discussed the bacterial biological mass related to loading,

flows, retention times and general disinfection (usually in % reduction).

      The types of industrial waste I became involved with includes the

following- Canning and Food Processing, Beverage- carbonated and non-

carbonated , potato waste, Meat and Slaughter Waste, Paper and Pulp Waste

and finally Domestic Secondary Effluent.  I was soon to find out that

the controling parities did not want to know what levels of enteric

organisms may be present in their waste but wanted strictly just "coliform"

counts, if at all.  It was not until the states involved or Federal action

was indicated that the enteric information was wanted and then they wanted

a years study done in 30 days with no written reports unless it was in the

favor of the industry concerning their final discharge.  I am glad to report

that this was not the case concerning all industries I worked with but the

majority can be included.

      The following tables show the levels of enteric and indicator

organisms we found in these various waste.  The improvement in isolations

from water relates directly to the improved methods resulting from the use

of the membrane filter and media developed in the more recent years as

understanding of the microbes within the aquatic environment improved.

The items that are strongly indicated is  the  type of nutrient addition,

colloidal suspension, .»!.• bottom sludge development and  seed  organisms.

          1.  Canning and Food Processing Waste:

               a)  Initial stage of aerated  lagoons

                     Initial BOD, mg/1 - 1,400  (range: 160 -  2,400)

                     Flow rate - 125,000 gpd

                     Total coliform/100 ml -  320,000  (range:  2,000  to  1,500,000)

                     Fecal coliform/100 ml -  15,000  (range:  100  - 60,000)

                     (1) Results of Fecal Coliform Isolations


                         .E.  coli, type I -                35.0
                         Klebsiella pneumoniae            55.0
                         Enterobacter species             3.3
                         Pectobacterium                    6.0
                         *Salmonella species                0.7

               b)   Post-treatment effluent - discharged  without  chlorination

                     Final BOD,  mg/1 - 30  (range:  6.0 -  180), 97.8% reduction

                     Retention  time  in aerated  lagoons - 21  days

                     Total  coliform/100  ml - 3,000 (range: 800 - 7,000)

                     Fecal  coliform/100  ml - 140 (range: 20  - 500)

                      (1)  Results  of  Fecal Coliform  Isolations


                           £.• coli,  type  I                 32.1
                           Klebsiella pneumoniae          42.9
                           Enterobacter species             5.0
                           Pectobacterium                  9.3
                          *Salmonella species             10.7

           * Random isolates were confirmed as Salmonella typhimurium and
             12 isolates, confirmed  of Shigella sonnei.

2.  Beverage Waste-(carbonated and non-carbonated):

     a)  Initial stage of aerated lagoons

           Initial BOD, mg/1 - 1,600 (range: 800 - 13,000)

           Flow rate - 36,000 gpd

           Total coliform/100 ml - 7,500,000 (range: 15,000 - 64,000,000)

           Fecal coliform/100 ml - 50,000 (range:  2,500 - 750,000

           (1)  Results of Fecal Coliform Isolations


               IS. coli, type I                   5.6
               Klebsiella pneumoniae            68.0
               Enterobacter species             15.0
               Pectobacterium                    7.0
              *Salmonella species                4.4

     b)  Post treatment effluent - discharged without chlorination

           Final BOD, mg/1 - 75  (range  6.1 - 160), 95.3  % reduction

           Retention time in aerated lagoons - 14  days

           Total coliform/100 ml - 4,500  (range  15 - 230,000)

           Fecal coliform/100 ml - 1,700  (range  5 - 75,000)

           (1)  Results of Fecal Coliform Isolations


                !• coli, type I                  20.6
                Klebsiella pneumoniae            67.0
                Enterobacter species             4.7
                Pectobacteriutn                   5.8
               *Salmonella species                ].9
* Random isolates were  confirmed  as  Salmonella typhimurium.

          3.  Potato Waste:

                a)   Initial  stage  of  aerated  lagoons

                     Initial  COD,  mg/1  -  14,000 (range:  1,500 - 45,000)

                     Flow rage  -  75,000 gpd

                     Total  coliform/100 ml - 160,000,000 (range:  2,500,000 - 750,000,000)

                     Fecal  coliform/100 ml - 265,000 (ranpc:  18,000 - 850,000)

                      (1)   Results  of Fecal Coliform  Isolations


                           E_.  coli, type I                  0.9
                           Klebsiella pneumoniae           81.1
                           Emterobacter  species              9.4
                           Pectobacterium                   6.9
                          *Salmonella species               1.6

                b)   Post treatment effluent - discharged without chlorination

                      Final COD, mg/1 -  140 (range: 170 - 3,500),  99% reduction

                      Retention time in  aerated lagoons - 41 days

                     Total coliform/100 ml - 1,700 (range: 220 - 13,000)

                      Fecal coliform/100 ml - 45 (range: 7 - 11,000)

                      (1)   Results of Fecla Coliform Isolations


                          JL- coli, type  I                  15.5
                          Klebsiella pneumoniae            60.1
                          Enterobacter species              8.9
                         *Salmonella species                15.5
           * Random isolates were confirmed as Salmonella typhimurium and
             Salmonella St. paul.

A.  Meat and Slaughter Waste:

     a)  Initial stage of aerated lagoons

           Initial BOD, mg/1 - 26,000 (range: 2,600 - 45,000)

           Flow rate - 28,000 gpd

           Total coliform/100 ml - 670,000,000 (range: 3.5 mill. - 8.5 bill.)

           Fecal coliform/100 ml - 6,500,000 (range: 1.5 mill. - 1.2 bill.)

           (1)  Results of Fecal Coliform Isolations


                £• coli,  type I                 56.9
                Klebsiella pneumoniae           21.5
                Enterobacter species            13.8
                Pectobacterium                   0.5
                *Salmonella species               7.3

     b)  Post-treatment effluent - discharged without chlorlnation

           Final BOD, mg/1 - 400 (range  150 - 2,200), 98.5%  reduction

           Retention time in aerated lagoons - 36 days

           Total coliform/100 ml - 250,000 (range 17,000 - 1,300,000)

           Fecal coliform/100 ml - 18,700 (range 7,800 - 65,000)

           (1)  Results of Fecal Coliform Isolations


                JL- coli.,  type I                 65.8
                Klebsiella pneumoniae           24.1
                Enterobacter species             6.9
                Pectobacterium                   0.9
                *Salmonella species               2.3
 * Initial  random  isolates were  confirmed  as  Salmonella  typhimurium and
  St. paul.   Resampling  confirmed  these findings  and  added  var.  Copenhagen
  as well  as  Shigella  sonnei  and  flexneri.   Orders  were then passed that
  no more  Salmonella or  Shigella  isolations  should  be done  because of
  reports  to  NCCD.

     This  was a interesting case  in  that  wind  draft carried organisms from
 the aeration  lagoons at  times into the plant resulting  in loss of finished
 goods and  contamination  of entire  areas of  the plant  to allow processing
 to continue.

         5.  Paper  and Pulp Waste:

              This  phase  of work  resulted  in  some  real  challenges with  the  utnost

         use of  tact  and  diplomatic  relationship in  order  to  obtain  valid samples.

         It was  a question whether  any  of  the samples  representing  full production

               a)  Initial stage  of  aerated lagoon

                  Flow  rate -  unknown,  Initial BOD,  mg/1 - 2,200  (ranker 35-4,500)

                  Total  Coliform/100 ml -  1,500,000  (range:  500,000  -  5.7 nail.)

                  Fecal  Coliform/100 ml -  8,000 (range: 10 -  87,000)

                   (1)   Results of fecal coliform isolations.


                      —•  co^i»  type  i                    4.4
                      Klebsiella pneumoniac              85.0
                      Enterobacter species                9.5
                      Pectobacterium                     0.8
                     ^Salmonella species                 0.3

               b)  Post-treatment - discharged without  chlorination.

                   Final BOD,  mg/1 - 3,600  (range:  400  - 6,000), approx. 64% increase

                   Retention time in aerated lagoon - 2 days (claimed),  4'  discharge pipe

                   Total Coliform/100 ml -  57,000,000 (range:  2-79 mill.)

                   Fecal Coliform/100 ml -  130,000  (range:  60 - 750,000)

                   (1)  Results of Fecal Coliform Isolations


                      ,E. coli, type I                     0.4
                      Klebsiella pneumoniae              92.3
                      Enterobacter species                 6.7
                      Pectobacterium                      0.6
                     *Salmonella species                  0.008
          * Random isolates confirmed as Salmonella typhimurium.

    Field survey work has been done on one of the rivers used by

the industry for discharge of the paper and pulp waste.  This resulted

in demonstrated fish kills due to lowered DO in 1969 and 1970, COD from

12 to 260, Total Coliform/100 ml ranges from 10 to 171,000 with Fecal

Coliform/lOOml ranged from 3 to75,000 and Fecal Strep./100 mi ranged from

6 to 1,350.  Eleven type of pathogenic bacteria were found and confirmed

by CDC including Klebsiella pneumonia, Shigella sonnei, Salmonella typhirmurium,

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

We found one isolate of Klebsiella pneumonia above the major discharge area

while recovering types 1^, 58, 21, 22, 31, 33, 35, 65, 7, 56 and 67 up to

and including 27 river miles below the outfall.  Within the river we

found active "colonized" zones of Klebsiella pneumonia types 1^, 58, 21,

22, .and 31•  These types were tested for mouse pathogenicity and were shown

to be positive.  In the zones of "colonization" bottom rocks were recovered

that had pure cultures of Klebsiella pneumonia growing on the surface edges.

The chemistry factors of interest in these areas were- decreased DO, none

for some 7 miles, decreased pH of the water and presence of reducing and

hexose sugars.  Salmonella was found once when the fecal coliform was

3/100 ml and always when the fecal coliform was above  ^0/100 ml.  The

fecal strep, to fecal coliform ratio-ranged from 1:1^.6 to 325'1-  Bottom

samples below area of discharge ranged from 50,000 to  5i600,000 in total

coliform; fecal coliform ranged from 300 to 23,000, both test related to

100 mis of interface water samples.  In all bottom samples E. coli,  type I,

Klebsiella pneumonia and Salmonella typhirmurium were  demonstrated.

          6.   Secondary  unchlorinated  effluent:

               Total  coliforms/100 ml        581,000

               Fecal  coliforms/100 ml         32,500

               (1)   Results  of Fecal Coliform Isolations


                    !E.  coli, type I                 62.0
                    Klebsiella pneumoniae           18.0
                    Enterobacter species            14.3
                    Pectobacterium                   3-6
                    Salmonella species               2-1

               This waste has been used in disinfection and fish effects from tanks

          receiving the various effluents.  Presently the data is being processed

          and reviewed for publication by staff members at a later date.

          7.  Antibiotic Sensitivity Results on Random Selected Klebsiella pneumoniae

              Isolates are shown on the attached tables.

               Pseudcmonas aeruginoaa - Test were conducted for this organism

          resulting finding in all cases ranging from 3/100 mis. to 6,700/100 mis.

          This area became a major work load due to isolation and confirmation with

          a limited staff.  Hektoen Agar  (King 1968) was used which many times

          resulted in a coin toss as to what the colony was really indicating.  We

          have found Pseudomonas aeruginosa present in  low numbers where fecal

          coliforms were not detected and also have found the fecal coliforms and

          Salmonella when Ps.  aeruginosa was not found.   I agree  about  some  concern

          related  to this organism but  prefer  to think  of it  as  a working part  of

          the  total picture  and  not just  the "only" indicator.

# 36>d-3
Sec. Effl.ST
# 363-e-3
Sec. Effl.ST
# 365-b-3
# 365-h-3
# 366-k-3
# 371-C-3
# 426-g-3
Ozone Sec.Effl.
# 426-h-3
Ozone Sec.Effl.
# 429-C-3
Sec. Effl.
# 489-3
Lake Superior
Duluth Intake
# 1-6
Paper & Pulp
# 24-7
Paper & Pulp
# 24-12
Paper & Pulp
# 31-15
Paper & Pulp
# 31-16
Paper & Pulp
KLeb. Type
(if known)

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC





0.25 rag















10 meg















2.0 mg















5 meg















100 meg















             R = Resistant, I = Intermediate and S =  Sensitive

# 363-d-3
# 363-e-3
# 365-b-3
# 365-h-3
# 366-k-3
# 371-C-3
# ^26-g-3
Ozone Sec.Effl.
# ^26-h-3
Ozone Sec.Effl.
# 429-C-3
Sec. Effl.
# 429-X-3
Sec. Effl.
# ^89-3
Lake Superior
Duluth Intake
# 1-6
Paper & Pulp
# 2^-7
Paper & Pulp
# 2^-12
Paper & Pulp
# 31-15
Paper & Pulp
# 31-16
Paper & Pulp

KLeb. Type
(if known)
at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC

at CDC





Polymyxin B
300 units
















= Resistant, I =
30 meg
















Intermediate and
30 meg
















S = Sensitive
30 meg

















10 meg


















    Our experience seems to indicate that the bacterial flora of receiving

waters and the behavior of intestial track flora may have some interesting

relations.  The addition of too much nutrients and substrate can totally

disrupt the normal human natural flora balance.  Infants and adults when

fed cheese or apple cider in excess have resulting problems.  Kendall(l9l6)

discussed this nutritional effect.  High quality water is also extremely

low in total coliforms and void in many areas of fecal coliforms, except

below beavor ponds, shore bix-ds, etc.  These enterics found usually do not

survive long in cold, high quality fresh water without proper substrates.

    The adverse effects by nutrient and waste addition are clearly shown

by Gallagher,  (1970), Arthur,, Anierson,,

USDI.FWPCA  (1967), US HEW,PHS  (1966 & 1965) relating to waste in Puget

Sound, Slime Growth in Pulp Waste, Combined Waste, Pollution of Mobile

Bay and Pollution on  the Red River of the North.  One must remember that

these are just some of the numberous reports already published.

    The coliform areas have been well reviewed and tested since 190^

by Geldreich, (some 16 major articles in my file), Van Donsel,

Gordon (1970), Smith and Twedt  (1971), Senn, (1963) and many

others.  Tho text entitled "Sanitary Significance of Fecal Coll forms

In The Environment by Geldreich (1966) continues to answer questions

each time I review this reference.

    We are presently under planned, well organized criticism by  some  of

the major industrial and other large volume effluent nources.  Tho  time

for EPA microbiologist to become well organized  ic long  overdue.   We  must

work together, exchange information, have a excellent  working  knowledge

of literature reviews and above all obtain facts with a cool head.

Review of J3. coli isolations showed that 97.2 % of these were E_. coli,

type I.  In all cases when fecal coliform counts were greater than 100 per

100 mis. Salmonella was recovered.  Eickhoff (1972) on KLebsiella points

out the critical need for epidemiology work on PCLebsiella pneumonia in

recreational waters.  A proposal of this type was submitted by Dr. John

Matsen in 1971, #HMA and has been lost in Washington.  The need for such

is present even greater now.

    The developing legal actions across the nation seem to indicate the

need for a bacterial identification unit in EPA.  We must be able to

determine more often what constitutes many of the organisms found in

the various coliform counts.

    The plain fact remains at present microbiologist must become well

organised and work closely with the chemist, biologist, engineers,

adminstration and ourselves.  I basically believe EPA has the start

of a great  team therefore we should make full use of any and all facilities

and talent  at our finger tips.

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     WATER  POLLUTION", Annals of the Entomological  Society  of Americaj.Vol.  64,
     No.  6, p. 1467-1468.

         Water Resources Bulletin, Vol. 6, No. k, pp. ^73-^75, AMICl-591*.

     69.  Thorn, B.T.  (1970) ."KLE3STET.T.A IN FASCES", The Lancet 2:1033.

     70.  USDI, FWPCA and Washington  State Pollution  Control Commission  (1967),
         kjk pages.

         NORTH", Robert A.  Taft Sanitary Engineering Center, Cincinnati,  Ohio.

         THE RED RIVER OF  THE NORTH  (Minnesota-North Dakota)'/ Robert A. Taft
         Sanitary  Engineering Center, Cincinnati, Ohio.

     73.  Van Donsel, Dale  J.  and Edwin E. Geldreich  (1971), "RELATIONSHIPS  OF
         Vol. 5, pp. 1079-1087.

              APPENDIX D


Identification of Total Coliform Colonies
Selected Industrial Wastes
Concentration Colony Identification

Colony" Total Klebsiella- Other Non
Coliforms*/100 ml Descrip- No. Enterobacter Entero- Entero- EC
Source Total
Sugar Waste
Bone char filter
Washings 11,000
Sugar Waste
Treatment Plant 8,000
Fecal tion Colonies JjjcnxLfaciens bacteraceae bacteraceae (+)
<67 Green 24 18 4 22
<67 Green 23 21 0 21
Pulp Mill
Process Water
23,000     >60,000   Metallic
     Typical colonies on M-Endo and M-FC Agar
     M Endo Medium

                 TABLE II
    Identification of Various Colonies
From the Fecal Coliform Membrane Filter Test
        Selected Industrial Wastes
Goliforms*/100 ml
Source Total Fecal
Sugar Waste
Filter Washings 11,000 <67
Sugar Waste
Treatment Plant 8,000 <67
Sugar Waste
Treatment Plant
Pulp Mill
Process Water 23,000 >60,000
Pulp Mill
Process Water
San Joaquin R.
Stagnant Area <67 >6,000
Pulp Mill
Cooling Water <67 <67
* Typical colonies on M-Endo and M-FC
0 M-FC Medium

Colony0 Total
Descrip- No.
tion Colonies

Gray-white 18
Gray-white 14
Blue 7
Pink 24
Blue 22
Gray-blue 24
White 23

Colony Identification
Klebsiella- Other Non
Enterobacter Entero- Entero-
liquifaciens bacteraceae bacteraceae
15 3 0
14 0 0
6 1 0
7 1 16
3 3 16
1 3 20
0 18 5

C+) (+)

1 18
0 14
: 7
4 15
5 19
0 8
0 3



                                   FIGURE I
          .  10

        _J  10
                      '0/18     10/19    10/20    10/21

                         DATE   SAMPLED

                             FIGURE 2
             Composition of Total Coliform Population Found in Pulpraill Wastes
                     COUNTS  per 100 ML
Phmary  influent-   total colifoms
primary  influent Klebsielia
primary  effluent   total conforms
primary  effluent Klebsielia
 secondary influent  tofal  coh'forms
 secondary influent  Klebsielia
 .secodary  effluent   total coliforms
 secondary effluent  Klebsielia

                              FIGURE 3
              temp. 2O c
 24        36


       FIGURE 4
            7100 Ml.
pine need
Fe cea 1








                          44.5      45-0     45-5


   V. P






    (4.45 C)
                               FIGURE .
dork blue
~ -- _--
' —
light blu