A <-;*,/''
EPA-600/2-77-052
August 1977
Environmental Protection Technology Series
Office of Research and Development
U.S. Environmental Protection Agency
Ohio 45268
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U S Environmental
Protection Agency, have been grouped into nine series These nine broeid cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The nine series are'
1 Environmental Health Effects Research
2 Environmental Protection Technology
3 Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8 "Special" Reports
9 Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-77-052
August 1977
NEW MICROBIAL INDICATORS OF DISINFECTION EFFICIENCY
by
Richard S. Engelbrecht
Elaine F. Severin
Mark T. Masarik
Shaukat Farooq
Sai H. Lee
Charles N. Haas
Ajit Lalchandani
University of Illinois
Urbana, Illinois 61801
EPA-IAG-D4-0432
Project Officer
C. W. Chambers
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
This study was conducted
in cooperation with
U.S. Army Medical Research and Development Command
Washington, D.C. 20314
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U. S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.
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FOREWORD
The Environmental Protection Agency was created because of
increasing public and government concern about the dangers of pollution to
the health and welfare of the American people. Noxious air, foul water,
and spoiled land are tragic testimony to the deterioration of our natural
environment. The complexity of that environment and the interplay between
its components require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention, treat-
ment, and management of wastewater and solid and hazardous waste pollutant
discharges from municipal and community sources, for the preservation and
treatment of public drinking water supplies, and to minimize the adverse
economic, social, health, and aesthetic effects of pollution. This publi-
cation is one of the products of that research; a most vital communications
link between the researcher and the user community.
This paper provides new methodology enabling the investigator to
quantitate alternative bacterial indicators of pollution. This provides
a much more meaningful measure of the effectiveness of disinfection of
wastewater and water supplies, thereby limiting the introduction of con-
taminants into our waters and helping to protect the quality of our water
supplies.
Francis T. Mayo
Director
Municipal Environmental Research Laboratory
1 n
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ABSTRACT
Since the coliform group of organisms is less resistant to chlor-
ine than many pathogens, including viruses, the utility of both yeasts and
acid-fast organisms as indicators of disinfection efficiency was evaluated.
The geometric mean densities of acid-fast organisms, yeasts, and fecal
coliforms in domestic wastewater were 1.5 x 104, 5.3 x 104 and 3.9 x 106
organisms/100 mt, respectively. Full scale chlorination of trickling
filter and activated sludge effluents reduced the densities of these organ-
isms by 0.5, 3.0, and 5.0 logs, respectively. A mean density of 2.2 x 10$
fecal col i forms/gin wet feces was found in 30 different samples of fecal
matter, and that of yeasts was 562/gm in 29 of 30 samples. A mean density
of less than 100 acid-fast organisms/gm was found in only 13 of 30 fecal
samples. Four yeasts, Candida paJia.pi>-Ltot>, C. feAoie/c, T'^cJiobpoian
and RkodototLnJOjO. fiubsia; and three acid-fast organisms, Mycobac-
M. phJLuA. and M. .6megma£c4 were found to occur commonly
in domestic wastewater. The resistance to free chlorine was: acid-fast
organisms > yeasts > poliovirus type 1 Mahoney strain > S&tmonejtla
typk-lmu>vium > E&c.k&iic}ua. coti using mixed cultures, including two acid-
fast organisms and four yeasts at pH 6, 7, and 10, and 5° and 20°C. The
resistance to inorganic chloramines (5:1 wt ratio Cl2:NH3-N) was M.
kofLtuA-tum > C. panapA-iZoA-ift > M. p/i£e/c > E. coti at pH 7 and 20°C.
C. poAap-4xL£c4-c6 appeared to be more resistant to ozone than E. coti at
room temperature.
This report was submitted in fulfillment of Contract No. DADA 17-
72-C-2125 (U.S. Army Medical Research and Development Command) under the
partial sponsorship of the U.S. Environmental Protection Agency through an
interagency agreement (EPA-IAG-D4-0432). This report covers a period from
May 1, 1972 through April 30, 1975.
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CONTENTS
Foreword iii
Abstract iy
Figures vi
Tables viii
Acknowledgment 1X
1. Introduction 1
Overall Objectives of the Study 1
Summary of Previous Work 1
Background and Objectives of Report 4
2. Conclusions 6
3. Recommendations 8
4. Materials and Methods 9
Enumeration Technique for Yeasts - Field Samples 9
Enumeration Technique for Yeasts - Laboratory
Samples 10
Enumeration Technique for Acid-Fast Organisms -
Field Samples 10
Enumeration Technique for Acid-Fast Organisms -
Laboratory Studies 16
Enumeration Technique for Fecal Coliforms,
Ebchnru-ckia coLL, Salmon&aJLta and Poliovirus . 16
Occurrence of Acid-Fast Organisms, Yeasts,
and Fecal Coliforms in Wastewater 19
Occurrence of Acid-Fast Organisms, Yeasts,
and Fecal Coliforms in Fecal Matter 19
Chlorination Experiments 20
Chloramine Experiments 20
5. Results and Discussion 25
Occurrence of Acid-Fast Organisms, Yeasts,
and Fecal Coliforms in Wastewater 25
Occurrence of Acid-Fast Organisms, Yeasts,
and Fecal Coliforms in Fecal Matter 28
Identification and Ecology of Acid-Fast
Organisms and Yeasts Common to Wastewater . . 30
Chlorination Experiments 31
Chloramine Experiments 45
Ozone Studies 55
6. Work in Progress 61
References 63
Appendix 65
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FIGURES
Number Page
1 Enumeration Technique for Yeast Organisms 11
2 Enumeration Technique for Acid-Fast Organisms 17
3 Occurrence of Acid-Fast Organisms in East Side Urbana-Champaign
Wastewater Treatment Plant and Saline Branch of Salt Fork River,
Illinois 27
4 Response of Test Organisms in Mixed Culture to 0.4 mg/l Free
Available Chlorine Residual at pH 6 and 5°C 32
5 Response of Test Organisms in Mixed Culture to 0.9 mg/l Free
Available Chlorine Residual at pH 6 and 5°C 33
6 Response of Test Organisms in Mixed Culture to 0.5 mg/£ Free
Available Chlorine Residual at pH 6 and 20°C 34
7 Response of Test Organisms in Mixed Culture to 0.45 mg/l Free
Available Chlorine Residual at pH 7 and 5°C 35
8 Response of Test Organisms in Mixed Culture to 0.48 mg/l Free
Available Chlorine Residual at pH 7 and 5.1°C 36
9 Response of Test Organisms in Mixed Culture to 1.0 mg/l Free
Available Chlorine Residual at pH 7 and 5°C 37
10 Response of Test Organisms in Mixed Culture to 0.55 mg/l Free
Available Chlorine Residual at pH 7 and 24°C 38
11 Response of Test Organisms in Mixed Culture to 0.5 mg/l Free
Available Chlorine Residual at pH 10 and 5°C 39
12 Response of Test Organisms in Mixed Culture to 1.0 mg/l Free
Available Chlorine Residual at pH 10 and 5°C 40
13 Response of Test Organisms in Mixed Culture to 1.0 mg/l Free
Available Chlorine Residual at pH 10 and 20°C 41
14 HOC1 Concentration vs. Percent Survival of the Yeast Group
of Organisms 43
VI
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FIGURES (continued)
Number Page
15 HOC1 Concentration vs. Percent Survival of the Acid-Fast Group
of Organisms ........................... 44
16 Response of E&chojiichJjCL co£/c to Chloramine (5:1 of Clo:NhU-N),
pH 7 and 20°C .................... ....... 46
17 Response of Candida panap-!>-ito^-if> to Chloramine (5:1 of
C12:NH3-N), pH 7 and 20°C ..................... 47
18 Response of Mycobact&fviuin faita-itum to Chloramine (5:1 of
C12:NH3-N), pH 7 and 20°C ..................... 48
19 Response of Myc.oba&t&u.um pki^i to Chloramine (5:1 of
CL2:NH3-N), pH 7 and 20°C ..................... 49
20 Time vs. Concentration of Free Chlorine or Chloramine to
Achieve 99.9 Percent Inactivation of M. fioKtuAtum, M. pht&i,
C. pastapAiloAiA , and E. coti at pH 7 and 20°C ........... 51
21 Inactivation of C. pa>vzpt>JJtot>jj, and E. coti by Ozone with a
Detention Time of 12 Sec in Buffered, Demi nerali zed Water ..... 57
22 Equilibrium Kinetics of C. panapAitoAiA Inactivation by Ozone
in Buffered, Demi nerali zed Water ................. 58
23 Effect of Initial Density of C. paAap* LLobiA on Degree of
Inactivation for a Given Concentration of Ozone .......... 59
24 TOC of Various Densities of C. poAap^^o^^u ............ 60
vn
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TABLES
Number Page
1 Growth of Acid-Fast Isolates, M. pkl&i and M.
After 3 Days of Incubation in Various Media ........... 12
2 Composition of Various Media Evaluated for the Growth of
Acid-Fast Organisms ....................... 13
3 Percent Relative Growth of Acid-Fast Isolates, M. pkla-i and
M. faita-itum with Respect to Test Media No. 1 (Control) ...... 14
4 Growth of Acid-Fast Organisms in Wastewater Effluent Expressed
in Terms of Percentage Based on the Number of Colonies Found
in Test Media No. 1 (Control) .................. 15
5 Densities of Indicator Organisms at the Champaign-Urbana
Sanitary District East Side Plant and Its Receiving Water .... 26
6 Occurrence of Indicator Organisms in Fecal Matter ........ 29
7 Estimated Reaction Order of Concentration for Inactivation of
Selected Organisms by Free Chlorine and Chloramine at pH 7
and 20°C ............................ 52
8 Concentration of Disinfectant Needed to Achieve 99.9 Percent
Inactivation of Selected Organisms in 30 Minutes at pH 7
and 20°C ............................ 53
9 Time to Achieve 99.9 Percent Inactivation of Selected Organisms
with a Residual of 1.0 mg/l of Free Chlorine or Chloramine
at pH 7 and 20°C ........................ 53
vm
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ACKNOWLEDGMENTS
This study was performed in the Environmental Engineering Laboratories
of the Department of Civil Engineering, University of Illinois at Urbana-
Champaign and was funded jointly by the United States Environmental Pro-
tection Agency and the United States Army Medical Research and Development
Command. The helpful and inspirational suggestions provided by Dr. David
Foster, especially during the earlier portion of the study period covered
by this report, are gratefully acknowledged. Much of the data and
analysis of the results of the mixed culture chlorination study were pro-
vided by Mrs. Feyza S'urucu. The cooperation of the personnel of the
Urbana-Champaign Sanitary District is appreciated.
The critical comments, worthwhile suggestions, and encouragement
provided by Mr. Cecil W. Chambers, EPA Project Officer, and Colonel Leroy
H. Reuter of the United States Army during the course of the study are
greatly appreciated.
IX
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SECTION 1
INTRODUCTION
OVERALL OBJECTIVES OF THE STUDY
The major objective of chlorination of either wastewater efflu-
ents or water supplies is to produce a finished product that is acceptable
from a public health standpoint. In order to assess the efficiency of
chlorination in terms of destruction of pathogens, there is a need for a
reliable bioindicator which is at least as resistant to chlorine as the
most chlorine resistant pathogens. The bioindicator should also be
rapidly and unambiguously quantifiable in chlorinated effluents by simple
and easily applied techniques. Since it would appear that coliforms, the
most commonly applied group of bioindicators, do not meet the criterion
of being as resistant to chlorine as the most resistant pathogens, the
suitability of coliforms for judging the efficiency of chlorination may
be seriously questioned. This is particularly true where wastewater
reuse schemes are being considered, since, in these situations, protec-
tion of the public health is paramount.
The coliform organisms have been quite helpful in the past in
providing information on the potential presence of bacterial pathogens
in many types of waters. However, it would seem appropriate, in light
of their inability to satisfy the public health needs in many situations,
to reevaluate their application on a case-by-case basis. It is suggested,
therefore, that the indicator organisms should be selected on the basis
of the purpose and information required, e.g., the inactivation of resis-
tant pathogens by chlorination. It was with this purpose in mind that
this study was undertaken.
Representatives of two groups of organisms, yeasts and acid-fasts
have been isolated from treated and untreated chlorinated wastewater.
Studies to date have indicated that these groups of organisms are sub-
stantially resistant to chlorine and, as a result, should be considered
as possible indicators of chlorination efficiency. It is the purpose of
this project to critically analyze these groups of organisms for their
utility as indicator organisms.
SUMMARY OF PREVIOUS WORK
In a previous report to the Office of Research and Development
of the U.S. Environmental Protection Agency, dated February 1974, a
detailed literature review concerning the applicability of the currently
used fecal coliform index for determining disinfection efficiency in
terms of pathogenic organisms was presented.1 The basic conclusions
drawn from this literature review are restated here briefly.
1
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The literature indicates that the presently used coliform
group of organisms is far too sensitive to chlorine to be reliable in
indicating the potential presence of chlorine resistant pathogenic
agents, such as viruses. Although the response of viruses to chlorine
appears to vary widely, the existing data suggest that, with few excep-
tions, viruses are more resistant to chlorine than coliforms by a con-
siderable margin. In comparing the resistance of vegetative bacterial
pathogens to that of coliforms, it was reported that the relative resis-
tances were quite variable. For the majority of vegetative bacteria,
however, it appears as if their resistance to chlorine and chloramine
is essentially the same or less than that of the coliform bacteria.
Spore forming bacteria were found to be considerably more resistant to
chlorine than vegetative cells. The resistance of cyst forming, patho-
genic protozoans was also reported to be greater than coliforrn organisms
in laboratory studies. The cysticidal chlorine dose, however,, is highly
dependent upon the density of cysts used. Since the density of cysts
encountered in the field is normally much less than the densities used
in many of the laboratory experiments, it is likely that reported cysti-
cidal doses were greater than those required in the field. In cases
where acid-fast bacilli are of concern fromapublic health standpoint,
such as with My co bacterium tub&icuJLoAiA, the biochemical nature of the
mycolic acid and lipid structure of the cell wall of acid-fast organisms
appears to confer a degree of chlorine resistance greater than that
associated with the enteric bacteria. It was the basic conclusion of
this literature review that coliform organisms are not acceptable as
indicators of the disinfection efficiency in terms of the more chlorine
resistant pathogenic organisms.1
Studies to establish a new indicator of wastewater chlorination
efficiency were initiated with the isolation of 135 pure cultures of
organisms from various sources. Most of these organisms were obtained
from chlorinated secondary effluent, although three cultures were isolated
from chlorinated tap water and 13 cultures were obtained from the Depart-
ment of Microbiology at the University of Illinois at Urbana-Champaign.
Details of the isolation techniques have been reported previously.1
Isolates from the secondary wastewater effluent included both gram posi-
tive and gram negative rods, cocci, and mycelia with white, orange and
yellow pigments. Many of the bacteria isolated were gram positive spore
forming rods. These spore formers were deemed unsuitable as indicator
organisms. Because of the wide variety of enrichment media and incubation
temperatures used, as well as the duration of this phase of research over
several seasons, it was felt that the chlorine resistant isolates obtained
were probably representative of what might be present in chlorinated
wastewater effluents.
To further screen the isolates, chlorination experiments
using free chlorine at pH 7 were performed in chlorine demand free
water. The criteria selected for this initial screening of isolates
was that, to be considered resistant, the organism must exhibit some
degree of survival at concentrations and times up to 1 mg/£ free chlor-
ine for 30 minutes contact time.
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Using this criterion, the non-spore forming isolates which
showed the highest degree of chlorine resistance were yeasts and acid-
fast bacilli. Of these, one yeast, previously reported as isolate No. 30,
and two acid-fast bacilli, previously reported as isolates No. 132 and
No. 134, were selected for further study because of their superior
resistance to chlorine. Isolates No. 30, 132, and 134 have since been
identified as Candida. paJiapA , Mt/cobacte/w-uw pkl.&i and Myc.obact&uum
respectively.
More refined experiments with free chlorine were performed at
various concentrations ranging from 0.1 to 2.0 mg/£ free chlorine in pH 7
chlorine demand free phosphate buffer, at 20°C, using these organisms.
It was determined that C. pasiapt>
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A third selective method evaluated was a membrane filter
staining technique. The staining procedure used included Brook's carbol
fuchsin-dimethylsulfoxide stain and Brook's malachite green stain con-
taining acid-alcohol decolorizer.
It was determined that a combination of the oxalic acid pre-
treatment, Cohn and Middlebrook 7H9 selective medium with a combination
of the antibiotics, followed by the Brook's carbol-fuchsin staining pro-
cedure, gave satisfactory results for acid-fast enumeration. The method
currently used for acid-fast enumeration is discussed later.
BACKGROUND AND OBJECTIVES OF REPORT
This study was initiated 1 December 1969 under a contract
(17060 EYZ) from the U.S. Environmental Protection Agency. A report,
covering the period 1 December 1969 through 30 April 1972, was submitted
on 26 September 1972 and subsequently published by the U.S. Environmental
Protection Agency in February 1974.1 During the period covered by this
report, 1 May 1972 through 31 April 1975, the study was funded equally by
the U.S. Environmental Protection Agency and the U.S. Army Medical Research
and Development Command.
Work covered in the previous report1 finalized the initial phase
of the study which involved a search for a valid indicator organism of
disinfection efficiency. This phase of the project has been reviewed
briefly under "Summary of Previous Work" of this report and includes a
discussion of the isolation and screening for resistance to chlorine of
two groups of organisms commonly found in wastewater, acid-fast bacilli
and yeasts. Also included in this initial phase of study was the prelimi-
nary development of selective enumeration procedures for the detection
of acid-fast and yeast organisms in wastewater and surface water.
The second phase of the study, as discussed in this report,
includes the final work on the development of reliable enumeration tech-
niques. A detailed study of acid-fast organisms, yeasts, and fecal coli-
forms in wastewater to ensure the omnipresence of these organisms in
domestic waste streams and a study of the occurrence of acid-fast organ-
isms and yeasts in fecal matter to help define their source in wastewater
have been included. From these studies it was found that yeasts and acid-
fast organisms were consistently found in domestic wastewater. However,
while fecal matter was a fairly consistent and reliable source of yeasts,
fecal matter could not account for the high densities of acid-fast organ-
isms in wastewaters. To further define the source of these organisms in
wastewater, as well as in surface water, several of the more common acid-
fast organisms and yeasts isolated from wastewater were identified as to
genus and species.
To avoid the dangers associated with comparing the chlorine
response of indicator organisms and pathogenic organisms from separate
experiments, performed under possibly differing conditions, mixed culture
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experiments with free chlorine and varied pH and temperatures were
performed to study the relative resistance of yeasts and acid-fast
organisms along with &>cJn.
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SECTION 2
CONCLUSIONS
Additional information has been presented regarding the potential
use of acid-fast organisms and/or yeasts as indicators of chlorination
efficiency.
In determining the densities of acid-fast organisms, yeasts and
fecal coliforms in raw and treated wastewater, it was found that, while
fecal coliforms had a geometric mean density of 3.9 x 10^ organisms/100 mi
in raw wastewater, acid-fast organisms and yeasts consistently ranged 2
logs less, averaging 1.5 x 104 and 5.3 x 104 organisms/100 mi, respectively.
In treated effluents, fecal coliforms averaged 6.2 x 105 and 1.1 x 105,
acid-fast organisms averaged 5.8 x 103 and 4 x 103, and yeasts averaged
1.3 x 104 and 5.4 x 103 in trickling filter and activated sludge effluent,
respectively. In these cases, the yeast densities appeared to be less
variable than both acid-fast organisms and fecal coliforms. This investi-
gation was performed over a sufficient period of time to conclude that
acid-fast organisms and yeasts are omnipresent in wastewater and may, as
a result, be useful as an indicator organism without artificial seeding.
No substantial seasonal variation was noted.
Full scale chlorination of secondary wastewater effluent was
observed to reduce the densities of acid-fast organisms, yeasts and fecal
coliforms 0.5, 3.0 and 5.0 logs, respectively. These data provide further
evidence of the higher chlorine resistance of the proposed indicator
organisms when compared to the frequently used fecal coliform group.
Examination of 30 different samples of fecal matter indicated
that fecal coliforms were present in all of the samples with a geometric
mean value of 2.2 x 10^/gm wet feces. The density of yeasts varied over
a 4 log range with a mean value of 562/gm wet feces in 29 out of 30 samples.
Only 13 of 30 samples were positive for acid-fast organisms, averaging less
than 100/gm wet feces. It appears that the presence of acid-fast organisms
in wastewater may not be due entirely to fecal matter. On the other hand,
fecal matter appears to be a major source of the yeasts found in domestic
wastewater. Investigation of other possible sources needs to be made to
justify yeasts as an indicator of fecal pollution, however.
Four yeasts and three acid-fast organisms, commonly found to
occur in domestic wastewater, were identified as Candida.
Candida knuAnA., TsiickoApotion fieAme-ntanA, and RhodototLttla siubia;
tn^ium ^ofita-Uum, M. phi&i, and M. AmzgmatiA. A brief survey of the
literature revealed that, while the species of yeasts identified could
be commonly found in feces and the gastrointestinal tract of man, no
definite insight into the source of the acid-fast organisms in wastewater
could be made.
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Chlorination experiments performed using a mixed culture of
yeasts, acid-fast organisms, Ebchesu.?' .ia. coll A., Satmonatta. typki-muJviuw ,
and poliovirus type 1 Mahoney strain at several pH values and at 5° and
20°C in buffered chlorine demand free water confirmed the superior
resistance of the acid-fast and yeast groups of organisms to the other
organisms studied. In all experiments the order of resistance to free
residual chlorine was acid-fast organisms > yeasts > polio > SaJtmoneMLa >
E. CD Li, except at pH 10 where the response of the acid-fast organisms
and yeasts was similar. With increasing hypochlorous acid concentration,
the survival of yeasts was less for a given contact time; however, sur-
vival of the acid-fast organisms was still high. Increasing the ratio
of hypochlorous acid to hypochlorite ion by changing the pH had more
effect on the yeasts than on the acid-fast organisms. The response of
poliovirus to free chlorine in all experiments was more comparable to
that of the yeasts than to the acid-fast organisms.
The response of C. pajiap£-Ltot> , M. ^ofita-U^am, M. pkLaA., and
E. coti to chloramines in pH 7 buffered chlorine demand free water, using
pure cultures of the organisms, suggests that the yeasts and acid-fast
organisms are 1.5 to 5.6 times more resistant than E. coLL, considering
the concentration needed to inactivate 99.9 percent of the organisms in
30 minutes. The yeasts and acid-fast organisms were found to be between
20 and 100 times more resistant than E. coti. when considering the time
of contact needed to inactivate 99.9 percent of the organisms when a
residual of 1.0 mg/£ is used.
From a comparison of the relative resistance of the acid-fast
organisms and yeasts to hypochlorous acid, hypochlorite ion, and chlora-
mines at pH 7, a generalized observation can be made. Both the yeasts
and acid-fast organisms are highly resistant to all forms of chlorine;
however, the difference in resistance to hypochlorous acid, hypochlorite
ion, and chloramines is not as great as reported for other organisms.
A study has been initiated to find the inactivation response
of the proposed indicators to ozone. Preliminary studies have shown
that C. p(Vw.p&-iLo& is more resistant than E. coti at ambient tempera-
ture and pH 7. It has also been found that the degree of inactivation
is profoundly affected by the initial density of yeast in the feed
solution. Presently, there are not sufficient data to draw definite
conclusions about the response of the proposed indicators to ozone.
Information to date concerning the resistance of the proposed
new indicator organisms, yeasts and acid-fast organisms to various forms
of chlorine and the distribution of these organisms in wastewater indi-
cates their potential use as indicators of wastewater chlorination
efficiency.
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SECTION 3
RECOMMENDATIONS
1. To more fully evaluate yeasts as a potential indicator of disin-
fection efficiency, and perhaps fecal pollution, information should
be collected as to their source in wastewaters and surface waters
through a detailed literature and field study. The origin of
acid-fast organisms in wastewaters and surface waters should also
be reevaluated.
2. A detailed field study should be performed to determine the vari-
ations in the densities of yeasts and acid-fast organisms that
can be expected throughout a wastewater treatment plant and its
receiving stream over long periods of time. The question of
possible regrowth and/or die-off of these organisms should be
studied in natural waters.
3. The removal of yeasts and acid-fast organisms in batch scale unit
processes, such as those utilized in water supply treatment and
advanced wastewater treatment, should be investigated.
4. The selective growth media for the enumeration of acid-fast organ-
isms and yeasts should be reexamined with the objective of reducing
the incubation period required. Preferably, the incubation time
should be reduced to 24 hours or less.
5. A direct comparison of the resistance of mixed cultures of yeasts
and acid-fast organisms to selected species of pathogenic organisms
to various disinfectants, i.e., organic and inorganic chloramines,
ozone, and iodine, etc., should continue to be made.
6. A literature study should be undertaken to determine whether acid-
fast organisms as a group are sufficiently resistant to disinfection
to provide a margin of safety in cases where Mt/cobac£e/u.ujn
may present a threat to public health.
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SECTION 4
MATERIALS AND METHODS
Considerable time was spent in developing acceptable enumera-
tion techniques for acid-fast and yeast organisms in highly contaminated
samples, i.e., fecal matter, wastewater, and surface water (field samples).
In this section, developments in the selective methods since those last
reported1 shall be briefly discussed, as well as the enumeration tech-
niques for acid-fast and yeast organisms currently in use. In studies
where "clean systems" of pure isolates were used, i.e., in the chlorine
and chloramine experiments, the extensive procedures for enumeration of
acid-fast organisms and yeasts employed with the field samples were found
to be unnecessary. Several modifications of yeast and acid-fast enumera-
tion procedures have been made to facilitate work done in "clean systems"
and will be discussed. Also included in this secion are the enumeration
techniques for fecal coliforms, E. doli, SahnoneJULa., and poliovirus as
they pertain to the various experiments in which these organisms were
studied. The detailed procedures used to detect and enumerate these
organisms are given in Section IX, Appendix.
Detailed methods used in the preparation and performance of the
chlorine and chloramine experiments will also be covered in this section;
however, the procedures for the experiments with ozone will be dealt with
individually along with the results and discussion of the ozone experiments,
Enumeration Technique for Yeasts - Field Samples
Several different enrichment media, all adjusted to approxi-
mately a pH of 3.5, were examined for the enumeration of yeasts. The
pour plate and membrane filtration method were both employed and evalu-
ated from which it was determined that the membrane filtration method was
superior. Attempts to eliminate contaminating organisms included the
incorporation of various chemicals into the media and the creation of an
oxygen limiting environment. One such technique evaluated to create an
oxygen limiting environment was the use of an overlay of a 1:1 vaseline-
mineral oil mixture. This method, however, proved to be difficult to
manipulate. Another technique was explored and proved to be acceptable.
Briefly, the method consists of filtering a sample using a black membrane
filter; the use of a black membrane was found to increase visual contrast.
The filter is then placed in an inverted position on a thin layer of
solidified yeast extract-malt extract (YMA) medium in a culture dish.
The filter is then covered with a layer of softened medium. The inverted
filter technique inhibits the growth of filamentous fungi and antibiotics
inhibit aerobic spore forming bacilli. Colonies may be counted from the
bottom of the plate after an incubation time of 30 hours at 25°C. Incu-
bation at room temperature (18-22°C) may take somewhat longer to produce
countable colonies. Incubation at temperatures higher than 20°C, while
providing a more rapid growth rate, resulted in a lower recovery of yeasts.
-------
The use of rose bengal as a mold suppressant was also evaluated. Using
a pure culture of Candida paJia$&-ULoi>u>, a concentration greater than
0.005 percent rose bengal was found to restrict the colony size. A
concentration of 0.0015 percent rose bengal was, therefore, selected as
optimum for the recovery of pure cultures of C. pasiapA-ULoA-U. Adapta-
tion of the enumeration technique for use with raw wastewater necessi-
tated an increased concentration of rose bengal (0.02 percent) for
efficient elimination of mold contaminants. Figure 1 is a diagram
showing the sequence used in the enumeration of the yeasts.
Enumeration Technique for Yeasts - Laboratory Studies
The above enumeration procedure for yeasts is suggested for
field samples, i.e., for stools, wastewater, and surface waters. Several
modifications of this procedure were made to facilitate experiments
utilizing pure culture isolates, i.e., experiments with chlorine and
chloramines.
This method of enumerating yeasts consists of filtering the
samples through a black membrane filter and placing the filter in an
upright position on a thin layer of solidified YMA medium containing
various antibiotics. The incubation period required varied with the
temperature of the room. Generally, colonies could be counted after
30 hours with an incubation temperature of 25°C, or 48 hours with
incubation at 20°C. It should be pointed out that the basic difference
between the laboratory and field enumeration techniques for yeasts is in
the use of the rose bengal, pH adjustment of the medium to pH 3.6-3.7,
and the agar overlay. In the pure system studies, these procedures were
found to be unnecessary. The antibiotics which were incorporated suf-
ficiently inhibited the growth of other organisms on YMA. The anti-
biotics used were chloroamphenicol and penicillin.
Enumeration Technique for Acid-Fast Organisms - Field Samples
To find the best medium for the enumeration of acid-fast
organisms, various combinations of additives to Middlebrook and Cohn 7H9
mineral base were evaluated, using primarily sodium propionate as the
carbon source and malachite green as a bacteriostatic agent against non-
acid-fast organisms. Three concentrations of malachite green were
studied in combination with three different concentrations of propionate
in Middlebrook and Cohn 7H9 mineral agar base. A two-step selective and
enrichment technique was also evaluated. The results of these studies
are shown in Table 1. The growth of Mt/cobac^e/u-tun ^ontvJMm and Mt/co-
bact&iium phJL&i using these media was compared with that using Middlebrook
and Cohn 7H10 agar containing glycerol as the carbon source as the
control media. Growth of the acid-fast organisms was observed after 3
days on media containing 0.2 percent sodium propionate and 2.5 x 10~5,
10"4, and 10"3 percent malachite green, respectively. No growth was
observed until after 5 days on Middlebrook and Cohn 7H9 agar media con-
taining 0.4 percent sodium propionate and all three concentrations of
10
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Sample
Homogenization
Waring Blender (30 sec)
Dilution
Phosphate buffer, pH 7.0 (Standard
Filtration
Membrane (Standard MeJhodt,, Sec. 408A)
Cultivation
Yeast extract-Malt extract agar
(plus antibiotics and 4% rose bengal)
Inverted black membrane
Agar overlay
Incubation
30 hr - 25°C
Enumeration
Read from bottom of plate
Figure 1. Enumeration Technique for Yeast Organisms
11
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TABLE 1
Growth of Ac id-Fast Isolates, M. pkioA, and M.
After 3 Days of Incubation in Various Media
Average Number of Colonies per Plate
Media M" pkleA. M. fio/itwitwm
7H9 mineral base
plus
0.2% glycerol carbon source (control) 192 182
0.2% sodium propionate 163 151
0.4% sodium propionate --a
1.0% sodium propionate
7H9 mineral base with 0.2% sodium propionate
plus c K
2.5 x 10-5% MGb 162 138
1 x 10-4% MG 189 152
1 x 10'3% MG 144 154
7H9 mineral base with 0.4% sodium propionate
plus
2.5 x 10-5% MG
1 x 10-4% MG
1 x 10'3% MG
7H9 mineral base with 1.0% sodium propionate
plus
2.5 x 10-5% MG
1 x 10'4% MG
1 x 10-3% MG
a-, Dash indicates no growth following 3 day incubation.
MG, malachite green.
12
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malachite green. Growth was not observed on any of the media containing
1.0 percent sodium propionate. Thus, a concentratin of sodium propio-
nate above 0.2 percent inhibited the growth of the known acid-fast
organisms while malachite green concentrations as high as 10~3 percent
did not inhibit growth. When M. fiofctuAtwm and M. ph£&l were grown on
Middlebrook and Cohn 7H10 agar with 0.2 percent sodium propionate and
1:10 Middlebrook and Cohn medium with oleic acid-albumin-dextrose cata-
lase (OADC) enrichment, visible colonies appeared after 2-1/2 days. A
limited study of secondary effluent with this medium showed that waste-
water acid-fast organisms do not appear until after 3 to 4 days of
incubation. Media with four different combinations of sodium propionate,
mineral base 7H9, malachite green, and OADC were also studied and com-
pared to the results obtained with single strength 7H10 mineral base
and 2.5 x 10~^ percent malachite
are shown in Table 2 and referred to
, and 5. A two-step selective and
using media Nos. 4 and 5 with M.
step consisted of incubating the
with 7H9 propionate broth; after 24
transferred to 7H9 propionate solid media,
results are shown in Table 3. Growth of the
plus 0.2 percent sodium propionate
green. Composition of these media
as media Nos. 1 (control), 2, 3, 4;
enrichment technique was evaluated
faoKtii-itujn and M. phta-i. The first
membrane filter on a pad saturated
incubation, the pad was
media Nos. 4 or 5. The
hours
i.e.
acid-fast organisms with the two-step procedure was found to be inhibited,
TABLE 2
Composition of Various Media Evaluated for the
Growth of Acid-Fast Organisms
Media
Number Mineral Base
1 (Control)
2
3
4
5
7H10
7H9
7H9
7H9
7H9
Strength
of
Mineral Base
Single
Single
Double
Single
Double
Sodium
Propionate
(*)
0.2
0.1
0.2
0.1
0.2
Malachite a
Green OADC
(%)
2.5 x 10"5
10~4
io-4
10"4 1:10
IO"4 1:5
a
OADC - oleic acid-albumin-dextrose catalase-
13
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TABLE 3
Percent Relative Growth of Acid-Fast Isolates, M. pkini and
M. 6ovtu
-------
TABLE 4
Growth of Acid-Fast Organisms in Wastewater Effluent Expressed
in Terms of Percentage Based on the Number of
Colonies Found in Test Media No. 1 (Control)
Item
Incubation
Number of
Time (days)
Colonies per
1
(Control)
6
2210
2
5
7373
Media
3
6
3973
4
5
9590
5
5
5003
mi of Effluent
Percent of Colonies 100 334 180 434 226
Found in Control
acid was increased to 2.5 percent or greater. Increasing the contact
time of the 1.25 percent oxalic acid pretreatment step to 20 minutes
did not prove effective since spore forming bacteria survived the pre-
treatment. The possibility of prefiltering the water sample through a
5y membrane filter before pretreating the stream sample with oxalic acid
was investigated. However, since acid-fast organisms are known to
adhere to surfaces, e.g., suspended solids present in the water samples,
prefiltering with a 5y membrane filter would mean a loss of those acid-
fast organisms attached to solids; this would result in a low count in
enumerating acid-fast organisms. Attention was then given to the incor-
poration of combinations of antibiotics into the acid-fast enrichment
medium. The antibiotics studied included penicillin and nalidixic acid,
which inhibit gram-positive and gram-negative bacteria, and mycostatin
and actidione, which inhibit yeasts and molds. Penicillin, mycostatin,
and actidione were suspended directly in sterile water while nalidixic
acid was first dissolved in 1 N NaOH and subsequently diluted in sterile
water. The incorporation of these antibiotics was accomplished after the
acid-fast medium had been sterilized and cooled to 45°C. When samples of
activated sludge effluent and stream water were pretreated with oxalic
acid-NaOH, membrane filtered and the membranes cultured on Middlebrook
and Cohn 7H9 propionate, and malachite green medium containing various
combinations of antibiotics, a significant decrease in the number of
non-acid-fast organisms including spore forming bacteria was observed.
Examination of various combinations of antibiotics indicated that few, if
any, non-acid-fast organisms were present when the combination of anti-
biotics was as follows: penicillin, 25 units/m£; nalidixic acid, 25 yg/m£;
mycostatin, 50 pg/m£ of medium. The concentration of penicillin appeared
to be the major inhibiting antibiotic for spore forming bacteria. Unfor-
tunately, it is also the same antibiotic which inhibits acid-fast organisms
15
-------
which form colonies similar to M. pktz-i. It was concluded, however, that
the final decision as to the optimum combination of antibiotics had to be
based on the control of non-acid-fast organisms. Studies with stream
samples indicated that virtually all spore forming bacteria could be
eliminated when the acid-fast enumeration medium contained the concen-
trations of penicillin, nalidixic acid, and mycostatin indicated above.
After three days of incubation, both types of acid-fast organisms, i.e.,
colonies similar to those produced by M. fanta-itum and M. phteA, can be
detected in this antibiotic medium but most of the colonies appear beige-
colored. With extended incubation, a very high density of yellow-colored
acid-fast colonies similar to M. pkteA. appear on the membrane filters.
The present technique of enumerating acid-fast organisms in
wastewaters consists of pretreating samples with 2.5 percent oxalic acid
for 10 minutes and neutralizing with 2 percent NaOH. The samples are then
membrane filtered with the membranes being placed on enriched Middlebrook
and Conn 7H9 growth medium in a culture dish and incubated at 37°C for
approximately 72 hours. The filters are then removed from the agar sur-
face, heat dried, and stained by means of the Brooks acid-fast stain. The
conventional staining procedure was modified for use with membrane filters
by reducing the concentration of the dyes and the contact times. Pink
to red colonies, consisting of acid-fast organisms, are then counted.
Figure 2 is a diagram of the enumeration procedure for acid-fast organisms.
Enumeration Technique for Acid-Fast Organisms - Laboratory Studies
It was found that several modifications of the acid-fast enum-
eration technique for highly contaminated samples could be made to facili-
tate the handling of samples containing pure cultures of organisms, such
as employed in the chlorine and chloramine experiments. For the enumer-
ation of acid-fast organisms in these experiments, Middlebrook and Cohn
7H9 medium with OADC enrichment plus added dextrose and malachite green
with 1.5 percent agar was used as the growth medium. The membranes used
to filter the samples were incubated at 37°C for approximately 72 hours.
The pretreatment step (oxalic acid-NaOH) and the Brooks carbol-fuchsin
staining procedure, as described for enumerating acid-fast organisms in
the field studies, were not necessary in that the two species of acid-fast
organisms and the interfering organisms could be easily identified.
Enumeration Technique for Fecal Col i forms, E&c.h&u.dkui coti, ScUtmoniMa,
and Poliovirus
- The technique used for fecal col i form enumera
tion in the stool and wastewater investigations was that used by Buras
and Kott (1972).2 In this method, 25 mg of rosolic acid is dissolved in
5 ml of sterile 0.2 N NaOH. Two mi of this rosolic acid solution is
diluted with 98 mi of sterile water; 3.7 gm of Bacto m FC broth (Difco
Laboratories, Detroit, Michigan) is then added. The vessel containing
the medium is placed in boiling water for 10 minutes. The medium is
cooled and dispensed onto filter pads in 2 mi quantities. Membranes,
16
-------
Sample
Homogenization
Waring Blender (30 sec)
Pretreatment
1.25% Oxalic acid (10 min)
2.0% NaOH (neutralize)
Dilution
Phosphate buffer, pH 7.0 (Standard
Filtration
Membrane (Standard
, Sec. 408A)
Cultivation
7H9 Middlebrook and Cohn
(plus OADC, propionate, and antibiotics)
Incubation
72 hr - 37°C
Staining
Heat fix colonies on membrane
Brook's carbol fuchsin (primary stain)
10% oxalic acid rinse
Brook's malachite green (counterstain)
Distilled water rinse
Enumeration
Dark pink to red colonies
Figure 2. Enumeration Technique for Acid-Fast Organisms
17
-------
following filtration of the samples, are then placed on the pads and
incubated at 37°C for 24 hours. Fecal col i forms appear as dark blue
colonies, while pink, red and olive green mucoid colonies represent the
Aeiobac^eA, SaJbnoniMa. and Ktubbiztta. groups of organisms. The tech-
nique is basically the same as the Standard M&thodA3 technique for
fecal col i form enumeration except that 37°C is used for incubation
instead of 44.5°C. This technique eliminates the need for precise
control of temperature, i.e., 44.5°C, and allows differential enumera-
tion of other organisms of interest.
coti - The enumeration procedure used for E. aoti
in the chlorine and chloramine studies was essentially the same as that
outlined for the fecal coliform enumeration discussed above. Instead of
the use of the filter pads, however, 1.5 percent agar is added to Bacto
m FC broth and the filter membrane is placed on the solidified medium.
Culture plates were then submerged in plastic bags in a 44.5°C water
bath. After an incubation period of 24 hours, E. c.oti appear as dark
blue colonies.
SalmoneJLta - The method of choice for the enumeration of
5. ttjph-imuAA-um was with Bacto bismuth sulfite agar (Difco Laboratories ,
Detroit, Michigan). The samples were membrane filtered; the membrane
filters were then placed in an inverted position on solidified agar.
Yeasts had been observed to produce colonies with black pigmentation
characteristics of S. typfU-muA-iam in this medium. Therefore, to reduce
possible error in discriminating between yeasts and SciimoneJLla colonies,
the inverted filter technique was used. Black pigment produced by
yeasts is non-diffusible while the pigment produced by SaJLmon&lia dif-
fuses through the filter to the surrounding medium. To further reduce
yeast contamination, 50 units/m£ of mycostatin was added after auto-
claving. Incubation for SalmoneMa enumeration was at 37°C for 48 hours.
?otio\)-ift(ji& - Prior to enumeration on African Green Monkey
Kidney cells (BGM), samples containing viruses were treated with a mixture
of penicillin, streptomycin, mycostatin, and neomycin (0.5-0.75 m£/100 m£
sample). Surviving polioviruses were then enumerated on monolayers of
BGM cells. The monolayers were washed with serum- free Gibco M199 medium
(Grand Island Biological Company, Grand Island, New York). After washing,
0.5 m£ of a dilution of the sample containing virus, suspended in serum-
free media, was inoculated onto the monolayer. Incubation was for 2 hours
at 37°C, with manual agitation every 15 minutes to allow for uniform
adsorption. Excess fluid was then withdrawn, and the monolayer was
covered with 6 m£ of M199 medium containing MgCl2, calf serum (5 percent),
and 0.9 percent agar. After 2-3 days of incubation at 37°C, a 3 mt
overlay, consisting of M199 medium containing agar, serum, and 10 percent
neutral red, was added. On the same day as the addition of the overlay,
and for the following two days, plaques yielding non-stained areas in the
monolayer were counted. The counting and incubation, subsequent to the
neutral red overlay addition, were performed under minimal lighting con-
ditions inasmuch as neutral red has been reported to undergo photo-
decomposition to yield products toxic to tissue cultures. Cell controls
and poliovirus controls were used throughout to ensure the integrity of
18
-------
the assay. A detailed description of the preparation and maintenance
of the BGM cell line as well as the preparation of the antibiotic mixture
is given in Section IX, Appendix.
Occurrence of Acid-Fast Organisms, Yeasts, and Fecal Coliforms in
Hater
Using the techniques described for the enumeration of acid-
fast organisms and yeasts in field samples, a detailed survey of the
densities of acid-fast organisms and yeasts was undertaken at the East
Side Wastewater Treatment Plant of the Urbana-Champaign Sanitary Dis-
trict and its receiving stream, the Saline Branch of the Salt Fork
River. No direct comparison to pathogens was made but fecal coliform
densities were determined.
The East Side Plant consists of primary treatment followed by
parallel secondary treatment, activated sludge and trickling filter units.
To facilitate comparison between densities of organisms through the two
units, it was assumed that the flow through the plant was divided equally
between the two units. This assumption may not be entirely valid.
Initially eight stream sampling points were used in order to
establish a general pattern of distribution of the organisms in the
receiving stream. After analyzing the early data, it was decided that
the number of sampling points could be reduced to four without sacri-
ficing the value of the study. The first sampling point was located
100 feet upstream from the treatment plant outfall. The second sampling
point was the wastewater effluent outfall. Beginning in September 1973,
the effluents of the activated sludge and trickling filter portions of
the treatment plant were mixed and chlorinated such that a total chlorine
residual of 1 mg/£ remained after 15 minutes contact. The sampling
station at the outfall was, therefore, the chlorinated effluent of the
treatment plant after September 1973. Stations 5 and 2 were located
3 and 6 miles downstream from the treatment plant, respectively. It
should be noted that the presence of drainage tile, septic tank over-
flows, and agricultural runoff may contribute somewhat to the micro-
biology of the stream below the treatment plant, i.e., Stations 5 and
2. During the survey, samples of raw wastewater, trickling filter
effluent, and activated sludge effluent were also collected. Grab
samples were taken and in some cases filtered in the field; the filters
were placed on selective media as appropriate for the three groups of
indicators (yeasts, acid-fast organisms, and fecal coliforms) under
investigation using a Millipore portable water laboratory. Samples
were brought directly to the laboratory for analysis during cold weather.
Occurrence of Acid-Fast Organisms, Yeasts, and Fecal Coliforms in Fecal
Matter
The presence of yeasts in human faces was determined in earlier
studies associated with this project and reported in 1974.1 At that time,
19
-------
a suitable technique for acid-fast organism enumeration was not fully
complete. With the development of a reliable technique for enumerating
acid-fast organisms, a study was performed to determine the presence
and relative density of acid-fast bacilli in feces. The enumeration of
yeasts was also included in this study to confirm earlier findings. For
comparison, the density of fecal coliforms was also determined.
Freshly collected stool samples (10-20 g) were weighed into a
container and 99 mi of pH 7 phosphate buffer added. The samples were
then completely mascerated to provide a homogeneous suspension. A 100-
fold dilution of the suspension was prepared using phosphate buffer.
Fecal coliforms were enumerated using the modified fecal coli-
form technique previously discussed. Yeasts were detected by the pour
plate method employing YMA medium. A 5 ml aliquot of the 100-fold dilu-
tion was inoculated into an equal volume of softened medium, poured into
100 x 15 mm culture dishes and mixed by swirling. Once this layer
solidified another 10 ml of softened medium was added as an agar overlay.
The plates were incubated at room temperature for 48 hours at which time
yeast colonies were counted. Because of the particulate matter in the
fecal suspensions, it was necessary to eliminate the use of membrane
filters. The density of acid-fast organisms was determined using the
modified Middlebrook and Cohn 7H9-OADC medium containing antibiotics.
An aliquot of the 100-fold dilution of the fecal suspension was pre-
treated with an equal volume of 2.5 percent oxalic acid for 10 minutes
and then neutralized with 2 percent NaOH. A 5 mi aliquot of the pre-
treated suspension was inoculated into a suitable volume of the softened
medium and poured into 100 x 15 mm culture dishes containing a layer of
solidified medium. The agar was allowed to solidify and the culture
dishes incubated at 37°C until sufficient growth appeard for identifi-
cation of colonies. Colonies which were morphologically similar to
those known to be formed by acid-fast bacilli were sampled, streaked
on slides, and acid-fast stained for positive identification.
Chiorination Experiments
It has been stated that direct comparison of pathogens and
indicator organisms under the same, simultaneous conditions is the only
exact way in which the relative resistances of different organisms to
chlorine may be established. The inherent dangers of comparing data
presented by several investigators, collected under possibly dissimilar
conditions, should be apparent. With this in mind, an experimental
system was designed in which a "mixed culture" of organisms could be
tested for resistance to chlorine in one experiment. Included in the
mixed culture of organisms were poliovirus type 1 Mahoney strain,
E4c.keju.ch.-ia coli, Salmonella typlu.muAium, and a mixed culture of acid-
fast and yeast organisms isolated from wastewater, i.e., Myc.obac.teAA.um
pkl&i, Myc.obacteAA.um fioJituitum, Candida poAflp6>t£o4-c6, Candida ksiuA&i,
Rhodotoiula Jiubsw., and T^icko* potion fieAme.ntant>. Experiments were per-
formed at 5°C + 0.5°C and 20°C + 4°C with free chlorine residuals of
approximately 0.5 and 1 mg/£. The pH of the systems was adjusted to
20
-------
6, 7, or 10 to provide a wide distribution of hypochlorous acid and
hypochlorite ion concentrations. The buffered chlorine solutions
were prepared in the following manner. All buffers were prepared using
chlorine demand free water. Chlorine demand free water (CDFW) is
prepared by chlorinating deionized water through the addition of sodium
hypochlorite to a concentration of 3.5 mg/£ free chlorine residual,
followed by storage at room temperature for one week to insure the
oxidation of impurities which cause chlorine demand. One day before
the experiment, the CDFW is transferred to sterile, 5£ flasks, and the
pH adjusted by buffer. After the addition of buffer, the water is
boiled for 20 minutes to insure sterilization and the pH measured.
The solution is then dechlorinated by exposing it to ultraviolet light
for 24 hours. Three different buffer solutions were used, depending
upon the circumstances. The pH 6 buffer consisted of 3.05 gm of
NaH2P04-H20 plus 0.44 gm of Na2HP04 in 4£ CDFW. The pH 7 buffer con-
sisted of 1.7 gm of anhydrous NaH2?04 plus 0.3 gm NaOH in 5£ CDFW.
The pH 10 buffer was made from 25 m£ of a 0.1 M^ boric acid solution
plus 36.6 mi of a 0.1 M NaOH solution made up to 4£ in CDFW. Prior to
each experiment, a solution having the desired chlorine concentration
was prepared using the appropriate chlorine demand free buffer and
adding a calculated amount of a standard chlorine stock solution. The
stock solution was prepared from Clorox bleach (Clorox Company, Oakland,
California) and deionized water.
The experimental system consisted basically of three 600 mi
stainless steel beakers, each containing 400 mi of the desired reaction
solution, placed in a plexiglass tank. Beaker covers used to minimize
contamination were made from aluminum foil. Mixing in the beakers was
provided by a Phipps-Bird flocculator (Phipps-Bird Company, Richmond,
Virginia) outfitted with glass stirring rods. Constant temperature
conditions were maintained by pumping constant temperature water from
a water bath to the plexiglass tank. All beakers were allowed to equil-
ibrate to the desired temperature before the experiment continued. The
equilibration took approximately 15-20 minutes. Two of the stainless
steel beakers were designated for use as control reactors. The third
beaker was used as the experimental reactor. One control consisted of
an organism control, having no chlorine; the other was a chlorine control
containing only chlorine. Both of these controls were sampled at the
beginning and end of each experiment. With the organism control system,
it was possible to detect a change in the density of the organisms not
associated with the effect of chlorine. In all the experiments, no
significant decrease or increase was ever detected in the density of the
organisms in this control system. The chlorine control system was used
to determine the loss of chlorine residual associated with solution
preparation; it also enabled any chlorine demand associated with the
organisms to be determined. Chlorine residual was measured in the exper-
imental reactors at the beginning and end of each experiment, and reported
as the final measurement. A decrease in chlorine residual of greater than
about 5 percent during the experiment was considered significant and the
experiment aborted. Chlorine residuals were measured with a Wallace and
Tiernan amperometer (Wallace and Tiernan Company, Belleville, New Jersey).
21
-------
The preparation of the mixed culture of organisms consisted
of several steps. In order to reproduce the diversity of yeasts expected
to be found in field situations, four yeast species, C. pajia.p-t,4,to^^ ,
C. kAn&^i, R. fi.(nb>ta, and T. fatwe.Yita.Yii>, were inoculated at the same time
into a single culture flask containing nutrient broth and grown at
22°-24°C for 14-16 hours on a shaker. It was determined that an approx-
imately even distribution of these organisms could be obtained if the
yeasts were grown by this method.
Two species of acid-fast organisms, M. {,ositu.-i3:.um and M.
were used for these experiments. Due probably to the high lipid content
associated with acid-fast organisms, it was found that often up to 3 or
more logs of cells/ml were lost in the harvesting of the cells. This
loss in cell density seemed to be due to the aggregation of the cells
into clumps which tended to "float" at the air-water interface of the
centrifuge tube during the washing step. Because of this, the two acid-
fast organisms were grown and harvested separately to assure a more even
distribution in the final "mixed culture" used in the chlorine experiments,
The acid-fast organisms were cultured in a Middlebrook and Conn 7H9 broth
enriched with OADC plus 0.1 percent propionate. No antibiotics were
added to this broth. Cells were grown for 16-24 hours on a shaker at
room temperature.
The techniques for the growth of E. aoti and 3. typhimu/iium
cultures were similar. Nutrient agar slants were inoculated and grown
approximately 24 hours at 37°C. The growth on the slant was then care-
fully washed off with about 2-5 ml of sterile CDFW, buffered at pH 7.
The bacteria and yeast suspensions were then washed to remove
chlorine demand associated with any excess media. The washing procedure
consisted of centrifugation for 10 minutes using a clinical centrifuge,
followed by resuspension of the pellet in sterile chlorine demand free
phosphate buffer, pH 7. This washing procedure was repeated three times.
After the final wash, the pellet was resuspended in chlorine demand free
buffer and the optical density was read on a Bausch and Lomb Spectronic
20 spectrophotometer at 660 nm. Using predetermined extinction co-
efficients, approximate cell densities could be measured.
Poliovirus type 1 Mahoney strain was cultured by infecting
monolayers of African green monkey kidney (BGM) cells (obtained from
Dr. Gerald Berg, EPA, Cincinnati, Ohio). The monolayers thus infected
were incubated overnight at 37°C, and associated culture media were then
purified using the procedure of Scarpino oJi aJi.^ After two repeated
freezings at -70°C and thawings, the crude preparation was centrifuged
for 30 minutes at 2000 rpm at 5°C to remove the cell debris; the centri-
fuge was a Beckman ultracentrifuge Model L2-65B with a TI-60 rotor.
Pooled supernatant was frozen at -70°C, thawed, and centrifuged for 21
hours at 19,000 rpm at 5°C to pellet the virus. The pellet was then
washed with sterile buffered CDFW and harvested as above. The demand
free virus suspension was diluted to a known volume with CDFW, and
aliquots dispensed and stored at -70°C for use in experiments.
22
-------
To avoid the problem of chlorine demand caused by excessive
densities of organisms in the mixed culture inoculum, care was taken to
limit the total number of organisms in the inoculum. A 1 m£ volume of
the mixed culture was added to the reaction beakers. The approximate
density of organisms after inoculation in each 400 ml reactor was as
follows: yeasts and acid-fast organisms (each), 5 x 10^ organism/m£,
Salmonella and E. coti (each), 1 x 105/m£, and poliovirus, 1.5 x
The single mixed culture inoculum with densities of organisms large
enough to supply these numbers of organisms in the experimental reactors
was prepared by the addition of the correct number of each organism to a
single test tube and subsequent dilution to a final volume of 10 ml.
This procedure provided a workable density of organisms such that a 1 ml
sample volume at the final sampling time still contained sufficient viable
organisms to provide a statistically reliable plate count representing
an inactivation of 99.9 percent
After inoculation of the organisms into the reactor beakers,
samples were taken at frequent time intervals. Approximately 14.5 ml
of sample were withdrawn from the experimental reactors by sterile pipets
and rapidly added to sterile screw cap culture tubes containing 1.0 ml
of 0.05 Nl sodium thiosulfate (Fisher Scientific Co., Fair Lawn, New Jersey).
Sodium thiosulfate was used to destroy the chlorine residual. Samples
from the beaker used as the organism control were taken at the beginning
and the end of each experiment.
Chiorami ne Experiments
Studies were included to determine the response of the yeasts
and acid-fast organisms to chloramine. The experiments were designed as
closely as possible to follow the procedures used in the studies with
free chlorine and isolates No. 30, 132, and 134 as described by Engelbrecht
&t al.1 For the most part, these experiments were performed in a manner
similar to the "mixed culture" studies with free chlorine described in
the previous section. In these chloramine experiments, C. panjapA-iloAlA,
M. ph£exl and M. ^osutuitum (isolates No. 30, 132, and 134, respectively)
and E. toll were studied individually using pH 7 buffered CDFW and a
temperature of 20°C. Solutions of chloramines were prepared using a 5:1
weight ratio of chlorine to ammonia nitrogen in CDFW. The desired chlora-
mine solution, i.e., 5:1 weight ratio of Cl2:NH3-N, was made by adding
the correct amount of a stock ammonium chloride solution (200 mg/£ NHs-N)
to pH 7 buffered CDFW, followed by the addition of the appropriate amount
of stock chlorine solution. All solutions of chloramines were allowed
to react for 20 minutes to stabilize. The DPD titration method (N,N-
diethyl-p-phenylenediamine), as introduced by Palin,5 was used to deter-
mine the combined chlorine residual before and after the experiment.
Three notable procedure changes were made for these experiments
over the procedure described for the chlorine experiments. Firstly,
experiments were performed in M Erlenmeyer flasks rather than 600 ml
beakers. Mixing was by hand. Secondly, E. coli. was cultured in nutrient
broth on a shaker at room temperature rather than on nutrient agar slants.
23
-------
Finally, since the concentrations of chloramines ranged up to 10 mg/l,
varied amounts (1-4 mi) of 0.05 N sodium thiosulfate were used to destroy
the chlorine residual before enumerating the surviving organisms. Enum-
eration techniques for the acid-fast organisms, yeasts, and E. coii, were
as described for the modified laboratory methods. The culturing of the
acid-fast organisms and the yeasts was as described in the procedures
for the experiments with free chlorine.
24
-------
SECTION 5
RESULTS AND DISCUSSION
Occurrence of Acid-Fast Organisms, Yeasts, and Fecal Coliforms in
Wastewater
To permit a meaningful evaluation of disinfection efficiency,
it is essential that an indicator organism always be detectable in suf-
ficient numbers. Therefore, a study of the East Side Wastewater Treat-
ment Plant of the Champaign-Urbana Sanitary District and a short stretch
of the Saline Branch of the Salt Fork River which receives the treated
effluent was undertaken to find the densities of yeasts, acid-fast
organisms and fecal coliforms.
The densities of the three indicator groups were fairly con-
sistent in raw wastewater, while the activated sludge and trickling
filter effluents varied over a fairly narrow range (Table 5). Densities
observed in the receiving water were highly variable reflecting, in
part, the fluctuations in stream conditions. Figure 3 depicts the approx-
imate density of acid-fast organisms in the various wastewater samples
and at the three sampling points in the stream. High densities of
acid-fast organisms were found in the wastewater and in the treated
effluents. Acid-fast bacilli were present in very low numbers upstream
from the treatment plant, indicating a relatively small number is con-
tributed by urban and rural runoff. Averaging the acid-fast density
data for the activated sludge and trickling filter effluents and com-
paring this density with those found in the chlorinated effluent, a 13
percent survival of acid-fast organisms was observed (Table 5). It
should be noted that all the data given in Figure 3 and Table 5 were
collected following initiation of effluent chlorination in September
1973. This calculation assumes that flow from the activated sludge and
trickling filter plants was equal, an assumption which may not be entirely
valid. The actual survival of acid-fast bacilli following chlorination
may differ from the calculated value. However, the calculated value
indicates considerable survival of the acid-fast organisms following
chlorination. All acid-fast organisms observed in this study were cul-
turally and morphologically similar to M. fiofituAtum. The addition of
antibiotics to the medium apparently inhibited organisms similar to
M. pht&i or they did not survive the chlorination process. The densities
of yeast in the wastewater and stream samples at the above mentioned
treatment plant are presented in Table 5. The yeast density data are
not statistically as promising as that for the acid-fast organisms.
Calculations as those mentioned above for the acid-fast organisms show
a 0.2 percent survival of yeast in the chlorinated effluent. A high
degree of reduction in fecal coliform density occurred as a result of
chlorination, since they were not detectable in 50 mi sample volumes
(Table 5). An average Slog reduction in fecal coliform densities was
observed while comparable reductions for acid-fast organisms and yeasts
were approximately 0.5 and 3 logs, respectively. These data provide
25
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further evidence of the higher chlorine resistance of the proposed
indicator organisms when compared to the conventional coliform group.
Occurrence of Acid-Fast Organisms, Yeasts and Fecal Coliforms in
Fecal Matter
To assure the presence of an indicator in wastewater, a reliable
source of the organisms is necessary. Fecal matter will always be present
in domestic wastewater and provide a source of indicator organisms if the
organisms are of fecal origin. Urine will also be a constant component
of domestic wastewater but its microbial population is generally much less
than that of fecal matter and, for this reason, is a less reliable source
of indicator organisms.
The presence of yeasts in human feces, determined earlier in
this study, has been previously reported.1 More reliable enumeration
procedures for acid-fast organisms have since been developed, permitting
a more reliable assessment of their presence in feces. It was for this
reason, as well as to substantiate the data previously presented for
yeasts, that a study of the densities of acid-fast organisms, yeasts,
and fecal coliforms in fecal matter was undertaken.
The results of this microbiological examination of fecal matter
are presented in Table 6. Fecal coliforms were detected in all 30 samples
with a geometric mean value of 2.2 x 10^/gm wet feces. This average density
closely approximates values reported in the literature. The density of
acid-fast and yeast organisms in feces was generally low in comparison
to the density of fecal coliforms. Yeasts were detected in all samples
except one. The density of yeasts varied over a 4 log range with a mean
value of 562/gm wet feces. The range of densities observed were com-
patible with values determined in the earlier study. Less promising
results were obtained with the acid-fast organisms. Only 13 of 30 samples
were positive for acid-fast organisms. Moreover, the densities of acid-
fast organisms in the different samples were all less than 100/gm wet
feces. The mean value was 18.9/gm wet feces. Several factors might
account for the low numbers of acid-fast organisms detected in the stool
samples. The enumeration technique itself may be the limiting factor.
It may be that acid-fast organisms in wastewater are of fecal origin but
carried only by a certain proportion of the population. Also, it is
possible that the acid-fast organisms enter wastewater by a route other
than feces, e.g., urine, surface runoff, laundry or bathing water.
The investigation to determine the source of the potential
indicator organisms in wastewater has been partially successful. The
occurrence of yeasts in feces insures their presence in domestic waste-
water. However, the origin or source of acid-fast organisms in waste-
water has not yet been identified. Because of the low percentage of
these organisms detected in stool samples, it would appear that fecal
matter cannot be considered a significant source of acid-fast organisms.
It is for this reason that, while acid-fast organisms may represent a
valid indicator of chlorination efficiency, they are not a valid indicator
28
-------
TABLE 6
Occurrence of Indicator Organisms in Fecal Matter
Sample
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Geometric
Subject
Sex Age
F
M
F
M
M
M
M
M
M
M
F
M
F
M
M
F
F
F
M
M
M
M
F
M
F
F
M
F
M
M
mean
27
20
6
27
25
22
1 mo
20
8.5
25
2
23
25
25
21
86
91
80
53
85
90
92
21
7
36
4
40
30
23
28
Fecal a
Col i form
1.02 x 106
3.84 x 105
1.6 x 106
1.4 x 10?
2.58 x 106
5.85 x 107
1.38 x 109
6.0 x 107
2.8 x 10
2.8 x 106
1.0 x TO;?
2.0 x 10b
7.6 x 107
1.0 x 105
2.0 x 105
9.77 x 105
1.71 x IflS
5.87 x 107
2.0 x 107
2.31 x 10'
2.04 x 105
3.0 x 104
2.1 x 106
1.2 x 105
1.0 x 10b
1.0 x 105
2.0 x lof
9.25 x 105
6.8 x 107
1 .0 x 105
2.215 x 106
Yeasts3
2.69 x 103
5.0 x 101
5.0 x 10°
1.56 x 102
5.63 x 103
1.05 x 103
0
4.99 x 10!:
2.8 x 103
5.0 x 102
1.04 x 104
2.0 x 10'
2.0 x 102
6.55 x TO"5
4.06 x 104
2.04 x 104
1.7 x 102
7.1 x 10
2.0 x 101
5.0 x 102
6.0 x 101
1.0 x 102
1.2 x 105
2.0 x 10]
2.0 x 101
2.15 x 104
6.0 x 103
6.4 x 102
4.0 x 101
5.1 x 103
5.62 x 102
Fecal Col i form
Yeasts
3.79 x 102
7.68 x 102
3.2 x 105
8.97 x 103
4.58 x 102
5.57 x 104
__
1.2 x 105
1.0 x 104
5.6 x 103
9.6 x 10?
1.0 x 104
3.8 x 10?
1.53 x I0j
4.9 x 10°
4.79 x 101
1.0 x 106
8.27 x 10J>
1.0 x 106
4.6 x 104
3.4 x 103
3.0 x 102
1.75 x 101
6.0 x 103
5.0 x 104
4.6 x 10°
3.3 x 10°
1.44 x 103
1.7 x 106.
1.96 x 101
2.69 x 103
Acid-fasta
Organisms
40
0
35
0
0
0
0
0
0
0
0
0
33
60
0
0
10
0
0
20
13
6
60
0
0
30
10
5
0
10
18.9
b
b
b
b
b
b
c
c
c
b
c
b
b
c
c
b
b
b
b
b
b
b
c
c
b
c
c
b
b
c
Values are number of organisms detected per gram of wet feces.
Samples freshly collected and assayed same day.
c Samples collected were refrigerated overnight before being assayed.
29
-------
of fecal pollution much in the same way that fecal coliforms are a
valid indicator of fecal pollution but not a valid indicator of waste-
water chlorination efficiency.
Identification and Ecology of Acid-Fast Organisms and Yeasts Common to
Wastewater
In the case of determining efficient chlorination of wastewater,
a reliable source of any potential indicator organism is necessary to
ensure its presence in detectable numbers. Efforts to establish the
origin of these potential indicators were made by determining the identity
of representative isolates of the yeasts and acid-fast groups of organisms
and by considering their ecology. Morphologically distinct colonies from
both groups of potential indicators were isolated from various wastewater
samples. Ten such distinct acid-fast cultures were sent to the Illinois
Department of Public Health in Springfield, Illinois. Of these ten cul-
tures, five were identified as Mycoba.cteAA.um fiatta-itum, four as Mycobac-
teA-Lum pktzA., and a single culture as Mycoba.cteAA.um .&m&gma£u>. The three
identified species of acid-fast organisms are rapid growers and belong
to Group IV of the mycobacteria according to Runyon's classification.
M. phleA, is a saprophytic organism widely distributed in nature. It is
found in soil, dust, hay, on plants and similar sources.6'7 M.
is constantly present in human smegma and occurs in soil and dust.6'7
is also a saprophytic organism found in soil and water.7
Five distinct yeast cultures were sent to the Central! bureau
voor Schimmel cultures in the Netherlands for identification. Four of the
cultures isolated from wastewater were identified as Candida pcuia.pt>JJLot>JJ> ,
Cand-ida. kAuA&i, Rhodotofia.ta tiutbna, and TtiLcko&ponon {^cjimnntan^ . The
fifth isolate, which was from a fresh stool sample, was identified as
C. paAap^A.lo^-L& . C. fe^uAe/c is found in the feces of adults and children,
the alimentary tract of animals and is a part of the normal flora in the
vagina and the respiratory tract of man.9 C. paMap&JJLo&'U* is commonly
found on healthy skin as well as in cutaneous and mucosal lesions.9
R. tiuhia is one of the most common species of Rhodotohu&a. found in clin-
ical specimens, especially from the gastro-intestinal tract of man.
T. lojimantanA has been isolated from wood pulp and leather.9
The occurrence of various yeasts in fecal matter, in the vaginal
tract, and on healthy skin, would indicate their constant presence in
domestic wastewater. However, a similar analysis gives little insight
as to the source of acid-fast organisms in wastewater.
Identification of the potential indicator organisms as to genus
and species is also important in determining the possible pathogenicity
of these organisms to man. Of the several species of acid-fast organisms
and yeasts identified, M. fiofita-itum and C. paAa.pA may be pathogenic
under certain cases. M. fiotttu-itum is an opportunistic organism and can
produce cutaneous and deeper infection following trauma. Several docu-
mented case studies confirm the isolation of M. fioAtu-ltw from lesions
and abscesses following some form of trauma.8 C. pcuia.pi>-Uioi>-U> has been
30
-------
isolated as the cause of heart valve infection, especially in drug addicts.10
Chlorination Experiments
Experiments were designed to determine the chlorine resistance of
poliovirus type 1 Mahoney strain, E. coti, S. typhA.muAium, and mixed cul-
tures of yeasts and acid-fast organisms in the same system. The yeast
species used were C. poAapA-itoAiA, C. fe/ioiex., R. fiubfia, T. ^zAmnntdYti*, and
the acid-fast organisms used were M. pkteJ- and M. ^ofitnitum. Experiments
were conducted at two different temperatures, 5° + 0.5°C and 20° ± 4°C.
Free chlorine residuals of approximately 0.5 and 1 mg/£ were studied at
pH values of 6, 7, and 10. With these pH values, it was possible to study
the response of these organisms to a wide range of ratios of hypochlorous
acid to hypochlorite ion. At pH 6, approximately 98 percent of the free
chlorine residual is in the form of hypochlorous acid, whereas at pH 10,
hypochlorous acid is only approximately 0.4 percent of the free available
chlorine, depending upon the temperature of the system.11
The response of the "mixed culture" of organisms to free available
chlorine is shown in Figures 4 through 13. Results for E. c.oLi at the more
severe conditions, i.e., lower pH, higher chlorine concentration and
temperature, are not shown since more than 3 logs inactivation occurred
in 30 seconds. S. typkimuA^um, which was used in these experiments, showed
similar inactivation to chlorine as E. co£/c, although in one experiment
(Figure 12) it showed a somewhat higher resistance than E. coti. Except
for one experiment (Figure 13), E. aoti showed a lower resistance to
chlorine than poliovirus. The resistance of poliovirus to free chlorine
was noticeably less than the yeasts and the acid-fast organisms. However,
in most experiments, it showed a higher degree of resistance than either
E. cati or S. fypk-unuJiLwrn. Acid-fast organisms were observed to have the
highest degree of resistance to free chlorine in all the experiments. The
response of yeasts appeared to approximate that of poliovirus more than the
acid-fast organisms, however. The data presented for the response of the
mixed cultures of yeasts and acid-fast organisms to free chlorine clearly
demonstrate the superior resistance of both of these groups of organisms
over that of the other organisms studied.
In all cases, the results with the experiments performed at
approximately the same concentration of free chlorine and the same pH,
but at different temperatures, showed the expected trend, i.e., for all
the organisms studied, a higher temperature gave a higher degree of
inactivation with a constant contact time.
Hypochlorous acid is generally accepted as being a more potent
disinfectant than hypochlorite ion in the inactivation of microorganisms.
The most common test organism used to date to evaluate the efficiencies
of hypochlorous acid and hypochlorite ion has been E. c.oti. Fair &t a£.n
31
-------
100
10
(T3
>
>
i-
U
S-
O)
Q-
0.1
0.05
A Yeast
O Acid-fast
a Poliovirus
10
Figure 4.
20 30 40
Time (min)
50
Response of Test Organisms in Mixed Culture to
0.4 mg/£ Free Available Chlorine Residual at
pH 6 and 5°C
32
-------
100
10
(O
s-
3
uo
c
O)
O
S_
O)
Q-
0.1
0.05
A Yeast
O Acid-fast
o Poliovirus
10
Figure 5.
20
30
Time (min)
40
50
Response of Test Organisms in Mixed Culture to
0.9 mg/£ Free Available Chlorine Residual at
pH 6 and 5°C
33
-------
100
10
(0
s-
3
I/O
u
S-
O)
Q.
0.1
0.05
Yeast
O Acid-fast
0 Poliovirus
10
Figure 6.
20
30
Time (m1n)
40
50
Response of Test Organisms in Mixed Culture to
0.5 mg/t Free Available Chlorine Residual at
pH 6 and 20°C
34
-------
$-
3
oo
cu
o
0)
a.
Yeast
O Acid-fast
o Poliovirus
Salmonella
0.05
20
30 40
Time (min)
50
Figure 7.
Response of Test Organisms in Mixed Culture to
0.45 mg/£ Free Available Chlorine Residual at
pH 7 and 5°C
35
-------
>
•l~»
1-
CU
(J
0)
Q-
A Yeasts
o Acid-fast
n Poliovirus
0.05
Figure 8.
30 40
Time (min)
Response of Test Organisms in Mixed Culture to
0.48 mg/£ Free Available Chlorine Residual at
pH 7 and 5.1°C
36
-------
100
10
to
I/O
C
(U
o
O)
Q-
0.1
0.05
10
20
30
Time (m1n)
40
50
Figure 9. Response of Test Organisms in Mixed Culture to
1.0 mg/l Free Available Chlorine Residual at
pH 7 and 5°C
37
-------
TOO
to
oo
4->
o
i.
OJ
CL.
A Yeast
o Acid-fast
a Poliovirus
Salmon&Lta
Figure 10.
30
Time (min)
Response of Test Organisms in Mixed Culture to 0.55 tng/£
Free Available Chlorine Residual at pH 7 and 24°C
38
-------
100
10
>
3
<~n
-»->
c
-------
03
C/5
01
u
cu
0.
E. c.oJU
A Yeast
O Acid-fast
Q Poliovirus
Sahnonnlta.
0.05
20
30
Time (min)
40
50
Figure 12.
Response of Test Organisms in Mixed Culture to
1.0 mg/£ Free Available Chlorine Residual at
pH 10 and 5°C
40
-------
100
10
(O
J-
3
oo
c
QJ
0
Ol
Q_
0.1
0.05
E. coti.
A Yeast
0 Acid-fast
Ponovirus
SalmoneMa
20
30
Time (min)
40
50
Figure 13.
Response of Test Organisms in Mixed Culture to
1.0 mg/t Free Available Chlorine Residual at
pH 10 and 20°C
-------
in citing other references, reported that the inactivation efficiency of
hypochlorous acid was approximately 80 times greater than that of hypo-
chlorite ion in the inactivation of E. c.oti. Since the process of inac-
tivation of a cell by chlorine is hypothesized as consisting of 1) pene-
tration of the cell membrane, and 2) the reaction of the disinfectant
with a vital process of the organism, it becomes apparent that inactiva-
tion efficiencies of hypochlorous acid and hypochlorite ion are dependent
upon the nature of the organism tested. Figures 4 through 11 demonstrate
that with the concentrations of free chlorine used in these experiments,
the difference in the inactivation of E. co£/c and S. typtu.muAA.ujm at pH 6
and at pH 10 was significant. It was observed that, as the pH of the
system increased, the rate of inactivation of these organisms decreased.
In the experiments performed at pH 6 and 7 with 0.5 +_ 0.1 to 1 +_ 0.1 mg/£
free chlorine, E. uoLi was destroyed completely in less than 30 seconds.
This explains the absence of E. cjoti in Figures 4 through 10. However,
at pH 10, 1.8 percent of the viable cells of E. co&t survived 1 mg/£ free
available chlorine after a contact time of 10 minutes at 20°C (Figure 13).
A similar response was observed in the case of S. typhA,muA-ium. Although
it was completely inactivated in 30 seconds by a free available chlorine
concentration of 0.9 mg/£ at pH 6 and 5°C (Figure 5), 0.1 percent of the
5. typkAjmusiLwm survived 1 mg/£ of free available chlorine for 19 minutes
at pH 10 and 5°C (Figure 13).
Figure 14 shows the effect of increase in concentration of
hypochlorous acid on the inactivation of yeasts. The concentration of
hypochlorous acid as determined by the distribution of free chlorine
species at the different pH values of the various experiments, ranged
from 0.003 to 0.885 mg/£. The highest percent survival occurred when
the concentration of hypochlorous acid was minimum, indicating that the
inactivation of yeasts was primarily due to the germicidal action of the
hypochlorous acid. As the hypochlorous acid concentration increased, a
decrease in percent of survival was observed, which was quite noticeable
when the chlorine contact time was 30 or 40 minutes (Figure 14). The
curve representing a contact time of 10 minutes does not show any sig-
nificant decrease in the percent survival until the hypochlorous acid
concentration reaches approximately 0.4 mg/£. Apparently 10 minutes
contact time is too short to allow for significant inactivation of yeasts.
In contrast to the response of the yeasts to the increase in
hypochlorous acid concentration, the acid-fast bacilli were not affected
to the same degree. It can be observed in Figure 15 that, as the concen-
tration of hypochlorous acid increases, the percent of survival of the
acid-fast organisms decreases, indicating the higher inactivation effi-
ciency of hypochlorous acid. However, even with a contact time of 40
minutes, the decrease in percent survival with an increase in the con-
centration of hypochlorous acid is slight.
By comparing Figures 14 and 15, it may be concluded that in
the inactivation of yeasts and acid-fast organisms, hypochlorous acid is
more potent than hypochlorite ion. However, in the inactivation of
yeasts, the ratio of inactivation efficiency of the hypochlorous acid to
that of the hypochlorite ion was observed to be greater than in the case
42
-------
100
10
ITJ
C/)
c
OJ
o
O)
o.
0.1
0,
001
D 10 min chlorine contact time
A 30 min chlorine contact time
O 40 min chlorine contact time
0.01
0.1
HOC1 (mg/l)
Figure 14.
HOC! Concentration vs. Percent Survival of the
Yeast Group of Organisms
43
-------
100
10
(O
s_
3
c
Ol
o
O)
ex.
Q 10 min chlorine contact time
A 30 min chlorine contact time
o 40 min chlorine contact time
0.1
0.001
0.01
0.1
HOC1 (mgAt)
Figure 15.
HOC1 Concentration vs. Percent Survival of the
Acid-fast Group of Organisms
44
-------
of the acid-fast organisms. Estimates of the relative efficiency of
hypochlorous acid and hypochlorite ion were made by estimating the con-
centration of free chlorine necessary to produce 90 percent inactivation
of yeasts and acid-fast organisms with a constant contact period of 100
minutes for the three pH values investigated. The approximate free
chlorine residuals needed to produce this level of inactivation were then
evaluated with respect to the concentration of hypochlorous acid and
hypochlorite ion present using the reported ionic distribution for each
pH. By setting the total disinfecting capacity of the free chlorine
residual at any pH equal to the concentration of hypochlorous acid plus
an efficiency factor times the amount of hypochlorite ion present,
average values for the relative efficiencies of hypochlorous acid and
hypochlorite ion were found. By employing this method of analysis, it
was calculated that, in the inactivation of the yeasts, the hypochlorous
acid is approximately 5 to 20 times more effective than the hypochlorite
ion. In the case of the acid-fast organisms, inactivation by hypochlorous
acid is nearly as effective as inactivation by hypochlorite ion. It was
calculated that hypochlorous acid is only 1.1 to 2.5 times more effective
than the hypochlorite ion. It must be stated, however, that these low
values reported are based on extrapolations from only several data points.
These estimates were also for 90 percent inactivation of the yeasts and
acid-fast organisms. In this region, the inactivation curves presented,
for the most part, are still in the region of an apparent lag phase of
the death response; thus, the results may not be representative of the
response of the organisms to hypochlorite ion and hypochlorous acid at
higher degrees of inactivation. Nevertheless, the generalization can be
made that the acid-fast organisms and the yeasts appear to be highly
resistant to all forms of free chlorine, but that the difference in
resistance to the two forms, hypochlorite ion and hypochlorous acid, is
not as significant as that found for E. coti reported by Fair &t aJL.11
when discussing the results of Butterfield &t dt.12
Chloral-nine Experiments
Data for the response of yeasts and acid-fast organisms to free
chlorine have been presented by Engelbrecht &t aJi.1 It was the purpose
of this phase of experimentation to describe the differences of inacti-
vation of the new indicator organisms by free chlorine and chloramine.
Experiments were performed using stable, preformed solutions of chlora-
mines at pH 7 and 20°C. The test organisms were C. paAapt>-ULo-t>, M.
fiowtuAtum, M. phlzA and E. coti. Figures 16 though 19 represent the
results of experiments performed with chloramines. The response of E.
c.oLi to chloramine is shown in Figure 16. These data indicate that 99.9
percent inactivation of E. coti was achieved with 0.47, 0.30, and 0.18
mg/l chloramine in contact times of approximately 3.4, 4.8, and 8.8
minutes, respectively. C. pcuia.p£JJLQ&,ti> exhibited a 99.9 percent inacti-
vation with a residual of 5.74 and 4.0 mg/£ with contact times of approx-
imately 15 and 30 minutes, respectively (Figure 17). The response of
C. paMipAAloAAb to lower chloramine residuals is also shown in this
figure. In these experiments, C. paAap^-itoi,^ showed 11.5 percent sur-
vival for 30 minutes when exposed to a 2.83 mg/l residual and approximately
45
-------
100
10
(O
I/)
C.
0)
(J
i.
O)
Q.
1.0
0.1
0.01
0.001
0.47 mg/£
v
0.30 mg/£
0.18 mg/l
6 8 10 12
Time (min)
14
Figure 16. Response of EAc.hdSu.ii.kia c.ot-1 to Chloramine
(5:1 of C12:NH3-N), pH 7 and 20°C
46
-------
-------
TOO
10
to
3
C
OJ
(J
s-
1.0
0.1
0.01
10
20
50
30 40
Time (min)
Figure 18. Response of MUC.O bacterium ^ofutaitim to Chloramine
(5:1 of C12:NH3-N), pH 7 and 20°C
48
-------
100
10
^ 1
c
Ol
u
OJ
a.
0.1
0.01
Figure 19.
10
20
50
30
Time (min)
Response of Hyc.obac.tMA.um phl^i to Chloramine
(5:1 of C12:NH3-N), pH 7 and 20°C
49
-------
32 percent survival for 50 minutes when exposed to a residual of 0.9 mg/£.
Figure 18 shows the response of M. ^on^uL-itim to various concentrations
of chloramine. Inactivation to 99.9 percent required residuals of 6.4,
6.22, and 4.65 mg/£ for contact times of approximately 13, 18, and 42
minutes, respectively. M. ^onta-itim also exhibited approximately 6 per-
cent survival for 60 minutes when exposed to 3.25 mg/£ chloramine. The
response of M. phJL&i to chloramine is shown in Figure 19. Slightly more
than 0.11 percent of the original culture was observed to survive for
12 minutes with a residual of 5.5 mg/£. With a residual of 3.8 mg/-£,
0.3 percent was observed to survive after 15 minutes. Inactivation to
99.9 percent was found when M. pkl&i was exposed to 1.5 mg/l chloramine
for 28 minutes.
Figure 20 is a log-log plot of concentration of free chlorine
or chloramine vs. time to produce 99.9 percent inactivation of E. c.oLi,
C. paA&pAllo&iA , M. {tOfttuJ-tum, and M. ph£e/c at pH 7 and 20°C. Data
representing the response of the organisms to free chlorine were discussed
in an earlier report from this project.1 In several cases, data for 99.9
percent inactivation with free chlorine or chloramine were not available.
In such cases, data for 99.9 percent inactivation were extrapolated.
From Figure 20, several empirical statements concerning the disinfection
of these organisms can be made. One common method of analysis of disin-
fection data is in terms of the empirical expression, Cnt = constant.
This relation is used to linearize the data for constant temperature, pH,
organism and percent inactivation. In this expression, C - concentration
(mg/£) , t - time, and n = unitless value. On a log-log plot of concen-
tration vs. time, the slope of the curve, -1/n, is easily found. Knowl-
edge of the value of n is of interest because an estimate of the relative
order of reaction of concentration and time of disinfection can be made.11
For n > 1, disinfection is more dependent upon concentration of disinfec-
tant than upon contact time. For n < 1, disinfection depends more upon
contact time than upon concentration.
Values for n have been compiled for M. phJLeA., M. faoJutuuitum, C.
and E. coti in Table 7. From the discussion of the meaning
of the n value, perhaps the most striking feature of these results is the
difference in the empirical reaction order, n, with respect to the use of
free chlorine or chloramine. When free chlorine is used as the disin-
fectant, the n values for M. pktai-, M. faofvtu.^u> are
nearly the same. These values are all slightly above 1, indicating that
the inactivation by free chlorine of these organisms is slightly more
dependent upon concentration than upon time of contact. In the case of
chloramine, the n values for M. ^on^tu/Mm and C. paAapii-iio^^ are 2.1
and 1.9, respectively. These values indicate that, with chloramine, the
inactivation of these two organisms is much more dependent upon concentra-
tion than upon time of contact than it is for free chlorine.
The empirical reaction order of concentration in the case of
M. pkieA. shows the opposite trend with respect to a change in the disin-
fectant species than do the other two organisms. For free chlorine, the
n value is 1.15 while, for chloramine, it is 0.65, indicating that M. pkl&i
is more dependent upon time of reaction than upon the concentration
50
-------
CD
C
o
(O
S-
o
c
o
c
(O
U
O)
M. pkleA
Chloramine
E. coti
Chloramine
10
1000
Time (min)
Figure 20.
Time vs. Concentration of Free Chlorine or Chloramine
to Achieve 99.9 Percent Inactivation of M. j5o-^tactum,
M. pktZA., C. poAapixctoi^i, and E. coti at pH 7 and 20°C
51
-------
TABLE 7
Estimated Reaction Order of Concentration for
Inactivation of Selected Organisms by Free
Chlorine and Chloramine at pH 7 and 20°C
Organism
M. iovtu^um
M. pkteA.
C. pOAO.pt, JJtOt>Aj>
E. coti
n n
Free Chlorine Chloramines
1.1 2.1
1.15 0.65
1.1 1.9
1.0
of the disinfectant. It was previously stated that, with free chlorine
as the disinfectant, concentration was the more important factor. This
basic difference in the response of two species from the same genus,
M. phtie. and M. fiosutuAtum, to a change in disinfectant species should
serve as a warning in attempting to define standards of disinfection
efficiency for a whole group of organisms by studying the responses of
only one or two species under laboratory conditions.
Tables 8 and 9 show the relative effectiveness of chloramine
and chlorine at pH 7 and 20°C. The data in Table 8 were produced by
assuming a constant contact time of 30 minutes and determining the con-
centration of free chlorine or chloramines necessary to produce 99.9
percent inactivation. The data in Table 8 were produced by assuming a
residual of free chlorine or chloramine and then finding the time required
to inactivate 99.9 percent of M. phl&i, M. ^ofitdUnm, C. pojia.pt>JJiot>-u> and
E. co-tt. Data are also represented for E. coti as found by Butterfield
it oJL.12 and Butterfield and Wattie.13
From Table 8, it can be seen that, for a 30 minute contact
time, a free chlorine residual of between 1.0 and 3.5 mg/£ is needed to
provide for 99.9 percent inactivation of the proposed indicator organisms.
Data presented by Butterfield at at.12 and Butterfield and Wattie13
indicate that a free chlorine residual of 0.03 mg/l is required to
totally inactivate E. coti. Thus, for a 30 minute contact time, M. phtui
and C. p
-------
TABLE 8
Concentration of Disinfectant Needed to Achieve
99.9 Percent Inactivation of Selected Organisms
in 30 Minutes at pH 7.0 and 20°C
Organism
M. lovtwubm
M. phJiQA.
C. p-ii.
E. doti
E. doU*
Chloramine
(min)
4000
37
450
1.5
15
Free Chlorine
(min)
120
24
24
-
1.2
Ratio
Chloramine/Chlorine
33.0/1
1.5/1
18.7/1
-
12.5/1
From Butterfield at at.12 and Butterfield and Wattie1.3
53
-------
resistant than E. co£/c. C. paJiapAi2oA, chloramine is apparently only 1.4, 1.45 and 4.0 times less
effective than free chlorine under these conditions. This would indi-
cate that, for a 30 minute contact time with these three organisms,
disinfection to 99.9 percent inactivation with free chlorine produces
approximately the same effect as with chloramines. This can be explained
for the moment by examining Figure 20. This figure presents data for
the inactivation of M. pkteA., M. {,oJvtuuitum, C. poAap-i-ULo^-u and E. coti
with free chlorine and chloramine at pH 7 and 20°C. Notice that if the
curve for the inactivation of M. ^o^ta-itam with free chlorine is extended,
it will intersect the curve for 99.9 percent inactivation with chloramine
at the point corresponding to approximately a 15 minute contact period
and a disinfectant concentration of 7.0 mg/£. This suggests that, with
short times of contact and high dosages of chlorine, M. ^o^ita-itiMn may be
more resistant to free chlorine than it is to chloramine. Using the same
argument, it may be pointed out that, if the inactivation curves for
M. phleA. are extended over long periods of contact and small dosages of
disinfectant, M. pkl&i may also be more resistant to free chlorine than
it is to chloramine. It must be remembered, however, that no experimental
data are available over the extended areas of the curves used in this
analysis. It may very well be that these curves are not linear in these
regions. Only more research can resolve this question.
Table 9 uses a constant 1 mg/l chlorine residual as the criter-
ion to compare the effectiveness of chloramine and chlorine. To achieve
99.9 percent inactivation of M. ph£&i, M. fiofctuitum and C. p
-------
and C. paJtapt>iJLoi>JJ> are approximately TOO, 20, and 20 times, respectively,
more resistant than E. coti when using 1 mg/l as the criterion for disin-
With residuals in the form of chloramine, the above three
respectively, 260, 2.4, and 30 times more resistant than
comparison is based upon the data reported by Butterfield
It can also be seen that, using these same conditions,
. phJteA., C. pafiapt>jJLoi,
-------
In initial experiments, the resistance of C. pasiapt>i£oA4A and
E. CLoLi to various ozone concentrations was determined. If the log of the
percent of organism survival for a constant detention time of 12 seconds
is plotted against the log of total applied ozone (Figure 21), a straight
line results. Here detention time refers to the mean contact time in the
continuous flow system. It should be pointed out that the reactor is
operated for at least 6 equivalent volume turnovers for each detention
time so as to achieve steady state before enumerating the surviving organ-
isms. Preliminary experiments were carried out to determine the time
required to reach steady state for a detention time of 5 minutes. Figure 22
shows that steady state is reached within 5 minutes or with one volume
turnover.
It was observed in the preliminary experiments that for a given
concentration of ozone, the degree of inactivation is profoundly affected
by the initial density of organisms. Therefore, three different experi-
ments were performed with three different initial densities of C. paAap^-i-
lo&iA, e.g., 1.55 x 10?, 1.25 x 106, and 1.35 x 105 cells/me, respectively.
It can be seen in Figure 23 that, for an applied ozone concentration of
0.14-0.15 mg/£, a 4 log reduction occurred when the initial density of
yeasts was 1.35 x 10^ cells/m£ while no observable inactivation took place
when the initial density was 1.55 x 107 cells/iu£. With a detention time
of 1 minute, about 15 percent survival was observed when the initial density
of C. pana.pA was 1.25 x 106 cells/m£. The concentration of total
organic carbon (TOC) was also determined for three different initial den-
sities of the yeast in the feed solution in order to confirm the different
degrees of inactivation with respect to TOC for a given concentration of
ozone. Figure 24 shows the TOC values for the different densities of
C. p-it>. The TOC was about 120 mg/£ for an initial yeast density
of 1.55 x 107/m£, compared to approximately 4 mg/£ for a yeast density of
1.35 x 105/m£. A comparison of Figures 23 and 24 would suggest that there
is a correlation between yeast inactivation by ozone and organism density
and/or TOC. It would appear that there is an increased TOC with an increase
in yeast density. Presumably the increased TOC caused an increased ozone
demand which, in turn, led to less inactivation. Thus, 0.15 mg/£ of
ozone was not sufficient to inactivate yeasts when the density was approx-
imately 107/m£, TOC of 120 mg/£, while the same concentration of ozone
gave 4 log reduction in yeasts when the density was I05/m£, TOC of 4 mg/£.
This preliminary study indicates that ozone is an efficient
disinfectant. The major part of inactivation occurs in the first few
seconds of contact time and then it stabilizes. The initial density of
yeast in the feed solution has a substantial effect on the degree of
inactivation due to its high ozone demand. Results of one study show
that C. paAapAJJLo&'ti, was more resistant than E. coti when both were mixed
together in feed solution. Studies of a similar nature will be expanded
to include mixed cultures of yeasts, acid-fast organisms, Saj&nonatta.,
E. colsi and poliovirus to determine the response of these organisms to
ozone. If the yeasts and acid-fast organisms prove to be as resistant
to ozone as they are to chlorine, the utility of these organisms as indi-
cators of disinfection efficiency will be greatly enhanced.
56
-------
100
10
S-
3
4-1
c
d)
(J
JUiot>JJ> and E. coti
by Ozone with a Detention Time of 12 Sec. in
Buffered, Demineralized Water
57
-------
s_
3
O)
Q.
uu
90
80
70
60
50
40
30
20
10
Fi
\
\
\
\
\
.
Ozone oonc. = 0.14-0.15 mg/£
Detention time = 5 min
Efflu
, r oa
Tempe
— —
ent pH = 6.9
Aapi> = 1.25 x 10 /ml—
rature = 22°C
— £
^— — -
— <
— — •—
0123 45
Volume turnovers
gure 22. Equilibrium Kinetics of C. p&na.p&-U.oA-u>
Inactivation by Ozone in Buffered,
Demi nerali zed Water
58
-------
100
10
s-
3
oo
o
OJ
D-
0.1
0.01
Ozone cone. = 0.14-0.15 mg/l
Effluent pH = 6.9
Temperature = 22°C
Initial Densities
1.55 x 107/m£
1.25 x 106/m£
1.35 x 105/m£
0.001
Figure 23.
1
3 4
Detention time (min )
Effect of Initial Density of C.
on Degree of Inactivation for a Given
Concentration of Ozone
59
-------
10C
10'
O)
CO
c
CD
to
(O
2 io6
10 TOO
TOC (mg/£)
Figure 24. TOC of Various Densities of C.
1000
60
-------
SECTION 6
WORK IN PROGRESS
For any new indicator organism(s), e.g., wastewater yeasts or
acid-fast organisms, to be of value in determining disinfection effi-
ciency, the indicator(s) must satisfy certain criteria. One of these
criteria is that the indicator organism(s) must be detectable whenever
the pathogens of concern are present. If the indicator organisms are
to be widely used, they must be present in wastewater and receiving
streams on a worldwide basis. Therefore, a re-examination of the avail-
able literature to ascertain the reported occurrence of yeast organisms
is underway, with special consideration being given to their presence
in wastewaters outside the continental United States. Attention is also
being given to the examination of alternate enumeration methods for
yeasts with emphasis being placed on reducing the presently required
incubation time of 30 hours. At the same time, the literature is being
reviewed for more information relevant to acid-fast organisms as a poten-
tial new indicator of disinfection efficiency. In this review, the
occurrence of acid-fast bacteria in wastewater and raw water is being
considered; however, the emphasis will be on acid-fast microorganisms,
as a class, and their resistance to disinfecting agents, particularly
with reference to Myc.obact&su.wm tu.baAcu£ot>-l(>. This review will assist
in determining whether or not the use of acid-fast organisms, as indi-
cators of disinfection efficiency, will provide a margin of safety in
those cases where Myc.obact&tiun\ tubeAc.utot>JJ> is felt to be of some con-
cern as a waterborne pathogen.
Work previously reported as a result of this study has demon-
strated the occurrence of yeasts and acid-fast organisms in raw wastewater,
and in effluent following treatment (activated sludge, trickling filter,
chlorination), using a single wastewater treatment plant, i.e., the East
Side Treatment Plant of the Urbana-Champaign Sanitary District. To be
useful as a new indicator of disinfection efficiency, the proposed indi-
cators must be consistently present in wastewater effluents in the
absence of adequate disinfection and in surface waters below outfalls,
particularly if the receiving stream is used as a raw water supply source.
Monitoring of a single system over a period of at least a year is now
underway to ensure that the indicator organisms originating from a waste-
water treatment plant are present in the receiving water and through a
water treatment plant in sufficiently high densities to render the new
indicator organisms useful. The system under investigation is a 35 mile
stretch of the Salt Fork of the Vermin ion River in Champaign and Vermillion
Counties, Illinois. Upstream, the community of St. Joseph, Illinois, has
a well operated contact stabilization wastewater treatment plant. Down-
stream, the community of Oakwood takes river water and treats it by con-
ventional coagulation-flocculation, etc. A semi-monthly analysis of the
wastewater treatment plant, its receiving stream, and the water treatment
plant is made for fecal coliform, acid-fast organisms and yeasts. In
61
-------
addition to being able to determine the temporal variation of the
indicator organisms in a field situation, attention is also being given
to possible regrowth or non-point input of the proposed new indicator
organisms. Monitoring the indicator organisms following various unit
operations and processes at both the wastewater and water treatment
plants will facilitate the observation of any selective removal of
either of the proposed new indicator organisms. It may be noted that
previous work at the East Side Treatment Plant of the Urbana-Champaign
Sanitary District indicated that the yeast organisms are less variable
in their occurrence throughout a wastewater treatment plant than either
the acid-fast or fecal coliform organisms; the current work is capable
of verifying this observation.
In addition to using the selected stream for the study out-
lined above, it is also being used as a source of water in performing
laboratory investigations on the removal capabilities of various treat-
ment units. That is, the removal of the proposed indicator organisms by
processes employed in the treatment of water supplies and by advanced
wastewater treatment is being evaluated. Raw water samples, obtained
from the stream and containing a natural population of the proposed
indicator organisms, as well as coliform organisms, will be subjected to
batch experiments involving various processes. Through these studies,
it will be possible to confirm the field observations as to the removal
of the proposed indicator organisms by water and advanced wastewater
treatment processes, as well as to observe any preferential removal of
acid-fast organisms, coliform organisms, or yeasts.
Later, it is expected that studies concerning the behavior of
the proposed new indicators to iodination will be initiated. Since
iodine may be used in an emergency situation or in the disinfection of
individual or small water supplies, the response of the proposed new
indicators to iodine is significant.
Depending upon the results of the literature review on the
relative resistance of acid-fast organisms and Myzobact&iium ;tubetcu£c4-u
laboratory studies on the relative sensitivity of the acid-fast bacteria
and the M. tu.bejL^> organism to chlorine may be undertaken.
Additional work on the response of the proposed new indicators
to ozonation is continuing.
62
-------
REFERENCES
1. Engelbrecht, R. S., Foster, D. H., Greening, E. 0., and Lee, S. H.,
"New Microbial Indicators of Chlorination Efficiency," EPA Report
670/2-73-082 (1974).
2. Buras, N., and Kott, Y., "A New Approach in E. coLi Identification,1
P/ioc. 6th InteAncuU-onat Wat&i Pott. R&seoAcA Con^., Pergamon Press
(1972).
3. Standard Mzthodb fan. the. Examination o£ WateA and WoAtewat&L, 13th
ed., Amer. Pub. Health Assn., Amer. Water Works Assn., and Water
Poll. Control Fed., Amer. Public Health Assn. Publication Office,
Washington, D. C. (1971).
4. Scarpino, P. V., Berg, G., Chang, S. L., Dahling, D., and Lucas,
M., "A Comparative Study of the Inactivation of Viruses in Water
by Chlorine," Wat&L Ru>e.a>ich, 6, 959-965 (1972), Pergamon Press,
Great Britain.
5. Pal in, A. T., "The Determination of Free and Combined Chlorine in
Water by the use of Diethyl-p-phenylene Diamine," Joan. Am&t. Wat&i
., 49, 873 (1957).
6. Breed, R. S., Murray, E. G. D., and Hitchens, A. P.,
Manual oft V&t&uninative. Bacteriology, 6th ed., Williams and Wilkins
Co. , Baltimore (1948).
7. Joklik, W. K. , and Smith, D. T. (eds.), 2t!>: A Taxonomy Study, 2nd ed., Delft,
The Netherlands (1970).
10. Laskin, A. I., and Lechevalier, H. A. (eds.), Handbook o
b^iotoQij, Vol. 1, Organismic Microbiology, CRC Press, Cleveland,
Ohio (1973).
11. Fair, M. F., Geyer, J. C., and Okun, D. A., WateA and
, Vol. 2, John Wiley and Sons, Inc., New York (1968).
12. Butterfield, C. T., Wattie, E., Megrarian, S., and Chambers, C. W.,
"Influence of pH and Temperature on the Survival of Col i forms and
Enteric Pathogens when Exposed to Free Chlorine," Pubtic Health
R&povt, 63, 51, 1837 (1943).
63
-------
13. Butterfield, C. T. , and Wattle, E., "Influence of pH and Tempera-
ture on the Survival of Col i forms and Enteric Pathogens when
Exposed to Chloramine," Pabtic. He,aJttk Re.po/i&>, 61, 157 (1946).
14. Pavoni , J. L., Tittlebaum, M. E., Spencer, H. T. , Fleischman, M.,
Nebel , C., and Gotschling, R. , "Virus Removal from Wastewater
Using Ozone," WatoA. and Sewage. WotfexS, 59, 119 (Dec. 1972).
15. Gomella, C., "Ozone Practices in France," JOUA. AmeA. Wate-t
., 64, 39 (1972).
16. Fetner, R. H., and Ingols, R. S., "A Comparison of the Bacterial
Activity of Ozone and Chlorine against E. coti at 1°," JouA. Gun.
. , 75, 381 (1956).
17. Scott, D. B. M., and Lesler, E. C., "Effect of Ozone on Survival
and Permeability of E. coti, " JOUA.. Gen. HicAoblol. , 85, 567 (1963)
18. Shechter, H., "Spectrophotometric Method for Determination of
Ozone in Aqueous Solutions," WateA ReAzasick, 7, 729 (1973).
64
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APPENDIX
LABORATORY APPARATUS FOR ENUMERATION OF YEASTS, ACID-Fast ORGANISMS
FECAL COLIFORMS, E. co£t, AND SalmonMa
All glassware and other apparatus required in using the membrane
filter technique should be sterilized as described in "Washing and Sterili-
zation," Section 403, Standard Me.thodt> (13th ed.).
1. Bottles for collection of field samples.
2. 20 mi screw cap test tubes for collection of laboratory samples.
3. Sterile containers for phosphate buffer (e.g., 16 oz prescription
bottles).
4. Pi pets and graduated cylinders.
5. Containers for storing culture medium.
6. Culture dishes: disposable, sterile plastic petri dishes, 60 x 15 mm.
7. Membrane filtration unit, including filter funnel and a 1.0 £ filtering
flask.
8. Vacuum source.
9. Filter membranes, 47 mm diameter with 0.45y pore size and grids (black
or white membrane filters).
10. Forceps.
11. 95% ethyl or absolute methyl alcohol for sterilizing the forceps by
ignition.
12. Bunsen burner.
13. Incubators which can provide a temperature of 37°C and maintain a high
level of humidity.
14. Wide field dissecting microscope.
15. Autoclave.
16. Water bath at a temperature of 44.5°C.
17. Refrigerator.
18. Waring blender (for field samples only).
65
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ENUMERATION TECHNIQUE FOR YEASTS - FIELD STUDIES
A. Laboratory Apparatus
Same as described under Laboratory Apparatus for the Enumeration
of Yeasts, Acid-Fast Organisms, Fecal Coliforms, E. coti and SalmoYieJUta
B. Materials and Culture Media
Media and reagents should be sterilized in an autoclave at 121°C
for 15 minutes.
1. Phosphate buffer: Dissolve 1.7 g potassium dihydrogen phosphate,
KH2P04 and 0.3 g sodium hydroxide, NaOH in 5 I deionized or dis-
tilled water. The pH should be approximately 7.0.
2. 1 N HC1
3. Yeast extract-malt extract agar (YMA) medium
Yeast extract 3.0 g
Malt extract 3.0 g
Peptone 5.0 g
Dextrose 10.0 g
Agar 15.0 g
*Chloramphenicol (Sigma Chem Co.) 5.0 g
*Penicillin (Sigma Chem Co., 1595 units/mg 1.0 g
**Rose bengal, 4% aq. sol. 5.0 mt
Deionized or distilled water 1000 mt
*Antibiotics used to inhibit bacterial contamination
**Rose bengal used to suppress mold growth.
C. Samples
Samples should be collected in sterile containers and refrigerated
not more than 5 hours before use.
D. Procedure
1. Melt and store previously prepared and sterilized YMA medium in
water bath at 45°-50°C.
2. Adjust pH of YMA medium to PH 3.6 to 3.7 with 1 N HC1.
66
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3. Pour 5.0 mt YMA medium into a culture dish and solidify.
4. Blend wastewater samples in a Waring blender for 30 seconds to
disperse any clumps.
5. Select the size of the sample which will result in the growth of
at least 10 and not more than 80 yeast colonies per culture dish.
Use phosphate buffer solution for preparing dilutions.
6. Accomplish filtration as described in "Standard Total Coliform
Membrane Filter Procedure," Section 408A, StcwdaAd
7. After filtration place the membrane filter in an inverted position
on the top of the agar in the previously prepared YMA plates.
8. Pour 6.0 mt of the prepared YMA medium over the membrane filter
and solidify.
9. Incubate at 37°C and/or room temperature. See comment below for
recommended incubation time.
10. Examine growth for white or pigmented yeast colonies from the
bottom of the culture dish using a dissecting microscope. Some
yeast colonies may appear pink due to the rose bengal .
Comments
Generally, it has been found that yeasts grow more rapidly at 37°C
than at room temperature. However, the recovery of yeasts is generally
higher when incubated at room temperature as compared to incubation at 37°C.
Yeast can be detected after 20 hours when incubated at 37°C and after 30
hours when incubation is carried out at room temperature.
67
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ENUMERATION TECHNIQUE FOR YEASTS - LABORATORY STUDIES
A. Laboratory Apparatus
Same as described for Enumeration of Yeasts, Acid-Fast Organisms,
Fecal Coliforms, E. c.oti and SaimoneJLta.
B. Materials and Culture Media
Media and reagents should be sterilized in an autoclave at 121°C
for 15 minutes.
1. Phosphate buffer pH 7, same as described in Enumeration Technique
for Yeasts - Field Samples.
2. Yeast extract-malt extract agar (YMA) medium
Yeast extract 3.0 g
Malt extract 3.0 g
Peptone 5.0 g
Dextrose 10.0 g
Agar 15.0 g
*Chloramphenicol (Sigma Chem Co.) 5.0 g
*Penicillin (Sigma Chem Co.) 1.0 g
Deionized or distilled water 1000 mi
*Antibiotics used to inhibit bacterial contamination.
3. 0.05 N sodium thiosulfate solution in deionized water.
C- Samples
Samples from the chlorine and chloramine experiments should be
immediately neutralized in 0.05 N sodium thiosulfate.
D. Procedure
1. Select the size of the sample which wi"1! result in the growth of
at least 10 and not more than 80 yeast colonies per culture dish.
Use phosphate buffer solution for preparing dilutions.
2. Accomplish filtration as described in "Standard Total Coliform
Membrane Filter Procedure," Section 408A, Standard M&tkod*.
68
-------
3. Place membrane filter right side up on solidified agar.
4. Count colonies after 48 hours incubation at room temperature.
69
-------
ENUMERATION TECHNIQUE FOR ACID-FAST ORGANISMS - FIELD SAMPLES
A. Laboratory Apparatus
Same as described for Enumeration of Yeasts, Acid-Fast Organisms,
Fecal Coliforms, E. c.otl and SaimontMa.
B. Materials and Culture Media
1. Phosphate buffer, pH 7. Same as for Enumeration Technique for
Yeasts - Field Samples.
2. Oxalic acid (2.5%) for pretreatment.
3. NaOH (2%) for neutralizing oxalic acid.
4. Phenolphthalein indicator solution (0.5% alcoholic solution).
5. Acid-fast bacillus medium
Middlebrook and Cohn 7H9 mineral base 4.7 g
(BBL, Cockeysville, Maryland)
Malachite green 1.0 mg
Sodium propionate 1.0 g
Agar 15.0 g
Deionized or distilled water 1000 mi
Autoclave the above for 15 min at 121°C; cool then add
Middlebrook and Cohn OADC enrichment 100 ml
(BBL, Cockeysville, Maryland)
Antibiotic stock suspension 10 mi
6. Antibiotic stock suspension
Penicillin (Sigma Chem Co.) 2500 units/m£
Naladixic acid (Sigma Chem Co.) 2500 pg/m£
Mycostatin (Gibco Chem Co.) 5000 units/m£
7. Brook's acid-fast stain (modified)
a) Stock Brook's carbol fuchsin
Basic carbol fuchsin 4 g
70
-------
Phenol 12 g
Ethyl alcohol 25 ml
DMSO (dimethyl sulfoxide) 25 mi
Glycerol 25 mi
Dilute to 160 mi with distilled water and filter through
0.45y membrane filter prior to use.
b) Stock Brook's malachite green and decolorizer
Glacial acetic acid 30 mi
Glycerol 50 mi
Malachite green, 2% aq. sol. 220 mi
c) Ethyl alcohol
Filter solution through 0.45y membrane filter prior to use.
*
d) Acid decolorizer, oxalic acid, 10% aq. sol.
C. Samples
Wastewater samples should be collected in sterile containers and
refrigerated not more than 5 hours before use.
D. Procedure
1. Prepare acid-fast bacillus medium, sterilize and cool in water
bath at 45°C (avoid reheating medium).
2. Asceptically add 10 ml Middlebrook and Cohn OADC enrichment to
100 mi acid-fast bacillus medium and mix gently to avoid air
bubbles.
3. Pour the above medium into a culture dish and solidify.
4. Extra culture dishes with media may be stored in a refrigerator
for subsequent use.
5. Blend the sample for 30 sec to disperse any clumps.
6. Pretreatment of sample to eliminate non-acid fast organisms.
a) React equal volumes of sample and 2.5% oxalic acid for 10 min.
b) Neutralize with 2% NaOH using phenolphthalein as indicator.
7. Select the size sample which will result in the growth of at least
10 and not more than 80 acid-fast colonies per culture dish. Use
phosphate buffer solution for preparing dilutions.
3% HC1 can also be used; results are compatible to using 10% oxalic acid.
71
-------
8. Accomplish filtration as described in "Standard Total Coliform
Membrane Filter Procedure," Section 408A, Standard M&tkodd.
9. After filtration place the membrane filter in culture dish contain-
ing the acid-fast selective medium.
10. Incubate at 37°C in humidified incubator.
11. When colonies appear, membrane filter is removed from surface of
medium and acid-fast stained (colonies generally appear after 3
days incubation).
a) Staining procedures
1) Dilute stock Brook's carbol fuchsin 1:5 in distilled
water.
2) Dilute stock Brook's malachite green decolorizer solu-
tion 1:20 in ethyl alcohol.
3) Place diluted stains into separate petri dishes.
*4) Dispense 25 ml of 10% oxalic acid into two petri dishes.
5) Dry and heat fix colonies by gently heating membrane
filter over a low flame.
6) Stain the dried filter for 1 min in 1:5 carbol fuchsin
solution.
7) Rinse in distilled water to remove excess stain.
8) Decolorize the filter in 10% oxalic acid solution for
1 min.
9) Rinse the filter in water.
10) Repeat steps 8) and 9) using the second petri dish with
oxalic acid.
11) Counterstain the filter for 1 min in 1:20 malachite green
decolorizer.
Rinse the filter in water to remove excess stain.
12. Observe stained membrane filter while moist under a dissecting
microscope. Count darkly stained pink and/or red colonies which
represent positive acid-fast bacilli. Non-acid-fast colonies may
be colorless, green or very faint pink.
Same procedure is followed when using 3% HC1
72
-------
ENUMERATION TECHNIQUE FOR ACID-FAST ORGANISMS - LABORATORY STUDIES
A. Laboratory Apparatus
Same as described for Enumeration of Yeasts, Acid-Fast Organisms,
Fecal Coliforms, E. co-U and 5o£mone££a.
B. Materials and Culture Media
Reagents (1, 2 and 3) and culture media should be sterilized in an
autoclave at 121°C for 15 min.
1. Phosphate buffer, pH 7. Same as for Enumeration Technique for
Yeasts - Field Samples.
2. Acid-fast bacillus medium
Middlebrook and Cohn 7H9 mineral base 4.7 g
(BBL, Cockeysville, Maryland)
Malachite green 1.0 mg
Sodium propionate 1.0 g
Agar 15.0 g
Deionized or distilled water 1000 ml
Autoclave the above for 15 min at 121°C, then add
Middlebrook and Cohn OADC enrichment 100 ml
(BBL, Cockeysville, Maryland)
Mycostatin (Gibco Chem Co.) 100,000 units
3. 0.05 N sodium thiosulfate solution in deionized water
C. Samples
Samples from chlorine and chloramine experiments should be immedi-
ately neutralized with 0.05 N sodium thiosulfate.
D. Procedure
1. Prepare acid-fast bacillus medium, sterilize and cool in water bath
at 45°C (avoid reheating medium).
2. Aseptically add 10 ml Middlebrook and Cohn OADC enrichment and 10 g
dextrose to 100 ml of acid-fast bacillus medium and mix gently to
avoid air bubbles.
73
-------
3. Pour the above medium into a culture dish and solidify.
4. Extra culture dishes with media may be stored in a refrigerator
for subsequent use.
5. Select the size sample which will result in the growth of at least
10 and no more than 80 acid-fast colonies per culture dish. Use
phosphate buffer solution for preparing dilutions.
6. Accomplish filtration as described in "Standard Total Coliform
Membrane Filter Procedure," Section 408A, Standard
7. After filtration place the membrane filter in culture dish contain-
ing the acid-fast selective medium.
8. Incubate at 37°C in humidified incubator.
74
-------
ENUMERATION TECHNIQUE FOR E. caJU AND FECAL COLIFORMS
A. Laboratory Apparatus
Same as described for Enumeration of Yeasts, Acid-Fast Organisms,
Fecal Coliforms, E. coti and SaimoneJLia..
B. Materials and Culture Media
Media and reagents should be sterilized in an autoclave at 121°C
for 15 min.
1. Phosphate buffer, pH 7. Same as for Enumeration Technique for
Yeasts - Field Samples.
2. 0.05 N sodium thiosulfate solution in deionized water.
3. Fecal coliform medium
Bacto mFC agar 52 g
Sterile deionized water 1000 ml
0.2 N NaOH 10 mi
Rosalie acid 100 mg
C. Samples
Samples from the chlorine and chloramine experiments should be
immediately neutralized in 0.05 N sodium thiosulfate.
D. Procedure
1. Suspend 52 g of mFC agar in 1000 mi deionized or distilled water.
2. Heat to boiling or until agar melts. DO NOT AUTOCLAVE. Cool to
55°C.
3. Dissolve 100 mg rosalic acid in 10 mi 0.2 N NaOH. Add the solution
to 1000 mi fecal coliform medium and mix gently to avoid air
bubbles.
4. Pour 5.0 mi fecal coliform medium into a culture dish and solidify.
5. Select the size of the sample that will result in growth of at
least 10 and not more than 80 colonies per culture dish. Use
phosphate buffer for preparing dilutions.
75
-------
6. Accomplish titration as described in "Standard Fecal Coliform
Membrane Filter Procedure," Section 408B, Standard Method*.
1. After filtration, place the membrane filter in culture dish con-
taining the fecal coliform selective medium.
8. Submerge culture dishes in plastic bags in 44.5°C waterbath.
9. Count dark blue colonies after 24 hours incubation.
76
-------
ENUMERATION TECHNIQUE FOR Salmonella
A. Laboratory Apparatus
Same as described for Enumeration of Yeasts, Acid-Fast Organisms,
Fecal Coliforms, E. coti and SalmoneJLla.
B. Materials and Culture Media
Media and reagents should be sterilized in an autoclave at 121°C
for 15 min.
1. Phosphate buffer, pH 7. Same as for Enumeration Technique for
Yeasts - Field Samples.
2. 0.05 N sodium thiosulfate solution in deionized water.
3. Bismuth sulfite agar
Difco bismuth sulfite agar 52 g
Deionized water 1000 ml
*Mycostatin (Gibco Chem Co.) 5000 unts/100 ml
*Mycostatin is added to inhibit the growth of yeasts.
C. Samples
Samples from chlorine experiments should be immediately neutralized
with 0.05 N sodium thiosulfate.
D. Procedure
1. Bring agar to boil to dissolve the agar, being cautious not to
overheat. Overheating destroys the selectivity of the medium.
2. Disperse warm agar into culture dishes, twirling slightly to keep
the flocculent suspension dispersed.
3. Select the sample size that will result in the growth of at least
10 and not more than 80 colonies per culture dish. Use phosphate
buffer solution for preparing dilution.
4. Accomplish filtration as described in "Standard Total Coliform
Membrane Filter Procedure," Section 408A, Standard M&tkod*.
11
-------
5. Place membrane filters upside down on solidified agar.
6. Count black colonies from the top of the culture dish after 24
hours incubation at 37°C.
78
-------
VIRAL ENUMERATION TECHNIQUE AND TISSUE CULTURE MAINTENANCE
A. Laboratory Apparatus
All glassware and other apparatus required in the enumeration of
poliovirus and the maintenance of the BGM cell line should be washed
and sterilized as described in "Washing and Sterilization," Section
403, Standard M&thod* (13th ed.).
1. 20 mi screwcap test tubes for sample collection.
2. Sterile containers for phosphate buffer storage (e.g., 16 oz
prescription bottles).
3. Pi pets and graduated cylinders.
4. Containers for storing culture medium.
5. Disposable, sterile, tissue culture flasks, 30 and 250 mi.
6. Vacuum source.
7. Bunsen burner.
8. Incubators which can provide a temperature of 37°C and are adapt-
able for COo if tissue culture dishes are used.
9. Tissue culture microscope.
10. Autoclave.
11. Refrigerator
12. Freezers, -20°C and -70°C.
13. Laminar flow hood or UV-sterilized cabinet for tissue culture work.
B. Materials and Culture Media
1. Maintenance Medium (MM)
Ml99 10X (Gibco Chem Co.) 50 mi
Fetal calf serum (Gibco Chem Co.) 50 mi
NaHC03 (7.5%) 10 mi
79
-------
Antibiotics (Gibco Chem Co., penicillin, 5 ml
fungizone, streptomycin, 100X)
Sterile distilled water 385 mi
2. Serum Free Medium (SFM)
Same as in MM, omitting fetal calf serum and increasing water
to 435 ml.
Double Strength Medium (DSM)
Ml99 10X 20 ml
Fetal calf serum 4 ml
NaHCOa (7.5%) 4 ml
MgCl2 0%) 2 ml
Antibiotics (as in MM) 2 ml
Sterile distilled water 68 ml
3. Staining Medium (SM)
Ml 99 10X 20 ml
Neutral red (0.1%) 10 ml
NaHCOs (7.5%) 4 ml
Antibiotics (as in MM) 2 ml
Sterile distilled water 64 ml
Notes
1. All solutions are either purchased sterile or are sterilized by
filtration through a 0.45p membrane filter.
2. All solutions and media containing neutral red should be stored in
the dark to prevent photo-decomposition.
3. Antibiotics, M199, and fetal calf serum can be purchased from Grand
Island Biological Co. Antibiotics and fetal calf serum are stored
at -20°C until used.
C. Maintenance of Cell Line
African green monkey kidney cells (BGM) were obtained from Dr. G.
Berq (EPA, Cincinnati) and are stored frozen at -70°C. When needed,
ampoules containing BGM cells can be aseptically broken and inoculated
into tissue culture flasks containing medium (MM). The medium is with-
drawn and replaced with fresh MM on the third day following inoculation.
When microscopic examination confirms the existence of a confluent mono-
layer, usually on day six or seven, the spent medium is discarded. Five
ml of Trypsin-EDTA (Gibco) is then added to the flasks to remove sorbed
medium. A second 5 ml rinse with Trypsin-EDTA is accomplished, leaving
0.5-1.0 ml in each flask (250 ml tissue culture bottles are used.) The
flasks are then placed in a 37°C incubator for approximately 5 min,
until detachment of the monolayer is evident.
80
-------
Ten mi of MM is then added to each flask to stop the trypsinization
process. The concentration of cells is then determined in the following
manner:
1. To a test tube, 0.1 mi of cell suspension is added to 0.9 ml of a
0.5 percent solution of trypan blue.
2. Viable cells, which fail to stain with trypan blue can be enumer-
ated microscopically using a hemocytometer.
To each large (250 mi) flask, 40 mi of MM is added along with suffi-
cient volume of cell suspension to achieve an initial cell concentra-
tion of 3 x 104/m£. These large bottles are then incubated (37°C) for
304 days, or until the medium color changes from red to orange-yellow.
The medium is then decanted and replaced with fresh MM. A confluent
monolayer generally appears within 3-4 days after medium replacement.
To individual small flasks (30 mi), 6 mi of MM containing 8 x TO4 cells/
mi is added. Medium is replaced within 1-2 days when dictated by the
color change from red to orange-yellow in the indicator system. Con-
fluence occurs 1-2 days after medium replacement, at which time these
small bottles should be used for the virus assay.
To ensure that BGM cells are always available, a portion of the
cells are stored at -70°C. Young cultures, generally those which have
just achieved confluence, are used. Monolayers are trypsinized as
described above. Cell suspensions from several flasks are pooled and
centrifuged in a clinical centrifuge at 1000 rpm for 5 min. Super-
natant medium is then decanted and the volume of the cell pack esti-
mated. Cells are suspended in 20 volumes of MM medium containing 20
percent fetal calf serum and 15 percent dimethylsulfoxide (DMSO), and
mixed by repeating pipetting. One mi aliquots are delivered into
sterile ampoules which are sealed with a torch, and rapidly frozen at
-70°C, where they can be stored until needed.
D. Viral Enumeration
Samples from chlorination experiments should be immediately neutra-
lized with 0.05 N sodium thiosulfate. To 10 mi of the sample, 0.5-0.7
mi of an antibiotic-antimycotic solution containing 10,000 units
penicillin/m£ , 10,000 meg streptomycin/m£ and 25 meg Fungizone/m£ is
added. This mixture can be purchased from Grand Island Biological Co.,
Grand Island, New York. The treated sample is then frozen at -70°C
until the assay is to be performed. In making the assay, the sample
should be thawed and decimal dilutions made using SFM.
Confluent cell monolayers prepared as described are drained of
the spent medium. The cells are then inoculated with 0.5 mi of
sample per 30 mi culture flask. The flasks are then incubated at 37°C
for 2 hours, with gentle manual agitation every 15 min, to allow for
uniform viral attachment.
The excess fluid is then decanted, and 6 mi of an overlay
81
-------
consisting of equal volumes of DSM and melted 1.8 percent Bacto-Agar
(Difco) is gently added to each flask. The overlay should be maintained
at 43°C prior to addition, to keep the agar from solidifying and yet not
raise the overlay temperature too high so as to damage the cells. When
the overlay solidifies, the flasks are incubated upside down at 37°C
for 3 days.
On the third day, an additional 3 mi of media consisting of equal
volumes of SM and melted 1.8 percent Bacto-Agar is added to each flask.
After the addition of the staining layer, and on the fourth and fifth
days of incubation, plaques consisting of unstained areas on the cell
monolayer can be counted.
The number of pfu/m£ is thus the total number of plaques visible
during the three-day period, corrected for the initial dilution factor.
82
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORTNO.
EPA-600/2-77-052
3. RECIPIENT'S ACCESSIOWNO.
4. TITLE AND SUBTITLE
NEW MICROBIAL INDICATORS OF DISINFECTION EFFICIENCY
5. REPORT DATE
August 1977 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(s)Richard s. Engelbrecht, Blaine F. Severin,
Mark T. Masarik, Shaukat Farooq, Sai H. Lee, Charles N,
A jit. Lai r.handani
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
University of Illinois
Urbana, Illinois 61801
10. PROGRAM ELEMENT NO.
IBB 043
11. CONTRACT/GRANT NO.
EPA-IAG-D4-0432
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory--Cin.,OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final May 1972-April 1975
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
This project was jointly funded under Contract No. DADA 17-72-C-2125 with the U.S. Army
Medical Research and Development Command, Washington, D.C. 20314, and through an inter-
agency agreement (EPA-IAG-D4-0432) with the U.S. Environmental Protection Agency
16. ABSTRACT
Since the coliform group of organisms is less resistant to chlorine than
many pathogens, including viruses, the utility of both yeasts and acid-fast organ-
isms as indicators of disinfection efficiency was evaluated. Densities of acid-
fast organisms, yeasts and fecal coliforms in domestic wastewater averaged
1.5 x 104, 5.3 x 104, and 3.9 x 106 organisms/100 mi, respectively. Full scale
chlorination of trickling filter and activated sludge effluents reduced the density
of these organisms by 0.5, 3.0, and 5.0 logs, respectively. A mean density of
2.2 x 106 fecal coliforms/gm wet feces was found in thirty different samples of
fecal matter while that of yeasts was 562/gm in 29 of 30 samples. A mean density
of less than 100 acid-fast organisms/gm was found in only 13 of 30 fecal samples.
Four yeasts and three acid-fast organisms were found to occur commonly in domestic
wastewater. The resistance to free chlorine was: acid-fast > yeasts > poliovirus
type I Mahoney strain > SaLmoneMa. typk+.muAi.um > E&ch > M. pht E. coti at pH 7 and 20°C. C. pajia.p*<
appeared to be more resistant to ozone than E. coti at room temperature.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Disinfection
Chlorination
Indicator species
Mycobacterium
Ozonization
Wastewater
Water treatment
Yeasts
Acid-fast organisms
6M
DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport/
Unclassified
21. NO. OF PAGES
93
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
83
ft U.S.GO«lll«IIB(TPIIimi(6 OFFICE. 1977-757-056/6476
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