EPA-670/2-73-082
February Environmental Protection Technology Series
New Microbial Indicators
of Wastewater
Chlorination Efficiency
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office ot Research and
Monitorinq. Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
14. Environmental Monitoring
5. Socioeconomic Environmental studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
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 guality
standards.
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EPA 670/2-73-082
February 1974
NEW MICROBIAL INDICATORS OF WASTEWATER
CHLORINATION EFFICIENCY
by
Richard S. Engelbrecht
David H. Foster
Elaine 0. Greening
Sal H. Lee
University of Illinois
Urbana, Illinois 61801
Project 17060 EYZ
Program Element 1BB043
Project Officer
C. W. Chambers
U. S.^Environmental Protection Agency
National Environmental Research Center
Cincinnati, Ohio 45268
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Document!, U.S. Government Prmtmi Office. Waihtafton, D.C. 30WJ - Price »1.15
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EPA Review Notice
This report has been reviewed by the Environmental Protection Agency
and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the Environ-
mental Protection Agency, nor does mention of trade names or commer-
cial products constitute endorsement or recommendation for use.
11
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ABSTRACT
The conform group of indicator organisms has a relatively low resistance
to chlorine when compared to pathogens such as enteric viruses and proto-
zoan cysts. Consequently, an effort has been initiated to find a new
chlorine resistant bioindicator of wastewater chlorination efficiency.
Organisms surviving chlorination of wastewater effluents to a free chlor-
ine residual were collected for study by plating on various growth media.
This procedure provided 135 chlorine resistant isolates for further
examination. A yeast and two different acid-fast bacilli were found to
be resistant to chlorination in the range considered necessary for the
inactivation of pathogens, including viruses. One acid-fast bacillus
survived 2.0 mg/£ free chlorine for 67 min while the other survived 1.0
mg/t free chlorine for 15 min. The yeast resisted 1.0 mg/£ free chlorine
for 20 min. In comparison, a pure culture of E. coLi failed to survive
5 min contact with 0.03 mg/£ free chlorine. Yeasts were found to be easily
cultured on acidified yeast extract-malt extract agar. Acid-fast staining
of colonies on membrane filters incubated on mineral-propionate medium,
following sample pretreatment with oxalic acid and NaOH provided a selec-
tive assay technique for acid-fast organisms. Significant quantities of
yeasts and acid-fast organisms were found in wastewater effluents and
yeasts have been isolated from stools. The chlorine resistance and other
characteristics of the yeasts and acid-fast bacilli under study suggest
that they show promise as bioindicators for wastewater chlorination
efficiency for the more chlorine resistant pathogens. This report was
submitted by the University of Illinois at Urbana-Champaign, Department
of Civil Engineering, in fulfillment of Project Number 17060EYZ under the
sponsorship of the Environmental Protection Agency.
This report was submitted in fulfillment of Project Number 17060 EYZ by
the University of Illinois under the sponsorship of the Environmental
Protection Agency. Work was completed as of July 3, 1973.
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CONTENTS
Section Page
I Conclusions 1
II Recommendations 3
III Introduction 5
Relative Resistance of Conforms and Viruses 5
to Chlorine
Relative Resistance of Conforms and Vegetative 6
Bacterial Pathogens to Chlorine
Resistance of Cysts to Chlorine 6
Resistance of Acid-Fast Bacilli to Chlorine 7
Prevalence of Yeasts and Acid-Fast Organisms in 8
Fecal Material and Wastewater
Objectives of this Study 10
IV Materials and Methods 13
Measurement of Chlorine Residuals 13
Isolation of Chlorine Resistant Organisms 13
Culturing of Chlorine Resistant Isolates 14
Screening of Isolates for Chlorine Resistance 14
Detailed Chlorlnation Studies 16
Enumeration of Potential Indicator Organisms 16
in Human Fecal Material and Wastewater
V Results and Discussion 23
Isolates Examined for Chlorine Resistance 23
Further Examination of Isolates from Chlorinated 23
Wastewater
Enumeration of Yeasts 1n Fecal Material and 37
Wastewater
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CONTENTS (continued)
Section Page
Selective Techniques for Enumeration of 50
Acid-Fast Bacilli
VI Acknowledgements 59
VII References 6'!
VIII Publications and Patents 67
VI
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FIGURES
Page
1 Response of Isolate No. 30 to Free Chlorine Residuals 27
Ranging from 0.1 to 1.0 mg/Z
2 Response of Isolate No. 30A to Free Chlorine Residuals 28
Ranging from 0.1 to 1.0 mg/£
3 Response of Isolate No. 82 to Free Chlorine Residuals 29
Ranging from 0.1 to 1.0 mg/£
4 Response of Isolate No. 132 to Free Chlorine Residuals 30
Ranging from 0.1 to 1.0 mg/2,
5 Response of Isolate No. 134 to Free Chlorine Residuals 32
Ranging from 0.5 to 2.0 mg/£
6 Free Chlorine Residuals and Contact Times Necessary for 33
99.9 Percent Kill for Isolates No. 134, No. 30A, No. 132,
and No. 30 as well as for Organisms Compiled by Berg
7 Comparison of Response of E. co-tt ATCC-11229 to Free 35
Chlorine with Data from Butterfield et o£.
8 Chiorination Study of Isolate No. 30 Seeded in Diluted 38
Secondary Effluent
VII
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TABLES
No. Page
1 Conditions of Isolation and Properties of 24
Representative Isolates
2 Chlorination Stucfy of Isolate No. 30 Seeded in 36
Secondary Effluent
3 Examination of Fecal Samples for Total Plate Count 39
and Yeasts
4 Statistical Analysis of Fecal Yeast Densities 40
5 Yeast Densities in Raw Municipal Wastewater 42
6 Yeast Densities in Activated Sludge Effluent 43
7 Yeast Densities in Trickling Filter Effluent 44
8 Total Bacteria and Coliform Content in Raw 45
Municipal Wastewater
9 Total Bacteria and Coliform Content in Activated 46
Sludge Effluent
10 Total Bacteria and Coliform Content in Trickling 47
Filter Effluent
11 Pre-Screening for Chlorine Resistance of Yeasts 49
Isolated from Unchlorinated Wastewater Samples or Stools
12 Effect of Selective Pretreatment Methods on Growth 52
of Acid-Fast Isolates
13 Effect of Selective Media on Growth of Acid-Fast 54
Isolates
14 Effect of Sulfa Drugs in Selective Media on Growth 56
of Acid-Fast Isolates
viii
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SECTION I
CONCLUSIONS
1. EAcAe/u.c/u.a c.oti is not sufficiently resistant to chlorine to be
a useful bioindicator of wastewater chlorination efficiency for chlorine
resistant pathogens such as viruses.
2. Contact of wastewater for 5 min with sufficient chlorine to satisfy
a portion or all of the chlorine demand is a feasible means of screening
out chlorine sensitive organisms from wastewater. Moderately to highly
chlorine resistant organisms surviving this procedure may then be col-
lected for detailed study.
3. The degree of chlorine resistance of organisms collected in the
screening procedure is not well correlated with the chlorine dosage used.
4. The majority of chlorine resistant organisms collected as potential
new indicator organisms were gram positive sporeformers; these were not
studied further due to the dependence of the sporeforming property on
generally uncontrollable biological factors.
5. A yeast, Isolate No. 30, and two acid-fast bacilli, Isolate Nos. 132
and 134, were found to be sufficiently resistant to free chlorine resid-
uals in chlorine demand free water to be potentially useful as bioindi-
cators of wastewater chlorination efficiency.
6. Isolate Nos. 30, 132 and 134 show resistance to free chlorine equal
to or greater than that reported for resistant pathogens such as Coxsackie
A2 virus; the isolates are able to withstand 1.0 mg/£ free chlorine in
chlorine demand free water for up to 15, 15, and 60 min contact time,
respectively.
7. Yeast organisms could be selectively cultured from secondary efflu-
ents on yeast extract-malt extract glucose agar adjusted to pH 3.5 and
incubated at 20°C; however, growth of acidophilic molds caused some inter-
ference in enumeration.
8. Yeasts were isolated from human fecal material in concentrations up
to 10^ yeasts per g in 80 percent of 31 samples tested.
9. Yeasts were isolated from wastewater in average concentrations of
1500, 220, and 420 yeast per mi of raw wastewater, activated sludge and
trickling filter effluents, respectively.
10. Use of sample pretreatment by acidification with 2.5 percent oxalic
acid for 5 to 10 min followed by neutralization with 2 percent NaOH con-
siderably reduced the number of contaminating organisms for the enumeration
of acid-fast bacteria in wastewater.
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11. A method for acid-fast staining of membrane filters was developed
and, v/hen combined with oxalic acid-NaOH pretreatment and selective media,
provided a sensitive and selective method for enumeration of acid-fast
bacteria in wastewater.
12. Limited studies of raw wastewater indicated the presence of acid-
fast bacteria in concentrations of 137 to 500 per mfc.
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SECTION II
RECOMMENDATIONS
1. Chlorine resistant organisms isolated thus far require further
evaluation as to their resistance in a variety of wastewater effluent
types.
2. The membrane filter technique for enumerating acid-fast bacilli
should be thoroughly studied as to its sensitivity, reliability and
accuracy in highly contaminated samples such as wastewater.
3. A method for inhibiting mold growth in assaying wastewater efflu-
ents for chlorine resistant yeasts is needed.
4. The quantities of proposed indicators in fecal material, and
various types of wastewater effluents, including AWT type effluents,
in a variety of climatic and geographical situations, should be
determined.
5. Taxonomic studies of the proposed indicators should be carried out.
6. Proposed indicators should be evaluated under field conditions in
a variety of water quality situations for appropriate sampling methods,
the need for and type of sample concentration techniques, and proper
sample storage procedure.
7. Laboratory scale studies of the response of the proposed indicator
organisms to AWT processes is necessary to provide information on their
usefulness in water reuse schemes.
8. The response of the proposed indicators to disinfectants other
than chlorine should be studied.
9. The resistance of the proposed indicator to disinfectants should
be directly compared to that of resistant pathogens; comparison of the
relative survival rates of the indicators and pathogens in receiving
water is needed.
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SECTION III
INTRODUCTION
The presence of pathogenic organisms in the effluents of conventional
secondary and advanced wastewater treatment operations which may be
resistant to the widely used chlorine disinfection process is a matter
of increased concern. It is presently anticipated that direct reuse
of reclaimed wastewater effluents for potable water supplies will be
necessary in the foreseeable future in many areas of this country due
to rising population and increased per capita water demand. A reliable
biological indicator of the efficiency of the wastewater disinfection
step is one approach that can be used to protect the health of popula-
tions using reclaimed wastewater for potable water supplies and also to
alleviate public health fears of people using waters receiving chlorinated
waste effluents for other purposes such as contact recreation.
RELATIVE RESISTANCE OF COLIFORMS AND VIRUSES TO CHLORINE
The literature indicates that the presently used coliform group of
organisms is far too sensitive to chlorine to be of much value in
indicating the potential presence of chlorine resistant infective
agents such as viruses. At low temperatures, i.e. 0° to 6°C, polio 1
and Coxsackie A2 viruses are much more resistant to chlorine than
EAch&U.chj,a, cati while adenovirus 3 is apparently more sensitive (1, 2).
Nupen (3) concluded that coliform findings should not serve as a measure
of the virus contamination in water and sewage. Liu vt at. (4) studied
the chlorine resistance of 20 human enteric viruses seeded in Potomac
River water. The least chlorine resistant virus, reovirus I, required
only 2.7 min contact with 0.5 mg/Jl free chlorine for a 99.99 percent
kill, assuming first order kinetics. The most chlorine resistant viruses
were poliovirus 2 and Coxsackie B5 which required 40 min to reach the
same kill as the reovirus under the same conditions and assumptions.
Experimentally, Coxsackie A5 and echovirus 12 survived these conditions
for 53.5 and more than 60 min, respectively. Kruse' (5) compared the
chlorine resistance of bacterial virus f2 and E. coti under a variety
of experimental conditions. E. co&t was found to be considerably more
sensitive to inorganic and organic chloramines at 0° to 4°C than the
model virus. For example, the density of E. coJU decreased nearly 5
logs in less than 1 min while the model virus f2 was inactivated to
slightly more than 1 log following 10 min contact with 10 mg/£ chlorine
in the presence of 10~3 M NHaCl. The work of Kruse' demonstrated that
inactivation rates are considerably reduced for both E. (Loti and virus
when the combined chlorine residual is organic chloramine as opposed
to inorganic forms. Kjellander and Lund (6) have reported similar
findings for E. cjoti and poliovirus 3. Burns and Sproul (7) found that
bacteriophage T2 was considerably more resistant to free chlorine than
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E, c.oti. The resistance of viruses to chlorine appears to vary widely.
Thus, if a full assessment of the relative value of an indicator organism
is to be made, the chlorine resistance of all enteric viruses must be
studied. The data that do exist suggest that, with few exceptions,
viruses are more resistant to chlorine than coliforms by a considerable
margin.
RELATIVE RESISTANCE OF COLIFORMS AND VEGETATIVE BACTERIAL PATHOGENS TO
CHLORINE
The resistance of wastewater pathogens other than virus to chlorine is
quite variable. Kabler (8) compared the chlorine resistance of E. c.oti,
keAjobacteA a&Loge,neA, ?&o.u.domoYia& a&iu.&ino&a, SatmoneJtJta typho&a, and
SklQoJUUi dyAe.ntVu.ae. and Aonn&L. At pH 7 and 20° to 25°C, resistance
to free chlorine was approximately the same for E. coti and ?. aeAu.g4.noAa
but was less than that found for S. typho&a and Sh^igelta dyAe.nteJu.ae..
Resistance to combined chlorine was comparable for E. a>tL, P. a&suiQ4.noAat
Skig<a AonneA., and S. typkoAa, while Skigetta dyAe.nteAA.ae. was more
sensitive than the others. A. aeAogeneA was more resistant to both com-
bined chlorine and free chlorine than the other bacteria studied. Tonney
were 5 to 20 times more
resistant to chlorine than coliforms. Favero and his associates (11. 12)
showed Staph. awieuA to be more resistant to chlorine than either coli-
forms or P. aeAag
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. and species of the HoA^ane££a-Waeg£eAta group are pathogenic
protozoans of man capable of being transmitted by the water route (16).
Chang (17) reported that cysts were 160 times more resistant to HOC1
than E. coti and 9 times more resistant than the hardier entero viruses.
Hypochlorous acid residuals at 1.2 mg/£ for 30 min contact were required
to kill 100 percent of E. ki&tofytica. cysts at 23° C in one study (18).
Stringer (16) compared the cysticidal properties of 2 mg/JZ, each of free,
combined inorganic and combined organic chlorine at pH 7 and 27°C and
observed that a 10 min and 40 min contact time was necessary for a 99
percent kill for the first two categories, while organic chloramines
failed to destroy more than 50 percent of the cysts even after 100 min
contact. Cysticidal activity was greatest at low pH. Although the high
degree of resistance which amoebic cysts have to chlorine should be a
matter of concern, it should be noted that they are generally effectively
removed by coagulation-sedimentation and filtration (19). In addition,
they occur in cyst contaminated waters at levels not likely to be greater
than 5 cyst per ma (20). Since the cysticidal chlorine dose greatly
depends on the quantity of cysts present, and, since most investigations
have employed cyst concentrations greatly in excess of those that might
be expected to occur naturally, it is likely that reported cysticidal
doses are greater than those required in the field.
RESISTANCE OF ACID-FAST BACILLI TO CHLORINE
spp. and NocaAdia. spp. are acid-fast organisms which have
been recovered from fecal material and wastewater (21, 22). Hycabac.-
teAiw tubvicu£oA4A is of major concern from a public health standpoint
while other members of the acid-fast genera may be pathogenic depending
on various biological factors. Mycobacteria differ from other vegeta-
tive bacteria in that they have protective layers of mycolic acid and
lipids in their cell walls (23). These cell wall constituents confer
a degree of resistance of mycobacteria to chemical agents such as chlor-
ine that are generally not found in other vegetative bacterial groups.
Musehold (24) found 17 mg/£ was insufficient while 340 mg/£ was suffi-
cient to kill tubercle bacilli following a 2 hr contact in sewage
samples. Kabler (25) found that up to 2 hr contact with 1 to 2 mg/£
chlorine was necessary to achieve a 99 percent inactivation of M. -tubeA-
cjuJto&iA. Greenberg and Kupka (26) reviewed about 15 different studies
which led them to conclude that a chlorine dosage of 20 mg/Jl with a 2
to 3 hr contact time or a chlorine residual of at least 1 mg/JJ, after
1 hr contact were the conditions necessary to destroy all tuberculosis
organisms. If these organisms are of concern from a public health
standpoint, then higher chlorine residuals and longer contact periods
are required to inactivate tuberculosis organisms in comparison to those
needed to destroy enteric bacteria.
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PREVALENCE OF YEASTS AND ACID-FAST ORGANISMS IN FECAL MATERIAL AND WASTEWATER
Two groups of organisms which this study has shown to have potential as
chlorine resistant bioindicators are yeasts and acid-fast bacilli.
Studies of their occurrence in fecal material and wastewater are rather
limited, and knowledge of acid-fast organisms in these materials has
focused principally on the tubercle bacilli.
Cooke (27) reviewed the occurrence of yeasts in a variety of habitats
and reported that they occur commonly in the alimentary tracts of higher
animals which ingest them with their food. Saprophytlc yeasts are able
to pass through the digestive tract relatively unscathed by the acid
conditions in the stomach. Asporoo/enous type yeasts appear to pre-
dominate in human feces. While fecal yeasts are generally retained no
longer than ingested food, pathogenic strains of Candida. atbwJtop&t Candida, and TxAchoApoJton frequently
encountered.
8
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Add-fast organisms are of interest because of their reported chlorine
resistance and the possible pathogenicity of some .Voco/tdca and Ito/cobac-
tutAW species. Reports of tubercle bacilli in feces date back to at
least 1900. Reports of add-fast bacilli in feces other than the
tubercle bacilli are quite sparse. Klose (33) found acid-fast bacilli
in the stools of 55 of 60 patients showing sputum positive for tubercle
bacilli. No acid-fast bacilli were found in 12 fecal specimens from
persons with sputum negative for tubercle bacilli. Alexander (34)
found acid-fast organisms 1n stools of 38 of 45 pulmonary tubercu-
losis patients. Laird vt ad. (35) reviewed similar studies and reported
that the majority of patients showing positive sputum specimens also
provided stool specimens which contained add-fast organisms. Acid-fast
organisms were not frequently found 1n the feces of healthy persons.
Wilson and Rosenberger (36) detected acid-fast organisms 1n 21 percent
of over 1000 fecal samples from apparently healthy individuals. Laird
it aJL. (34), on the other hand, found acid-fast organisms in the feces
of only 2 of 54 persons with negative sputum. It should be noted that
these findings are quite outdated in terms of techniques employed. It
1s quite possible that the use of more modern methods not specifically
aimed at finding tubercle bacilli but more generally towards isolating
all potential acid-fast organisms would show a greater percentage of
the healthy population to be excreters of acid-fast organisms,
Several investigations on the presence of tubercle bacilli 1n wastewater
have appeared 1n the literature. Musehold (24) added tubercle bacilli
to river water, sewage, and sewage sludge and exposed the samples to
prevailing environmental conditions. Viable bacilli were recovered from
river water and sewage sludge even after 5 months and 6 1/2 months
exposure to these conditions, respectively. Bacilli were also demon-
strated 1n the effluent from a secondary wastewater treatment plant.
Jensen and Jensen (21) were consistently able to recover viable M.
tubvicutjo&ii> 1n effluents from sanatoria following biological treatment,
even when the populations of aerobic enteric bacteria had been reduced
to 0.2 percent of their original numbers. These authors also determined
that 10 of 10 samples each from raw sewage and activated sludge effluent
from a sanitorlum treatment plant were positive for tubercle bacilli
while only 1 of 10 samples from the chlorinated effluent contained
detectable quantities of these organisms. Dosage was 10 mg/i chlorine
with a contact in excess of 3 hr. Pramer vt aJL. (22) reported that
avian tubercle bacilli seeded into raw wastewater was not removed by
6 hr of settling. Chemical coagulation with ferric chloride at pH 5.0
resulted 1n removal of 99 percent of the bacilli as did sand filtration.
On the other hand, aeration of the wastewater for 24 hr in laboratory
biological treatment units had little effect on the density of tubercle
bacilli. Free chlorine residual disinfection of the wastewater was neces-
sary to achieve significant reduction of the bacilli. The authors noted
that free residuals and dosages used were considerably higher than that
normally maintained for disinfection of ordinary municipal wastewater.
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The authors studied a tuberculosis sanitorium trickling filter plant and
found 1500 M. tub&iculoA organisms per mi in the raw waste which were
reduced to 10 per mi in the chlorinated plant effluent. However, the
raw sludge had 100,000 organisms per m£ indicating that while removal
may have appeared to be efficient, tremendous quantities of viable path-
ogens had been concentrated in the sludge. Digestion of the sludge was
only able to reduce the M. tubeAculoAit* density to 10,000 per mfc. Mu'ller
(37) reported that raw wastewater contained 5 to 100 tubercle bacteria
per liter compared to 100 times as many SaAnonztla. organisms in the same
volume. She stated that tubercle bacteria must be considered to be a
regular component of urban wastewater, even without known sanitaria
sources. The investigator found 90 percent of the raw wastewater and
50 percent of the settled wastewater samples to be positive for tubercle
bacilli. On the other hand, sludges subjected to 30°C under septic con-
ditions showed no tubercle bacilli. Sludge samples taken from drying
beds used for waste activated sludge were 44 percent positive even after
30 days drying time. It would appear from these studies that while
biological treatment may be counted on for some removal of acid-fast
organisms, both the effluent and the resulting sludges will contain
viable organisms. Chlorination of the effluent with high dosage at
extended contact periods may provide a safe effluent.
OBJECTIVES OF THIS STUDY
The major objective of Chlorination of wastewater effluents is to pro-
duce an effluent that is acceptable from a public health standpoint.
In order to assess the efficiency of wastewater 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 resis-
tant pathogens. The bioindicator should also be rapidly and unambigu-
ously 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 wastewater 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 re-
evaluate their application on a case-by-case basis. It is suggested,
therefore, that the indicator organisms should be selected for use on
the basis of the purpose and information required, e.g., the inactiva-
tion of resistant pathogens by Chlorination. It was with this purpose
in mind that this study was undertaken.
10
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It need not be emphasized that the majority of results obtained in this
study are of a negative nature. This is to be expected in a search study
of this type. It may be pointed out that several decades were required
to develop the coliform test to its present state of refinement, with
many false leads and pitfalls along the way. The negative results pre-
sented here may help other investigators to avoid the expenditure of
research efforts in areas that show little promise of a successful outcome.
On the other hand, it is felt that several promising developments towards
the realization of a new indicator of wastewater chlorination efficiency
have arisen from this study. The original objective of the study to find
an organism(s) that is as resistant to chlorine as the most resistant
pathogens and to develop rapid, simple, sensitive and selective procedures
for their enumeration in secondary and advanced waste treatment effluents
has been met at least in part. Further study will be required to refine
techniques and to test the validity of the proposed wastewater indicators
under a variety of field conditions. The techniques developed in this
study have provided several candidate indicator organisms for measurement
of chlorination efficiency and, thus, have considerably expanded the
available alternatives in assessing the public health quality of chlori-
nated effluents.
11
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SECTION IV
MATERIALS AND METHODS
MEASUREMENT OF CHLORINE RESIDUALS
The measurement of chlorine residuals in secondary effluents was found
to be most satisfactory when the DPD (N,N-dimethyl-p-phenylene-diamine
monohydrochloride obtained from Eastman Kodak Company, Rochester, New
York) colorimetric method (38,39,40,41) was used because both free and
total chlorine residuals can be determined in the same samples within a
2 min period. It should be noted that high levels of chloramines may
interfere with this procedure, yielding a higher apparent free chlorine
residual than is actually present. Oxidation-reduction potential measure-
ments were employed to confirm the reliability of this method.
It is generally accepted that the amperometric method of measuring chlo-
rine residuals is the preferred procedure in studying the response of
microorganisms to chlorine when precise information is desired (7,39,42).
Burns and Sproul (7) conducted a study in which chlorine residuals
determined by amperometric titration showed a constant relationship to
organism destruction whereas orthotolidine-arsenite (OTA) residuals
did not. Chambers (43) found that if the amperemeter was "sensitized"
before use by repeated chlorine residual measurements, i.e. 15 times,
using a solution having approximately the same concentration of chlorine
as the test solution, chlorine concentrations as low as 0.005 mg/Jl could
be detected. An amperometric titrater (Wallace and Tiernan, Inc.,
Belleville, New Jersey) was used to measure chlorine residuals in the
controlled chlorination studies carried out with chlorine demand free
water. The procedure for amperometric titrations described in Standard
M&tkod& (44) was followed.
ISOLATION OF CHLORINE RESISTANT ORGANISMS
Several activated sludge and trickling filter effluent samples from the
East Side Treatment Plant of the Urbana and Champaign Sanitary District
and a sample of oxidation pond effluent from the Decatur Sanitary District
Sewage Treatment Plant were the main sources of potential indicator
organisms. These samples were filtered through several layers of cheese-
cloth and stored at 10°C for never more than 5 hr before chlorination.
The different effluent samples were chlorinated either to satisfy part
of the demand by adding from 6 to 63 mg/Jl of chlorine or to reach a free
chlorine residual of 0.15 to 8.0 mg/Jl by adding from 75 to 176 mg/£ of
chlorine as sodium hypochlorite for contact periods of 5 to 30 min at
either room temperature or 20.5°C. Following dechlorination with an
excess of 0.05N_ sodium thiosulfate, aliquots were plated on several types
of media. These included nutrient agar and tryptone-glucose agar
13
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from Difco Laboratories (Detroit, Michigan) and trypticase-soy agar, WL
Differential agar, and Middlebrook and Cohn 7H10 agar from BBL (Cockneys-
vilie, Maryland). Media prepared in the laboratory included starch agar
(0.5 percent peptone, 0.5 percent yeast extract, 0.3 percent soluble
starch, 1.5 percent agar), nitrate agar (1 percent peptone, 0.5 percent
yeast extract, 0.4 percent potassium nitrate, 1.5 percent agar), and
succinate agar (0.2 percent NH^N03, 0.1 percent KgHPO*, 0.02 percent
MgS04, 0.5 percent sodium succinate, 1.5 percent agar;. The agar was
obtained from Difco Laboratories and the other chemicals from Mallinckrodt
Chemical Works (St. Louis, Missouri). Double strength nutrient agar
adjusted separately to pH 3, 5, 7, and 9 was also used. The streaked
plates were incubated at 10°, 20°, 37°, and 45°C for 24 to 96 hr.
CULTURING OF CHLORINE RESISTANT ISOLATES
Organisms that grew on the originally inoculated plates, following chlor-
ination of the effluent, were examined by a phase-contrast microscope for
morphological characteristics, Gram stained, and subcultured to obtain
pure cultures. Stock cultures were made on nutrient agar slants and
stored at 5°C. Subcultures collected for detailed study were periodically
transferred to fresh agar slants.
In preparation for controlled chlorinatlon experiments, the extinction
coefficient of each isolate was determined. This was accomplished by
growing each organism in nutrient broth on a shaker and, after an appro-
priate incubation period, the culture was centrifuged and the cells
washed twice and resuspended in phosphate buffered water at pH 7. The
optical density of the suspension was measured at 660 n.m. in a Spectronic
20 spectrophotometer and various dilutions were plated in nutrient agar
pour plates. The data obtained from pour plate colony enumeration were
correlated with the optical density to obtain the extinction coefficient.
SCREENING OF ISOLATES FOR CHLORINE RESISTANCE
Pure cultures of isolates were examined as to their specific resistance
to chlorine. Several types of water were considered for use as the basic
suspending menstrum, including 1) raw ground water aerated to precipitate
ferric iron, membrane filtered, and buffered to pH 7; 2) water prepared
in the same manner as No. 1 but autoclaved after pH adjustment; 3) auto-
claved distilled water buffered to pH 7; and 4) physiological saline pre-
pared from deionlzed water buffered to pH 7. However, for various reasons
these waters were found to be unsatisfactory. On the other hand, it was
found that the requirements for these studies could be met best by using
the chlorine demand free water as developed by Butterfield e£ at. (45),
the complete preparation of which was described by Megregian (46).
The glassware was washed with Haemosol (Melnecke and Company, New York)
14
-------
and then soaked in a strong chlorine solution (75-150 mg/fc) for at
least 2 hr before rinsing at least 10 times with deionized water and
finally with chlorine demand free water (43). Glassware used with
chemicals, potentially toxic to the organisms under investigation,
was soaked overnight in cleaning acid (35 m£ of saturated sodium di-
chromate in 1 a of concentrated sulfuric acid) and then rinsed with
water. Sterilization was accomplished in a dry hot air oven at 190°C
for 2 hr.
Prior to a chlorination experiment, the isolate to be examined was
grown overnight in an aerated nutrient broth culture. The washed sus-
pension was then diluted on the basis of cell concentration as deter-
mined by optical density measurements and 1 m£ aliquots were added to
the test flasks so as to have a final cell concentration of 103 to
104/m£ in the flasks.
The response of the various isolates to chlorine was determined using
essentially the same procedure described by Butterfield &t oJL. (45).
A determined amount of chlorine and a test organism were added to a
number of sterile 1 £ erlenmeyer flasks, each containing 500 to 700 m£
of sterile chlorine demand free water. Two control flasks were treated
in the same manner except that one received only chlorine and the other
only the test organisms. The flasks were incubated in a water bath at
20.5°C. The preliminary screening was originally performed using
chlorine dosages of 0.1, 0.5, and 1.0 mg/&, but was later changed to a
single dosage of 0.5 mg/fc so that three or more isolates could be
screened at one time. Later to facilitate the screening of isolates,
chlorination experiments were done with a mixture of morphologically
similar organisms in one reaction flask. Those isolates showing some
resistance at 0.5 mg/Jl free chlorine residual were then further screened
as to chlorine resistance with the three chlorine concentrations men-
tioned above. The free chlorine residuals were measured by amperometric
titration after 5 and 30 min contact. In this phase of study, 1 ma
aliquots were removed from the test flasks at 5, 15, and 30 min intervals,
dechlorinated with excess 0.025 N thiosulfate in a fivefold dilution,
diluted tenfold further in pH 7 phosphate buffer, and plated on agar
pour plates using an appropriate nutrient medium. Replicate plates of
each dilution were made and incubated for 24 to 48 hr. Promising iso-
lates were subjected to more detailed studies with chlorine dosages of
0.1, 0.3, 0.5, 0.7, and 1.0 mg/Jl with removal of 1 mfc aliquots at 5, 10,
15, 20, and 30 min intervals. Further studies employed higher chlorine
concentrations and longer exposure times depending on the chlorine resis-
tance of the particular isolate. The entire selection procedure from
the chlorination of effluent to the final chlorination experiments using
high chlorine dosages in chlorine demand free water, provided a means of
continually eliminating organisms which were unsuitable due to low chlo-
rine resistance and other undesirable properties.
15
-------
DETAILED CHLORINATION STUDIES
Using the most promising organisms, six separate sets of experiments were
carried out to obtain further information concerning their properties,
The purpose of one group of experiments was to determine the length of
time and concentration required for 99.9 percent kill of the test organism.
The contact times were increased to as much as 200 min and the chlorine
concentrations up to 2 mg/£.
A culture of E. wti ATCC-11229, obtained from the National Environmental
Research Center in Cincinnati, Ohio, was examined for its resistance using
concentrations of free residual chlorine of 0.01 to 0.04 mg/Jl at a cell
concentration of Kr/mJl. Samples were taken following 1, 5, 10, 15, 20,
and 30 min of exposure to chlorine. This organism was employed due to
its previous use by other investigators so as to allow comparison of results.
Mixed culture chlorination experiments were carried out using yeast isolate
No. 30 and E. c.oti ATCC-11229 to study survival at chlorine concentrations
of 0.02, 0.1, and 0.5 mg/A. These two organisms were added together in
the same reaction flasks; the concentration of E. coti was 5000/mA and
that of No. 30 was 1000/mJi. Organism survival was determined after 1, 5,
10, 15, 20, and 30 min contact with the chlorine. To distinguish between
the two organisms, samples were plated on EMB agar on which only E. cjoti
gives the lactose reaction and on nutrient agar adjusted to pH 3.5 with
d-tartaric acid (J. T. Baker Chemical Company, New Jersey) for detection
of Isolate No. 30.
The appropriateness of a potential new indicator organism as applied to
wastewater chlorination was evaluated through studies of its Chlorine
resistance when seeded in secondary effluent. In experiments involving
the use of yeast Isolate No. 30, chlorine residual measurements were made
using the DPD method. The apparent free chlorine residuals measured in
these experiments may have been due, in part, to interference caused by
high concentrations of chloramines. However, the alternative ampero-
metric method was found to produce unreliable results due to an indis-
tinct endpolnt when used for chlorine measurements with the wastewater
samples. In order to reduce the chlorine demand and the production of
chloramines, wastewater effluents were diluted tenfold prior to chlorina-
tion. This dilution probably provided a quality water that might be
similar to an AWT type effluent. Chlorine dosage could thus be markedly
reduced while still allowing the attainment of free chlorine residuals.
Thus, meaningful survival curves for resistance to free chlorine could be
obtained.
ENUMERATION OF POTENTIAL INDICATOR ORGANISMS IN HUMAN FECAL MATERIAL
AND WASTEWATER
For a test organism to be a suitable indicator of fecal contamination, it
16
-------
should be routinely present in human fecal material and also in domestic
wastewater. Therefore, both stool samples and wastewater were examined
for the presence of yeasts and acid-fast bacteria, two groups of isolates
which showed chlorine resistance at levels considered necessary for inac-
tivation of pathogens, including viruses.
Yeast Enumeration in Fecal Material and Wastewater
Because the growth of the majority of bacteria and actinomycetes is
inhibited at pH values less than 4.5 while yeasts and molds are not, a
selective enrichment medium yeast extract-malt extract glucose agar (YMA)
(yeast extract 0.3 percent, malt extract 0.3 percent, glucose 1 percent,
peptone 0.5 percent, agar 2 percent) which had been adjusted to pH 3.5-
4.0 with 1 N HC1 was used as the medium of choice for yeast enumeration
in both fecal samples and wastewater. Initially the acid medium used
was potato dextrose agar adjusted to pH 3.5 with 10 percent tartaric
acid. Wickerham (47) recommended that yeast extract-malt extract glucose
agar (YMA) be used as the medium of choice for yeast enrichment when the
pH is lowered to the acid range (pH 3.5-4.0). This medium was evaluated
using yeast Isolate Nos. 30 and 30A. After daily examination for the
growth of yeasts in three acid media, i.e. nutrient agar, potato-dextrose
agar, and YMA, all adjusted to pH 3.5, it was concluded that the rate of
yeast growth in YMA was the most favorable.
Stool samples were obtained primarily from two sources. Stools from
individuals, not using any form of therapeutic drugs but confined to a
nursing home for reasons other than infectious medical problems, were
used. In addition, stools from healthy university student volunteers
were obtained for study. One to four "wet" grams of human feces were
suspended in sterile phosphate buffered water (pH 7) to yield a 1 to 10
percent suspension and macerated with glass beads on a reciprocal shaker
until the samples were homogeneously suspended. Serial tenfold dilu-
tions were made aseptically. Again, pour plates using YMA-acid media
and diluted stool samples were made; the yeast colonies present after
3 to 5 days incubation at room temperature and 37°C were counted.
Various samples of wastewater were obtained at the East Side Treatment
Plant operated by the Urbana and Champaign Sanitary District. The major-
ity of wastewater samples, obtained at the plant, were initially filtered
through several layers of cheesecloth at the time of sampling in the
field and stored at 5°C for not more than 5 hr before being examined.
In addition, in order to determine the effect of sample preparation on
the apparent densities of organisms present in wastewater, alternate
sample treatment methods were compared to cheesecloth filtration.
Samples were taken without filtration directly to the laboratory and a
portion of each sample was filtered through several layers of cheese-
cloth and vigorously shaken. The remaining portion of the sample was
divided into two parts, one of which was homogenized by blending for
about 30 sec while the other was vigorously shaken approximately 25
17
-------
times as recommended in StundaAd M&tkodt* (44) in order to break the
bacterial clumps. The yeast densities for each sample preparation
method were then determined and compared.
Enumeration of yeasts in wastewater was accomplished by the pour plate
technique using replicate plates of YMA-acid media and diluted wastewater.
The yeast colonies present after 3 to 14 days incubation at 20°C and 37°C
were determined.
Development of Selective Techniques for Yeast Enumeration in Fecal
Material and Wastewater
Since YMA-acid medium also permits mold growth, difficulty was experienced
in counting discrete yeast colonies. Several means of eliminating inter-
fering contaminants were investigated.
A mold inhibitor, sodium propionate, was added at a concentration of
0.25 percent to suppress mold growth. Sodium propionate was found to
be not entirely satisfactory in this regard. An anaerobic incubation
method with a parafin layer on YMA-acid medium was also evaluated. To
eliminate occasional acidophilic bacterial growth on YMA-acid plates,
an antibiotic, oxytetracycline (Terramycin, Chas. Pfizer Co.) was incor-
porated into the medium. Ten m£ of a 0.1 percent solution, freshly
prepared and sterilized by membrane filtration, was added to 100 m£ of
melted 45°C YMA-acid medium at pH 3.5.
In order to compare the relative presence of indicator organisms with
"normal" wastewater and fecal organisms, a total plate count was made
in addition to yeast enumeration by preparing duplicate nutrient agar
pour plates at each of several dilutions and counting the colonies present
after a minimum 2 days incubation at 37°C.
Enumeration of Acid-Fast Bacilli in Fecal Material and Wastewater
Samples of wastewater and stools for the determination of acid-fast
bacilli densities were obtained and treated essentially the same as for
yeasts as outlined above.
Development of Selective Techniques for Enumeration of Acid-Fast Bacilli
in Fecal Material and Wastewater
A similar problem of contaminants as in the yeast studies was encountered
when stools and wastewaters were examined for acid-fast bacteria. Three
approaches were taken to develop selective techniques for enumeration of
acid-fast bacilli. First, several methods of pretreating samples prior
to culturing were examined as to their relative efficacy in removing
18
-------
contaminants. A second approach was to use various selective media for
acid-fast bacilli. To some of the selective media various chemical agents
were added in order to suppress growth of contaminats. Lastly, a tech-
nique was developed for the acid-fast staining of membrane filters for
positive identification and enumeration of acid-fast organisms. The
selective methods were evaluated for known acid-fast bacilli obtained
from wastewater prior to study of fecal and wastewater samples.
Methods for the Pretreatment of Samples
Since sputum samples have been routinely examined for the presence of
acid-fast tubercle bacilli for many years, the selective technique
applied to sputum was investigated. The alkaline sputum treatment
method (48) using 1, 2, 6, and 10 percent NaOH treatment was examined
with known cultures of acid-fast bacilli obtained from secondary efflu-
ents. Samples were contacted with alkali for various time periods
and then neutralized with dilute HC1.
In addition, pretreatment of sample with quaternary ammonium compounds
was evaluated to determine the ability of this pretreatment to suppress
growth of contaminants and to assess the ability of acid-fast organisms
to survive this treatment. Four quaternary ammonium compounds (QAC),
Arquad T-2C-50, Armeen C, Armeen 12D, and Roccal, were first dissolved
in phosphate buffer and a predetermined amount added to cell suspensions
for exposure periods of either 5 or 10 min. These suspensions were
centrifuged to remove the cells from the QAC; the cells were then resus-
pended in buffer and plated in Middlebrook and Cohn 7H10 agar. Armeen
C and Armeen 12D dissolved more completely in 40 percent ethanol than
in buffer; thus, further experiments with these QAC were carried out
using 40 percent ethanol solutions.
Pretreatment of cultures containing both acid-fast bacteria as well as
potentially interfering microorganisms with 5 percent oxalic acid for
30 min followed by neutralization with 4 percent NaOH was recommended
by David (49). Evaluation of this pretreatment was undertaken first
by treating Isolate Nos. 132 and 134 with oxalic acid-NaOH to determine
their sensitivity. In order to evaluate oxalic acid-NaOH pretreatment
for wastewater and fecal samples, contact time and the oxalic acid con-
centration were varied.
Development of Selective Media for Enumeration of Acid-Fast Bacilli
Middlebrook and Cohn 7H10 agar is generally considered to be a selective
medium for acid-fast organisms. However, in highly contaminated speci-
mens, such as wastewater and feces, the selectivity of this medium was
not sufficient to overcome the growth of other, non-acid-fast organisms.
Moreover, the use of oxalic acid-NaOH and the other pretreatment methods
19
-------
was found not to be sufficient because a number of spore forming
organisms were observed to survive treatment in wastewater and fecal
samples. A satisfactory selective medium for acid-fast bacteria is
necessary due to their low concentration in wastewater in comparison
to contaminant organisms.
Incorporation of chemotherapeutic agents and various dyes into an
enriched acid-fast bacteria medium was investigated. Two acid-fast
bacilli, Isolate Nos. 132 and 134, and samples of secondary effluent
were studied separately in Middlebrook and Cohn 7H10 agar containing
penicillin at concentrations from 10 to 250 units per mfc of medium as
well as malachite green and Eosin Y at concentrations of 0.005 to 0.0075
percent. In addition, several other drugs were tested against the two
acid-fast bacteria using sensitivity discs impregnated with various
quantities of the drugs. Impregnated sensitivity discs were placed on
pour plates and the presence or absence of a zone of inhibition was
noted. Studies of the relative sensitivity of wastewater organisms
and the two acid-fast bacilli to eight antibiotics (chloromycetin,
neomycin, erythromycin, kanamycin, novobiocin, penicillin, streptomycin,
and tetracycline with 2, 5, 10, or 30 meg per disc) and eight sulfa
drugs (sulfamethacypyridazine, elkosin, gantrisin, sulfadiazine, sulfa-
merazine, sulfa thiazole, thiosulfil and triple sulfa with 50 or 300
meg per disc) were carried out to determine the usefulness of their
incorporation into selective media using the sensitivity disc method.
Additionally, five sulfa drugs (kynex, gantrisin, elkosin, sulfathiazole
and sulfamethizole) were suspended in phosphate buffer, then later in
40 percent ethanol, and a predetermined amount was incorporated into
Middlebrook and Cohn 7H10 agar. The resistance of the two acid-fast
bacilli and activated sludge effluent organisms was examined separately
and quantified in terms of survival at various drug concentrations as
compared to control plates containing no antibiotics or sulfa drugs.
Since many selective culturing techniques are based on information gen-
erated from knowledge of the microbial metabolic pathways, a selective
culturing medium for acid-fast bacilli using organic acids and other
metabolites as the sole carbon source was investigated. Mineral salt
medium (NaCl, 0.1 g; CaCl2> 0.1 g; FeClo, 0.02 g; MgS04-7H20, 0.2 g;
CNHJ2S04, 0.7 g; KF^POa, 1.0 g; agar, 15 g; distilled water, 1000 ml)
containing a parafin hydrocarbon as the sole carbon source was evaluated
as a selective medium for acid-fast bacilli. Additional carbon utiliza-
tion studies, including 15 carbon sources; acetate, benzoate, pyruvate,
lactate, propionate, malate, oxalate, citrate, tartrate, glycerol,
succinate, glucose,maltose, lactose, and mannose, were carried out with
the acid-fast cultures. A buffered mineral medium containing the single
carbon source under investigation was used to culture the acid-fast
bacilli. Pour plates were inoculated with stock cultures of the acid-
fast isolates and growth noted during a 2 weeks incubation period at 37°C.
20
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Development of an Acid-Fast Staining Procedure for Membrane Filter
Enumeration of Acid-Fast Bacilli
Another approach taken to the problem of selectively enumerating acid-
fast bacilli in wastewater was the development of a membrane filtration
technique. The general procedure for membrane filtration of bacteria
in wastewater, outlined in Standard Method* (44), was modified by using
an enrichment broth and medium specific for acid-fast bacilli, e.g.,
Middlebrook and Cohn 7H10 agar. The plates containing the membranes
were incubated in a humidified incubator at 37°C until the colonies
were visible but not so large that they were easily washed off the fil-
ter when subjected to the staining procedure. A filter was removed
from the agar with a forceps and carefully dried above a flame so as
to prevent charring the filter. The filters (Type HA Mi Hi pore filters,
Mi Hi pore Corp., Bedford, Mass.) were stained by a modified acid-fast
staining procedure. The solution used in the procedure included 0.5 m£
Brook's carbol fuchsin-dimethylsulfoxide stain in 20 ml water and 0.5 m£
Brook's malachite green stain containing the decolorizer (acid-alcohol)
in 20 m£ water; both reagents were obtained from Curtin Scientific (St.
Louis, Missouri). The two dye solutions, water for washing the filters,
and absolute ethanol were placed in regular sized petri dishes. The
filter was immersed in the carbol fuchsin solution for 30 sec and then
rinsed completely in water by gently swishing the filter in water with
the forceps. The membrane was decolorized by immersion in ethanol and
stained in the malachite green solution for 30 sec. After rinsing in
water, the membrane was allowed to dry and colonies were observed under
a dissecting microscope at a magnification of 50 X.
Since no single selective method was completely satisfactory for the
routine isolation of acid-fast bacteria from mixed microbial populations
such as in wastewaters and feces, methods combining the most satisfactory
pretreatment and culturing techniques of those studies were evaluated.
Since the samples should be homogenous, large clumps were dispersed by
the use of a blender. Samples were then centrifuged to further separate
particulates from the liquid suspension. The centrifuged particles were
resuspended in sterile phosphate buffer and the procedure repeated if
significant quantities of solid particles were observed. The homogeneous
liquid suspension was then treated with 2.5 percent oxalic acid for 5 to
10 min, followed by neutralization with 2 percent NaOH. The sample was
then diluted with sterile phosphate buffer prior to application of the
membrane filter technique. After 2 to 3 days incubation on a selective
medium or when visible colonies were evident, the filter was stained
with the modified acid-fast staining procedure and examined for pink
colonies indicating positive acid-fast bacilli.
21
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SECTION V
RESULTS AND DISCUSSION
ISOLATES EXAMINED FOR CHLORINE RESISTANCE
Pure cultures of 135 isolates were collected for chlorination resis-
tance studies. Although most of these organisms were isolated from
secondary effluents, three bacterial cultures were obtained from chlori- .
nated tap water and thirteen pure cultures of known species were
obtained from the Department of Microbiology, University of Illinois.
The known stock cultures included the genera: F£avobact&tx.ujTi, Pieacfoinonai,
S&inatia., MicA.oc.oc.cu:>, Sa^cina, C/itomofaac^eAxxuri,, Stapky£acjOC.c.uA, \McAo-
monobpoM., and S&i&ptomy. Isolates were also established by sub-
culturing from selected colonies surviving on the assay plates in the
preliminary chlorine screening tests. It was postulated that these sur-
vivors might have greater resistance to chlorine than the parental isolates
Conditions of isolation and properties of representative isolates are
given in Table 1. For isolation, this includes the source of each iso-
late, the amount of chlorine added to the effluent and, in some cases,
the free and total chlorine residuals remaining after 5 or 30 min, the
medium, and temperature of incubation. The properties reported in Table 1
are morphology, and Gram reaction. Many of the bacteria found after
chlorination of secondary effluents were Gram positive spore forming rods.
Only a few of these were retained for further study because of the depen-
dence of their spore forming properties on environmental conditions which
makes them generally unsuitable as indicator organisms. Many different
morphological types of organisms were isolated, primarily at incubation
temperatures from 20° to 37°C. Isolates included both Gram positive and
negative rods, cocci, and mycelia with white, orange and yellow pigments.
Organisms were found when as much as 126 mg/i of chlorine was added to
an effluent sample to give 25 mg/£ total and 17 mg/£ free chlorine resid-
ual. Acid-fast bacteria, yeasts and actinomycetes were the most unusual
isolates. The isolation of organisms was conducted over a sufficiently
long period to account for any seasonal variation in the wastewater
microbial population. Considering the number of isolation experiments
and the period of examination it is believed that the isolates obtained
were representative of those that might be commonly found in effluents.
FURTHER EXAMINATION OF ISOLATES FROM CHLORINATED WASTEWATER
Although the isolates survived various degrees of secondary effluent
chlorination {Table 1), most of the organisms isolated were too sensi-
tive to chlorine to be useful bioindicators, according to the criteria
selected for this study; that is, some degree of survival at concentra-
tions and contact times up to 1 mg/Jl free residual chlorine for 30 min.
23
-------
Table 1
Conditions of Isolation and Properties of Representative Isolates
Isolate
No.
5
27
30
32
37
62
67
70
75
82
87
88
Source
AS
AS
AS
AS
AS
AS
AS
AS
TF
TF
TF
TF
Chlorine Free
Added Residual
mg/t mg/l
30.6
42.2
15.2
15.2
44.0
63.0
113.4 2.0
113.4 2.0
126.0 17.0
25.2
25.2
100.8 4.3
ISOLATION
Total
Residual 2
mg/£ Medi a
NA
NA
NA
NA
NA
31.5 SA
NA
ST
25.0 NA
4.6 NA
4.6 TS
19.0 ST
Temp.
°C
20, 37
37
37
37
20
37
20
37
37
20
20
37
PROPERTIES
Morphology Gram Reaction
rust, rods, spores +
yellow rods
oval budding yeast +
6 range mycelium +
oval cocci
yellow rods
yellow rods
curved rods
short rods
round budding yeast +
gold, rods, spores +
yeast, true mycelium +
-------
Table 1 (continued)
ro
01
ISOLATION
I sol ate
No.
90
106
114
124
128
132
134
Source
TF
TF
OP
AS
TF
TF
OP
Chlorine Free
Added Residual
mg/£ mg/t
12.6
37.8
30.0
30.0
30.0
30.0
30.0
Total
Residual
mg/t
6.0
24.0
22.7
23.0
25.0
25.0
22.7
Media2
NA
NA
NA
(pH 9)
7H10
NA
7H10
7H10
Temp.
°C
37
37
37
25
10
25
25
PROPERTIES
Morphology Gram Reaction
granular rods
orange mycelium +
large cocci in yellow +
packets
orange rods
yellow coccobacilli
yellow rods, acid fast
thin rods, acid fast
'Effluents -AS, Activated Sludge; TF, Trickling Filter; OP, Oxidation Pond
BMedia - NA, Nutrient Agar; SA, Succinate Agar; TS .Trypti case-soy Agar; ST, Starch; 7H10, Middlebrook and Cohn
-------
There was no significant correlation found between the chlorine resis-
tance of the isolates as determined in chlorine demand free water and
the chlorine dosage used in the isolation experiments. This may be
ascribed to organism protection by clumping and by extraneous materials
in the chlorinated effluent. None of the tap water isolates nor the
known stock cultures tested were sufficiently chlorine-resistant to
satisfy the initial screening criteria.
Excluding spore-forming bacilli, the isolates which showed the most
chlorine resistance were yeasts and acid-fast bacilli which were all
able to grow on nutrient agar as well as on several other types of media
at 37°C. The general shape of the inactiyation curves followed a pattern
frequently reported by various authors, with a lag phase followed by an
increasing rate which finally leveled off. However, the length of each
section of the curve as well as its rate varied with the chlorine con-
centration, the organism being studied, and other undefined experimental
conditions, e.g., those relating to the growth of the culture in the
nutrient broth.
The first promising isolate found was No. 30, an oval-shaped, asporo-
geneous, budding yeast that forms pseudomycelia. Curves indicating its
survival to chlorination (Figure 1) show that 0.08 percent of the cells
survived a free chlorine residual of 1.0 mg/£ for 20 min while survival
after 30 min exposure at 0.1 mg/£ free residual chlorine was greater
than 80 percent.
In studying Isolate No. 30, one of the colonies which appeared on the
assay plate following exposure to 1.0 mg/£ free residual chlorine for
20 min was subcultured as Isolate No. 30A. Studies on No. 30A showed
that it was more resistant to chlorine than the parent culture, No. 30,
with nearly 0.2 percent of the initial inoculum surviving a free chlo-
rine residual of 1.0 mg/£ for 30 min (Figure 2). However, in later
experiments the chlorine resistance of No. 30A appeared to decrease
somewhat, approaching that of Isolate No. 30. This may have been due
to an unstable mutation to higher resistance or to some function of
cultivation of the organism.
A spherical budding yeast, Isolate No. 82, was also more resistant to
chlorine than most of the bacteria examined (Figure 3); however, because
its chlorine resistance was much lower than that of No. 30, organism
No. 82 was not studied in any further detail. Only about 2 percent of
the cells survived a free chlorine residual of 1.0 mg/& after a 5 min
exposure.
Isolate No. 132 is an acid-fast yellow pigmented rod which showed
promising chlorine resistance. Figure 4 shows 63 percent survival
after 30 min contact of 0.1 mg/t free residual chlorine and 0.05 per-
cent survival after 15 min of 1.0 mg/£. Although always remaining at
quite a high level, the resistance of this orqanfsm was more variable
than that of the other isolates in comparing different experiments.
26
-------
o
3
(ft
"c
u
I
FIG. I
0.
10
20
30 40 50
Contact Time, min.
60
RESPONSE OF ISOLATE NO. 30 TO FREE CHLORINE
RESIDUALS RANGING FROM O.I TO 1.0 mg/J
27
-------
3
cn
c
0)
O
k.
Q>
Q.
0.1
10
20 30 40 50
Contact Time, min.
60
70
FIG. 2
RESPONSE OF ISOLATE NO. 30A TO FREE CHLORINE
RESIDUALS RANGING FROM O.I TO 1.0 mg/J?
28
-------
o
>
3
(A
20 30 40 50
Contact Time, min.
FIG. 3
RESPONSE OF ISOLATE NO. 82 TO FREE CHLORINE
RESIDUALS RANGING FROM 0.1 TO 1.0 mg/J
29
-------
o
0)
O
ai
10
20 30 40 90
Contact Time, min.
60
7Q
FIG. 4
RESPONSE OF ISOLATE NO. 132 TO FREE CHLORINE
RESIDUALS RANGING FROM O.I TO 1.0 mg/J
30
-------
The most resistant organism isolated was No. 134, another acid-fast
bacillus. To obtain satisfactory survival curves for Isolate No. 134,
it was necessary to use higher dosages of chlorine than those used in
experiments with the other isolates (Figure 5). The lowest concentra-
tion used was 0.5 mg/£ and 2.0 mg/£ the highest, although 1.5 mg/£ was
the highest dosage applied in most experiments. In one test, 2 percent
of the cells survived a free chlorine residual of 2.0 mg/fc for 67 min.
The free chlorine residuals and contact times necessary to achieve 99.9
percent kill of the four test organisms have been extrapolated from the
data in Figures 1, 2, 3, 4, and 5 and are presented for comparison with
data compiled by Berg (50) from chlorination studies on E. call, adeno-
virus, poliovirus 1, and Coxsackie virus A2 (Figure 6). The isolates
collected from wastewater in the current study show a definite trend
toward increased chlorine resistance over that of the traditional coli-
form indicator. Isolate Nos. 30, 30A, and 132 show resistance compa-
rable to or greater than that reported for resistant pathogens such as
Coxsackie A2 virus. Isolate No. 134 shows a significantly greater
resistance than the pathogens. It must be pointed out that there is
an inherent danger in comparing data presented by several investigators
collected under possibly dissimilar conditions. Nevertheless, the data
do suggest that it is possible to obtain vegetative bacteria with suf-
ficient chlorine resistance to make them suitable as indicators of the
efficiency of chlorination in destroying resistant pathogens.
Examination of Figure 6 might suggest that Isolate No. 134 could be
too chlorine resistant to be useful as an indicator organism. This is
not the case for two reasons. First, it is desirable that an indicator
organism provide a reasonable margin of safety in indicating the effi-
ciency of chlorination in destroying pathogens. Secondly, the pathogens
cited in Figure 6 represent only a few of the chlorine resistant patho-
gens found in wastewater and, therefore, might not be representative of
the most chlorine resistant pathogens present in wastewater. Indeed,
Liu (4) recently showed, in a study of 20 viral pathogens, that resis-
tance depended on the type of data used to judge resistance. For
example, the most resistant viruses, using percent survival at 5 min
contact with 0.5 mg/Jl free chlorine as a judgment criterion, were not
the same as those found to require the longest time to reach 99.99 per-
cent destruction, assuming first order kinetics (4). In either case,
Isolate Nos. 134 and 30A show more resistance to chlorine than the most
resistant viral pathogens studied by Liu. Isolate Nos. 30 and 132 show
superior survival at 5 min and a similar contact time necessary to
achieve a 99.9 percent destruction. It should be pointed out that the
experimental conditions used by Liu were different from those used in
studying the organisms isolated in the current study. Direct comparison
of pathogens and the indicator organism under the same conditions is the
only exact manner in which relative resistance to chlorine may be
established.
31
-------
o
0.1
10
20 30 40 50
Contact Time, min.
60
70
FIG. 5 RESPONSE OF ISOLATE NO. 134 TO FREE CHLORINE
RESIDUALS RANGING FROM 0.5 TO 2.0 mg/jp
32
-------
10
55
£
Polio Virus
Type I
Coxsaekie A2 Virus
(Clark* SKablflr,
(Weldenkopf,
1958)
E.Col
(Butterfield ft
prg. No
134
Adenovirus
Type 5
(Clarke, Stevenson
Org.No.30
ft Kobler, 1956)
0.01
10 100
Contact Tim* (minutes)
1000
FIG. 6 FREE CHLORINE RESIDUALS AND CONTACT TIMES NECESSARY
FOR 99.9 PERCENT KILL FOR ISOLATES NO. 134, NO. 30A,
NO. 132 and NO. 30 AS WELL AS FOR ORGANISMS COMPILED
BY BERG (50)
33
-------
The results of screening E. cjsti ATCC-11229 with free residual chlorine
concentrations of 0.01 to 0.04 mg/£ (Figure 7) showed acceptable agree-
ment with similar data collected by Butterfield et at. (42) for the same
E. coti strain. The free chlorine residuals obtained by Butterfield et at.
(45) decreased in the reaction flasks from 0.03 to 0.02 and from 0.02 to
0.01 mg/Jl after 60 min while those in the present study remained constant
throughout the experiment. The chlorine resistance of this E. c.oti strain
is considerably less than that observed for pathogens studied by Liu (4)
or those compiled by Berg (50).
In the mixed culture chlorination experiments using Isolate No. 30 and
E. coti. ATCC-11229, the percentage survivals of both organisms were less
than those observed when each organism was examined alone. The reason
for this discrepancy was not readily apparent. It could have been due
to organism antagonism in the reaction flask or even growth interference
in the agar plates.
Results of experiments with Isolate Nos. 132 and 134 using Tween 80 and
mixing to remove the effects of possible clumping of these organisms on
their chlorine resistance showed no significant differences from results
obtained in previous studies when Tween 80 was not used and mixing was
not employed. Thus, clumping either was not a factor in these experiments
or was not eliminated when Tween 80 was used in nutrient broth medium
for the growth of Isolate Nos. 132 and 134. Examination of organism
resistance to chlorine under static conditions appeared to be as valid
as when the tests were performed with mixed reaction systems.
Several experiments were carried out to determine the efficiency of
chlorination in destroying Isolate No. 30 in wastewater (Table 2). Samples
of activated sludge effluent were collected in July 1971 and seeded with
Isolate No. 30 and studied in controlled chlorination experiments. In
general, even though the chlorine dosages employed in these experiments
did not satisfy the total chlorine demand, Isolate No. 30 failed to con-
sistently survive even 5 min contact in 2 of the 3 experiments. In the
experiment where the yeast isolate did exhibit significant survival
(7/20/71), the total available chlorine residual at 5 min contact was
less than that in the experiments where Isolate No. 30 failed to survive.
Samples of secondary effluent were diluted tenfold, seeded with Isolate
No. 30, and studied in controlled chlorination experiments. These experi-
ments were designed to allow use of lower chlorine doses to achieve a
desired total chlorine residual and to provide a situation similar to
that which might be seen in AWT type effluents. However, the results
may differ from those obtained in chlorinating actual AWT effluents since
the concentrations and types of organic matter and nitrogenous compounds
in such effluents could differ from those in diluted secondary wastewater
effluents. In addition, the concentration of suspended matter in diluted
secondary effluent may not be truly representative of AWT effluents.
Survival curves obtained for diluted wastewater samples are presented
34
-------
o
>
C
4)
0)
Q.
0,02 -*-0,OI mg/i
(Butterfield fllol, 45)
O.OE mq/Jt
0,03-»-0,OEmg/X
(Butterfield stall
15 20 25
Contact Time, min.
FIG. 7 COMPARISON OF RESPONSE OF E, coli ATCC-II229 TO
FREE CHLORINE WITH DATA FROM BUTTERFIELD et a[ (45)
35
-------
TABLE 2
Chlorination Study of Isolate No. 30 Seeded in Secondary Effluent
CO
cr>
Exp.
Date
1971
77TJ
7/20
7/23
Chlorine
Dosage
(mg/A)
47.1
47.5
48.0
33.8
34.4
4.8
5.2
Apparent
Chlorine
Residuals
(mg/A)
1.60
0.73
1.10
0.10
0.12
1.02
1.35
5 min
Total b
Available
Chlorine
Residuals
(mg/a.)
4.60
8.10
9.30
2.56
2.69
3.46
3.40
Contact Time (min)
15 min
Percent
Survival
0
0
0
73.1
67.7
0
0
Apparent
Chlorine
Residuals
(mgA)
1.30
0.25
0.18
0.10
0.12
0.40
0.70
Total
Available
Chlorine
Residuals
(mgA)
2.60
4.60
7.30
2.54
2.65
1.74
1.50
Percent
Survival
0
0
0
11.7
14.9
0
0
Apparent
Chlorine
Residuals
(mgA)
1.00
0.17
0.18
0.12
0.10
0.25
0.60
30 min
Total
Available
Chlorine
Residuals
(mg/£)
2.20
4.00
0.90
2.49
2.50
1.22
0.96
Percent
Survival
0
0
0
0.5
1.1
0
0
aFree chlorine residual determined within 2 min by the DPD method in the presence of combined chlorine;
interference by combined chlorine may be present and measured as free chlorine
Total available chlorine measured by the DPD-potassium iodide method
-------
in Figure 8. Survival (Isolate No. 30) was measurable at 30 nrin contact
but varied widely with survivals of 0.6 and 49.9 percent for experiment with
2.75 and 1.4 mg/i total available chlorine, respectively,at 30 min. Although
not shown in Figure 8, survival amounted to 0.01 percent with a total chlor-
ine residual of 4.80 mg/£ and a 30 min contact time.
The low survival of Isolate No. 30 in the data of Table 2 and Figure 8 is
not surprising when one considers the high chloramine concentrations
involved in these studies. High chloramine concentrations can be an effec-
tive disinfectant with the contact periods employed with these data. In
addition, a transitory free chlorine residual most likely will be present
immediately after the addition of chlorine and prior to the reaction of
chlorine with wastewater organic and inorganic amines. At the high chlor-
ine dosages employed in these experiments a considerable level of free
chlorine would be present in the reaction flask for a period of time.
ENUMERATION OF YEASTS IN FECAL MATERIAL AND WASTEWATER
Fifty-four fecal samples from healthy human volunteers were examined for
total plate count bacteria on nutrient agar and yeast densities using
acidified nutrient media, i.e. nutrient agar or YMA. The data (Table 3)
indicates little or no correlation between the number of organisms deter-
mined by total plate count and the yeasts present in feces. The majority
of the stool samples, at both incubation temperatures (room ternp^ature,
approximately 25°C, and 37°C), showed 0 to 50 yeasts/g on a wet basis
(Table 4). In samples numbered 24 to 54, one-fifth of the stools exam-
ined showed yeast densities in the high range of 1001 to 10,000/g of
feces. These latter stool samples were tested using a more concentrated
suspension of fecal material than used in samples 1 to 23. This appears
to have improved sensitivity. Approximately 41 percent of samples 1 to
23 showed the presence of yeasts, while 69 percent of samples 24 to 54
showed yeasts with incubation at 37°C. Use of room temperature for
incubation of samples also improved results. Over 80 percent of the
samples were positive when plated on pour plates incubated at room tem-
perature (approximately 25°C). When the arithmetic means for room tem-
perature and 37°C data are compared for those samples where paired data
exists, room temperature data indicate an advantage over 37°C incubation
by 1,481 to 1,360 yeasts/g of feces. Of the 28 possible sets of data
paired, 16 show greater values for room temperature incubation and only
six for 37°C, with six giving an equal count at both temperatures of
incubation. It has been reported that the pH of the medium, its com-
position and the temperature of incubation have a significant effect on
the enumeration of yeasts. With respect to temperature, 21°C or 25°C
has been suggested to be optimum (51); this is in agreement with the
findings of this study. Therefore, room temperature with YMA acid
medium at 10 percent or higher concentration of fecal material seems to
be the technique of choice for examination of stool samples for yeasts.
From this limited study of 54 fecal samples it may be concluded that an
37
-------
o
>
c
«
u
FIG. 8
12345
Total Available Chlorine Residuals, mg/l
CHLORINATION STUDY OF ISOLATE NO. 30 SEEDED
IN DILUTED SECONDARY EFFLUENT
6
38
-------
CO
TABLE 3
Examination of Fecal Samples for Total Plate Count and Yeasts
Total1
Plate
Sample Count
Number 37°C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
33
15
36
18
1.6
5.5
13
102
67
35
0.3
0.25
2
9
1.2
1.6
1.8
4.1
Yeasts2
25°C 37°C
0
0
0
4,000
_
100
0
0
400
0
0
0
1,150
250
0
0
250
100
Samp! e
Number
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Total1
Plate
Count
37°C
1.8
13
8
29
320
280
2
170
40
65
2.7
61
3.6
6.6
3.9
38
35
25
Yeasts2
25°C 37°C
30
75
620
12
0
140
0
1,040
1,160
0
3
0
25
0
0
200
0
150
-
38
440
12
0
150
0
1,120
-
0
0
0
19
Sample
Number
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Total1
Plate
Count
37°C
62
11
1.7
1.6
2
18
55
5.2
800
140
470
400
480
170
230
1.7
6.7
330
Yeasts2
25° C 37° C
18
0
11,000
6
1
2,400
72
36
2
12
11,200
5,900
5
10
-
5,000
2,310
1,610
15
1
10,000
5
0
2,230
68
41
0
0
10,000
4,300
290
0
13,500
5,000
2 800
l',600
1
6
Number x 10 u per wet gram feces
2Number per wet gram feces; samples 1 and 2 were plated on acidified potato dextrose agar; samples
3, 7 and 8 were plated on acidified nutrient agar; all others were plated on acidified YMA;
samples 1-23 were macerated at 1 percent concentrations in phosphate buffer while 4 to 10 percent
suspensions were used for samples 24-54.
-------
TABLE 4
Statistical Analysis of Fecal Yeast Densities
A.
Frequency Distribution of Fecal Yeast Densities
Yeast per
gram feces
0
1-50
51-100
101-500
501-1,000
1,001-10,000
>10,000
Samples 1 - 232
37°C
59.1
0.0
9.1
22.7
0.0
9.1
0,0
Samples
Room Temp.4
16.7
40.0
6.7
3.3
3.3
23.3
6.7
24 - 543
37°C
31,0
24.3
3.5
10.3
0.0
20.6
10.3
B.
Statistical Values for Fecal Yeast Densities
Samples 1 - 23 Samplf 24 " 54 All Samples
37°C Room Temp.4 37°C 37°C
Mean
Median
Percent Positive
300
0.3
40.9
1420
27.5
83.3
1720
37.8
69.0
1141
15
52.9
Values are percent of total number of samples.
2
Includes 1 percent dilution of fecal material and incubation
using either nutrient agar, potato dextrose agar, or YMA media.
3
Includes 4 to 10 percent dilution of fecal material and incubation
using only YMA medium (pH 3.5-3.9).
Approximately 25°C.
40
-------
encouragingly high percentage of the population show yeasts in their
stools, and in concentrations up to ICr/g of fecal material.
In research complementary to the objectives of this study but not sup-
ported directly by this grant, Panswad (52) studied yeast densities in
raw wastewater and wastewater effluents of the East Side Treatment Plant
of the Urbana and Champaign Sanitary District. He found that there was
a variation in the number of yeasts detected at 20° and 37°C. After 3
days of incubation more yeasts were observed by colony formation at 37°C
than at 20°C in general but, as incubation time increased, this observa-
tion was reversed, i.e. more yeast colonies were detected at 20°C.
Mossel at at. (51) also observed that incubation at 20° to 22°C gave
much higher yeast counts, Based upon visual inspection, more rapid
colony development of yeasts occurred at 37°C for no growth was evident
at the lower temperature (20°C) until after 3 days of incubation.
A mold inhibitor, sodium propionate, added into YMA medium was found to
inhibit yeasts as well as interfering organisms in wastewater and was
not useful for that reason. When 100 ug/mfi, oxytetracycline was incor-
porated into the medium to inhibit acidophilic bacteria, some degree of
yeast suppression was indicated by a lower yeast colony count. Thus,
no adjustment other than lowering the pH value of YMA medium was used
to provide a selective technique for yeast enumeration.
The yeast densities found in the Urbana and Champaign Sanitary District
wastewater samples are presented in Tables 5, 6, 7, 8, 9, 10, and 11.
It should be noted that this treatment plant is somewhat unique in that
the raw wastewater flow is split and a portion treated by the activated
sludge process and part by trickling filters. This permits direct com-
parison of two secondary treatment processes treating the same raw
waste. These data are classified to show the effects of pretreating
the samples by homogenization (shaking or blending) with that of filtra-
tion plus shaking.
The pretreating of samples was designed to disrupt any clumps present 1n
the samples and, therefore, release any yeasts imbedded in clumps so
that these yeasts could be enumerated. Filtering through cheesecloth, a
common practice 1n studying wastewater microorganisms, would remove many
clumps and any yeasts associated with them, while vigorously shaking the
samples 25 times or blending for 30 sec should break up the clumps. The
pretreatment appeared to have Its greatest effect on raw wastewater and
the least effect on activated sludge effluent samples. Using data for
6 days of Incubation, the ratios between filtered and shaken, shaken only,
and blended samples were 1:5:4, 1:0.9:1, and 1:2.1:1.8 for raw sewage,
activated sludge, and trickling filter effluents, respectively. Appar-
ently many yeasts are associated with clumps affected by the pretreatment
1n raw wastewater, fewer 1n trickling filter effluents, and very few, 1f
any, 1n activated sludge effluents.
41
-------
ro
TABLE 5
Yeast Densities in Raw Municipal Wastewater
(Acidified YMA Medium at 20°C)
Date
10/05/71
10/12/71
10/21/71
10/27/71
12/07/71
12/14/71
12/16/71
12/17/71
Average
Avg.
Wastewater
Flow
(mgd)
11.9
9.5
8.8
9.1
13.1
14.5
16.4
16.4
Value
Filtered
and
Incubation
4 6
*
370
290
300
930 1
240
260
250
500
400
450
80
500
290
340
400
Shaken
, days
10
*
500
630
100
*
300
450
500
Yeast Count per mil
Shaken
Incubation, days
4 6 10
3500 4000 4300
800 1000 1100
500 800 1000
1400 1900 2100
Blended
Incubation,
4 6
2300 3000
400 500
650 1000
1100 1500
days
10
3200
600
1250
1700
Enumeration was not possible because of the spreading of mold growth on the medium surface,
-------
co
TABLE 6
Yeast Densities in Activated Sludge Effluent
(on Acidified YMA Medium at 20°C)
Avg.
Wastewate
Flow
Date (mgd)
10/05/71
10/12/71
10/21/71
10/27/71
12/07/71
12/14/71
12/16/71
12/17/71
Average Val
11.9
9.5
8.8
9.7
13.1
14.5
16.4
16.4
ue
Yeast Count per mJl
ir Filtered and Shaken Shaken
Incubation,
4 6
201
95
280
30
180
120
95
180
*
130
310
80
40
400
175
140
220
Blended
days Incubation, days Incubation, days
10 4 6 10 4 6 10
210
400
150
60
630 220 300
215 120 150
200 100 140
310 147 200
* 175 300 600
160 150 170 215
190 100 180 350
140 220 370
Enumeration was not possible because of the spreading of mold growth on the medium surface.
-------
TABLE 7
Yeast Densities in Trickling Filter Effluent
(on Acidified YMA Medium at 20°C)
Date
10/05/71
10/12/71
10/21/71
10/27/71
12/07/71
12/14/71
12/16/71
12/17/71
Average
Avg.
Wastewater
Flow
(mgd)
11.9
9.5
8.8
9.7
13.1
14.5
16.4
16.4
Value
Filtered and
Incubation
4 6
330
100
300
95
110
210
130
90
140
*
170
500
160
190
250
200
150
200
Yeast Count per ma
Shaken Shaken
, days Incubation, days
10 4 6 10
200
750
210
270
350 300 600 690
200 180 240 380
200 250 370 400
280 . 240 400 490
Bl ended
Incubation, days
4 6 10
270 550 800
280 350 360
260 350 400
270 420 520
Enumeration was not possible because of the spreading of mold growth on the medium surface.
-------
cn
TABLE 8
Total Bacteria and Coliform Content in Raw Municipal Wastewater
(Incubated at 37°C)
Date
9/19/71
9/23/71
9/30/71
10/21/71
10/27/71
12/14/71
12/16/71
12/17/71
Average Val
Avg.
Flow
(mgd)
10.8
10.3
11.6
8.8
9.7
14.5
16.4
16.4
ue
Total Count x 10 per mH
Filtered and Shaken Shaken Blended
Incubation,
2 4
3.00
2.30
2.30
1.70
0.90
3.70
1.79
1.60
2.00
3.12
2.90
2.30
1.80
6.80
2.60
2.30
2.80
days Incubation, days Incubation, days
6246 246
2.40
1.90
7.40 5.20 9.10 10.80 4.30 6.70 7.20
2.65 5.70 6.10 7.00 4.20 5.00 5.20
2.40 3.60 4.70 5.10 5.00 6.50 7.75
3.00 4.60 6.60 7.60 4.50 6.10 6.70
*
Col i forms
x 10 6
per m£
0.33
0.60
0.15
0.36
Filtered and shaken samples
-------
TABLE 9
Total Bacteria and Coliform Content in Activated Sludge Effluent
(Incubated at 37°C)
Date
9/19/71
9/23/71
9/30/71
10/21/71
10/27/71
12/14/71
12/16/71
12/17/71
Average
Avg.
Flow
(mgd)
10.8
10.3
11.6
8.8
9.7
14.5
16.4
16.4
Value
Total Count x 10 per m£
Filtered and Shaken Shaken Blended
Incubation, days Incubation, days Incubation, days
24 6246 246
1.00
1.40
1.60
1.80
1.00
1.40
3.20
2.40
1.70
2.30
2.20
2.60 2.80
1.50 1.50
2.80 3.10 2.60 3.20 4.40 2.60 4.70 5.30
4.40 4.60 4.00 4.30 4.50 4.20 5.20 5.80
3.20 3.80 2.80 3.70 4.20 2.70 3.30 4.10
2.60 3.20 3.10 3.70 4.30 3.00 4.40 5.00
*
Coli forms
x 10 6
per ma
0.42
0.96
0.26
0.55
Filtered and shaken samples
-------
TABLE 10
Total Bacteria and Coliform Content in Trickling Filter Efflent
(Incubated at 37°C)
Date
9/19/71
9/23/71
9/30/71
10/21/71
10/27/71
12/14/71
12/16/71
12/17/71
Average
Avg.
Flow
(mgd)
10.8
10.3
11.6
8.8
9.7
14.5
16.4
16.4
Value
Total Count x 10 per m£
Filtered and Shaken Shaken Blended
Incubation, days Incubation, days Incubation, days
246246 246
1.14
1.74
0.62
0.84
0.85
0.45
0.80
1.50
0.95
2.72
0.90
1.24 1.50
1.20 1.25
0.80 1.20 1.05 1.30 1.40 2.40 5.30 5.50
1.60 1.85 0.90 1.30 1.42 1.34 2.15 2.50
1.75 1.90 0.73 1.08 1.25 1.90 2.40 2.80
1.40 1.60 0.89 1.23 1.35 1.85 3.28 3.60
Coli forms
x 10 6
per m&
0.17
0.19
0.12
0.14
0.15
Filtered and Shaken Samples
-------
A comparison of the average data of blended samples in Tables 5, 6, and
7 shows a reduction in number of yeast organisms present following sec-
ondary treatment, with a greater reduction occurring in the activated
sludge process than in the trickling filter treatment. In Tables 8, 9,
and 10, the total bacterial count in samples subjected to various pre-
treatment methods - filtration plus shaking, shaking, and blending alone,
is compared. Pretreatment by blending seemed to be the best method of
sample pretreatment according to the data presented. The plate counts
after blending the sample were higher in general than those after the
other two pretreatment methods. It can be seen from Tables 8, 9, and 10
that the conform content in raw wastewater, activated sludge, and trick-
ling filter effluents ranges from 0.15 to 0.60 x 106/m£, 0.26 to 0.96
x 106/m£, and 0.12 to 0.19 x 106/m£, respectively. It is apparent that
the activated sludge process used at the Urbana and Champaign Sanitary
District is less efficient in removing coliform organisms than the trick-
ling filter treatment.
A comparison of the limited data available on yeast densities in waste-
water as compared to total plate count bacteria and coliforms yields
approximate ratios for these organisms. Total plate count bacteria are
nearly 4000, 13,500, and 7000 times more numerous than yeasts in raw
wastewater, activated sludge, and trickling filter effluent samples,
respectively. Coliforms were approximately 200, 1500, and 290 times more
prevalent, respectively, in the same samples. Since comparative data for
total plate count bacteria, col i forms, and yeasts exist only for filtered
and shaken samples which will not reflect organisms associated with large
clumps, these ratios should be regarded only as approximations.
Examination of stool samples and wastewater provided about 47 yeast
isolates in addition to those obtained from chlorination screening of
wastewater. These cultures were randomly selected from assay plates to
provide a variety of morphological types and were by no means inclusive
of all yeast colonies found on the assay plates, Identification of yeasts
is generally based on morphological, physiological, biochemical, cultural
and other observations, e.g., shape, size, maximum and minimum, as well
as optimum, temperature for budding and sporulation (53). Morphologically
these 47 yeast cultures were categorized into four main groups. The first
group, and the most frequently encountered, were spherical to oval budding
yeasts forming white smooth colonies that exhibit sedlmented growth 1n
liquid medium. The second group of yeasts were spherical to oval budding
yeasts forming beige colonies and producing a pellicle in liquid medium.
The third group of yeasts were elongated to oval budding organisms forming
creamy white colonies and producing sedimented growth in liquid media,
The last group of yeasts were spherical to oval budding organisms that
formed creamy to mucold pink colonies and yielded sedlmented growth 1n
liquid media.
Thirteen of these fecal or wastewater yeast isolates were Individually
prescreened with 0.5 mg/£ free chlorine using chlorlne-demand-free water
under controlled conditions (Table 11). Four of the cultures tested
48
-------
TABLE 11
Prescreening for Chlorine Resistance of Yeasts Isolated^9'
from Unchlorinated Wastewater Samples or Stools
Culture
No.
17 T
35 T
2 T
36 T
36 T
53 RT
48
48
15
10
12
44 RT
52
(b)
(b)
(b) .
(b)
(b)
(b)
(c)
(c)
(c)
(c)
(c)
(b)
(c)
5 min
92.5
0.18
91.7
45.8
90.5
76.8
>90
0
63.4
6.0
99.9
52.0
2.6
Time
15 min
4.3
0
58.7
0
3.7
0.9
2.0
0
11.0
0
25.2
0.6
0.1
30 min
0.04
0
0.17
0
4.0
0
0
0
0
0
0.16
0
0
(a) All values were determined at 20°C, pH 7, using 0.5 mg/A free
chlorine; results are expressed in percent survival compared to
a control
(b) Cultures isolated from wastewaters
(c) Cultures isolated from stools
49
-------
showed moderate to strong resistance to chlorine. The remainder did not
survive 30 min contact time. Representative of each of the four morpho-
logical-cultural groups described above were included in these chlorination
experiments.
A heterogeneous collection of another 24 yeast isolates was examined in
a single mixed culture for chlorine resistance. Nutrient agar slants
streaked with pure cultures of yeast were sampled with an inoculating
loop and placed in four flasks containing nutrient broth. Yeasts placed
in each of the flasks were of a single morphological type based on the
morphological classification discussed above. Broth cultures were grown
at 25°C for 24 hr on a shaking table. Following growth, the four cultures
were sampled volumetrically, the samples combined, and washed several
times in sterile phosphate buffer. The washed mixed cells were then used
as the inoculum for the chlorination experiment. A free chlorine dosage
of 1.05 mg/£ was applied. Approximately 1 percent of the total popula-
tion of yeasts survived after a 5 min contact time. Surviving colonies
on YMA were retained for further study. This study indicated that the
mixed population of yeasts present in wastewater may be sufficiently
resistant to chlorine to provide a basis for a new indicator group for
determination of wastewater chlorination efficiency, as opposed to the
use of a single species or morphological type.
SELECTIVE TECHNIQUES FOR ENUMERATION OF ACID-FAST BACILLI
Since the acid-fast characteristics of Isolate Nos. 132 and 134 had
definitely been established, selective techniques to isolate and to
enumerate these two cultures were based principally on methods suitable
for Mt/cobacvte^cum isolation. The possibility that these isolates could
be members of the genus Wocotctca was recognized although a limited deter-
minative study indicated that the cultures belonged to the genus
Isolate No. 132 is a strongly acid-fast bacillus producing yellow, smooth
convex subsurface colonies in pour plates. In liquid media, such as
nutrient broth, the organism grows homogeneously. The cells appear to be
pleomorphic and are arranged singly or in aggregates, large and small.
The optimum growth temperature of this isolate was observed to be 37° C,
although the isolate was also capable of growing at 10°, 20°, 25°, and
45°C. The isolate was found to be catalase positive but negative for
the hydrolysis of casein, gelatin and starch. In addition, the culture
utilized acetate, succinate, citrate, malate, oxalate, propionate and
pyruvate as a sole carbon source.
Isolate No. 134, the most chlorine resistant isolate, usually grew as
thin rods forming small branch-like aggregates, exhibiting a moderate
acid-fast characteristic. It produced irregular buff-colored subsurface
colonies in pour plates. The optimum growth temperature for this isolate
50
-------
appeared to be 37°C but growth also occurred at 10°, 20°, 25°, and 45°C.
Due to the formation of cellular aggregates, suspensions of this isolate
were particulate in nature rather than homogeneous. Addition of Tween 80
to liquid cultures resulted in some degree of dispersal of the cell sus-
pensions. Isolate No. 134 was found to be capable of utilizing acetate,
succinate, propionate, citrate, malate, oxalate, and pyruvate as sole
carbon sources. Both Isolate Nos. 132 and 134 failed to grow on media
containing benzoate and lactate as the sole carbon source. Isolate No.
134 was observed to be catalase positive but negative for casein, gelatin,
and starch hydrolysis. Further physiological studies of these two isolates
are needed to provide definitive information as to their identity.
A number of studies were carried out in an effort to develop a rapid, sen-
sitive and selective technique for the isolation and enumeration of these
acid-fast organisms. These studies included examination of methods of
sample pretreatment, study of selective media for acid-fast organisms,
and development of a membrane-filter, acid-fast staining technique. The
objective was principally to eliminate growth of nonacid-fast organisms
in assaying highly contaminated samples such as fecal material and waste-
water.
The alkaline sputum treatment procedure generally used as a sample pre-
treatment method for clinical identification of the tubercle bacilli in
sputum specimens (48) was applied to samples of secondary effluents.
Samples treated with 1 percent sodium hydroxide showed survival of spore-
formers and a few acid-fast bacilli. To eliminate growth of sporeformers
on assay plates, the NaOH dosage was increased to 2, 6, and 10 percent.
Although growth of sporeformers was suppressed, acid-fast bacilli were
also sensitive to this treatment.
Quaternary ammonium compounds (QAC) have been used widely as disinfectants
in bacteriology laboratories. Various QAC were, therefore, examined to
determine if their use as a sample pretreatment chemical would result in
suppressing growth of nonacid-fast contaminants present in wastewater
samples. Isolate Nos. 132 and 134 were treated with various QAC at
different concentrations and contact times (Table 12). Neither acid-
fast isolate survived treatment with Arquad T-2C-50. Both organisms
showed some survival to treatment with Armeen C and Isolate No. 134 sur-
vived 5 min contact with Armeen 12D. These two QAC, i.e. Armeen C and
12D, were examined further to determine how effectively they suppressed
growth of contaminating wastewater organisms. Trickling filter effluent
samples were collected, split, and portions treated separately with
0.005 percent of each QAC for 5 min contact. An untreated portion of
the effluent was inoculated into Middlebrook and Cohn 7H10 agar pour
plates. This untreated sample served as a control. Treatment of efflu-
ent organisms with Armeen C resulted in a 50 percent reduction of organ-
isms growing on 7H10 agar as compared to the control. Armeen 12D sup-
pressed growth of 95 percent of the effluent organisms as compared to
the controls, A limited number of colonies surviving treatment with
51
-------
TABLE 12
Effect of Selective Pretreatment Methods
on Growth of Acid-Fast Isolates
Pretreatment
Chemical and
Contact Time
Arquad T-2C-502
(5 min)
(10 min)
2
Armeen C
(5 min)
(10 min)
Armeen 12D2
(5 min)
(10 min)
Oxalic Acid-NaOH
(5 min)
(5 min)
(30 min)
Colonies Colonies
Concentration
0.005%
Control
0.1%
Control
0.005%
Control
0.1%
Control
0.005%
Control
0.1%
Control
2.5% Oxalic Acid
Control
5.0% Oxalic Acid
Control
5.0% Oxalic Acid
Control
per %
Plate Survival
0 0
190
0 0
90
54 0
190
0 0
90
0 0
190
0 0
90
47 94
50
15 29
50
1040 59
1770
per %
Plate Survival
0 0
180
0 0
250
150 87
180
0 0
250
120 66
180
0 0
250
79 30
260
23 9
260
190 19
990
' M« JJ1 A.L. »A A 1. ~ « _l f*«* t* M ^Ll 1 rt *« M •* u% !.••*.** i i*» ** *j •+ r+ 4* L* *«. f*\**f\l.l 4-t* mf\ f\ llim T M Ti 1 1
experiments.
QAC was dissolved in phosphate buffer (pH 7); suspensions were centri-
fuged immediately after the contact time with QAC and the cells separa-
ted from the QAC.
52
-------
Armeen 12D were randomly selected from the pour plates and were examined
for acid-fast properties; 3 of 5 colonies studied were found to be acid-
fast positive. Although both Isolate Nos. 132 and 134 showed some resis-
tance to Armeen C, this QAC did not suppress growth of wastewater organ-
isms adequately. While Armeen 12D, on the other hand, suppressed growth
of nonacid-fast organisms quite well, Isolate Nos. 132 and 134 were
generally sensitive to this QAC. Therefore, the use of QAC as sample
pretreatment agents did not appear promising.
However, since it was found that the QAC dissolved more completely in
ethanol than in water, further experiments with QAC were carried out
using 40 percent ethanol-QAC solutions. Low survival of secondary efflu-
ent organisms was obtained when secondary effluent was treated with
either 0.005 or 0.05 percent Armeen 12D for 5 min periods. A few col-
onies examined from the Armeen 12D pretreated assay plate show acid-
fastness. Isolate Nos. 132 and 134 were pretreated with ethanol-Armeen
12D solutions and qualitatively appeared to be able to withstand this
treatment to some degree.
Use of acid treatment of samples followed by neutralization was studied
as a means for reducing the quantity of nonacid-fast organisms on pour
plates (Table 12). Incubation of Isolate Nos. 132 and 134 for 30 min
in 5 percent oxalic acid, followed by neutralization with 4 percent
NaOH, prior to plating in 7H10 agar, gave encouraging results as a sample
pretreatment procedure (Table 12). Both treatment contact time and the
oxalic acid concentration were varied. Use of 2.5 percent oxal^' acid
for a contact period of 5 min, followed by neutralization with 2 percent
NaOH, resulted in nearly complete recovery of Isolate No. 132. A
limited study of Isolate Nos. 132 and 134 seeded in secondary effluent
showed that virtually all of the test organisms could be recovered after
the oxalic acid-NaOH treatment (2.5 percent oxalic acid for 5 min contact
time) while only 0.36 percent of the microorganisms normally present in
the effluent survived the same treatment. Therefore, this pretreatment
method in isolating acid-fast organisms has been adopted tentatively for
analysis of wastewater samples.
Since other microorganisms, especially sporeformers, may survive the
oxalic acid-NaOH pretreatment, additional selectivity in the technique
is necessary and could be accomplished through the use of a culture
medium which is suitable for growth of only the acid-fast organisms
present in wastewaters. A search for an appropriate selective medium
was undertaken by incorporating various chemotherapeutic agents and dyes
into the enriched MycobacteJuwn medium, Middlebrook and Conn 7H10 agar.
Results of survival of Isolate Nos. 132 and 134 on media incorporating
penicillin, Eosin-Y, and malachite green are presented in Table 13. Both
cultures showed good recoveries when plated in a medium containing Eosin-Y,
However, when a sample of a secondary effluent was inoculated on Eosin-Y
medium, virtually the same survival of effluent organisms was seen as
in control plates containing no Eosin-Y. Since Isolate No. 132 failed to
grow in a medium incorporating malachite green, no further study of its
53
-------
TABLE 13
Effect of Selective Media on Growth of Acid-Fast Isolates
Selective
Agent
in Media
Penicillin
Eosin-Y
Malachite
green
Penicillin
Eosin-Y
Penicillin
Eosin-Y
Isolate No. 132
Concentration
500 units /m£ agar
1000 units /mfi, agar
Control
0.005% in agar
Control
0.005% in agar
0.0075% in agar
Control
10 units/mJl agar
Control
0.005% in agar
Control
(10 units /ma agar)
(0.005% 1n agar)
Control
Colonies
per Plate
0
0
1200
380
310
0
0
280
1
150
150
150
0
150
%
Survival
0
0
100
0
0
<0,7
100
0
Isolate No. 134
Colonies
per Plate
4
0.5
1220
130
170
740
630
800
650
700
710
700
710
700
%
Survival
0.4
0.1
76
93
80
93
100
100
Selective agents were incorporated into Middlebrook and Cohn 7H10 agar
at concentrations indicated.
54
-------
selectivity was carried out.
Penicillin treatment at concentrations of 25, 50, 100, and 250 units/m£
of 7H10 agar, as well as a combination of heat and penicillin treatment,
all resulted in the growth of many sporeformers. Isolate Nos. 132 and
134 apparently could not survive the concentrations of penicillin used.
Thus, it appears that the use of penicillin is not feasible as a selec-
tive agent.
Evaluation of 8 antibiotics with sensitivity discs indicated that, as a
whole, antibiotics were not suitable since most inhibited Isolate Nos.
132 and 134. The sulfa drugs, on the other hand, showed some promise
since both isolates were observed to tolerate the drug concentrations
impregnated on the discs. Solutions of 5 different sulfa drugs were
therefore incorporated separately into Middlebrook and Cohn 7H10 agar
and tested against the acid-fast cultures as well as activated sludge
effluent. Results obtained are presented in Table 14. Isolate No. 132
was generally sensitive to all the sulfa drugs tested. Since the sulfa
drugs did not dissolve completely in the phosphate buffer used, the
actual concentration of the drugs in the agar was not precisely known.
The concentration of sulfa drugs in the vicinity of the sensitivity
discs on agar plates depends on the rate of diffusion of the sulfa drugs
into the media and was, therefore, not precisely known. Thus, the
results using the sensitivity discs, and the studies using sulfa drug
suspensions should not be compared. In summary, the use of these
chemotherapeutic agents did not appear feasible since the actual concen-
tration of drugs could not be controlled accurately.
Since the carbon utilization studies indicated that the acid-fast isolates
under study were able to grow on simple mineral media with a simple carbon
source, it was felt that this knowledge might be applied to the develop-
ment of a selective culture medium for these organisms. Since Isolate
Nos. 132 and 134 grew on mineral salt medium containing paraffin as the
sole carbon ^source, this medium was tested for its selectivity for acid-
fast bacilli in wastewater samples. Difficulty was encountered in dis-
persing the medium in pour plates although its selectivity appeared to
be satisfactory. Acetate and propionate were also selected for use in
single carbon source media. These organic compounds were incorporated
into Koser's citrate medium as a replacement for citrate as the carbon
source. Selectivity for acid-fast bacilli was demonstrated in that growth
of sporeformers was suppressed. Results using raw sewage and acetate
media showed a variety of morphological and colonial forms. Those cul-
tured on propionate appeared to be morphologically similar to acid-fast
Isolate Nos. 132 and 134. Moreover, the selective medium using propio-
nate showed greater inhibition of sporeformers and other nonacid-fast
wastewater organisms than the acetate medium. Colonies were randomly
picked from propionate medium plates inoculated with raw wastewater and
streaked individually on Middlebrook and Cohn 7H10 agar medium. Pure
cultures produced on this medium were sampled and tested for acid-fast
55
-------
TABLE 14
Effect of Sulfa Drugs in Selective Media on Growth of Acid-Fast Isolates
en
Apparent Cone.
of Drug
Chemotherapeutic in Water
Agents (mg/£)
No drug present
Kynex
Sulfathiazole
Gantrisin
Sulfamethiozole
Elkosin
Control
1
5
10
1
5
10
1
5
10
1
5
10
1
5
10
Isolate No. 132
Counts/
Plate
72
22
0
0
0
0
0
0
0
0
0
0
0
54
" 0
0
Survival
%
30.6
0
0
0
0
0
0
0
0
0
0
0
75
0
0
Isolate No. 134
Counts/
Plate
290
330
310
330
39
1
0
310
290
290
320
280
13
310
310
310
Survival
%
100
100
100
13.5
0.3
0
100
100
100
100
89.8
4.1
100
100
100
Microorganisms in
Activated Sludge Effluent
Counts/
Plate
310;280
280
160
140
200
120
73
250
150
160
250
130
93
230
210
160
Survival
%
93
52
47
66
40
25
84
51
55
85
44
31
76
71
54
-------
organisms. Virtually all colonies found on propionate-mineral medium
were acid-fast.
The possibility of using the membrane filtration technique after a
selective pretreatment to determine the presence of acid-fast bacilli
was investigated. Determination of acid-fast bacilli was done by means
of a modified acid-fast staining procedure. The result is applicable
here because only acid-fast organisms can retain the primary stain
after the acid-alcohol decolorization treatment. Samples were pre-
treated with oxalic acid, incubated, and neutralized as described above.
While the resulting data were limited, selective culturing of test
samples on membrane filters using a propionate-mineral growth medium
following oxalic add-NaOH pretreatment provided the potential selec-
tivity and sensitivity necessary to allow the use of acid-fast organisms
as a possible bioindicator on a routine basis. The examination of
several different raw sewage samples indicated that there were signi-
ficant quantities of acid-fast bacilli present. Counts of 137 to approx-
imately 500 acid-fast bacilli/nut of raw wastewater were observed using
this procedure. Limited studies of wastewater effluent samples did not
provide reliable results due to considerable quantities of finely
divided suspended matter present on the filters. Several types of
colonies were present including beige, dry-rough colonies, two types
of creamy smooth colonies, and yellow smooth colonies. Creamy and
yellow colonies were found to consist of organisms which were strongly
acid-fast. While the beige type colonies indicated organisms which
were definitely acid-fast, they showed a weaker reaction. The yellow
colonial type appears to be similar to Isolate No. 132. The beige
variety in pure culture showed a significant percentage of survival
after 30 min contact with 1 mg/Jl free chlorine. This variety showed
certain resemblances to Isolate No. 134. The interference by suspended
matter in enumerating effluent samples may have been due to insufficient
dilution of samples since the suspended matter seemed to show a limited
degree of positive acid-fast staining. The literature indicates that
M?/c0bae£e/uuuri is not Inactivated during conventional wastewater treatment (22,
24, 37) and therefore should be present in the effluent in concentrations
approximating those found in raw sewage. The dilutions used in effluent
studies were 100 fold lower than those employed for raw sewage. As a
result the few assay plates obtained were probably overgrown with acid-
fast organisms.
The combination of a selective pretreatment and a selective culturing
method in the order of oxalic acid-NaOH pretreatment, membrane filtra-
tion, incubation of the filter on propionate-mineral medium and add-
fast staining of the filter appears to be promising for the isolation
and enumeration of acid-fast organisms from wastewater, but further
study is necessary to refine the methods and to more fully evaluate
its accuracy.
57
-------
SECTION VI
ACKNOWLEDGEMENTS
This project was conducted under the auspices of the Environmental Engi-
neering Program of the Department of Civil Engineering, University of
Illinois, Urbana-Champaign Campus. The helpful suggestions and ideas
of the Environmental Engineering Laboratory personnel, particularly Mr.
Parviz Anrirhor, are gratefully acknowledged. The data on yeast densities
in wastewater collected by Mr. Thongchai Panswad in a supplementary study,
not directly supported by this grant, provided a valuable input to this
project. The cooperation of the personnel of the Urbana-Champaign Sani-
tary District is appreciated.
The many suggestions, encouragement, and critical evaluation of the
project data provided by Mr. Cecil W. Chambers, EPA Project Officer,
is gratefully acknowledged.
59
-------
SECTION VII
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of Viruses by Water Treatment Procedures," Heo&tfi LoboAotoJur
Science, 3, No. 3, pp 170 (1966).
51. Mossel, D. A. A., Visser, M. , and Mengerink, W. H. J., "A Comparison
of Media for the Enumeration of Moulds and Yeasts 1n Foods and
Beverages," Laboxatony PMLvUte., 11, pp 109 (1962).
52. Panswad, T., "Occurrence of Yeasts 1n Municipal Wastewater Treatment,"
M. S. Special Problem, University of Illinois, Department of Civil
Engineering, Urbana (1972).
53. Prescott, S. C., and Dunn, C. G., InduAtMaJi fAicnobAology, 3rd ed.,
McGraw-Hill, New York (1959).
65
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SECTION VIII
PUBLICATIONS AND PATENTS
Greening, E. 0., "Microblal Indicators for the Biological Quality of
Treated Wastewater Effluents," M.S. thesis, Department of Civil Engineering,
University of Illinois at Urbana, 1971.
Greening, E. 0., Lee, S. H., and Engelbrecht, R. S., "Microbial Indicators
for the Biological Quality of Chlorinated Wastewater Effluents," presented
at 72nd Annual Meeting of American Society for Microbiology, April 28, 1972.
67
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Report No.
w
NEW MICROBIAL INDICATORS OF WASTEWATER
CHLOR1NATION EFFICIENCY
Engelbrecht, R. S., Foster, D. H., Greening, E. 0.,
and Lee, S. H.
Department of C1v1l Engineering (Environmental
Engineering) at Urbana-Champaign, University of
Illinois, Urbana, Illinois
5. Report Date
6. ' ' ' . .
«. Petforaiuz Organbttion
Report No.
17060 EYZ
13. Typ* W Report and
Ptrtad Coveted
1BB043/21ASS/05
Environmental Protection Agency report number,
EPA-670/2-73-082, February 1974.
The coHform group of Indicator organisms has a relatively low resistance to
chlorine when compared to pathogens such as enteric viruses and protozoan cysts. Con-
sequently, an effort has been Initiated to find a new chlorine resistant b1o1nd1cator
of wastewater chloHnatlon efficiency. Organisms surviving chlorlnatlon of wastewater
effluents to a free chlorine residual were collected for study by plating on various
growth media. This procedure provided 135 chlorine resistant Isolates for further
examination. A yeast and two different add-fast bacilli were found to be resistant
to chlorlnatlon 1n the range considered necessary for the 1nact1vat1on of pathogens,
Including viruses, One add-fast bacillus survived 2.0 mg/£ free chlorine for 67 m1n
while the other survived 1.0 mg/£ free chlorine for 15 m1n. The yeast resisted 1,0
mg/£ free chlorine for 20 m1n. In comparison, a pure culture of E. cote failed to
survive 5 m1n contact with 0.03 mg/l free chlorine. Add-fast staining of colonies on
membrane filters Incubated on mineral-proplonate medium, following sample pretreatment
with oxalic add and NaOH provided a selective assay technique for add-fast organisms.
Significant quantities of yeasts and add-fast organisms were found 1n wastewater
effluents and yeasts have been Isolated from stools. The chlorine resistance and other
characteristics of the yeasts and add-fast bacilli under study suggest that they show
promise as b1o1nd1cators for wastewater chlorlnatlon efficiency for the more chlorine
resistant pathogens.
11a. Descriptors * *
B1o1nd1cators , bacteria, conforms, yeasts , chlorlnatlon , wastewater
treatment*, viruses, public health, water reuse, activated sludge, trickling filters,
resistance*, sewage bacteria
17b. Identifiers
Enteric viruses, chlorine resistance, pour plates, add-fast bacilli
17c. COWRR Field & Group 05A, 05D
WASHINGTON. D. C, 2024O
David H. Foster
'"»•••> Univ. of Illinois, Urbana. Illinois
«U.S. GOVERNMENT PRINTING OFFICE: 1974 546-318/336 1-3
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