METHODS FOR BACTERIOLOGICAL
EXAMINATION OF SOLID WASTE
AND WASTE EFFLUENTS
U. S. ENVIRONMENTAL PROTECTION AGENCY
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METHODS FOR BACTERIOLOGICAL EXAMINATION
OF SOLID WASTE AND WASTE EFFLUENTS
This publication (SW-*68r.of) was prepared
by MIRDZA L. PETERSON,
Senior Research Microbiologist
National Environmental Research Center—Cincinnati
U.S. ENVIRONMENTAL PROTECTION AGENCY
1972
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TABLE OF CONTENTS
SECTION A. Introduction and General Laboratory Procedures
1. Introduction
2. 'General Laboratory Procedures
2.1 Glassware Washing
2.2 Sterilization
2.3 Culture media
SECTION B. Collection and Preparation of Samples
1. Method for collection of solid waste
or semi-solid waste samples
2. Method for collection of liquid samples-
Quench and Industrial waters or Leachate
3. Method for collection of incinerator
stack effluents
4. Method for collection of dust samples
5. Method for preparation of solid and
semi-solid samples for analyses
SECTION C.
FIGURE 1
FIGURE 2
FIGURE 3
REFERENCES
Bacteriological Examination of Waste and
Related Materials
1. Method for preparation of decimal
dilutions of a solid, semi-solid or
liquid waste material
2. Methods for total viable bacterial
cell number
3. Methods for presence of numbers
of coliform group
4. Method to determine the presence of
viable heat-resistant spore-formers
5. Methods to detect enteric pathogenic
bacteria
6. Method for examination of stack effluents
7. Method for examination of dust
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SECTION A. Introduction and General Laboratory Procedures
1. INTRODUCTION
1.1 A goal of microbiological research has been the development of
methods for the detection and enumeration of pathogenic bacterial species
in solid waste and waste effluents. Attempts to isolate such organisms
from solid waste on a routine basis have not been fruitful due to low
initial numbers and/or to relatively short periods of survival. Patho-
genic microoganisms in waste are constantly subjected to such debilitating
environmental factors as chemical additives, drying, freezing, heat, and
pH extremes. These factors often affect cultivation of these organisms
in media originally designed for diagnostic purposes. For these reasons
attention was directed primarily toward the development of methods for
the detection and enumeration of a group of organisms of sanitary and
health significance. Three procedural lines of investigation were
undertaken:
1.1.1 To develop methods suitable for indicating the sanitary
quality of solid waste before and after processing or disposal.
1.1.2 To develop methods suitable for determining the efficacy
of operational procedures in removing or destroying the microorganisms.
1.1.3 To develop methods suitable for indicating the health
hazard of solid waste in which pathogenic species may be present in
small numbers.
1.2 An investigation was made to evaluate presently employed
bacteriological methods applicable to solid waste and related materials.
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This evaluation led to the establishment of reliable methods which
are best suited to routinely measure under practical conditions the
bacteriological quality of solid waste, incinerator residue, industrial
and quench waters, leachate, stack emissions, and dust in and around
waste processing areas. The methods described here will determine:
1.2.1 Total viable bacterial cell number
1.2.2 Total coliforms
1.2.3 Fecal coliforms
1.2.4 Heat-resistant spores
1.2.5 Enteric pathogens, especially Salmonella sp.
It should be remembered that minor changes in technical procedure
may result in marked changes in the validity of the data.
2. GENERAL LABORATORY PROCEDURES
2.1 Glassware washing
2.1.1 All glassware known to contain infectious material must
be sterilized by autoclaving before washing.
2.1.2 All glassware which is to be used in microbiological
tests must be thoroughly washed before sterilization using a suitable
detergent and hot water, followed by hot water and distilled water
rinses. Six to twelve rinses may be required to remove all traces
of inhibitory residues from the glass surface.
2.2 Sterilization
2.2.1 Dry heat is utilized for the sterilization of glass sampling
bottles, foil covered flasks, beakers, graduates, pipets packed tightly
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in sealed cans, or articles which are corrosively attacked by steam.
Recommended time-temperature ratio for dry heat sterilization is 170 C
for 2 hr.
2.2.2 Saturated steam under pressure (or autoclaving) is the
most frequently used sterilization method. Media, dilution water,
and materials (such as rubber, paper, cotton, cork, heat-stable
plastic tubes and closures) are sterilized by autoclaving at 121 e.
Sterilization time for media and dilution water for volumes up to
500 ml is 15 min; 1000 ml quantities are held for 20 min.; instruments -
15 min; gloves - 20 min.; and packs - 30 min., measured from the time
the autoclave temperature reaches 121 C.
2.2.3 Membrane filters are sterilized for 10 min at 121 C with
fast steam exaust at the end of the sterilization process.
2.2.4 Heat-sensitive carbohydrates and other compounds should
be sterilized by passage through a cellulose esther membrane or another
bacteria retaining filter.
2.3 Culture Media
2.3.1 The use of dehydrated media is recommended whenever possible,
as these products offer the advantages of good consistency from lot
to lot, requires less labor in preparation, and are more economical.
Each lot should be tested for performance before use.
2.3.2 Measurement of the final pH of a prepared culture medium
should be accomplished colorimetrically or potentiometrlcally after
autoclaving and cooling. Acceptable pH range is 7.0 ± 0.1.
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2.3.3 Media should be stored in a cool, dry, and dark place to
avoid dehydration, deterioration, and adverse light affect. Storage
in the refrigerator usually prolongs the shelf life of most media.
Media should not be subjected to long periods of storage because
certain chemical reactions may occur in a medium even at refrigerator
temperatures.
2.3.4 Many of the media referred to in Methods can be obtained
from commerical sources in a dehydrated form with complete information
on their preparation, therefore these media will be listed but not
described in this section. Described in this section are those media
which are formulated from ingredients or from dehydrated materials.
2.3.5 Tabulation of culture media
Bacto-agar
Bismuth sulfite agar
Blood agar
Brain heart infusion broth
Brilliant green lactose bile, 2%
Brilliant green agar
Coagulase mannitol agar
Dextrose
E. C. broth
Eosin methylene blue agar, Levine
Fluid thioglycollate medium
Gelatin
H-broth
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Indole nitrite medium
KCN medium
Lactose
Lactose tryptose broth
Lauryl tryptose broth
Lyslne decarboxylase medium
M-Endo broth
M-FC broth
MacConkey agar
Malonate broth, Ewing modified
Maltose
Mannitol salt agar
Mannltol
Methyl red-Voges Proskauer medium
Nitrate broth
Nutrient agar
Phenol red broth base
Phosphate buffer, APHA, pH 7.2
Sabouraud dextrose agar
Salmonella Shlgella agar
SB6 enrichment broth
Selen1te-F enrichment broth
SIM medium
Simmons citrate agar
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Sucrose
Triple sugar iron agar
Trypticase soy agar
Tryptone glucose extract agar
Urea agar base concentrate (sterile)
XLD agar
2.3.6 Culture media requiring preparation
2.3.6.1 Blood Agar -- Suspend 40 gms of trypticase soy
agar in a liter of distilled water. Mix thoroughly. Heat with
agitation and boil for one minute. After solution is accomplished,
sterilize by autoclaving for 15 min at 121 C. Cool agar to 45
to 50 C, and add 5 to 7 per cent sterile defibrinated sheep blood
mixing evenly throughout the medium. Pour into sterile Petri
dishes. After solidification invert dishes and incubate overnite.
2.3.6.2 Phenol Red Broth Base — Dissolve 15 gms in a
liter of distilled water. Add five to ten gms of desired carbohydrate.
Use Durham fermentation tubes for detection of gas formation. Arrange
tubes loosely in suitable containers and sterilize to 116 to 118 C for
15 min.
2.3.6.3 Phosphate Buffer Solution— To prepare stock phosphate
buffer solution, dissolve 34.0 g potassium dihydrogen phosphate, KH2PO,
in 500 ml distilled water, adjust to pH 7.2 with IN NaOH, and dilute to
] liter with distilled water.
Add 1.25 ml stock phosphate buffer solution to 1 liter distilled
water. Dispense in amounts that will provide 99 ± 2.0 ml or 9 ± o.2 ml
after autoclaving.
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SECTION B. Methods for Collection and Preparation of Samples
1. METHOD FOR COLLECTION OF SOLID WASTE OR SEMI-SOLID WASTE SAMPLES
1.1 Equipment and materials
1.1.1 Sample containers, specimen cups, sterile, 200-ml size
(Falcon Plastics, Los Angeles)
1.1.2 Sampling tongs, sterile (stainless steel, angled tips, 18" long)
1.1.3 Shipping container, insulated, refrigerated, 6"xl2" J.D.*
1.1.4 Disposable gloves
1.2 Procedure
1.2.1 Using sterile tongs collect 20 to 40 random 100 gm to
200 gm samples and place in sterile sampling containers. When
collecting samples from contaminated sources wear disposable gloves
and avoid contaminating the outside of the container.
1.2.2 Identify samples on tag and indicate time and date of
sampling. If incinerator residue samples are taken, record operating
temperatures of incinerator.
1.2.3 Deliver samples to laboratory. It is recommended that
the examination be started preferably within 1 hr. after collection;
the time elapsing between collection and examination should in no
case exceed 30 hours. Sample temperature is maintained as near that
at collection time as possible.
2. METHOD FOR COLLECTION OF LIQUID SAMPLES - QUENCH OR INDUSTRIAL
WATERS OR LEACHATE.
2.1 Equipment and materials
*If sample is shipped to a laboratory for analysis and examination
can not begin within one hour of collection, the container must be
insulated and sample maintained below 10°C during the maximum of 6 hours.
Such samples should be refrigerated upon receipt in the laboratory and
processed within 2 hours.
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2.1.1 Sample bottle, screw capped, 250 ml size, sterile, or
plastic bag, 16-oz. size, sterile.
2,2. Procedure
2.2.1 Collect sample in bottle or plastic bag leaving an air
space in the container to facilitate mixing of the sample prior to
examination. When collecting samples from contaminated sources
wear disposable gloves .and avoid contaminating the outside of the
container.
2.2.2 Identify and deliver samples to laboratory. When shipping
of samples to laboratory, protect containers from crushing or other
damage, and maintain temperature as near that at collection time as
possible* Examine within 4 hours. If water sample contains residual
chlorine a dechlorinating agent such as sodium thiosulfate is added
to collection bottles to neutralize any residual chlorine and to
prevent a continuation of the bactericidal action of chlorine
during the time the sample is in transit to the laboratory. The
sodium thiosulfate is added to the clean sample bottle before sterili-
zation in an amount to provide an approximate concentration of 100mg/£
in the sample.
3. METHOD FOR COLLECTION OF INCINERATOR STACK EFFLUENTS
3.1 Equipment and materials
3.1.1 Armstrong portable sampler (2), equipped with sampling
assembly (Fig. 1). The sampler is mounted on a steel plate (6 by 12
inches) and can be enclosed by a metal cover with a handle attached
(Fig. 1). On one side of the base is a vacuum pump with a 6-ft cord
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and switch. The pump is capable of drawing up to 1 cu. ft. per
min of air [vacuum of 5.6 inches (14.3 cm) of water]. On the other
side of the base, a 700 ml wide-mouth, Pyrex bottle contains 300 ml of
0.067 M phosphate buffered solution, (pH 7.2), prepared by standard methods(4).
The two-hole rubber stopper has a 1-inch (2.54 cm) piece of cotton-
plugged glass tubing in one of the two holes. The stopper, glass
tube, and contents of the bottle are maintained sterile. The bottle
is held to the base plate by three removable spring clips, which are
attached at the base and at a wire triangle slipped over the top of
the bottle. The sampling probe is made of stainless-steel tubing
of appropriate diameter [e.g., 0.25-inch inside diameter (0.64 cm)].
The probe end has a right-angle bend so that the opening faces the
stack gas current. The tubing must be long enough to reach all parts
of the stack. The tubing is coiled to permit additional cooling of
the gases and is straight for 1 or 2 ft (30.48 or 60.96 cm) at a
right angle to the other straight length. Before use, the sampling
probe is sterilized by dry heat sterilization. It is important to
keep the inside of the probe dry to minimize adsorption of micro-
organisms on the walls of the tubing. When sampling, the probe is
inserted into the stack at locations that will yield a representative
sample. The other end of the sterile probe is inserted through the
sterile rubber stopper to approximately 0.5 inch (1.27 cm) above
the buffered water. This is done to reduce the frothing that would
occur if the probe were inserted below the surface; enough froth
results to capture the microorganisms.
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3.2 Procedure
3.2.1 Draw stack effluent through the sterile stainless steel
tube by a 1.0 cfm vacuum pump, cooling the tube by a water jacket.
3.2.2 Obtain a 10 cubic foot sample by drawing the stack effluent
for 10 min.
3.2.3 Identify sample on tag and examine within 4 hours. The
Armstrong portable sampler provides a method for qualitative, non-
isokinetic sampling, adjustable to isokinetic conditions
4. METHOD FOR COLLECTION OF DUST SAMPLES
4.1 Equipment and materials
4.1.1 Andersen sampler (3)
4.1.2 Trypticase soy agar containing 5% sheep blood to trap
skin and respiratory tract bacteria (6 plates per sample).
4.1.3 Eosin methylene blue agar to enumerate intestinal tract
bacteria.
4.2 Procedure
4.2.1 Draw air through the sterile, assembled sampler at 1.0 cfm
with a vacuum of 15 inches of mercury.
4.2.2 Remove agar plates from the sampler, cover, and incubate
at 35±0.5 C. Use aseptic technique throughout the procedure.
5. METHOD FOR PREPARATION OF SOLID AND SEMI-SOLID SAMPLES FOR ANALYSES
5.1 Equipment and materials
5.1.1 Cold phosphate buffer, 0.067 M, (pH 7.2), sterile (4)
5.1.2 Blender, Waring (Model 1088), sterile
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5.1.3 Balance, with weights, 500 gm capacity
5.1.4 Tongs, sterile
5.1.5 Beakers, two, 5000 ml and 1000 ml sizes, sterile, covered
with aluminum foil before sterilization.
5.2 Procedure
5.2.1 Using aseptic technique composite all random samples
into a 5000 ml beaker. Mix well.
5.2.2 Weigh 200 gm of the subsample into a 1000 ml beaker
5.2.3 Transfer the weighed sample to a sterile blender
5.2.4 Add 1800 ml of sterile phosphate buffered solution to
the blender.
5.2.5 Homogenize for 15 sec. at 17,000 rpm ^16'
5.2.6 Prepare a series of decimal dilutions as described below.
[Section C 1.1-1.3]
Solid waste and residue samples for enteric pathogenic bacteria are
examined directly without homogenization.
SECTION C. Bacteriological Examination of Waste and Related Materials
(description of methods)
1. METHOD FOR PREPARATION OF DECIMAL DILUTIONS OF A SOLID, SEMI-
SOLID OR LIQUID WASTE MATERIAL
1.1 Immediately after homogenization of any sample [Section B
5.2.1 - 5.2.6] transfer 1 ml portion of the homogenate (10 dil.)
to a dilution bottle containing 99 ml of phosphate buffered solution.
Stopper and shake the bottle 25 times.
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1.2 Prepare dilutions as indicated in Figure 2.
1.3 Again shake each dilution vigorously 25 times after adding
aliquot of sample.
These dilutions are used to inoculate a series of selected culture
media for the detection of various groups of micro-organisms as
described in the following sections of this manual.
2. METHODS FOR TOTAL VIABLE BACTERIAL CELL NUMBER
The chief cultural method for determining total viable bacterial
densities has been the agar plate method. (4 - 6) Experience
indicates that an approximate enumeration of total number of viable
bacteria multiplying at a temperature of 35 C may yield useful in-
formation concerning the sanitary quality of the waste entering a
processing or a disposal site, and provide useful information in
judging the efficiency of procedures employed in solid waste processing
and/or disposal operations. The viable microbial count provides
valuable information concerning the microbiological quality of en-
vironmental aerosols existing in or around a waste processing plant
or a disposal site.
2.1 Equipment, materials and culture media
2.1.1 Pipettes, 1.1 ml with 0.1 ml and 1 ml graduations
2.1.2 Dilution blanks, phosphate buffered solution, 99 ml
±2 ml (cold)
2.1.3 Culture dishes (100 x 15 ml) plastic, sterile
2.1.4 Water bath for tempering agar, 45±1 C
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2.1.5 Incubator 35±0.5 C
2.1.6 Colony counter, Quebeck
2.1.7 Sterile glass spreader, bent rod
2.1.8 Tryptlcase soy agar with 7% defibrinated sheep blood
(TSA+blood)
2.1.9 Tryptone glucose extract agar (TGEA)
Prepare T6E agar as indicated on label and hold in a melted condition
in the water bath (45 C).
Dissolve ingredients of ISA and heat to boiling. Sterilize by
autoclaving at 121 C for 15 min. Cool to 45 C and add sheep blood.
Dispense in Petri plates and allow to solidify. Invert plates and
place them in incubator overnight to dry.
2.2 Procedure for bacterial count by pour plate
2.2.1 Pipette 1 ml, 0.1 ml or other suitable volume of the
sample into each of appropriately marked duplicate culture plates,
being sure to shake each dilution bottle vigorously 25 times to
resuspend material that may have settled out.
2.2.2 Add 10 to 12 ml of melted TGE agar to the sample in the
Petri plate
2.2.3 Mix dilution and the agar medium by rotating or tilting
the plate
2.2.4 Allow plates to solidify as rapidly as possible after
pouring
2.2.5 Invert plates and incubate them at 35 C ± 0.5 C for
24 ± 2 hour.
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2.2.6 Count all colonies using Quebeck colony counter, the
objective being to count plates with 30 - 300 colonies
2.2.7 Compute the colony count per gm of waste ( wet weight)
or related solid material, per 100 ml of water. The number of
bacteria should not include more than two significant figures.
2.3 Procedure for bacterial count by streak plate
2.3.1 Dispense 0.1 ml samples of the serially diluted homo-
genate (or liquid) on the surface of each of appropriately marked,
duplicate blood agar plates containing trypticase soy agar base
and 7% defibrinated sheep blood.
2.3.2 Using a sterile glass spreader and starting with the
highest dilution plates, spread the inoculum evenly over the agar
surface.
2.3.3 Invert plates and incubate them at 35 C for 24 hr ± 2 hr.
2.3.4 Count the number of colonies on plates with 30 to 300
colonies.
2.3.5 Select and mark colonies for further testing.
3. METHODS FOR PRESENCE OF MEMBERS OF COLIFORM GROUP
The coliform bacteria have long been used in the United States as
indicators of fecal pollution in sanitary bacteriology. Some members
of the coliform group organisms are found in the feces of warm-blooded
animals, in the guts of cold-blooded animals, in soils, and on many
plants. Studies have shown that warm-blooded animal feces from humans,
animals, or birds may at any time contain disease-producing micro-
organisms (7). It was pointed out that cold-blooded animal feces are
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quantitatively Insignificant as a source of pollution, but the
coliform bacteria from plants or soils that have been recently exposed
to fecal pollution have the same significance as those from feces; on
the other hand, the coliform bacteria deriving from soils or plants
that have not been exposed to recent fecal contamination has less
public health significance.
Adequate treatment of waste prior to disposal and proper operational
design of a waste processing plant should remove all coliform organ-
isms. Treated or processed waste containing coliform bacteria demon-
strates an inadequate treatment and should be considered of more or
less sanitary significance. The contamination of waste by fecal matter
may be one avenue of transmission of pathogenic microorganisms to the
environment and man.
The presence of fecal matter in waste and related materials is determined
by the standard tests for the coliform group described in Standard
Methods for the Examination of Water and Waste Water (4). The completed
Most Probable Number (MPN) procedure is employed. The testing method
includes the elevated temperature test (44.5 C) that indicates that
fecal or non-fecal origin of coliform bacteria. Comparative laboratory
studies conducted showed that the MPN estimate is the most suitable
method for achieving a representative enumeration of the coliform
organisms in solid waste and waste effluents (8).
3.1 Equipment and materials
3.1.1 Pipettes, sterile - deliveries to 10 ml, 1 ml (1.1 ml)
and 0.1 ml.
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3.1.2 Media prepared in ferraantation tubes:
Lauryl tryptose broth
Brilliant green lactose bile broth, 2%
Lactose tryptose broth
E. C. broth
3.1.3 Media for plating:
Eosin methylene blue agar plates
Nutrient agar slants
3.1.4 Dilution blanks, phosphate buffered solution, sterile,
99 ml or 90 ml amounts.
3.1.5 Incubator - adjusted to 35 C ± 0.5 C
3.1.6 Water bath - adjusted to 44.5 C ± o,5 C
3.2 Procedure for total coliform group
3.2.1 Presumptive test
3.2.1.1 Inoculate a predetermined volume of sample into 5
lauryl tryptose broth tubes. The portions of the sample used for
inoculation should be decimal multiples and submultiples of 1 ml.
3.2.1.2 Incubate the fermentation tubes at 35 ± 0.5 C for
24 ± 2 hours.
3.2.1.3 Examine for the presence of gas. If no gas is
formed incubate up to 48 ± 3 hours.
3.2*2 Confirmed test
3.2.2.1 Submit all presumptive test tubes showing any amount
of gas at the end of 24 and 48 hr incubation to the confirmed test.
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Using a sterile platinum loop, 3 ram in diameter, transfer one loopfiil
of medium from the presumptive test fermentation tube to a fermentation
tube containing brilliant green lactose bile broth.
3.2.2.2 Incubate the inoculated brilliant green lactose bile
broth tube for 48 ± 3 hours at 35 ± 0.5 C. The presence of gas in any
amount in the fermentation tubes of the brilliant green lactose bile
broth within 48 ± 3 hours indicates a positive confirmed test.
3.2.3 Completed test
3.2.3.1 Submit all confirmed test tubes showing any amount
of gas to the completed test. Streak an eosin methylene blue agar
plate from each brilliant green bile broth tube as soon as possible
after the appearance of gas.
3.2.3.2 Incubate the plates at 35 ± 0.5 C for 24 ± 2 hours.
3.2.3.3 Fish one or more typical or atypical colonies from
plating medium to lactose tryptose broth fermentation tubes and
nutrient agar slants.
3.2.3.4 Incubate the broth tubes and the agar slants at
35 ± 0.5 C for 24 ± 2 or 48 ± 3 hours
3.2.3.5 Prepare gram stained smears from the nutrient agar
slants if gas is produced in any amount from lactose broth
3.2.3.6 Examine smears under oil immersion. If no spores
are found on the slant the test may be considered "completed" and
the presence of col 1form organisms demonstrated.
3.3 Procedure for fecal coliform group (E. C. broth)
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3.3.1 Submit all gas positive tubes from the Standard Methods
presumptive test (lauryl tryptose broth) to the fecal coliform test.
Inoculate a E. C. broth fermentation tube with a 3 mm loop of broth
from a positive presumptive tube.
3.3.2 Incubate the broth tube in a water bath at 44.5 ± 0.5 C for
24 hours. All E.G. tubes must be placed in the water bath within
30 min. after planting.
3.3.3 Gas production in the E. C. broth fermentation tubes
within 24 hours is considered a positive reaction indicating fecal
origin.
3.4 Computing and Recording Most Probable Number (MPN)
The calculated estimate and the 95% confidence limits of the MPN have
been presented in the current (13th) edition of Standards Methods for
Examination of Water and Waste Water. (Ref. 4, Table 407, p. 673.)
This table is based on five 10 ml, five 1.0 ml, and five 0.1 ml
sample portions. When the series of decimal dilutions such as 1.0,
0.1, and 0.01 ml are planted, record 10 times the value in the table;
if a combination of portions of 0.1, 0.01, and 0.001 ml are planted
record 100 times the value in the table. MPN values for solid samples
are calculated per gram of wet weight; MPN for liquid samples are
recorded per 100 ml.
4. METHOD TO DETERMINE THE PRESENCE OF VIABLE HEAT-RESISTANT SPORE -
FORMERS
It was of importance to enumerate those heat-resistant, spore-forming
microorganisms in waste, incinerator residue, quench or industrial
waters which survive 80 C temperature for as long as 30 minutes. With
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respect to mere survival of heat most microorganisms in an actively
growing (vegetative) state are readily killed by exposures to
temperatures of around 70 C for 1 to 5 minutes (9). Cells inside of
solid material such as discarded meat products may escape heat longer
because the heat does not penetrate immediately into the center of
solid masses. Large masses of non-fluid solid matter require a long
time (1-1/2 to 2 hr), even in the autoclave (121 C) to be heated
thoroughly so that the center reaches a sporocidal temperature. Other
reports point out (10), that although internal air temperatures of
municipal incinerators usually range from 1200 to 1700 F (650 to 925 C)
in continuous operation, intermittent use and overcharging of the in-
cinerator and moisture content of the :*aste may interfere with sterili-
zation of the residue.
A test of this type reveals operational problems of a waste processing
plant and identifies unsatisfactory quality of waste effluents of a
municipal incinerator.
4.1 Equipment and materials
4.1.1 Test tubes, sterile - screw capped, 20 x 150 mm
4.1.2 Pipettes, sterile - graduated, 10 ml
4.1.3 Water bath - electrically heated, thermostatically controlled
at 80 ± 0.5 C, equipped with thermometer, range 0 to 110 C, NBS certified.
Volume of water should be sufficient to absorb cooling effect of rack
of tubes without drop in temperature greater than 0.5 C.
4.1.4 Test tube support - for holding tubes
4.2 Procedure
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4.2.1 Transfer 10 ml from each original sample and from each
successive dilution thereof to screw-capped test tubes, being careful
to avoid contaminating the lip and upper portion of tube with sample.
4.2.2 Place tubes in a rack
4.2.3 Place rack of tubes in water bath at 80 C for 30 min. Tubes
should be immersed so that the water line is approximately 1-1/2 inches
above the level of samples in the tubes.
4.2.4 At the end of the 30 min holding period, remove the rack
of tubes from the water bath and place in cold water for 5 min to cool
the tubes.
4.2.5 Determine viable heat-resistant spore count by agar pour
plate method (Section C, 2.2.1 - 2.2.7)
4.2.6 Report results as "viable heat-resistant spore count per
gm"
5. METHODS TO DETECT ENTERIC PATHOGENIC BACTERIA
An access of fecal pollution to environment by nontreated and improperly
disposed waste may add enteric pathogenic bacteria to a body of water
or a water supply. The most common type of pathogen which may be found
in untreated waste is Salmonella. The wide distribution of the many
types of Salmonella in many species of animals with which man has
contact or may use as food makes it difficult to prevent transmission
to man (11). Infections may occur through food, milk, or water con-
taminated with infected feces or urine, or by the actual ingestion of
the infected animal tissues (12). Salmonella has been found in many
water supplies (13), polluted waters (17 - 19), and raw municipal refuse
and in incinerator residue (14 - 20).
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The detection of enteric pathogenic bacteria such as Salmonella and
Shigella in municipal solid waste before and after a treatment and/or
disposal determines the microbiological quality of the material and
serves as a procedure to determine the efficacy of a waste treatment
process in removing or destroying the fecal-borne pathogens. Results
obtained in the testing may also be used for the design of epidemiological
studies in other programs.
The method described below has been tested in field and has been described
by Peterson and Klee (14) and Spino (20), using incubation temperatures
of 39.5 C and 41.5 C.
5.1 Equipment, materials and media
5.1.1 Incubator, 37 C
5.1.2 Water baths, constant temperature, 39.5 C and 41.5 C
5.1.3 Flasks, wide mouth, 500 ml size
5.1.4 Membrane filter holder
5.1.5 Flasks, vacuum, 2000 ml size
5.1.6 Balance, with weights, 100 gm capacity
5.1.7 Needle, inoculating
5.1.8 Media and reagents
Selenite brilliant green/sulfa enrichment broth
Selenite F enrichment broth
Eosin methylene blue (EMB) agar
Salmonella - Shigella (SS) agar
Bismuth sulfite (BS) agar
McConkey's agar
Brilliant green (BG) agar
Triple sugar iron (TSI) agar
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Urea medium
XLD agar
Salmonella antiserums
Shigella antiserums
Biochemical media (15)
5.1.9 Diatomaceous - earth (Johns-Manville, Celite 505), sterile
5.2 Procedure to detect pathogens in solid waste and incinerator
residue.
5.2.1 Add a previously weighed 30-gm sample to each of two flasks
containing 270 ml Selenite F enrichment broth and also to each of two
flasks containing 270 ml Selenite brilliant green/sulfa (SBG) enrichment
broth. Shake to mix.
5.2.2 Incubate one Selenite F and one SBG flask at 39.5 C and the
other two at 41.5 C for 16 to 18 hours.
5.2.3 After incubation streak one loopful from each enrichment
medium on each of four plates of Salmonella-Shigella and other selec-
tive enteric media.
5.2.4 Incubate the plates at 37 C for 24 - 48 hours, and pick
suspicious colonies to triple sugar iron agar slants.
5.2.5 Incubate the slants at 37 C for 24 hours and complete
identification by appropriate biochemical and serological test as
directed (15). Isolation and preliminary identification is described
in Figure 3.
5.3 Procedure to detect pathogens in quench or industrial waters
and in leachate
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5.3.1 Place a portion of sterile diatomaceous - earth on the
screen of a stainless steel membrane filter holder enough to form
a 1" layer.
5.3.2 Filter 800 ml sample through the earth layer
5.3.3 Remove one half of the diatomaceous - earth layer with
a sterile spatula and place into 90 ml of Selenite F enrichment broth;
place other half of the earth layer into 90 ml of Selenite brilliant
green/sulfa enrichment broth. Shake both flasks to mix.
5.3.4 Incubate both flasks in a water bath at 39.5 C for 16 to
18 hours.
5.3.5 Proceed as directed (Section C, 5.2.3. - 5.2.5)
6. METHOD FOR EXAMINATION OF STACK EFFLUENTS
6.1 As described in Methods for collection of stack effluents
using the Armstrong sampler, the microorganisms are impinged into
a 300 ml phosphate buffered soultion.
6.1.1 Filter 100 ml of the "inoculated" phophate buffered
solution through a 0.45jji HA membrane filter (4).
6.1.2 Transfer membrane filter with sterile forceps to a culture
plate containing trypticase soy agar.
6.1.3 Incubate culture plate under constant saturated humidity
for 20 hours (± 2 hrs) at 35 C.
6.1.4 After incubation, remove cover from culture plate and
determine colony count with the aid of a low-power (10-15 magnifi-
cations) binocular wide-field microscope. Characterize colonies
using specific isolation media.
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6.1.5 Remove a 10 ml portion of the "inoculated" phosphate
buffered solution and examine for viable heat-resistant spores as
directed (Section C, 4.2.1 - 4.2.6).
Microbioal counts are reported as organisms per 1 cu ft air.
If the sample is not taken under isokinetic conditions, the results
are qualitative. If the stack velocity is known and remains relatively
constant, however, the rate of flow of the sampler can be adjusted to
isokinetic conditions to yield quantitative results.
7. METHOD FOR EXAMINATION OF DUST
7.1 As described in Methods for collection of dust samples, the
Andersen sampler is used with two types of media, trypticase soy
agar (TSA-BBL product) containing 5% sheep blood and eosin methylene
blue agar (EMB-Difco product). The TSA/blood agar is used to isolate
a wider range of fastidious organisms such as Staphylococci, Strepto-
cocci, and Diplococci. The EMB agar is used to isolate gram-negative
bacteria. The plates are incubated aerobically at 35 C for 24 hours.
(Preliminary studies showed that few organisms in the dust would grow
under anaerobic conditions.) Enumeration of colonies is made with a
Quebec colony counter. Microbial count is reported as organisms per
1 cu ft air. At times when microbial counts are high, the sampling
time is 0.25 min, thus yielding 0.25 cu ft air.
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G= = =
rHOSFHATE BUFFER
Fio. 1. Portable sampler for microorganisms in
Incinerator stack emissions.
- 25 -
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FIGURE i Preparation of Decimal Dilutions
INJ
cn
Solid or semi-
solid waste
sample
Liquid
Sample
2Q6 gms
I
10 El
Blend with
1800 ml
buffered water
-------
FIGURE '3 Isolation and Preliminary Identification
Solid was^e or residue
Sample 30 gms
EMB
Agar
Selenite F
1
Selenite brilliant !
Green/Sulfa 270 ml
Inciibate at 39.5 C ail
d 41.5 C
SS
Agar
BS
Agar
BG
Agar
Triple Sugar Iron agar
Urea medium (Christensen)
2- to 4-hour reading of urea medium
Proteus group
(Reincubate negative
urea medium)
H2S
(TSI agar)
| M2S-
Salmuncllti polyvalent
antiserums
Macdonkey's
Agar
' x
4 plat<
s each
polyvalent antiserums
Salmonella polyvalent antiserums
Identify
confirm
+
serologically ;
jiochemically.
1
Do biochemical
tests.
r - 1
Identify serolopi-
cally; confirm
biochemically.
T "
Do biochemical
tests.
If not readily identifiable,
proceed to biochemical tests
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REFERENCES
1. Hanks, T. G. Solid Waste/disease relationships.
U. S. Dept. of Health, Education, and Welfare, PHS, BDPEC,
NCUIH, Solid Waste Program, Cincinnati, 1967
2. Armstrong, D. H. Portable sampler for Micro-organisms in
Incinerator stack emissions. Appl. Microbiology, Vol. 19,
No. 1, p. 204 - 205, 1970
3. Andersen, A. A. New Sampler for the collection, sizing and
enumeration of viable airborne particles. J. of Bacteriology
76:471 - 484, 1958
4. American Public Health Association, Standard methods for the
Examination of water and waste water. Am. Public Health
Association, New York, 1971
5. American Public Health Association, Inc. Standard methods
for the examination of Dairy products Microbiological and
Chemical. Am. Public Health Association, Inc, New York, 1960
6. Harris, A. H. and M. B. Coleman. Diagnostic procedures and
reagents. Am. Public Health Association, Inc. New York, 1963
7. Clark, H. F. and P. W. Kabler. Revaluation of the significance
of the coliform bacteria. Journal of Am. Water Works Assoc.,
p. 931 - 936, 1964
8. L. Smith. A brief investigation of two procedures for total
and fecal coliform determinations in municipal solid waste.
Research Services Laboratory, Div. of Research and Development,
1969, not published.
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9. Froblsher, M. Fundamentals of microbiology. 6th edition,
p. 151, 152, 1957.
10. Barbeito, M. S. and G. G. Gremillion. Microbiological safety
evaluation of an industrial refuse incinerator. Appl. Microbiology
16:291 - 295, 1968
11. Dauer, Carl C. 1960 Summary of Disease outbreaks and a 10-year
resume. Public Health Report, Vol. 76, No. 10, p. 915, Oct. 1961
12. Dubos, Rene. Bacterial and mycotic infections of man.
J. B. Lippincott, Philadelphia, 1958.
13. Weibel, S. R. F. R. Dixon, R. B. Weidner, and L. J. McCabe.
Waterborne-disease outbreaks 1946-1960. J. Am. Water Works
Association. Vol. 56, p. 947 - 58, August, 1964.
14. Peterson, M. L. and A. J. Klee. Studies on the detection of
Salmonellae in municipal solid waste and incinerator residue.
Intern. J. Environmental Studies. Vol. 2, p. 125-132, 1971.
15. Edwards, P. R. and W. H. Ewing. Isolation and grouping of
Salmonella and Shigella cultures. U. S. Dept. of Health,
Education, and Welfare, PHS, Communicable Disease Center,
Laboratory Branch, Atlanta, Georgia, p. 1 - 39, September 1962
16. Peterson, M. L. and F. J. Stutzenberger. Microbiological
evaluation of incinerator operations. Appl. Microbiological,
Vol. 18, No. 1, p. 8 - 13, 1969
17. Spino, D. F. Elevated-temperature techniques for the isolation
of Salmonella from streams. Appl. Microbiol., 14, 591, 1966.
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18. Scarce, L. E. and M. L. Peterson. Pathogens in streams
tributary to the Great Lakes. In Proc. Ninth Conf. on
Great Lakes Res., March 28-30, 1966, Chicago, p. 147.
Public No. 15, Ann Arbor, Univ. of Mich., 1966.
19. Peterson, M. L. The occurrence of Salmonella in streams
draining Lake Erie Basin. In Proc. Tenth Conf. on Great Lakes.
Res., Apr. 10-12, 1967, Toronto, p. 79. Ann Arbor, Univ. of
Mich., 1967.
20. Spino, D. Bacteriological study'of(,,the New Orleans East Incin-
erator, IJ.S". Environmental iProtect!on Agency,-Office of Research
and Monitoring, 1971.
ycr 72330rm
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