Chapter 8

Tests for Groups

of Microorganisms
J. F. Frank and A. E. Yousef
(J. F. Frank, Tech. Comm.)

his chapter includes tests for groups of microorganisms that are particu-
T larly significant to ensure the quality and safety of dairy products.
Microorganisms representing any given group may vary in significance,
and dairy processors may become interested in testing for an individual
microorganism rather than a group of microorganisms. Nevertheless, it is
easier and often faster to test for these microorganisms as groups and use
the results to make general inferences about the microbiological quality of
the product.
8.010 Psychrotrophic Bacteria (Class O)
Psychrotrophic bacteria are those bacteria that grow at refrigeration tem-
peratures (≤7°C) within 7-10 days regardless of their optimal growth tem-
perature. Psychrotrophic bacteria that are commonly isolated from dairy
products belong to a variety of genera that include but are not limited to
Pseudomonas, Flavobacterium, Alcaligenes, Acinetobacter, Klebsiella, Bacillus,
and Lactobacillus. The psychrotrophic bacteria most detrimental to dairy
product quality are the oxidative, gram-negative rods belonging to the
genus Pseudomonas. These are also the psychrotrophs most commonly iso-
lated from milk. Some pathogenic bacteria isolated from milk (e.g., Listeria
monocytogenes and Yersinia enterocolitica) are also psychrotrophic. With the
exception of Bacillus spp.,18 psychrotrophs are inactivated by pasteurization,
so their presence in processed milk indicates either improper pasteurization
or postpasteurization contamination. Growth of psychrotrophs in raw milk
(to ≥106 CFU/mL) can reduce the quality of the pasteurized product even
though the cells are inactivated.
1. Scope: The psychrotrophic count procedure is used to estimate the level
of microorganisms capable of growing during refrigerated storage and to
predict product shelf life. This method is applicable to raw and pasteur-
ized milk, cream, and cottage cheese.

227
 2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Psychrotrophic bacteria are those bacteria capable of produc-
ing colonies on the specified medium during incubation at 7°C for 10 days.
4. Principle: Samples are plated using a rich, nonselective agar medium.
Selection for psychrotrophic bacteria is accomplished by incubating the
plates at refrigeration temperature.
5. Diluents, Culture Media, and Reagents: See chapter 6, Standard Plate
Count Method (6.020).
6. Apparatus and Glassware: An incubator capable of being maintained at
7° ± 1°C. See chapter 6 for additional apparatus and glassware.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare the test sample and primary dilution and pour-plate
as described in chapter 6. Melted medium should be cooled to 45° ± 1°C
before pouring plates to prevent destruction of some psychrotrophic bac-
teria.25 Incubate plates at 7° ± 1°C for 10 days. At temperatures above 7°C,
other nonpsychrotrophic organisms may grow, possibly yielding mis-
leading results.
10. Expression of Results: Calculate results as described in chapter 6.
11. Precision: Precision parameters for this test are undefined.
12. Test Report and Interpretation: Specify the method used, results
obtained, and any circumstance or deviations that may have affected the
result. Report results as psychrotrophic bacteria count per milliliter or
per gram.
8.020 Gram-Negative Bacteria (Class B)
1. Scope: This method is applicable to raw and pasteurized milk, cream,
and cottage cheese.
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: For the purpose of this test, gram-negative bacteria are those
bacteria capable of producing colonies in the presence crystal violet.6
4. Principle: Samples are plated using a selective agar medium. Selection
for gram-negative bacteria is accomplished by including crystal violet in
the medium and incubating at 21° ± 1°C.
5. Diluents, Culture Media, and Reagents: See chapter 6, Standard Plate
Count and Associated Methods for diluents.

228 Standard Methods for the Examination of Dairy Products

 Crystal violet tetrazolium agar: Add 1 mL of 0.1% crystal violet (0.1
g/100 mL ethanol) to 1 L of molten standard method agar (SMA). Sterilize
the mixture at 120° ± 1°C for 15 minutes. Cool to 45° ± 1°C. Add 5 mL of
filter-sterilized 1% aqueous solution of 2,3,5-triphenyl tetrazolium chlo-
ride.
6. Apparatus and Glassware: An incubator capable of being maintained at
21°C ± 1°C.
See chapter 6 for additional apparatus and glassware.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare dilutions of the product and pour-plate [chapter 6].
Use crystal violet tetrazolium agar. Incubate plates at 21° ± 1°C for 48 ± 3
hours. Count red colonies.
10. Expression of Results: Calculate results as described in chapter 6.
11. Precision: Precision parameters for this test are undefined.
12. Test Report and Interpretation: Report as gram-negative CFU per mil-
liliter or gram.

8.030 Thermoduric Bacteria (Class O)

The test for thermoduric bacteria is also known as a laboratory pasteuriza-
tion count. High thermoduric counts are consistently associated with unhy-
gienic production practices. The thermoduric count indicates thoroughness
of equipment sanitation and assists in detecting sources of organisms
responsible for high counts in pasteurized milk products. Thermoduric bac-
teria contribute appreciably to high standard plate counts in pasteurized
milk and milk products since spores of psychrotrophic Bacillus spp. survive
pasteurization. Vegetative cells resulting from these spores may grow dur-
ing refrigerated storage of milk and adversely affect the shelf life of the pas-
teurized product.
1. Scope: This test is applicable to raw and pasteurized fluid milk products.
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Thermodurics are microorganisms (vegetative cells or
spores) that survive pasteurization treatment. Thermoduric bacteria can
survive exposure to temperatures considerably higher than their maxi-
mum growth temperatures. Thermoduric bacteria isolated from milk
usually include species of Micrococcus, Microbacterium, Streptococcus,
Lactobacillus, coryneform group, Bacillus, and Clostridium.24
4. Principle: Milk in test-tubes is heated at 62.8° ± 0.5°C for 30 minutes to
simulate the holding pasteurization method. Survivors are counted
using the standard plate count method.

Chapter 8 ■ Tests for Groups of Microorganisms 229

 5. Culture Media and Reagents: See chapter 6.
6. Apparatus and Glassware
1. Test tubes: Sterile, 20 × 125 mm, with rubber- or plastic-lined screw
caps.
2. Pipets: Sterile, graduated, for 5-mL or 11-mL delivery.
3. Thermometers: 0.1°C graduations. Thermometers used in the water
bath and in the pilot tube with milk should cover the critical range of
temperature during pasteurization. These should be checked at least
annually against a thermometer certified by NIST [chapter 2].
4. Water bath: Electrically heated, thermostatically controlled at 62.8° ±
0.5°C, equipped with stirrer and thermometer. The volume of water
should be sufficient to absorb the cooling effect of tubes placed in the
bath without a drop in temperature below 62.8°C.
5. Test tube rack: Metal, plastic, or wire.
7. Sampling: Normally, samples of milk from individual producers, trucks,
and tankers are obtained. Samples of finished products may also be ana-
lyzed.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Aseptically transfer 5 mL of a thoroughly mixed milk sample
to a sterile test tube. Be careful to avoid contaminating the lip and upper
portions of the tube with milk. During this step, all samples and filled
tubes should be kept in ice water at 0° to 4.4°C. A temperature control
tube containing a thermometer and 5 mL of milk should be placed in the
test tube rack with the other samples so the temperature can be easily
monitored. Place the rack of tubes in the pasteurizing bath at 62.8° ±
0.5°C. Immerse the closed, watertight tubes either completely or to
approximately 4 cm above the level of milk in the bath. The temperature
of the milk should reach 62.8°C within 5 minutes. If bubbles come from the
immersed tubes during heating, the heat treatment is suspect because
contaminants may be drawn into the tubes when they are cooled.
Start timing the 30-minute holding period when the temperature in
the pilot tube reaches 62.8°C. At the end of the holding period, immedi-
ately place the rack of tubes in an ice water bath and cool to 10°C or
lower. Determine the laboratory pasteurization count by using the SPC
procedure or an alternative method [chapter 6].
10. Expression of Results: Calculate results as CFU/mL as described in
chapter 6.
11. Precision: Precision parameters for this test are unknown.
12. Test Report and Interpretation: Report results as laboratory pasteuriza-
tion count per milliliter (LPC/mL). Where an alternative aerobic count
method is used, add the appropriate designation to the reported count
to identify the method.

8.040 Thermophilic Bacteria (Class O)

Thermophiles are usually spore-forming bacteria that enter milk from vari-
ous sources on the farm, or from poorly cleaned equipment in the process-

230 Standard Methods for the Examination of Dairy Products

 ing plant. These bacteria rapidly increase in numbers when present in milk
or dairy products that are held at high temperatures. Thermophilic bacteria
may accumulate in high-temperature short-time (HTST) pasteurizers or
vacuum-concentration systems in continuous operation for lengthy periods.
1. Scope: In the dairy industry, the term thermophilic bacteria applies to
microorganisms that grow in milk or milk products held at elevated tem-
peratures (≥55°C) including low-temperature long-time (LTLT) pasteur-
ization conditions.6
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Thermophilic bacteria are those bacteria capable of producing
colonies on the specified agar medium during incubation at 55°C.
4. Principle: Samples of dairy products are plated on SMA and incubated at
55° ± 1°C. Incubation at this temperature allows only for growth of ther-
mophiles.
5. Diluents, Culture Media, and Reagents: See chapter 6 for diluents.
Use 15 to 18 mL agar medium/plate.
6. Apparatus and Glassware: An incubator capable of being maintained at
55° ± 1°C.
See chapter 6 for additional apparatus and glassware.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare dilutions of the sample and pour-plate as described
in chapter 6. Incubate plates at 55° ± 1°C for 48 hours. Maintain humidi-
ty during incubation to prevent drying of agar medium.
10. Expression of Results: Calculate results as described in chapter 6.
11. Precision: There are no precision parameters for this test.
12. Test Report and Interpretation: Report results as thermophilic bacterial
count per milliliter or per gram (TBC/mL or /g).

8.050 Proteolytic Microorganisms

8.051 Skim Milk Agar Method (Class O)
Many bacteria responsible for spoilage of refrigerated dairy products are
highly proteolytic and can cause flavor defects. Proteolytic enzymes pro-
duced by psychrotrophic bacteria during growth in milk often remain active
after HTST and ultrahigh temperature (UHT) heat treatments, and thus
reduce the quality of stored, heat-treated products. Tests for proteolytic bac-
teria may be useful in identifying practices during handling and storage of
raw or processed milk that can cause contamination of products.
1. Scope: This test can be applied to raw and pasteurized fluid milk and
other unfermented dairy products.

Chapter 8 ■ Tests for Groups of Microorganisms 231

 2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: This method detects microorganisms that produce extracel-
lular proteases capable of hydrolyzing casein and producing colonies at
the incubation time and temperature used.
4. Principle: Skim milk is added to SMA to form skim milk agar. Proteolytic
microorganisms in the plated sample hydrolyze the casein surrounding
the colony. When colonies are flooded with acid, intact casein produces a
white precipitate, whereas hydrolyzed casein does not precipitate and
the agar remains translucent. Acid-producing organisms can cause false-
positive reactions on skim milk agar.
5. Diluents, Culture Media, and Reagents
A. Skim milk agar: Mix 100 mL of sterile skim milk (10% wt/wt solids)
with 1 L of melted standard methods agar (SMA) adjusted to 49° ±
1°C.
B. Acid solution: Prepare 1% HCL or 10% acetic acid.
C. Diluents as described in Chapter 6.
6. Apparatus and Glassware: See chapter 6.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Proceed as for the SPC method [chapter 6] using skim milk
agar. Incubate the poured plates for 48 to 72 hours at 32° ± 1°C or, to
increase the recovery of psychrotrophic bacteria, for 72 hours at 21° ± 1°C.
Flood the incubated plates for 1 minute with hydrochloric acid or acetic
acid. Pour off the excess acid solution and count the colonies surround-
ed by clear zones.
10. Expression of Results: Calculate results as indicated in chapter 6.
11. Precision: Precision parameters for this test have not been defined.
12. Test Report and Interpretation: Report as proteolytic colony count per
milliliter or per gram. Indicate the method used including medium,
incubation time, and temperature.

8.052 Standard Methods Caseinate Agar Method (Class B)

1. Scope: See 8.051.1.
2. References: See 8.051.2.
3. Definition: See 8.051.3.
4. Principle: Proteolytic microorganisms hydrolyze caseinate surrounding
the colony, causing the agar to clear. Caseinate agar is preferred over
skim milk agar for enumerating this group of bacteria because caseinate
agar is more sensitive when detecting weakly proteolytic organisms and
rarely produces false-positive reactions.16

232 Standard Methods for the Examination of Dairy Products

 5. Diluents, Culture Media, and Reagents
A. Diluents: See chapter 6.
B. Standard methods caseinate agar
1. Prepare standard methods caseinate agar using dehydrated SMA
as a base.
2. Prepare 0.015 M of trisodium citrate by dissolving 4.41 g of hydrat-
ed trisodium citrate in enough microbiologically suitable (MS)
water to make 1 L.
3. Dissolve 23.5 g of dehydrated SMA in 500 mL of the citrate solu-
tion. Dissolve 10 g of sodium caseinate in the other 500 mL of cit-
rate solution, using a blender to aid in dispersion. Mix the 2 solu-
tions and autoclave at 120° ± 1°C for 15 minutes.
4. Prepare a 1-M calcium chloride solution by adding 218.99 g of cal-
cium chloride hexahydrate to 1 L of MS water, and autoclave at
120° ± 1°C for 15 minutes.
5. Before pouring plates, add 20 mL of the sterile 1-M calcium chlo-
ride solution to the molten agar and mix.
6. Apparatus and Glassware: See chapter 6.
7. Sampling: See chapter 3.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Pour caseinate agar into plates so that the medium is 2 mm
thick (e.g., 12 mL for an 8.5-cm plate).
After the medium has hardened, plate 0.1-mL quantities of the sample
or diluted sample on the agar surface and spread evenly with a sterile,
bent-glass rod. To ensure absorption of the sample, allow inoculated
plates to dry for 15 minutes
Incubate plates (in inverted position) for 48 to 72 hours at 32° ± 1°C or
for 72 hours at 21° ± 1°C. Colonies surrounded by a white or off-white
zone of paracasein precipitate are proteolytic. Highly proteolytic bacteria
will also produce a clear inner zone.
Aerobic plate counts can be estimated using caseinate agar. However,
to measure the proportion of proteolytic organisms in the total microflo-
ra, only uncrowded plates (fewer than 80 colonies per plate) can be read
accurately. For best results, plates may have to be read twice—once at 48
± 3 hours to estimate the total count and again after 72 ± 3 hours to ensure
that slower-to-develop proteolytic colonies are included.
10. Expression of Results: Calculate results as indicated in chapter 6.
11. Precision: Precision parameters for this test have not been defined.
12. Test Report and Interpretation: Report as proteolytic colony count per
milliliter or per gram. Indicate the method used including medium,
incubation time, and temperature.

8.060 Lipolytic Microorganisms (Class B)11,23

1. Scope: This test can be applied to milk and high fat dairy products. It can
be used to investigate the cause of flavor defects associated with fat
hydrolysis.

Chapter 8 ■ Tests for Groups of Microorganisms 233

 2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: This method detects microorganisms that produce extracel-
lular lipase capable of hydrolyzing trybutyrin and able to produce
colonies at the incubation time and temperature used. Many psy-
chrotrophic bacteria as well as molds and some yeasts are lipolytic.
4. Principle: Sample dilutions are spread on spirit blue agar and plates are
incubated under conditions that allow for colony formation. Colonies of
lipolytic microorganisms hydrolyze the tributyrin, resulting in a clear
zone and/or color change. The lipase reagent contains tributyrin as a
substrate. It is the simplest triglyceride occurring in natural fats and oils.
Some microorganisms that hydrolyze tributyrin will not hydrolyze other,
more complex triglycerides or fats containing longer chain fatty acids.
The result can be a misleading count.
5. Diluents, Culture Media, and Reagents: See chapter 6 for diluents.
A. Spirit blue agar: Prepare spirit blue agar by hydrating the powdered
medium and sterilizing the mixture at 120° ± 1°C for 15 minutes as
instructed by the manufacturer. Cool the melted agar to 50° to 55°C and
add 3% lipase reagent with thorough mixing. If desired, use 1% to 2%
of the lipase reagent and sonicate the mixture to emulsify the reagent
uniformly in the agar. Pour 10 to 12 mL of the medium into a sterile petri
plate and allow the medium to solidify.
B. Lipase reagent.
6. Apparatus and Glassware: See chapter 6.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare 1:10 or other suitable dilutions of the product to be
tested. Spread 0.1 mL of the desired dilution over the surface of solidified
spirit blue agar.
Invert the plate and incubate at 32° ± 1°C for 48 ± 3 hours. To increase
the recovery of psychrotrophic bacteria, incubate for 72 ± 3 hours at 21°
± 1°C. The complete medium is light blue and translucent if it is not
emulsified, or lighter blue and opaque if it is emulsified. Colonies of
lipolytic microorganisms develop a clear zone and/or a deep blue color
around and under each colony.
10. Expression of Results: Calculate results as indicated in chapter 6.
11. Precision: Precision parameters for this test have not been defined.
12. Test Report and Interpretation: Report as lypolytic colony count per
milliliter or per gram. Indicate the method used including medium,
incubation time, and temperature.

234 Standard Methods for the Examination of Dairy Products

8.070 Lactic Acid Bacteria (Class B)
8.071 Total Lactic Acid Bacteria
Enumerating lactic acid bacteria in dairy products is useful when evaluating
product quality including the cause of acid defects and in evaluating lactic
starter cultures. There are numerous methods for enumerating lactic acid
bacteria in dairy products. This group of microorganisms is diverse with dif-
ferent growth requirements and sensitivities to selective agents. Therefore,
there is no growth medium that can be applied to all samples. In cases where
significant populations of nonlactic microflora are present, media having
greater selectivity may be required at the risk of inhibiting some of the lactic
microflora. Lactobacillus selection agar19 and MRS4 broth with added agar are
suitable when selectivity for lactobacilli is desired. Other useful media are
described by Gilliland et al.7 When selectivity is not required or when injured
cells may be present, SMA [chapter 6] may be used. However, lactic acid bac-
teria grow slowly on SMA and pin-point colonies are observed after incuba-
tion. Dye indicators and sodium azide should be used with caution because
some strains of lactic acid bacteria are inhibited by their presence.
Lactobacilli that survive pasteurization may be sublethally injured.
Consequently, lactobacilli in young cheese should be enumerated by using
a relatively nonselective medium followed by colony characterization.
1. Scope: The test for total lactic acid bacteria is applicable to products such
as fluid milk, cottage cheese, cheese, cultured milks, uncultured products
containing added cultures and lactic starter cultures.
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Lactic acid bacteria found in dairy products are a diverse
group consisting primarily of Streptococcus, Lactococcus, Leuconostoc, and
homofermentative and heterofermentative Lactobacillus species.
4. Principle: Sample dilutions are plated on lactic (Elliker) agar5 and incu-
bated at 32° or 37°C for 48 hours. Since this medium is not highly selec-
tive, nonlactic microorganisms may produce colonies. Composition of
this medium may be modified to aid in detecting acid-producing micro-
organisms. This is accomplished by adding 1 mL of a 1.6% alcoholic solu-
tion of bromcresol purple directly to 1 L of the medium before steriliza-
tion. Colonies producing a yellow halo are acid producers. To make SMA
or lactic agar more selective, add 0.02% sodium azide before sterilization.
Lactic acid bacteria are resistant to sodium azide, which inhibits
Enterobacteriaceae and aerobic organisms.
5. Diluents, Culture Media, and Reagents
1. Diluent: Use 0.1% peptone water sterilized at 120° ± 1°C for 15 min-
utes. Use of phosphate diluent [chapter 6] may reduce cell recovery.
2. Culture media: Lactic (Elliker) agar and MRS are commercially avail-
able.

Chapter 8 ■ Tests for Groups of Microorganisms 235

 6. Apparatus and Glassware: See chapter 6, Microbiological Count
Methods.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Samples must be handled with care because lactic acid bacte-
ria are easily injured by adverse environments. Prepare dilutions of prod-
ucts using peptone water.
Pour-plating of sample dilutions is done as described for the SPC
method [chapter 6], but using lactic or MRS agar. Colony development is
facilitated by either using an agar overlay or incubating plates in a
reduced-oxygen atmosphere.
Plates may be incubated for 48 ± 3 hours at either 32° or 37° ± 1°C,
depending on whether mesophilic or thermoduric organisms are being
enumerated.
Because lactic and MRS agar are not highly selective, individual
colonies should be tested for catalase reaction, gram reaction, and cell
morphology. Gram-positive, catalase-negative cocci, or rods that produce
acid are tentatively considered to be lactic acid bacteria.
10. Expression of Results: Report colony count as acid producers count per
milliliter or per gram if acid production was visualized, or as CFU/mL or
g if an acid indicator was not used.
11. Precision: Precision parameters for this test have not been defined.
12. Test Report and Interpretation: Report colony counts as in 8.071.10.
Specify the medium used, the incubation conditions, and the results of
colony testing.
Injured cells may not be recovered on media specifically designed for
growth of lactic acid bacteria. Acidified lactobacillus selection (LBS) agar
is more selective than MRS and lactic agar and should be used with
greater caution. However, MRS and lactic agar media are sufficiently
inhibitory to prevent the recovery of some heat-injured lactobacilli that
may be present in young cheese. SMA recovers injured lactobacilli, but
this medium is not selective.

8.072 Rods and Cocci in Yogurt

1. Scope: This method is used for differentiating Streptococcus thermophilus
and Lactobacillus delbrueckii in yogurt. It has not been evaluated for use on
other products.
2. References: See 8.071.2.
3. Definition: Rods and cocci are Lactobacillus and Streptococcus spp. used in
yogurt fermentation.
4. Principle: L. delbrueckii subsp. bulgaricus produces larger colonies and
more acid on this medium than does S. thermophilus. The greater acid
production results in a large white halo around colonies of L. delbrueckii
subsp. bulgaricus.
5. Diluents, Culture Media, and Reagents: See chapter 6 for diluents.
Culture media17: Prepare yogurt lactic agar as follows. Reconstitute

236 Standard Methods for the Examination of Dairy Products

 nonfat dry milk to 11% (wt/wt) solids and autoclave at 120° ± 1°C for 12
minutes. Add the warm sterile reconstituted milk, 7% (vol/vol), to tem-
pered lactic (Elliker) agar.
6. Apparatus and Glassware
1. Apparatus for producing a low oxygen and enhanced carbon dioxide
atmosphere.
2. Incubator capable of maintaining 37°C.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Pour plates and allow the surfaces to dry.
Prepare sample dilutions by aseptically blending 1 g of yogurt in 99 g
of chilled, sterile 0.1% (wt/vol) peptone water at high speed for 2 min-
utes. This procedure breaks up chains of cells for a more accurate analy-
sis.
Prepare serial dilutions in sterile peptone water and surface plate 0.1-
mL amounts onto poured, predried plates of yogurt lactic agar.
Incubate the plates in a low-oxygen, carbon dioxide-enhanced atmos-
phere at 37° ± 1°C for 48 ± 3 hours. Streptococcus thermophilus colonies are
small and white with no cloudy zone whereas L. bulgaricus colonies are
large and white with a white cloudy zone.
10. Expression of Results: Report results as colony-forming units of rods or
cocci per gram. Specify the medium used and whether results are con-
firmed by using gram stains.
11. Precision: Precision parameters for this test have not been defined.
12. Test Report and Interpretation: See 8.072.10.

8.080 Enterococci (Class B)

There are over 20 species in the genus Enterococcus. Enterococcus faecalis and
E. faecium are found in dairy products; these species are recognized as being
primarily of fecal origin and are the target microorganisms of this test. The
Enterococcus count is more reliable than the coliform count as an index of the
sanitary quality of churned butter since enterococci survive better than col-
iforms in the unfavorable microenvironment of salted butter.22 In addition,
the Enterococcus count may be a more reliable indicator of the sanitary qual-
ity of yogurt than the coliform count because coliforms are more readily
inactivated in the low pH environment than are enterococci.
While KF streptococcal agar has been accepted by most industry and reg-
ulatory agencies for the quantitative estimation of enterococci in nondairy
foods, more selective conditions are necessary in dairy products to reduce
background growth of other streptococci and lactobacilli.21 For example,
m-enterococcus agar is suggested for membrane filtration procedures, and
it has been used to select for enterococci in dried foods, including nonfat dry
milk. However, this medium may not inhibit the growth of Lactobacillus aci-
dophilus and Streptococcus bovis.
1. Scope: This method may be applied to all dairy products and environ-
mental samples.

Chapter 8 ■ Tests for Groups of Microorganisms 237

 2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Enterococci enumerated by this method are primarily of the
E. faecium and E. faecalis species.
4. Principle: This method employs citrate azide agar pour plates which are
overlayed and incubated at 37°C for 48 to 72 hours. Enterococci colonies
are stained blue by the tetrazolium blue in the medium.
5. Diluents, Culture Media, and Reagents
A. Citrate azide agar:
Base:
Yeast extract 10 g
Pancreatic digest of casein 10 g
Sodium citrate 20 g
Agar 15 g
MS water to make 1L
Heat to boiling to dissolve ingredients. Disperse in 100-mL
amounts and autoclave at 121° ± 1°C for 20 minutes.
Temper base medium 48°C and add 1 mL of 0.1% sterile (121° ± 1°
for 20 minutes) aqueous tetrazolium blue and 1 mL of 4% sterile (121°
± 1° for 20 minutes) aqueous sodium azide to each batch of 100 mL.
Final pH should be 7.0 ± 0.2 at 25°C.
B. Diluents as described in chapter 6.
6. Apparatus and Glassware: See chapter 6, Microbiological Count
Methods.
An incubator capable of maintaining 37° ± 1°C.
7. Sampling: See chapter 3, Sampling Dairy and Related Products. See also
chapter 13 for environmental samples.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Inoculate petri dishes with 0.1 or 1 mL of appropriate sample
dilutions.
Add 12 to 15 mL of sterile, citrate azide agar (tempered at 45°C) per
plate. The medium must be allowed to solidify while the plates are on a
level surface.
Add 3 to 4 mL of citrate azide agar as an overlay to cover the surface
of the solidified agar completely.
After the overlay has solidified, incubate the plates at 37° ± 1°C for 48
to 72 hours.
To facilitate the counting of colonies, a thin sheet of white tissue paper
may be placed under the petri dish on the illuminated colony counter.
This enhances the contrast between the colonies and the background.
Count only blue colonies.
10. Expression of Results: Calculate results as described in chapter 6.
11. Precision: Precision parameters for this test are undefined.

238 Standard Methods for the Examination of Dairy Products

 12. Test Report and Interpretation: Report as enterococci count per milli-
liter or per gram and specify the method used.

8.090 Aerobic Bacterial Spores (Class B)

1. Scope: This is a method for enumeration of psychrotrophic or mesophilic
aerobic bacterial spores. It can be applied to raw or heated fluid milk,
evaporated or other canned milk products, and milk and milk-derived
powders.
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Aerobic bacterial spores enumerated by this method are gen-
erally in the genus Bacillus. These spores can survive in products given
mild to severe heat treatments and can cause defects such as sweet-cur-
dling in pasteurized milk and coagulation of canned evaporated milk.
4. Principle: The sample is heated to 80° ± 1°C for 12 minutes to inactivate
vegetative cells and activate bacterial spores. After cooling, serial dilu-
tions of the sample are plated on SMA. These plates are incubated at
either 32° ± 1°C to enumerate mesophilic spores or at 7° ± 1°C to enu-
merate psychrotrophic spores.18
5. Diluents, Culture Media, and Reagents: SMA (chapter 6) with 0.1% sol-
uble starch added before sterilization.
Diluents as described in chapter 6.
6. Apparatus and Glassware
1. Erlenmeyer flasks: Sterile, 500-mL size, with rubber- or plastic-lined
screw caps.
2. Water or ethylene glycol bath: With thermostatic control for 80° and
82°C, equipped with shaker for constant agitation of flasks.
3. Thermometers: For use in water or ethylene glycol bath and pilot
flask.
4. Plating equipment and supplies [chapter 6].
5. Incubator capable of maintaining either 32° or 7°C.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Place 200 mL of each thoroughly mixed milk sample into a
sterile Erlenmeyer flask and screw on the cap. Seal the outside of the cap
with masking tape to prevent airborne contamination.
Dispense 200 mL of milk in the pilot flask and seal it with a rubber
stopper equipped with a thermometer.
Place the pilot and test flasks in a water bath at 82° ± 1°C and agitate
them throughout the heat treatment. When the contents of the control
flask reach 79°C, lower the temperature of the bath to 80°C, or move the
flasks to a second bath in which the water is set at 80° ± 1°C. After the

Chapter 8 ■ Tests for Groups of Microorganisms 239

 contents of the pilot flask reach 80°C, keep the samples in the bath for an
additional 12 minutes.
Cool the samples immediately in an ice bath.
Dispense samples in petri plates, pour on SMA with starch, and incu-
bate the plates, either at 32° ± 1°C for 48 hours for mesophilic spore
counts, or at 7° ± 1°C for 10 days for psychrotrophic spore counts.
To detect low levels of psychrotrophic contamination, the heated milk
can be incubated at 7° ± 1°C for 5 to 7 days before plating. If samples are
to be taken during preincubation, a separate flask may be used for each
sampling.
10. Expression of Results: Calculate results as described in chapter 6.
11. Precision: Precision parameters have not been determined for this test.
12. Test Report and Interpretation: Results should be reported as
mesophilic or psychrotrophic aerobic spore count per milliliter. The
preincubation time should be noted if applicable.
High numbers of spore-forming bacteria in milk may indicate unsan-
itary production practices and may cause product defects, as previously
noted. However, the initial mesophilic spore count of raw milk is not a
good indicator of the keeping quality of the heated product.

8.100 Anaerobic Sporeformers (Class B)

Anaerobic sporeforming bacteria found in dairy products are in the genus
Clostridium. No single method can provide an accurate estimate of anaero-
bic sporeformers, since some spores (C. sporogenes, C. botulinum, and related
types) require heat activation for germination whereas others (C. perfringens,
butyric anaerobes, and injured spores) are insufficiently heat resistant to
survive the activation process. When using this method, the analyst must
choose a spore activation heat treatment suitable for the population being
analyzed. The method presented here will enumerate mesophilic anaerobic
sporeformers and is not specific for C. botulinum or C. perfringens. Analysts
should be aware that cultures produced from the described method may
contain botulinum toxin, which is extremely toxic to humans. Botulinum
toxin is inactivated by autoclaving.
1. Scope: The method has been applied to milk and cheese. The method is
easily modified for determination of Clostridium tyrobutyricum, a com-
mon cause of late gas defect in cheese.3,9
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 7: Coliform and Other Indicator Bacteria.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: Anaerobic bacterial sporeformers are defined for the purpos-
es of this method as mesophilic Clostridium spp.
4. Principle: The most probable number (MPN) method gives more repro-
ducible results than colony count methods and is therefore recommend-
ed.20 Sample or sample slurry is heated to activate spores. Samples or

240 Standard Methods for the Examination of Dairy Products

 sample dilution is added to tubes of reinforced clostridial medium (RCM)
(for total anaerobic spores) or RCM modified by substituting sodium lac-
tate for glucose for estimating numbers of C. tyrobutyricum. Tubes are
sealed and incubated at 37° ± 1°C for 7 days. Positive tubes show gas pro-
duction. RCM and RCM-lactate are not highly selective media.
5. Diluents, Culture Media, and Reagents: See chapter 6 for diluents.
A. Reinforced clostridial medium.
B. Reinforced clostridial medium with lactate
Beef extract 10 g
Yeast extract 3g
Tryptone 10 g
Sodium lactate (72% solution) 28 g
Sodium acetate 5g
Cysteine hydrochloride 0.5 g
Soluble starch 1g
Agar 1g
MS water sufficient to make 1L
Adjust pH of the medium to 5.5 to 5.7 using 20% lactic acid.
Sterilize by autoclaving at 120° ± 1°C for 15 minutes.
C. Agar plug: Mix 20 g agar with 1 g sodium thioglycolate and dissolved
in sufficient MS water to make 1 L. Sterilize by autoclaving at 120° ±
1°C for 15 minutes.
6. Apparatus and Glassware
1. Narrow neck flasks or test tubes sufficient for 3-tube MPN [chapter
7]; no Durham tubes are needed.
2. Other glassware as listed in chapter 6.
3. Incubator capable of maintaining 37° ± 1°C.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Heat milk samples as described in 8.090.9 at 80°C for 10 min-
utes.
If presence of injured spores is suspected (such as in fresh cheese) the
heat treatment may be reduced to 62.5°C for 30 minutes.
If analyzing 10-g samples, use flasks or tubes containing 10 mL of
double strength medium. Otherwise add 1-g samples or sample dilutions
to 9 mL of medium.
Use RCM medium for total anaerobic sporeformers and RCM-lactate
for C. tyrobutyricum. Just before use, media should be boiled or steamed
for 10 to 20 minutes and quickly cooled to exclude oxygen.
Distribute sample and sample dilutions into flasks or tubes of media
as described for a 3-tube MPN [chapter 7]. Seal each tube or flask with an
agar plug by pouring a small volume of melted agar containing sodium
thioglycolate.
Incubate at 37° ± 1°C for 7 days. Positive tubes are those that show gas
production by raising the agar plug. Tubes may contain botulinum toxin,
so there must be no human contact with tube contents until after auto-
claving.

Chapter 8 ■ Tests for Groups of Microorganisms 241

 10. Expression of Results: Calculate results as MPN/g using the 3-tube
MPN table [chapter 7].
11. Precision: Determine confidence limits from the MPN table [chapter 7].
12. Test Report and Interpretation: Report as MPN anaerobic sporeform-
ers/gram or per milliliter. Give confidence limits associated with the
result. Specify the medium used and the spore activation treatment.

8.110 Yeast and Mold Counts

8.111 Antibiotic Plate Count Method (Class A2)
1. Scope: This method is suitable for use with all dairy products. The
method, however, may not recover osomotolerant fungi that are some-
times found in low water activity products.
2. References: Chapter 2: Laboratory Quality Assurance and Safety.
Chapter 3: Sampling Dairy and Related Products.
Chapter 4: Preparation of Media, Dilution Water, and Sterilization.
Chapter 6: Microbiological Count Methods.
Chapter 9: Microbiological Methods for Dairy Products.
3. Definition: For this method, yeasts and molds are defined as fungal cells
capable of forming colonies under the specified conditions. Slow-grow-
ing yeasts and molds may not be enumerated by this method.
4. Principle: Sample or sample dilutions are spread plated on culture
medium that employs antibiotics to inhibit growth of bacteria. Plates are
incubated at 25°C for 5 days under aerobic conditions and under suffi-
cient humidity to prevent the medium from dehydrating. The neutral pH
of the culture medium provided for greater recovery of yeasts and molds
than in the case of acidified media.14,15
5. Diluents, Culture Media, and Reagents: See chapter 6 for basic materi-
als and diluents.
A. Culture medium: See chapter 6, SMA.
B. Antibiotic solution: Suspend 500 mg each of chlortetracycline
hydrochloride and chloramphenicol (U.S. Biochemical Corp.,
Cleveland, OH; Sigma, St. Louis, MO) in 100 mL of sterile, phosphate-
buffered diluent (chapter 4). (Chloramphenicol is highly toxic; skin
contact should be avoided.) Filter-sterilization is generally not
required. The suspension can be stored for 2 months at 5°C without
loss of inhibitory activity.
6. Apparatus and Glassware: See chapter 6, Microbiological Count
Methods.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure
A. Preparation of medium: Prepare SMA according to the manufacturer’s
directions. Sterilize and cool to 45° ± 1°C. Add 2 mL of antibiotic solu-
tion per 100 mL of medium, and mix.

242 Standard Methods for the Examination of Dairy Products

 B. Plating, incubation, and counting: Prepare samples as for the SPC
[chapter 6]. Use the surface (spread) plating technique by spreading
0.1 or 0.2 mL of the sample or the sample dilution over predried plates
using a sterile, bent-glass rod. If agar surfaces are sufficiently dry, 1
mL of sample can be spread over 3 plates to increase the sensitivity of
the method. Do not invert the plates during incubation. Incubate at
25°C for 5 days.
10. Expression of Results: Calculate results as described in chapter 6, but
use plates that have between 15 and 150 colonies. If plates are outside
this range, estimated counts can be reported.
11. Precision: Precision parameters are outlined in chapter 1.
12. Test Report and Interpretation: Report as yeast and mold per gram or
per milliliter and specify the method used. If results must be obtained
from plates containing colony numbers not within the recommended
counting range, report the results as estimated yeast and mold per gram
or per milliliter.

8.112 Acidified Potato Dextrose (Glucose) Agar (Class O)

1. Scope: This method is recommended only for dairy products that contain
insignificant numbers of debilitated or injured fungal cells. These are
products in which the microbial population has not been subject to pro-
cessing stresses such as heat, acidification, or other antimicrobial treat-
ments.8
2. References: See 8.111.2.
3. Definition: See 8.111.3.
4. Principle: See 8.111.4.
The medium is acidified to pH 3.5 to inhibit the growth of bacteria.
5. Diluents, Culture Media, and Reagents: See chapter 6 for basic materi-
als and diluents.
A. Culture medium: Potato dextrose (glucose) agar (commercially avail-
able).
B. Acidifying agent: Sterile 10% tartaric acid. Sterilize the tartaric acid
solution at 120° ± 1°C for 10 minutes.
6. Apparatus and Glassware: See chapter 6, Microbiological Count
Methods.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure
A. Preparation of medium: Prepare potato dextrose (glucose) agar
according to the manufacturer’s directions. Sterilize and cool to 46°C.
Add enough sterile 10% tartaric acid to adjust the pH of the medium
to 3.5 ± 0.1.
B. Plating, incubation, and counting: Prepare and plate samples as
described in section 8.111.9. Incubate at 25° ± 1°C for 5 ± 0.5 days and
count plates [chapter 6] that have between 15 and 150 colonies.

Chapter 8 ■ Tests for Groups of Microorganisms 243

 10. Expression of Results: See 8.111.10.
11. Precision: See 8.111.11.
12. Test Report and Interpretation: See 8.111.12.

8.113 Yeast Extract-Glucose-Chloramphenicol Agar (Class B,

IDF Recommended Medium10):
1. Scope: See 8.111.1.
This medium is especially useful for samples that are expected to con-
tain predominantly yeasts or yeasts that have been injured.
2. References: See 8.111.2.
3. Definition: See 8.111.3.
4. Principle: See 8.111.4.
5. Diluents, Culture Media, and Reagents: See chapter 6 for basic materi-
als and diluents.
Culture medium:
Yeast extract-glucose-chloramphenicol agar.
Yeast extract 5g
Glucose 20 g
Chloramphenicol solution 1 mL
Agar 15 g
MS water to make 1L
Disperse ingredients in the water in the order listed, and boil or steam
for about 30 minutes to dissolve agar. Sterilize for 15 minutes at 120° ±
1°C. Adjust the pH after sterilization to 6.6 ± 0.1.
6. Apparatus and Glassware: See chapter 6.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare, inoculate, and count plates as described in 8.111.9
using yeast extract glucose chloramphenicol agar.
10. Expression of Results: See 8.111.10.
11. Precision: See 8.111.11.
12. Test Report: See 8.111.12.

8.114 Dichloran-Rose Bengal-Chloramphenicol (DRBC) Agar (Class B)

1. Scope: This method is suitable for all dairy products and environmental
samples. The method is especially useful for enumerating molds when
samples are expected to contain species of Rhizopus and Mucor, which
tend to rapidly spread over plates made from other media.12 Such plates
are often not countable because slower-growing molds are overgrown.
This medium may give lower yeast counts compared to yeast extract-
glucose-chloramphenicol medium.
2. References: See 8.111.2.
3. Definition: See 8.111.3.
4. Principle: See 8.111.4.

244 Standard Methods for the Examination of Dairy Products

 The addition or rose bengal and dichloran (2,6-dichloro-4-nitroaniline)
restricts the growth of spreading fungi without reducing fungal counts.
5. Diluents, Culture Media, and Reagents
See chapter 6 for basic materials and diluents.
Culture medium:
Dichloran-rose bengal-chloramphenicol (DRBC) agar
Glucose 10 g
Peptone, bacteriological 5g
Potassium dihydrogen phosphate 1 g
Magnesium sulfate—7H2O 0.5 g
Agar 15 g
MS water to make 1L
Rose bengal (0.5 mL of 5% wt/vol; 25 mg in
water)
Dichloran (1 mL of 2% wt/vol; 2 mg in ethanol)
Chloramphenicol 100 mg
Disperse ingredients in the water in the order listed and boil or steam
about 30 minutes to dissolve agar. Sterilize for 15 minutes at 120° ± 1°C.
Store prepared media in the dark to prevent decomposition of rose ben-
gal. Stock solutions of rose bengal and dichloran need no sterilization
and are stable for long periods.
6. Apparatus and Glassware: See chapter 6, Microbiological Count
Methods.
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: Prepare, inoculate, and count plates as described in 8.111.9.
10. Expression of Results: See 8.111.10.
11. Precision: See 8.111.11.
12. Test Report: See 8.111.12.

8.115 Dry Rehydratable Film Method1,2,13,26 (3M™ Petrifilm™ Yeast and Mold
Count) (Class A2)
1. Scope: See 8.111.1.
2. References: See 8.111.2.
3. Definition: See 8.111.3.
4. Principle: The 3M™ Petrifilm™ yeast and mold count plate is a ready-
made culture medium system that consists of 2 plastic films coated with
nutrients, antibiotics, a cold-water-soluble gelling agent, and a phos-
phatase indicator that facilitates colony enumeration. The plate is inocu-
lated with 1 mL of undiluted or diluted sample. The sample is spread
over approximately a 30 cm2 growth area. The gelling agent is allowed to
solidify and the plates are incubated and then enumerated.
5. Diluents and Reagents: See chapter 6.
6. Materials, Apparatus, and Glassware: Refer to product literature (3M
Microbiology Products, 3M Center, Building 275-5W-05, St. Paul, MN
55144-1000 USA) for use information.

Chapter 8 ■ Tests for Groups of Microorganisms 245

 A. Equipment and supplies
1. Petrifilm™ YM plates including plastic spreader (3M
Microbiology Products, 3M Center, Building 275-5W-05, St. Paul,
MN 55144-1000 USA).
2. Pipets: 1.0 mL or pipeter [chapter 6].
3. Diluent blanks: [chapter 6].
4. Incubator set at 25° ± 1°C.
5. Colony counters or tallies [chapter 6].
7. Sampling: See chapter 3, Sampling Dairy and Related Products.
8. Preparation of Test Sample: See chapter 6, Microbiological Count
Methods.
9. Procedure: See product package insert for additional details.
Place the Petrifilm™ YM plate on a level surface and label.
Lift the top film and inoculate 1 mL of sample to the center of the bot-
tom film with either a pipet or pipeter.
Drop the top film onto the innoculum.
Place the plastic spreader on the center of the plate.
Distribute the sample evenly by pushing downward on the center of
the plastic spreader. Do not slide the spreader across the film.
Remove spreader and leave the plate undisturbed for at least one
minute to permit the gel to solidify. Incubate plates in a horizontal posi-
tion at 20° to 25°C for 5 days with the clear side up in stacks of no more
than 20 plates.
10. Expression of Results: Report as yeast and mold per gram or per milliliter
and specify the method used. If results must be obtained from plates con-
taining colony numbers not within the recommended counting range,
report the results as estimated yeast and mold per gram or per milliliter.
Yeasts appear as blue-green or off-white and form small, defined
colonies. Mold colonies are often blue but may also assume their natural
pigmentation (for example black, yellow, green). Molds tend to be larg-
er and more diffuse than yeast colonies. Count and record the number
of yeasts and molds on each plate counted. Use films containing
between 15 and 150 colonies for calculating results.
The circular growth area is approximately 30 cm2. Estimates can be
made on plates containing greater than 150 colonies by counting the
number of colonies in 1 or more representative squares and determining
the average number per square. Multiply the average number per
square by 30 to determine the estimated count per plate.
Estimates may be noted after 3 days of incubation of samples that are
suspected to produce crowded plates or where sporulating molds may
interfere with accurate colony enumeration.
11. Precision: See chapter 1.
12. Test Report and Interpretation: See 8.115.10.

8.120 Cited References

1. BEUCHAT, L.R.; NAIL, B.V.; BRACKETT, R.E.; FOX, T.L. Evaluation of a culture film
(Petrifilm™ YM) method for enumerating yeasts and molds in selected dairy and
high-acid foods. J. Food Prot. 53:864-874; 1990.

246 Standard Methods for the Examination of Dairy Products

 2. BEUCHAT, L.R.; NAIL, B.V.; BRACKETT, R.E.; FOX, T.L. Comparison of the Petrifilm
yeast and mold culture film method to conventional methods for enumerating yeasts
and molds in foods. J. Food Prot. 54:443-447; 1991.
3. DASGUPTA, A.P.; HULL, R.R. Late blowing of Swiss cheese: incidence of Clostridium
tyrobutyricum in manufacturing milk. Austral. J. Dairy Technol. 44:84-87; 1989.
4. DEMAN, J.C.; ROGOSA, M.; SHARPE, M.E. A medium for the cultivation of lacto-
bacilli. J. Appl. Bacteriol. 23:130-135; 1960
5. ELLIKER, P.R.; ANDERSON, A.W.; HANNESSON, G.W. An agar culture medium for
lactic acid streptococci and lactobacilli. J. Dairy Sci. 39:1611-1612; 1956.
6. FRANK, J.F.; CHRISTEN, G.L.; BULLERMAN, L.B. Tests for groups of microorgan-
isms. In: MARSHALL, R.T., ed. Standard methods for the examination of dairy products,
16th ed. Washington, DC: APHA; 1992: 271-286.
7. GILLILAND, S.E.; SANDINE, W.E.; VEDAMUTHU, E.R. Acid-producing microor-
ganisms. In: SPECK, M.L., ed. Compendium of methods for the microbiological examination
of foods, 2nd ed. Washington, DC: APHA; 1984:184-196.
8. HENSON, O.E.; HALL, P.A.; ARENDS, R.E.; ARNOLD, E.A.,Jr.; KNECHT, R.M.; JOHN-
SON, C.A.; PUSCH, D.J.; JOHNSON, M.G. Comparison of four media for the enumer-
ation of fungi in dairy products: a collaborative study. J. Food Sci. 47:930-932; 1982.
9. HERLIN, A.H.; CHRISTIANSSON, A. Cheese-blowing anaerobic spores in bulk milk
from loose-housed and tied dairy cows. Milchwissenschaft 48:686-690; 1993.
10. INTERNATIONAL DAIRY FEDERATION. Method 94B. IDF-IDF; 1990.
11. INTERNATIONAL DAIRY FEDERATION. Standard method for the count of lipolyt-
ic organisms. FIL-IDF 41; 1966.
12. KING, A.D.; HOCKING, A.D.; PITT, J.L. Dichloran-rose bengal medium for enumer-
ation and isolation of molds from foods. Appl. Environ. Microbiol. 37:959-964; 1979.
13. KNIGHT, M.T.; NEWMAN, M.C.; BENZINGER, M.J.; NEUFANG, K.L.; AGIN, J.R.
Dry rehydratable film and conventional culture methods for enumeration of yeasts
and molds in foods. J AOAC International 80:806-823; 1997.
14. KOBURGER, J.A. Fungi in foods: 1. Effect of inhibitor and incubation temperature on
enumeration. J. Milk Food Technol. 33:433-434; 1970.
15. KOBURGER, J.A. Fungi in foods: 5. Response of natural populations to incubation
temperatures between 12° and 32°C. J. Milk Food Technol. 36:434-435; 1973.
16. MARTLEY, F.G.; JAYASHANKAR, S.R.; LAWRENCE, R.C. An improved agar medi-
um for the detection of proteolytic organisms in total bacterial counts. J. Appl.
Bacteriol. 33:363-370; 1970.
17. MATALON, M.E.; SANDINE, W.E. Improved media for differentiation of rods and
cocci in yogurt. J. Dairy Sci. 69:2569-2576; 1986.
18. MIKOLAJCIK, E.M.; SIMON, N.T. Heat resistant psychrotrophic bacteria in raw milk
and their growth at 7°C. J. Food Prot. 41:93-95; 1978.
19. ROGOSA, M.; MITCHELL, J.A.; WISEMAN, R.F. A selective medium for the isolation
and enumeration of oral and fecal lactobacilli. J. Bacteriol. 62:132-133; 1951.
20. ROSEN, B.; MERIN, U.; ROSENTHAL, I. Evaluation of clostridia in milk. Milchwis-
senschaft 44:355-357; 1989.
21. SARASWAT, D.S.; CLARK, W.S., Jr.; REINBOLD, G.W. Selection of a medium for the
isolation and enumeration of enterococci in dairy products. J. Milk Food Technol.
26:114-118; 1963.
22. SARASWAT, D.S.; REINBOLD, G.W.; CLARK, W.S., Jr. The relationship between
Enterococcus, coliform and yeast and mold counts in butter. J. Milk Food Technol.
28:245-249; 1965.
23. SHELLEY, A.W.; DEETH, H.C.; MACRAE, I. C. Comparison of a simple butterfat agar
medium with other media use for isolation and enumeration of lipolytic bacteria from
dairy products. J. Dairy Res. 54:413-420; 1987.
24. THOMAS, S.B.; DRUCE, R.G.; PETERS, G.J.; GRIFFITHS, D.G. Incidence and signifi-
cance of thermoduric bacteria in farm milk supplies: a reappraisal and review. J. Appl.
Bacteriol. 30:265-298; 1967.
25. VANDERZANT, C.; MATTHYS, A.W. Effect of temperature of the plating medium on
the viable count of psychrophilic bacteria. J. Milk Food Technol. 18:383-388; 1965.
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ating molds and yeasts in cheese and yogurt. J Food Prot. 57: 913-914; 1994.

Chapter 8 ■ Tests for Groups of Microorganisms 247

248 Standard Methods for the Examination of Dairy Products