USP 43 Microbiological Tests / (61) 6457

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(61) MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS:

MICROBIAL ENUMERATION TESTS
INTRODUCTION
The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under
aerobic conditions.
The tests are designed primarily to determine whether a substance or preparation complies with an established specification
for microbiological quality. When used for such purposes,follow the instructions given below, including the number of samples
to be taken, and interpret the results as stated below.
The methods are not applicable to products containing viable microorganisms as active ingredients.
Alternative microbiological procedures, including automated methods, may be used, providedthat their equivalence to the
PharmacopeiaI method has been demonstrated.

GENERAL PROCEDURES
Carryout the determination under conditions designed to avoid extrinsic microbial contamination of the product to be
examined. The precautionstaken to avoid contamination must be such that they do not affectany microorganisms that are to
be revealed in the test.
If the product to be examined has antimicrobial activity, this is, insofar as possible, removed or neutralized. If inactivators
are used for this purpose, their efficacy and their absence of toxicity for microorganisms must be demonstrated.
Ifsurface-active substances are used for sample preparation, their absence of toxicityfor microorganisms and their
compatibilitywith any inactivators used must be demonstrated.

ENUMERATION METHODS
Use the Membrane Filtration method or one of the Plate-Count Methods, as directed. The Most-Probable-Number (MPN)
Method is generallythe least accurate method for microbial counts; however, for certain product groups with verylow
bioburden, it may be the most appropriate method.

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 6458 (61) / Microbiological Tests USP 43

The choice of a method is based on factors such as the nature of the product and the required limitof microorganisms. The
method chosen must allow testing of a sufficient sample sizeto judge compliance with the specification. The suitability of the
chosen method must be established.

GROWTH PROMOTION TEST, SUITABILITY Of THE COUNTING METHOD AND NEGATIVE

CONTROLS

General Considerations
The ability of the test to detect microorganisms in the presence of product to be tested must be established.
Suitability must be confirmed if a change in testing performance or a change in the product that may affect the outcome of
the test, is introduced.

Preparation of Test Strains

Use standardized stable suspensions of test strains or prepare as stated below. Seed-lot culture maintenance techniques
(seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed
from the original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 7.
Table 1. Preparation and Use of Test Microorganisms
Suitability of Counting Method in the Pres-
Growth Promotion ence of Product
Preparation of Test Total Aerobic Total Yeasts and Total Aerobic Total Yeasts and
Microorganism Strain Microbial Count Molds Count Microbial Count Molds Count
Staphylococcus aureus such as Soybean-Casein Digest Agar Soybean-Casein Di- Soybean-Casein Dl-
ATCC 6538, NCIMB9518, or Soybean-Casein Digest ge~l Agar and Soy- gest Agar/MPN
ClP 4.83, or NBRC 13276 Broth bean-Casein Digest Soybean-Casein Di-
30°-35° Broth gest Broth
18-24 hours ::;100du ::;100 du
30°-35° 30°_35°
::;3days ::;3days
Pseudomonas aeruginosa such Soybean-Casein Digest Agar Soybean-Casein Di- Soybean-Casein Di-
as ATCC9027, NCiMB 8626, or Soybean-Casein Digest gest Agar and Soy- gest Agar/MPN
CIP 82.118, or NBRC 13275 Broth bean-Casein Digest Soybean-Casein Dl-
30°_35° Broth gest Broth
18-24 hours ::;100 du ::;100du
30°-35° 30°_35°
::;3days ::;3days
Bacillus subtilis such as ATCC Soybean-Casein Digest Agar Soybean-Casein Di- Soybean-Casein Di-
6633, NCiMB 8054, CIP or Soybean-Casein Digest gest Agar and Soy- gest Agar/MPN
52.62, or NBRC 3134 Broth bean-Casein Digest Soybean-Casein Di-
30°_35° Broth gest Broth
18-24 hours ::;100 du ::;100 du
30°_35° 30°-35°
::;3days ::;3days
Candida albicans such as ATCC Sabouraud Dextrose Agar or Soybean-Casein Di- Sabouraud Soybean-Casein Di- Sabouraud
10231, NCPF 3179, IP48.72, Sabouraud Dextrose Broth gest Agar Dextrose Agar gest Agar Dextrose Agar
or NBRC 1594 20°_25° ::;100 cfu ::;100 cfu ::;100du ::;100 du
2-3 days 30°_35° 20°-25° 30°-35° 20°-25°
::;5days ::;5days ::;5days ::;5days
MPN: not applicable
Aspergillus brasiliensis such as Sabouraud Dextrose Agar or Soybean-Casein Dl- Sabouraud Soybean-Casein Dl- Sabouraud
ATCC16404, IM1149007, IP Potato-Dextrose Agar gest Agar Dextrose Agar gest Agar Dextrose Agar
1431.83, or NBRC 9455 20°_25° 5-7 days, or until ::;100 du ::;100 cfu ::;100du ::;100 du
good sporulation is achieved 30°-35° 20°_25° 30°-35° 20°_25°
::;5days ::;5days ::;5days ::;5days
MPN: not applicable

Use Buffered Sodium Chloride-Peptone Solution pH 7.0 or Phosphate Buffer Solution pH 7.2 to maketest suspensions;to suspend
A. brasiliensis spores, 0.05% of polysorbate 80 may be added to the buffer. Usethe suspensions within 2 hours, or within 24
hours ifstored between 2° and 8°. As an alternativeto preparing and then diluting a fresh suspension of vegetative cells of A.
brasiliensis or B. subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension is used
for test inoculation. The stable spore suspension may be maintained at 2° to 8° for a validated period of time.

Negative Control
To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There
must be no growth of microorganisms. A negative control isalso performed when testing the products as described under
Testing of Products. Afailed negative control requires an investigation.

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 USP 43 Microbiological Tests / (61) 6459

Growth Promotion of the Media

Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from
the ingredients described.
Inoculate portions/plates of Soybean-Casein Digest Broth and Soybean-Casein Digest Agar with a small number (not more
than 100 du) of the microorganisms indicated in Table 1, using a separate portion/plate of medium for each. Inoculate plates
of Sabouraud Dextrose Agar with a small number (not more than 100 du) of the microorganisms indicated in Table 1, using a
separate plate of medium for each. Incubate according to the conditions described in Table 1.
For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized
inoculum. For a freshly prepared inoculum, growth of the microorganisms comparable to that previously obtained with a
previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible growth of the microorganisms
comparable to that previously obtained with a previously tested and approved batch of medium occurs.

Suitability of the Counting Method in the Presence of Product

PREPARATION OF THE SAMPLE

The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the
procedures described below can be demonstrated to be satisfactory, a suitable alternative procedure must be developed.
Water-Soluble Products-Dissolve or dilute (usually a 1 in 10 dilution is prepared) the product to be examined in Buffered
Sodium Chloride-Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soybean-Casein Digest Broth. If necessary,adjust
to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.
Nonfatty Products Insoluble in Water-Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered
Sodium Chloride-Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soybean-Casein Digest Broth. A surface-active agent
such as 1 g per L of polysorbate 80 may be added to assistthe suspension of poorly wettable substances. If necessary, adjust
to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.
Fatty Products-Dissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with the minimum
necessary quantity of sterile polysorbate 80 or another noninhibitory sterile surface-active reagent heated, if necessary, to not
more than 40° or, in exceptional cases, to not more than 45°. Mix carefully and if necessarymaintain the temperature in a water
bath. Add a sufficient quantity of the prewarmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully,
while maintaining the temperature for the shortest time necessaryfor the formation of an emulsion. Further serial 10-fold
dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another
non inhibitory sterile surface-active reagent.
Fluids or Solids in Aerosol Form-Aseptically transfer the product into a membrane filter apparatus or a sterile container for
further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested.
Transdermal Patches-Remove the protective cover sheets ("release liners") of the transdermal patches and place them,
adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a suitable sterile porous material (e.g.,
sterile gauze) to prevent the patches from sticking together, and transfer the patches to a suitable volume of the chosen diluent
containing inactivators such as polysorbate 80 and/or lecithin. Shake the preparation vigorously for at least 30 minutes.

INOCULATION AND DILUTION

Add to the sample prepared as directed above and to a control (with no test material included) a sufficient volume of the
microbial suspension to obtain an inoculum of not more than than 100 cfu. The volume of the suspension of the inoculum
should not exceed 1% of the volume of diluted product.
To demonstrate acceptable microbial recovery from the product, the lowest possible dilution factor of the prepared sample
must be used for the test. Where this is not possible due to antimicrobial activity or poor solubility, further appropriate protocols
must be developed. If inhibition of growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension
may be added after neutralization, dilution, or filtration.

NEUTRALIZATION/REMOVAL OF ANTIMICROBIAL ACTIVITY

The number of microorganisms recovered from the prepared sample diluted as described in Inoculation and Dilution and
incubated following the procedure described in Recovery of Microorganisms in the Presence of Product, is compared to the number
of microorganisms recovered from the control preparation.
If growth is inhibited (reduction by a factor greater than 2), then modify the procedure for the particular enumeration test
to ensure the validity of the results. Modification of the procedure may include, for example,
1. An increase in the volume of the diluent or culture medium;
2. Incorporation of a specific or general neutralizing agents into the diluent;
3. Membrane filtration; or
4. A combination of the above measures.
Neutralizing Agents-Neutralizing agents may be used to neutralize the activity of antimicrobial agents (see Table 2). They
may be added to the chosen diluent or the medium preferably before sterilization. If used, their efficacy and their absence of
toxicity for microorganisms must be demonstrated by carrying out a blank with neutralizer and without product.

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 6460 (61) / Microbiological Tests USP 43

Table 2. Common Neutralizing Agents/Methods for Interfering Substances

Potential Neutralizing
Interfering Substance Agents/Method
Glutaraldehyde, mercurials Sodiumhydrogen sulfite (Sodiumbisulfite)
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine
Quaternaryammonium compounds (QACs), piuahydroxybenzoates (para- Lecithin
bens), bis-biguanides
QACs, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, halogens,aldehydes Thiosulfate
EDTA (edetate) Mg orCa ions

Ifno suitable neutralizing method can be found, it can be assumed that the failure to isolatethe inoculated organism is
attributable to the microbicidal activity of the product. This information serves to indicate that the article is not likely to be
contaminated with the given species of the microorganism. However, it is possible that the product inhibits onlysome of the
microorganisms specified herein, butdoes not inhibitothers not included among the test strainsor those for whichthe latter
are not representative. Then, performthe test with the highestdilution factor compatiblewith microbial growth and the specific
acceptance criterion.
RECOVERY OF MICROORGANISMS IN THE PRESENCE OF PRODUCT
Foreach of the microorganisms listed, separate tests are performed. Only microorganisms of the added test strain are
counted.
Membrane Filtration-Use membrane filters having a nominal pore size not greater than 0.45 urn. The type of filter material
ischosen insuch a waythat the bacteria-retaining efficiency isnot affectedbythe components of the sampleto be investigated.
For each of the microorganisms listed, one membrane filter is used.
Transfer a suitable quantity of the sample prepared as described under Preparation of the Sample, Inoculation and Dilution
and Neutralization/Removal of Antimicrobial Activity (preferably representing 1 g of the product, or less if large numbers of du
are expected) to the membrane filter, filter immediately, and rinsethe membrane filterwith an appropriate volumeof diluent.
Forthe determination of total aerobic microbial count (TAMC), transferthe membrane filter to the surface of the Soybean-
Casein Digest Agar. Forthe determination of total combined yeasts and molds count (TYMC), transferthe membrane to the
surfaceof the Sabouraud Dextrose Agar. Incubate the plates as indicated in Table 7. Perform the counting.
Plate-Count Methods-Perform plate-count methods at least in duplicate for each medium, and use the mean count of the
result.
Pour-Plate Method-for Petri dishes 9 cm in diameter, add to the dish 1 mL of the sample prepared as described under
Preparation of the Sample, Inoculation and Dilution, and Neutralization/Removal of Antimicrobial Activity and 15 to 20 mL of
Soybean-Casein Digest Agar or Sabouraud Dextrose Agar, both media maintained at not more than 45°. If larger Petri dishes are
used, the amount of agar medium is increased accordingly. Foreach of the microorganisms listed in Table 7, at leasttwo Petri
dishes are used.
Incubate the plates as indicated in Table 7. Take the arithmetic mean of the counts per medium, and calculate the number
of du in the original inoculum.
Surface-Spread Method-for Petri dishes 9 cm in diameter, add 15 to 20 mL of Soybean-Casein Digest Agar or Sabouraud
Dextrose Agar at about 45° to each Petri dish, and allow to solidify. If larger Petri dishes are used, the volumeof the agar is
increased accordingly. Drythe plates, for example, in a laminar-airflow cabinet or in an incubator. Foreach of the
microorganisms listed in Table 7, at least two Petri dishes are used. Spread a measured volumeof not less than 0.1 mL of the
sample, prepared as directed under Preparation of the Sample, Inoculation and Dilution, and Neutralization/Removal of
Antimicrobial Activity over the surface of the medium. Incubate and count as directed for Pour-Plate Method.
Most-Probable-Number (MPN) Method-The precision and accuracyof the MPN Method is less than that of the Membrane
Filtration method or the Plate-Count Method. Unreliable results are obtained particularly for the enumeration of molds. Forthese
reasons, the MPN Method is reserved for the enumeration of TAMC in situationswhere no other method is available. Ifthe use
of the method is justified, proceed as follows.
Preparea seriesof at leastthree serial 10-fold dilutions of the product as describedfor Preparation of the Sample, Inoculation
and Dilution, and Neutralization/Removal of Antimicrobial Activity. From each level of dilution, three aliquotsof 1 g or 1 mL are
used to inoculate three tubes with 9 to 10 mL of Soybean-Casein Digest Broth. If necessary a surface-active agent such as
polysorbate 80, or an inactivator of antimicrobial agents may be added to the medium. Thus, ifthree levels of dilution are
prepared, nine tubes are inoculated.
Incubatealltubes at 30° to 35°for not more than 3 days. Ifreading of the results isdifficult or uncertainowingto the nature
of the product to be examined, subculture in the same broth or in Soybean-Casein Digest Agarfor 1 to 2 days at the same
temperature, and use these results. From Table 3, determine the most probable number of microorganisms per g or mL of the
product to be examined.

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USP 43 Microbiological Tests / (61) 6461

Table 3. Most-Probable-Number Values of Microorganisms

MPN per g or
Observed Combinations of Numbers of Tubes per mL of
Showing Growth in Each Set Product 95% Confidence Limits
Number of g or mL of Product per Tube
0.1 0.01 0.001
0 0 0 <3 0-9.4
0 0 1 3 0.1-9.5
0 1 0 3 0.1-10
0 1 1 6.1 1.2-17
0 2 0 6.2 1.2-17
0 3 0 9.4 3.5-35
1 0 0 3.6 0.2-17
1 0 1 7.2 1.2-17
1 0 2 11 4-35
1 1 0 7.4 1.3-20
1 1 1 11 4-35
1 2 0 11 4-35
1 2 1 15 5-38
1 3 0 16 5-38
2 0 0 9.2 1.5-35
2 0 1 14 4-35
2 0 2 20 5-38
2 1 0 15 4-38
2 1 1 20 5-38
2 1 2 27 9-94
2 2 0 21 5-40
2 2 1 28 9-94
2 2 2 35 9-94
2 3 0 29 9-94
2 3 1 36 9-94
3 0 0 23 5-94
3 0 1 38 9-104
3 0 2 64 16-181
3 1 0 43 9-181
3 1 1 75 17-199
3 1 2 120 30-360
3 1 3 160 30-380
3 2 0 93 18-360
3 2 1 150 30-380
3 2 2 210 30-400
3 2 3 290 90-990
3 3 0 240 40-990
3 3 1 460 90-1980
3 3 2 1100 200-4000
3 3 3 >1100

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 6462 (61) / Microbiological Tests USP 43

RESULTS AND INTERPRETATION

When verifying the suitability of the Membrane Filtration method or the Plate-Count Method, a mean count of any of the test
organisms not differing by a factor greater than 2 from the value of the control defined in Inoculation and Dilution in the absence
of product must be obtained. When verifying the sultablllty of the MPN Method, the calculated value from the inoculum must
be within 95% confidence limits of the results obtained with the control.
If the above criteria cannot be met for one of more of the organisms tested with any of the described methods, the method
and test conditions that come closest to the criteria are used to test the product.

TESTING OF PRODUCTS

Amount Used for the Test

Unless otherwise directed, use 109 or 10 mL of the product to be examined taken with the precautions referred to above.
For fluids or solids in aerosol form, sample 10 containers. For transdermal patches, sample 10 patches.
The amount to be tested may be reduced for active substances that will be formulated in the following conditions: the
amount per dosage unit (e.g., tablet, capsule, injection) is less than orequal to 1 mg, orthe amount perg or mL (for preparations
not presented in dose units) is less than 1 mg. In these cases, the amount of sample to be tested is not less than the amount
present in 10 dosage units or 109 or 10 mL of the product.
For materials used as active substances where the sample quantity is limited or batch size is extremely small (l.e., less than
1000 mL or 1000 g), the amount tested shall be 1% of the batch unless a lesseramount is prescribed or justified and authorized.
For products where the total number of entities in a batch is less than 200 (e.g., samples used in clinical trials), the sample
size may be reduced to two units, or one unit if the size is less than 100. .
Select the sample(s) at random from the bulk material or from the available containers of the preparation. To obtainthe
required quantity, mix the contents of a sufficient number of containers to provide the sample.

Examination of the Product

MEMBRANE FILTRATION

Use a filtration apparatus designed to allow the transfer of the filter to the medium. Prepare the sample using a method that
has been shown to be suitable as described in Growth Promotion Test and Suitability of the Counting Method, transfer the
appropriate amount to each of two membrane filters, and filter immediately. Wash each filter following the procedure shown
to be suitable.
For the determination of TAMC, transfer one of the membrane filters to the surface of Soybean-Casein Digest Agar. For the
determination of TYMC, transfer the other membrane to the surface of Sabouraud Dextrose Agar. Incubate the plate of Soybean-
Casein Digest Agar at 30° to 35° for 3 to 5 days and the plate of Sabouraud Dextrose Agar at 20° to 25° for 5 to 7 days. Calculate
the number of cfu per g or per mL of product.
When examining transdermal patches, separately filter 10% of the volume of the preparation described for Preparation of
the Sample through each of two sterile filter membranes. Transfer one membrane to Soybean-Casein Digest Agarfor TAMC and
the other membrane to Sabouraud Dextrose Agarfor TYMe.

PLATE-COUNT METHODS

Pour-Plate Method-Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion
Test and Suitability of the Counting Method. Prepare for each medium at least two Petri dishes for each level of dilution. Incubate
the plates of Soybean-Casein Digest Agar at 30° to 35° for 3 to 5 days and the plates of Sabouraud Dextrose Agar at 20° to 25°
for 5 to 7 days. Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for
TAMC and 50 for TYMe. Take the arithmetic mean per culture medium of the counts, and calculate the number of cfu per g
or per mL of product.
Surface-Spread Method-Prepare the sample using a method that has been shown to be suitable as described in Growth
Promotion Test and Suitabilityof the Counting Method. Prepareat least two Petri dishesfor each medium and each level of dilution.
For incubation and calculation of the number of cfu, proceed as directed for the Pour-Plate Method.

MOST-PROBABLE-NUMBER METHOD

Prepare and dilute the sample using a method that has been shown to be suitable as decribed in Growth Promotion Test and
Suitability of the CountingMethod. Incubate all tubes for 3 to 5 days at 30° to 35°. Subculture if necessary, using the procedure
shown to be suitable. Record for each level of dilution the number of tubes showing microbial growth. Determine the most
probable number of microorganisms per g or mL of the product to be examined from Table 3.

Interpretation of the Results

The total aerobic microbial count (TAMC) is considered to be equal to the number of cfu found using Soybean-Casein
DigestAgar; if colonies of fungi are detected on this medium, they are counted as part of TAMe. The total combined yeasts
and molds count (TYMC) is considered to be equal to the number of cfu found using Sabouraud Dextrose Agar; if colonies of
bacteria are detected on this medium, they are counted as part of TYMe. When the TYMC is expected to exceed the acceptance

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 USP43 Microbiological Tests / (62) 6463

criterion due to the bacterial growth, Sabouraud Dextrose Agar containing antibiotics may be used. If the count is carried out
by the MPN Method, the calculated value isTAMe.
When an acceptance criterion for microbiological quality is prescribed, it is interpreted as follows:
• 10' cfu: maximum acceptable count = 20;
• 102 cfu: maximum acceptable count =200;
• 103 cfu: maximum acceptable count = 2000;
and so forth.
The recommended solutions and media are described in Tests for Specified Microorganisms (62).

(62) MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS:

TESTS FOR SPECIFIED MICROORGANISMS
INTRODUCTION
The tests described hereafter will allowdetermination of the absence of, or limited occurrence of, specified microorganisms
that may be detected under the conditions described.
The tests are designed primarily to determine whether a substance or preparation complies with an established specification
for microbiological quality. When used for such purposes,follow the instructions given below, including the number of samples
to be taken, and interpret the results as stated below.
Alternative microbiological procedures, including automated methods, may be used, providedthat their equivalence to the
Pharmacopeial method has been demonstrated.

GENERAL PROCEDURES
The preparation of samples is carried out as described in Microbiological Examination of Nonsterile Products: Microbial
Enumeration Tests (61).
Ifthe product to be examined has antimicrobial activity, this is insofar as possible removed or neutralized as described in
Microbiological Examination of Nonsterile Products: MicrobialEnumeration Tests (61).
Ifsurface-active substances are used for sample preparation,their absence of toxicityfor microorganisms and their
compatibility with any inactivators used must be demonstrated as described in Microbiological Examination ofNonsterile Products:
Microbial Enumeration Tests (61).

GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE

TEST AND NEGATIVE CONTROLS
The ability of the test to detect microorganisms in the presence of the product to be tested must be established. Suitability
must be confirmed ifa change in testing performanceor a change in the product that may affectthe outcome of the test is
introduced.

Preparation of Test Strains

Use standardized stable suspensionsof test strainsas stated below. Seed-lotculture maintenance techniques (seed-lot
systems)are used so that the viable microorganisms used for inoculation are not more than five passages removed from the
original master seed-lot.
AEROBIC MICROORGANISMS
Groweach of the bacterial test strainsseparatelyin containers containing Soybean-Casein Digest Broth or on Soybean-Casein
DigestAgar at 30° to 35° for 18 to 24 hours. Grow the test strainfor Candida albicans separately on Sabouraud Dextrose Agar
or in Sabouraud Dextrose Broth at 20° to 25° for 2 to 3 days.

Staphylococcus aureus such asATCC6538, NCIMB 9518, ClP4.83, or NBRC 13276

Pseudomonas aeruginosa such asATCC9027, NCIMB 8626, CIP82.118, or NBRC 13275

Escherichia coli such asATCC 8739, NCIMB 8545, CIP53.126, or NBRC 3972

Salmonella enterica subsp. enterica serovarTyphimurium or, asan alternative, such asATCC 14028

Salmonella enterica subsp. enter/co serovarAbony such asNBRC 100797, NCTC 6017, or CIP80.39

Candida albicans such asATCC10231, NCPF 3179, IP48.72, or NBRC 1594

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