The International Pharmacopoeia - Eighth Edition, 2018 3.3.

1 Microbial enumeration tests

3.3.1 Microbial enumeration tests

This text is based on the internationally-harmonized texts developed by the Pharmacopoeial Discussion Group (PDG). Some
editorial modifications have been made in order to be in line with the style used in The International Pharmacopoeia.

The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi which 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, provided that their equivalence to the
pharmacopoeial method has been demonstrated.

The recommended test solutions and media are described in 3.3.2 Tests for specified microorganisms.

GENERAL PROCEDURES

Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined.
The precautions taken to avoid contamination must be such that they do not affect any microorganisms which 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.

If surface-active substances are used for sample preparation, their absence of toxicity for microorganisms and their compatibility
with inactivators used must be demonstrated.

ENUMERATION METHODS

Use the membrane filtration method or the plate-count methods, as prescribed. The most probable number (MPN) method is
generally the least accurate method for microbial counts; however, for certain product groups with very low bioburden, it may be
the most appropriate method.

The choice of a method is based on factors such as the nature of the product and the required limit of microorganisms. The
method chosen must allow testing of a sufficient sample size to 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 the product to be tested must be established.

Suitability must be confirmed if a change in testing performance, or the product, which 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 5 passages removed from the
original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 1.

Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer pH 7.2 to make test suspensions; to suspend A.
brasiliensis spores, 0.05% of polysorbate 80 may be added to the buffer. Use the suspensions within 2 h or within 24 h if stored
at 2–8 °C. As an alternative to 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 2–8 °C for a validated period of time.

Table 1. Preparation and use of test microorganisms

Suitability of counting method in the presence of

Growth promotion the product
Microorganism Preparation of test Total
strain aerobic Total yeasts and Total aerobic microbial Total yeasts and
microbial moulds count count moulds count
count

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

Staphylococcus Casein soya

aureus bean digest Casein soya bean digest
Casein soya bean agar and agar/MPN casein soya
  digest agar or casein soya
bean digest bean digest broth
casein soya bean
such as ATCC digest broth broth ≤ 100 CFU/
6538, NCIMB 30–35 °C ≤ 100 CFU/
30–35 °C
9518, CIP 4.83
or NBRC 18–24 h 30–35 °C
≤ 3 days
13276 ≤ 3 days

Pseudomonas Casein soya

aeruginosa bean digest Casein soya bean digest
Casein soya bean agar and agar/MPN casein soya
  digest agar or casein soya
bean digest bean digest broth
casein soya bean
such as ATCC digest broth broth ≤ 100 CFU/
9027, NCIMB 30–35 °C ≤ 100 CFU/
30–35 °C
8626, CIP
82.118 or 18–24 h 30–35 °C
≤ 3 days
NBRC 13275 ≤ 3 days

Casein soya
Bacillus subtilis bean digest
agar and Casein soya bean digest
  Casein soya bean agar/MPN casein soya
digest agar or casein soya bean digest broth
such as ATCC casein soya bean bean digest
digest broth broth ≤ 100 CFU
6633, NCIMB
30–35 °C ≤ 100 CFU
8054, CIP 30–35 °C
52.62 or NBRC 18–24 h 30–35 °C
≤ 3 days
3134
≤ 3 days

Casein soya bean digest

Casein soya Sabouraud-dextrose agar Sabouraud-dextrose
Candida Sabouraud-dextrose bean digest
albicans agar agar ≤ 100 CFU agar
agar or Sabouraud-
such as ATCC dextrose broth ≤ 100 CFU ≤ 100 CFU/
≤ 100 CFU
10231, NCPF 30–35 °C
20–25 °C
20–25 °C 20–25 °C
3179, IP 48.72 30–35 °C
≤ 5 days
or NBRC 1594 2–3 days
≤ 5 days ≤ 5 days
≤ 5 days
MPN: not applicable

Aspergillus Casein soya bean digest

Sabouraud-dextrose Casein soya
brasiliensis Sabouraud-dextrose agar Sabouraud-dextrose
agar or potato- bean digest
  agar agar
dextrose agar agar ≤ 100 CFU
20–25 °C ≤ 100 CFU ≤ 100 CFU
such as ATCC ≤ 100 CFU 30–35 °C
16404, IMI 20–25 °C 20–25 °C
5–7 days, or until 30–35 °C
149007, IP ≤ 5 days
good sporulation is ≤ 5 days ≤ 5 days
1431.83 or ≤ 5 days
achieved MPN: not applicable
NBRC 9455

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 is also performed when testing the products as described under Testing of
products. A failed negative control requires an investigation.

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 casein soya bean digest broth and casein soya bean digest agar with a small number (not more than
100 CFU) 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 CFU) of the microorganisms indicated in Table 1, using a

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

separate plate of medium for each. Incubate in 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, an 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 sterile pH 7.2 or casein soya bean digest broth. If necessary adjust to pH 6–8.
Further dilutions where necessary are prepared with the same diluent.

Non-fatty 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 pH 7.2 or casein soya bean digest broth. A surface-active agent such
as 1 g/l of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary adjust to pH 6–8.
Further dilutions where necessary are prepared with the same diluent.

Fatty products. Dissolve in isopropyl myristate R, sterilized by filtration, or mix the product to be examined with the minimum
necessary quantity of sterile polysorbate 80 or another non-inhibitory sterile surface-active reagent, heated if necessary to not
more than 40 °C, or in exceptional cases to not more than 45 °C. Mix carefully and if necessary maintain the temperature in a
water-bath. Add sufficient of the prewarmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully whilst
maintaining the temperature for the shortest time necessary for 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 liner") of the transdermal patches and place them, adhesive
side upwards, on sterile glass or plastic trays. Cover the adhesive surface with sterile porous material, for example, 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 min.

Inoculation and dilution

Add to the sample prepared as described above under Preparation of the sample and to a control (with no test material included)
a sufficient volume of the microbial suspension to obtain an inoculum of not more 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 above under Inoculation and dilution
and incubated following the procedure described below under Recovery of microorganism, 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 (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.

Table 2. Common neutralizing agents for interfering substances

Interfering substance Potential neutralizing method

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

Sodium hydrogensulfite (sodium

Glutaraldehyde, mercurials
bisulfite)
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine
Quaternary Ammonium Compounds (QACs), parahydroxybenzoates (parabens), bis-
Lecithin
biguanide
QACs, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, halogens, aldehydes Thiosulfate
EDTA (edetate) Mg2+ or Ca2+ ions

If no suitable neutralizing method can be found, it can be assumed that the failure to isolate the inoculated organism is
attributable to the microbial 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 only inhibits some of the
microorganisms specified herein, but does not inhibit others not included amongst the test strains or for which the latter are not
representative.

Then, perform the test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion.

Recovery of microorganism in the presence of product

For each 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 µm. The type of filter material is chosen in such a way that
the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For each of the
microorganisms listed one membrane filter is used.

Transfer a suitable amount of the sample prepared as described above under Suitability of the counting method in the presence
of product (preferably representing 1 g of the product or less if large numbers of CFU are expected) to the membrane filter, filter
immediately and rinse the membrane filter with an appropriate volume of diluent.

For the determination of total aerobic microbial count (TAMC) transfer the membrane filter to the surface of casein soya bean
digest agar. For the determination of total combined yeasts/moulds count (TYMC) transfer the membrane to the surface of
Sabouraud-dextrose agar. Incubate the plates as indicated in Table 1. 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 Suitability of the counting
method in the presence of product and 15–20 mL of casein soya bean digest agar or Sabouraud-dextrose agar, both media being
at not more than 45 °C. If larger Petri dishes are used the amount of agar medium is increased accordingly. For each of the
microorganisms listed in Table 1 at least 2 Petri dishes are used.

Incubate the plates as indicated in Table 1. Take the arithmetic mean of the counts per medium and calculate the number of CFU
in the original inoculum.

Surface-spread method

For Petri dishes 9 cm in diameter add 15–20 mL of casein soya bean digest agar or Sabouraud-dextrose agar at about 45 °C to
each Petri dish and allow to solidify. If larger Petri dishes are used the volume of the agar is increased accordingly. Dry the
plates, for example, in a laminar airflow cabinet or in an incubator. For each of the microorganisms listed in Table 1 at least 2
Petri dishes are used. Spread a measured volume of not less than 0.1 mL of the sample prepared as described under Suitability
of the counting method in the presence of product over the surface of the medium. Incubate and count as prescribed under Pour-
plate method.

Most-probable-number (MPN) method

The precision and accuracy of 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 moulds. For these reasons the MPN method is reserved for the

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

enumeration of TAMC in situations where no other method is available. If the use of the method is justified proceed as follows.

Prepare a series of at least three serial 10-fold dilutions of the product as described under Suitability of the counting method in
the presence of product. From each level of dilution 3 aliquots of 1 g or 1 mL are used to inoculate 3 tubes with 9–10 mL of
casein soya bean 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, if three levels of dilution are prepared nine tubes are inoculated.

Incubate all tubes at 30–35 °C for not more than 3 days If reading of the results is difficult or uncertain owing to the nature of the
product to be examined, subculture in the same broth, or casein soya bean digest agar, for 1–2 days at the same temperature
and use these results. Determine the most probable number of microorganisms per gram or millilitre of the product to be
examined from Table 3.

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 above under Inoculation and dilution in the
absence of the product must be obtained. When verifying the suitability 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 or 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 prescribed use 10 g 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 or equal to 1 mg or the amount per gram or millilitre (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 10 g or 10 mL of the product.

For materials used as active substances where sample quantity is limited or batch size is extremely small (i.e. less than 1000 mL
or 1000 g) the amount tested shall be 1% of the batch unless a lesser amount 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 2 units, or 1 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 obtain the 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 suitable as described in section 4 and transfer the appropriate amount to each of 2 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 casein soya bean digest agar. For the
determination of TYMC transfer the other membrane to the surface of Sabouraud-dextrose agar. Incubate the plate of casein
soya bean digest agar at 30–35 °C for 3–5 days and the plate of Sabouraud-dextrose agar at 20–25 °C for 5–7 days. Calculate
the number of CFU per gram or per millilitre of product.

When examining transdermal patches filter 10% of the volume of the preparation described under Preparation of the sample
separately through each of 2 sterile filter membranes. Transfer one membrane to casein soya bean digest agar for TAMC and the
other membrane to Sabouraud-dextrose agar for TYMC.

Plate-count methods

Pour-plate method. Prepare the sample using a method that has been shown to be suitable as described in section 4. Prepare for
each medium at least 2 Petri dishes for each level of dilution. Incubate the plates of casein soya bean digest agar at 30–35 °C for
3–5 days and the plates of Sabouraud-dextrose agar at 20–25 °C for 5–7 days. Select the plates corresponding to a given dilution
and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC. Take the arithmetic mean per culture
medium of the counts and calculate the number of CFU per gram or per millilitre of product.

Surface-spread method. Prepare the sample using a method that has been shown to be suitable as described in section 4.

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

Prepare at least 2 Petri dishes for each medium and each level of dilution. For incubation and calculation of the number of CFU
proceed as described 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 described
in section 4. Incubate all tubes for 3–5 days at 30–35 °C. 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 gram or millilitre of the product to be examined from Table 3.

Table 3. Most-probable-number values of microorganisms

Observed combinations of numbers of tubes showing growth in each set

Number of g or mL of product per tube MPN per g or per mL of product 95% confidence limits

0.1 0.01 0.001

0 0 0 Less than 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

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The International Pharmacopoeia - Eighth Edition, 2018 3.3.1 Microbial enumeration tests

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 More than 1100

Interpretation of the results

The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using casein soya bean digest
agar; if colonies of fungi are detected on this medium they are counted as part of TAMC. The total combined yeasts/mould 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 TYMC. When the TYMC is expected to exceed the acceptance 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 is the TAMC.

When an acceptance criterion for microbiological quality is prescribed it is interpreted as follows:

-101 microorganisms: maximum acceptable count = 20;

-102 microorganisms: maximum acceptable count = 200;
-103 microorganisms : maximum acceptable count = 2000, and so forth.

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