Document QAS/11.

409 FINAL
March 2012

3.3.1 MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS:

MICROBIAL ENUMERATION TESTS

Final text for addition to The International Pharmacopoeia

This monograph was adopted at the Forty-sixth WHO Expert Committee on

Specifications for Pharmaceutical Preparations in October 2011 for addition to the 4th
Edition of the International Pharmacopoeia

The text, reproduced with the permission of the European Pharmacopoeia with
appropriate editorial modifications, is one that has undergone pharmacopoeial
harmonization by the Pharmacopoeial Discussion Group (PDG) of the European
Pharmacopoeia (Ph.Eur), Japanese Pharmacopoeia (JP) and United States
Pharmacopeia (USP).

3.3.1 MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS:

MICROBIAL ENUMERATION TESTS

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.
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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, sterile, pH 7.0, TS or phosphate buffer,

sterile, pH 7.2, TS 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.
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Table 1. Preparation and use of test microorganisms

Micro Preparation Growth promotion Suitability of counting

organism of test strain method in the presence of
the product
Total Total yeasts Total Total yeasts
aerobic and moulds aerobic and moulds
microbial count microbial count
count count
Staphylococcus Casein soya Casein soya Casein soya
aureus bean digest bean digest bean digest
agar or agar and agar/MPN
such as ATCC casein soya casein soya casein soya
6538, NCIMB bean digest bean digest bean digest
9518, CIP 4.83 broth broth broth
or NBRC 30–35 °C ≤ 100 CFU/ ≤ 100 CFU/
13276 18–24 h 30–35 °C 30–35 °C
≤ 3 days ≤ 3 days
Pseudomonas Casein soya Casein soya Casein soya
aeruginosa bean digest bean digest bean digest
agar or agar and agar/MPN
such as ATCC casein soya casein soya casein soya
9027, NCIMB bean digest bean digest bean digest
8626, CIP broth broth broth
82.118 or 30–35 °C ≤ 100 CFU/ ≤ 100 CFU/
NBRC 13275 18–24 h 30–35 °C 30–35 °C
≤ 3 days ≤ 3 days
Bacillus subtilis Casein soya Casein soya Casein soya
bean digest bean digest bean digest
such as ATCC agar or agar and agar/MPN
6633, NCIMB casein soya casein soya casein soya
8054, CIP bean digest bean digest bean digest
52.62 or NBRC broth broth broth
3134 30–35 °C ≤ 100 CFU ≤ 100 CFU
18–24 h 30–35 °C 30–35 °C
≤ 3 days ≤ 3 days
Candida Sabouraud- Casein soya Sabouraud- Casein soya Sabouraud-
albicans dextrose agar bean digest dextrose agar bean digest dextrose agar
or agar ≤ 100 CFU agar ≤ 100 CFU/
such as ATCC Sabouraud- ≤ 100 CFU 20–25 °C ≤ 100 CFU 20–25 °C
10231, NCPF dextrose 30–35 °C ≤ 5 days 30–35 °C ≤ 5 days,
3179, IP 48.72 broth ≤ 5 days ≤ 5 days
20–25 °C
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or NBRC 1594 2–3 days MPN: not

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

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 5 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 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, sterile, pH 7.0, TS,
phosphate buffer sterile, pH 7.2, TS 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

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10 dilution is prepared) in buffered sodium chloride-peptone solution, sterile, pH 7.0 TS,

phosphate buffer , sterile, pH 7.2, TS 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,1 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 ten-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

1
New reagent.
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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

Glutaraldehyde, mercurials Sodium hydrogensulfite (sodium bisulfite)
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine

Quaternary Ammonium Compounds (QACs),

Lecithin
parahydroxybenzoates (parabens),
bis-biguanides

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
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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
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 ten-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
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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
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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. 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.
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Table 3. Most-probable-number values of microorganisms

Observed combinations of numbers of tubes MPN per g or 95%

showing growth in each set per ml of confidence
product limits
Number of g or ml of product per tube
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
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
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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.

***

New reagent to be added to Ph.Int.

Isopropyl myristate R. Propan-2-yl tetradecanoate. C17H34O2.

Description: A clear, colourless, oily liquid.
Miscibility: Immiscible with water, miscible with ethanol, with fatty oils, with liquid
paraffin. Relative density: About 0.853

***