European Pharmacopoeia 6.0 2.6.

12 Microbiological examination of non-sterile products

01/2008:20612

2.6.12 MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS

TOTAL COUNT OF VIABLE AEROBES

In this general chapter, two sets of tests are presented. The first set presents the
reference methods to determine compliance with the monographs. Therefore, the
reference to this chapter in a monograph implies compliance with the first set of
tests, unless the use of the second test set has been authorized. The tests in
the second set also constitutes official methods of the European Pharmacopoeia, and can
to be referred to as such, especially in the authorization requests for sale. It is intended
replace the first set with the second set once the relevant monographs
have been reviewed. The second set presents the tests developed in cooperation with
the Japanese Pharmacopoeia and the United States Pharmacopoeia to meet the requirements
harmonized.

A. EUROPEAN PHARMACOPOEIA METHOD

The tests described below will allow the quantitative enumeration of bacteria.
mesophilic and fungi that can grow under aerobic conditions.

The tests are designed primarily to determine if a substance mentioned in the

monograph in the Pharmacopoeia complies or does not comply with the specified microbiological requirements in the
monograph in question. When used for such purposes, follow the instructions that are
they indicate below, including the number of samples that must be taken, and interpret the
results as established below. The tests can also be used for testing
Effectiveness of antimicrobial preservatives (5.1.3) as described in the Pharmacopoeia. These
tests can also be used to monitor the quality of raw materials and can be used in
association with the guidelines on microbiological quality of pharmaceutical preparations
(5.1.4). When used for such purposes, for example by a raw material manufacturer
or finished product monitoring or for process validation, the execution of the tests,
including the number of samples to be taken and the interpretation of the results, are
matters of agreements between the manufacturer and the competent authority.

Carry out the determination under the designed conditions to avoid accidental contamination of the
product that will be examined. The precautions taken to avoid contamination must be
such that it does not affect any microorganism that is revealed in the test. If the product that is going to
If the examined substance has antimicrobial activity, it must be adequately neutralized. If used
inactivators for this purpose, their effectiveness and non-toxicity against must be demonstrated
microorganisms.

Determine the total count of viable aerobes using the membrane filtration method or
by the plate counting method, as indicated in the monograph.

The Most Probable Number (MPN) method is reserved for bacterial counts when not
It has another method. The selection of a method can be based on factors such as the nature
of the product and the expected number of microorganisms. Any of the methods that are
selection must be properly validated.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Cuando se usa junto con el capítulo5.1.3o5.1.4, puede usarse el método de vertido en placa, el
surface dispersion method and membrane filtration method.

SAMPLE PREPARATION

Sampling plan. The sampling of the product must follow a well-defined sampling plan. The plan
Sampling will depend on factors such as the batch size, health risks associated with
unacceptable and highly contaminated products, the characteristics of the product and the level of
expected contamination. Unless otherwise indicated, use samples of 10 g or 10 mL
from the substance or preparation that is to be examined, taken with the mentioned precautions
previously. Select the sample(s) randomly from the bulk material or from the containers
available for the preparation. If necessary, to obtain the required amount, mix the
content of a sufficient number of containers to obtain each sample, depending on the
nature of the substance or preparation that is to be examined.

An example of a sampling plan applicable to products where the homogeneity concerning the
The distribution of microorganisms can be a problem, it is the class three sampling plan. In
In this case, five samples from each batch are taken and investigated separately. The three classes
they are recognized as:

acceptable samples, that is, samples that contain less demUFC (colony-forming units of
colonies) per gram or milliliter, where the limit specified in the relevant monograph is;
(ii) marginal samples, that is, with more than 10 mUFC per gram or less.
milliliter
(iii) defective samples, that is, those containing more than 10 mUFC per gram or milliliter.

Water-soluble products. Dissolve or dilute 10 g or 10 mL of the product that is to be examined.

in buffered solution of sodium chloride-pepton pH 7.0 or in another suitable liquid. In
In general, a one in ten dilution is prepared. However, the characteristics of the product, or the
required sensitivity may need the use of other proportions. If it is known that the product
It has antimicrobial activity, an inactivator agent can be added to the diluent. If necessary,
adjust the pH to approximately 7 and prepare decimal dilutions in series using the same
diluent.

Non-fat products insoluble in water. Suspend 10 g or 10 mL of the product that is going to be

examined in buffered solution of sodium chloride-peptone pH 7.0 or in another suitable liquid.
In general, a one in ten dilution is prepared. However, the characteristics of some
products may require the use of larger volumes. A surfactant can be added
suitable, such as 1 g/L of polysorbate 80, to help suspend substances that do not
I am good. If it is known that the product has antimicrobial activity, an agent can be added.
inactivator to the diluent. If necessary, adjust the pH to about 7 and prepare dilutions
decimals in series using the same diluent.

Fat products. Homogenize 10 g or 10 mL of the product to be examined with no more

half of its weight of sterile polysorbate 80 or another suitable surfactant, heating, if it is
necessary, at no more than 40°C, in exceptional cases at no more than 45°C. Mix carefully and,
if necessary, maintain the temperature in a water bath or in an incubator. Add enough
buffered, preheated sodium chloride-peptone solution pH 7.0 for preparing a dilution
one in ten of the original product. Mix carefully while maintaining the temperature
during the shortest time necessary for the formation of an emulsion and in no case during
no more than 30 min. Decimal dilutions can be prepared in series using buffered solution

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

sodium chloride-peptone pH 7.0 containing an adequate concentration of polysorbate 80

sterile or another suitable surfactant agent.

Transdermal patches. Remove the protective covers ('peel off the backing') from ten patches of the
transdermal preparation, using sterile tweezers and placing them with the adhesive side facing up,
about sterile glass or plastic trays. If necessary, cover the adhesive surface with gauze
sterile (or monofilament polymer mesh type filter fabric), and transfer the ten patches to a
minimum volume of 500 mL of sodium chloride-peptone buffered solution pH 7.0 that
contain suitable inactivators, such as polysorbate 80 and/or lecithin. Shake vigorously the
Preparation for at least 30 minutes (Preparation A). Prepare another ten patches of the same.
put them in a minimum volume of 500 mL of medium D broth and shake vigorously
for at least 30 minutes (preparation B).

SAMPLE EXAMINATION

Membrane filtration. Use membrane filters that have a nominal pore size not
greater than 0.45 µm and whose effectiveness for retaining bacteria has been established. The type of
the filter material is selected in such a way that the bacteria retention efficiency is not affected
for the components of the sample to be investigated. The cellulose nitrate filters, for
for example, they can be used for aqueous, oily, and weakly alcoholic solutions and the filters of
cellulose acetate, for example, for strongly alcoholic solutions. The filtration device
It is designed to allow the transfer of the filter to the culture medium.

Transfer an appropriate amount of the prepared sample as described in the section

“Preparación de la muestra” (de preferencia representando 1 g del producto o menos, si se esperan
large numbers of colony-forming units) to each of two membrane filters, and
filter immediately. Wash each filter with three amounts, each of around 100 mL of
an appropriate liquid, such as a buffered solution of sodium chloride-peptone pH 7.0. To this
solution, surfactants such as polysorbate 80 or inactivators of agents can be added
antimicrobials. If validated, fewer than three washes may be applied. Transfer one of the
membrane filters, mainly intended for the enumeration of bacteria, on the surface
from an appropriate agar medium, such as medium B and the other, mainly intended for the
enumeration of fungi, on the surface of an appropriate agar medium, such as medium C. Incubate the
medium B agar plate at 30°C to 35°C and medium C agar plate at 20°C to 25°C for
five days, unless a reliable count is obtained in a shorter time. Select the
plates with the highest number less than 100 colonies, and calculate the number of units
colonial formers per gram or milliliter of the product.

When examining transdermal patches, filter 50 mL of preparation A separately, through

of each of two sterile membrane filters. Place a membrane in the agar B medium.
for the total count of aerobes and the other membrane in agar medium C for the count of
mushrooms.

Plate counting methods

a. Pouring method in plate. Using Petri dishes of 9 cm in diameter, add to each dish
1 mL of the prepared sample as described in the 'Sample Preparation' section, and 15 mL
20 mL of a liquefied agar medium suitable for bacterial culture (such as medium B), or 15
20 mL of a suitable liquefied agar medium for the cultivation of fungi (such as medium C) to not
more than 45°C. If larger Petri dishes are used, the amount of agar increases.
corresponding. Prepare at least two Petri dishes for each dilution level for each medium.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Incubate the plates at 30°-35°C (20-25°C for fungi) for five days, unless stated otherwise.
time to obtain a reliable count. Select the plates that correspond to a dilution and
that show the largest number of colonies less than 300 (100 colonies for the fungi). Use
the arithmetic mean of the counts and calculate the number of colony-forming units per
gram or milliliter.

b. Surface dispersion method. Using Petri dishes with a diameter of 9 cm, add 15-
20 mL of an appropriate liquefied agar medium for bacterial culture (such as medium B), or 15-
20 mL of a suitable liquefied agar medium for fungal cultivation (such as medium C) not exceeding
at 45°C for each Petri dish and allow to solidify. If larger Petri dishes are used, increase the
volume of agar accordingly. Dry the plates, for example in a flow hood.
in laminar air or in an incubator. Dispense a measured volume of no less than 0.1 mL of the
sample prepared as described in the section "Sample Preparation" on the surface
of the medium. Use at least two Petri dishes for each medium and dilution level. For incubation
and the calculation of the number of colony-forming units, proceed as described for the
plate casting method.

Method of the most probable number

The precision and accuracy of the most probable number (MPN) method is lower than that of the method
of membrane filtration or plate counting methods. Results are not obtained
reliable, particularly for the enumeration of fungi. For these reasons, the NMP method
it is reserved for the enumeration of bacteria 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 subsequent decimal dilutions of the product, as

describe in the section 'Sample Preparation'. Three aliquots are used from each dilution level.
from 1 g or 1 mL to inoculate three tubes with 9-10 mL of an appropriate liquid medium (such as broth
from medium A). If necessary, a surfactant such as the can be added to the medium
polysorbate 80 or an inactivator of antimicrobial agents. Thus, if three are prepared
dilution levels, nine tubes are inoculated. Incubate all the tubes for five days at 30°-
35°C. Record the number of tubes that show growth for each dilution level.
microbial. If reading the tubes is difficult or uncertain due to the nature of the product
examined, subcultured in the same broth or in an appropriate agar medium (such as the agar medium
B), for 18-24 h at the same temperature, and use these results. Using Table 2.6.12.-1,
determine the most probable number of bacteria per gram or milliliter of the examined product.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Table 2.6.12.-1. –Values of the most probable number of bacteria

Three tubes at each dilution level

Number of positive tubes NMP for Category* Confidence Limits
0.1 g 0.01 g 0.001 g gram 1 2 at 95 percent
0 0 0 <3 - -
0 1 0 3 x <1 17
1 0 0 3 x 1 21
1 0 1 7 x 2 27
1 1 0 7 x 2 28
1 2 0 11 x 4 35
2 0 0 9 x 2 38
2 0 1 14 x 5 48
2 1 0 15 x 5 50
2 1 1 20 x 8 61
2 2 0 21 x 8 63
3 0 0 23 x 7 129
3 0 1 38 x 10 180
3 1 0 43 x 20 210
3 1 1 75 x 20 280
3 2 0 93 x 30 390
3 2 1 150 x 50 510
3 2 2 210 x 80 640
3 3 0 240 x 100 1400
3 3 1 460 x 200 2400
3 3 2 1100 x 300 4800
3 3 3 1100 - -
Category 1: Normal results obtained in 95 percent of cases.
Category 2: Less likely outcomes obtained in only 4 percent of cases. These do not
They are used for important decisions. The results that are even less likely than
Those in category 2 are not mentioned and are always unacceptable.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

EFFECTIVENESS OF CULTURE MEDIA AND VALIDITY OF THE COUNTING METHOD

Cultivate the test bacterial strains separately in containers that have a liquid medium.
suitable (like half A broth) at 30°-35°C for 18 to 24 hours. Culture the test strains of
fungi separately on an appropriate agar medium (such as medium C without antibiotics) at 20-25°C
for 48 hours for Candida albicans, at 20-25°C for 7 days for Aspergillus niger.

Staphylococcus aureus such as ATCC 6538 (NCIMB 9518, CIP 4.83)

Escherichia coli such as ATCC 8739 (NCIMB 8545, CIP 53.126)

Bacillus subtilis like ATCC 6633 (NCIMB 8054, CIP 52.62)

Candida albicans such as ATCC 10231 (NCPF 3179, IP 48.72)

Aspergillus niger such as ATCC 16404 (IMI 149007, IP 1431.83)

Use buffered sodium chloride-peptone solution pH 7.0 to prepare suspensions of

Reference that contains around 100 colony-forming units per milliliter. Use the
suspension of each of the microorganisms separately as control of the methods of
counting, in the presence and absence of the product that is to be examined. When analyzing by the
membrane filtration method or by the plate count method, must be obtained a
count of any testing organisms that differs by no more than a factor of five from
calculated value of the inoculum. When analyzed by the most probable number method, the value
the calculated inoculum is within the 95 percent confidence limits of the results
obtained. To test the sterility of the medium and the diluent and the aseptic behavior of the
test, carry out the method using sterile sodium chloride-peptone solution pH 7.0 as
the preparation of the test. There should be no growth of microorganisms.

INTERPRETATION OF RESULTS

The bacterial count will be considered equal to the average number of colony-forming units.
found in the agar medium B. The fungal count will be considered equal to the average number.
of colony-forming units found in the agar medium C. The total count of
viable aerobes is the sum of bacterial count and fungal count as described
previously. If there is evidence that the same types of microorganisms grow in both
means, this can be corrected. If the count is done using the most number method
likely, the calculated value is the bacterial count.

When a limit is indicated in a monograph, it is interpreted as follows:

102microorganisms: maximum acceptable limit: 5 x 102,

103microorganisms: maximum acceptable limit: 5 x 103and so on.

If a sampling plan such as the class three sampling plan is used, proceed as follows:

Calculate the total viable aerobic count separately for each of the five samples. The
Substance or preparation passes the test if the following conditions are met:

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

none of the total counts of individual viable aerobes exceeds the prescribed limit in a
a factor of ten or more (that is, "unacceptable samples");

(ii) no more than two of the individual total viable aerobic counts are within the limit
prescribed and ten times its limit (that is, no more than two 'marginal samples').

The recommended solutions and culture media are described in the general chapter 2.6.13.

B. HARMONIZED METHOD: MICROBIOLOGICAL EXAMINATION OF PRODUCTS NOT

STERILE: MICROBIAL ENUMERATION TESTS

1. INTRODUCTION

The tests described below will allow for the quantitative enumeration of bacteria
mesophilic and fungi that can grow in aerobic conditions.

The tests are primarily designed to determine whether a substance or preparation

mentioned meets a set specification for microbiological quality. When it
use for these purposes, follow the instructions outlined below, including the
number of samples that should be taken, and interpret the results as established in
continuation.

The methods do not apply to products that contain viable microorganisms such as
active ingredients.

Alternative microbiological procedures can be used, including automated methods.

as long as its equivalence with the method of the Pharmacopoeia has been demonstrated.

2. GENERAL PROCEDURES

Carry out the determination under the designed conditions to avoid microbial contamination
extrinsic to the product that is going to be examined. The precautions taken to avoid the
contamination must be such that it does not affect any microorganism that is revealed in the test.

If the product to be examined has antimicrobial activity, it must be removed or

neutralized to the extent possible. If inactivators are used for this purpose, it must
demonstrate their effectiveness and lack of toxicity against microorganisms.

If surfactants are used for sample preparation, their absence must be demonstrated.
de toxicidad para los microorganismos y su compatibilidad con los inactivadores usados.

3. ENUMERATION METHODS

Use the membrane filtration method or the plate count method, as indicated. The
the most probable number (MPN) method is generally the least accurate method for the
microbial counts, however, for some product groups with a very low bioburden,
this may be the most appropriate method.

The selection of the method is based on factors such as the nature of the product and the required limit of
microorganisms. The selected method must allow for the testing of a sample size

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

sufficient to judge compliance with the specification. The suitability of the

selected method.

4. GROWTH PROMOTION TEST AND SUITABILITY OF THE METHOD

RECOUNT

[Link] CONSIDERATIONS

The ability of the test to detect microorganisms in the presence of must be established.
product that is going to be tested.

The suitability must be confirmed when a change is introduced in the execution of the test or in the
product, which could affect the outcome of the test.

4-2. PREPARATION OF TEST STRAINS

Use standardized stable suspensions of test strains or prepare them as indicated.

continuation. Techniques for crop maintenance of the planting lot are used (lot systems
seeding), in such a way that the viable microorganisms used for inoculation come from
no more than 5 passes taken from the original master seed lot. Grow each separately
test strain of bacteria and fungi, as described in Table 2.6.12:2.

Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffered solution.

pH 7.2 to prepare the test suspensions; to suspend the spores of A. niger, it can be
add 0.05 percent of Polysorbate 80 in the buffer. Use the suspensions within 2 hours or
within 24 hours if stored at 2 - 8 °C. As an alternative to prepare and then dilute a
fresh suspension of vegetative cells of A. nigero B. subtilis, a stable suspension is prepared
of spores and then an appropriate volume of the spore suspension is used for inoculation of
Test. The stable suspension of spores can be maintained at 2 - 8 °C for a period of time
validated.

4-3. NEGATIVE CONTROL

To verify the test conditions, a negative control is performed using the diluent.
selected instead of the test preparation. There should be no growth of microorganisms.

4.4. PROMOTION OF ENVIRONMENTAL GROWTH

Test each batch of semi-prepared material and each batch of semi-prepared from material.
dehydrated or of the described ingredients.

Inoculate portions/plates of digested casein soy broth and digested casein soy agar
with a small number (no more than 100 UFC) of the microorganisms indicated in the Table
2.6.12.-2, using a separate half-plate portion for each microorganism. Inoculate the
Sabouraud-dextrose agar plates with a small number (no more than 100 CFU) of the
microorganisms indicated in Table 2.6.12:2, using a separate plate of medium for each
one. Incubate under the conditions described in Table 2.6.12:2.

For solid media, the growth obtained should not differ by a factor greater than 2 from
of the calculated value for a standardized inoculum. For a freshly prepared medium, growth
The occurrence of microorganisms happens in a manner comparable to the previously obtained growth with

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

a batch of media that has been previously tested and approved. The liquid media are suitable if an occurs
Microorganism growth clearly visible comparable to the previous growth
obtained with a previously tested and approved batch of half.

4-5. SUITABILITY OF THE COUNTING METHOD IN THE PRESENCE OF PRODUCT

4-5-1. Sample preparation. The sample preparation method depends on the

physical characteristics of the product to be tested. If it cannot be demonstrated that none of
The procedures described below are satisfactory; a procedure must be developed.
alternative.

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 digested soybean casein broth. If necessary,
Adjust the pH to 6-8. When necessary, prepare additional dilutions with the same diluent.

Non-fat products insoluble in water. Suspend the product that is 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. It can
add a surfactant, for example 1 g/L of polysorbate 80, to help suspend the
substances that wet poorly. If necessary, adjust the pH to 6-8. When needed, it
prepare additional dilutions with the same diluent.

Fat products. Dissolve in isopropyl myristate, sterilized by filtration, or mix the

product that will be examined with the minimum necessary amount of sterile polysorbate 80 or other
non-inhibitory sterile surfactant agent, heated if necessary to no more than 40ºC, or in cases
exceptional at no more than 45ºC. Mix carefully, and if necessary, maintain the
temperature in a water bath. Add a sufficient amount of the preheated diluent
selected to prepare a 1 in 10 dilution of the original product. Mix carefully
while maintaining the temperature for the shortest time necessary for the formation of a
emulsion. Additional decimal dilutions can be prepared in series using the diluent
selected that contains an adequate concentration of polysorbate 80 or another surfactant
sterile non-inhibitor.

Fluids or solids in aerosol form. Aseptically transfer the product to a device with a filter.
from the membrane or to a sterile container to carry out the subsequent sampling. Use the content
total defined number of measured doses from each tested container.

Transdermal patches. Remove the protective cover ('take off the lining') of the patches.
transdermal patches, and place them with the adhesive side facing up, on sterile glass or
plastic. Cover the adhesive surface with a sterile porous material, such as gauze, to
prevent the patches from sticking to each other, and transfer the patches to an appropriate volume of
selected diluent containing inactivators such as polysorbate 80 and/or lecithin. Shake the
Preparation vigorously for at least 30 minutes.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Table 2.6.12.-2.– Preparation and use of test microorganisms

Microorganism Preparation of Promotion of growth Suitability of the contouring method in

the strain of product presence
test
Count Total count of Total count of Total count of
microbial of yeasts and microorganisms yeasts and
total aerobics mushrooms aerobics mushrooms
Staphylococcus If of If of When digested
golden digested from didgeridoo from of casein of
how: soy casein soy/broth of
ATCC 6538 the broth of the broth of digested from
NCIMB 9518 digerido from didgeridoo of - casein of -
CIP 4.83 soy casein soy casein soya/NMP
NBRC 13276 30-35 °C ≤ 100 UFC ≤ 100 UFC
18.24 h 30-35 °C 30-35 ºC
≤ 3 days ≤ 3 days
Pseudomronas If of If of If digested
aeruginosa digested from digested from of casein of
how: soy casein soy casein soy/broth of
ATCC 9027 the broth of the broth of didgeridoo of
NCIMB 8626 didgeridoo of didgeridoo of - casein of -
CIP 82.118 soy casein soy casein soya/NMP
NBRC 13275 30-35 °C ≤ 100 UFC ≤ 100 UFC
18.24 h 30-35 °C 30-35 ºC
≤ 3 days ≤ 3 days
Bacillus If of If from If digested
subtlety didgeridoo of digerido from of casein
like : soy casein soy casein soy/ broth of
ATCC 6633 the broth of the broth of didgeridoo of
NCIMB 8054 digerido from didgeridoo of - casein of -
CIP 52.62 soy casein soya/NMP
NBRC 3134 30-35 °C <= 100 UFC ≤ 100 UFC
18.24 h 30-35 °C 30-35 ºC
≤ 3 days ≤ 3 days
Candida If If If If digested Agar Sabouraud-
albicans Sabouraud- Sabouraud- Sabouraud of casein of dextrose
how: dextrose dextrose dextrose soy ≤ 100 UFC
ATCC 10231 the broth ≤ 100 UFC ≤ 100 UFC ≤ 100 UFC 20-25 °C
NCPF 3179 Sabouraud- 20-25 °C 20-25 °C 30-35ºC ≤ 5 days
IP 48.72 dextrose ≤ 5 days ≤ 5 days ≤ 5 days
NBRC 1594 20-25ºC MPN: I don't know

2-3 days apply

Aspergillus If If If If digested Agar Sabouraud-
nigercomo Sabouraud- Sabouraud- Sabouraud of casein dextrose
ATCC 16404 dextrose or agar dextrose dextrose soy ≤ 100 UFC
IMI 149007 dextrose ≤ 100 UFC ≤ 100 UFC ≤ 100 UFC 20-25 °C
IP 1431.83 20-25°C 20-25 °C 20-25 °C 30-35ºC ≤ 5 days
NBRC 9455 5-7 days, or until ≤ 5 days ≤ 5 days ≤ 5 days
achieve a MPN: I don't know

good apply
sporulation

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

4-5-2. Inoculation and dilution. Add to the prepared sample as described above.
(4-5-1) and in a control (without the test material included) a sufficient volume of the suspension
microbial to obtain an inoculum of no more than 100 CFU. The volume of the suspension of
The inoculum should not exceed 1 percent of the volume of the diluted product.

In order to demonstrate acceptable microbial recovery from the product, the factor must be used.
lowest possible dilution of the prepared sample for the test. When this is not possible due to
a la actividad antimicrobiana o a una solubilidad pobre, se deben desarrollar protocolos apropiados.
If the growth inhibition by the sample cannot be avoided by other means, the aliquot of the
Microbial suspension can be added after neutralization, dilution, or filtration.

4.5.3. Neutralization/removal of antimicrobial activity. The number of microorganisms

recovered from the diluted prepared sample as described in 4-5-2 and incubated in
agreement with the procedure described in 4.5-4, it is compared with the number of microorganisms
recovered from the control preparation.

If growth is inhibited (reduction by a factor greater than 2), modify the procedure to
the particular counting test, to ensure the validity of the results. The modification of
the procedure may include, for example, (1) an increase in the volume of the diluent or medium
of cultivation, (2) incorporation of specific or general neutralizing agents in the diluent, (3)
membrane filtration, or (4) a combination of the previous measures.

Neutralizing agents. Neutralizing agents can be used to neutralize activity.

of antimicrobial agents (Table 2.6.12-3). These can be added to the diluent or in the
selected medium, preferably before sterilization. If used, their efficacy and their absence
Toxicity to microorganisms must be demonstrated by performing a control with the neutralizing agent.
and without the product.

Table 2.6.12:3. – Common neutralizing agents for interfering substances

Interfering substance Neutralizing potential method

Glutaraldehyde, mercurials Sodium bisulfite
sodium bisulfite
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine
Quaternary Ammonium Compounds Lecithin
(CCAs), parahydroxybenzoates (parabens),
bis-guanidines
CCAs, iodine, parabens Polysorbate
Mercurials Thioglycerol
Mercurials, halogens, aldehydes Thiosulfate
EDTA (edetate) Iones Mg2+O Ca2+

If an adequate neutralizing method cannot be found, it can be assumed that the failure to
isolation of the inoculated microorganism is attributable to the microbicidal activity of the product. This
Information serves to indicate that it is unlikely for the product to be contaminated with the given species.
of microorganisms. However, it is possible that the product only inhibits some of the
microorganisms specified here, but does not inhibit other microorganisms not included among

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

the test strains or for which the latter are not representative. So, perform the test
with the highest dilution factor compatible with microbial growth and the criterion of
specific acceptance.

4-5-4. Recovery of microorganisms in the presence of the product. For each microorganism
list, separate tests are conducted. Only the microorganisms of the strain are counted.
added test.

4-5-4-1. Membrane filtration. Use membrane filters that have a pore size
nominal no greater than 0.45 µm. The type of filter material is selected in such a way that the
efficiency to retain bacteria is not affected by the components of the sample that is going to be
investigated. For each listed microorganism, a membrane filter is used.

Transfer an appropriate amount of the prepared sample as described in 4-5-1 to 4-5-3

(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 a volume
suitable diluent.

For the determination of the total aerobic microbial count

TAMC), transfer the membrane filter to the surface of the casein digested soy agar.
determination of the total combined count of yeasts/moulds
count, TYMC), transfer the membrane to the surface of Sabouraud-dextrose agar. Incubate the
plates as indicated in Table 2.6.12:2. Perform the counting.

4-5-4-2. Plate counting methods. Perform plate counting methods at least by

duplicate for each medium, and use the average count of the result.

4-5-4-2-1. Pouring method on plate

Para las cajas Petri de 9 cm de diámetro, agregar en la caja 1 mL de la muestra preparada como se
describe en 4-5-1 a 4-5-3 y 15-20 mL de agar de digerido de caseína de soya o agar Sabouraud-
dextrose, both media at a temperature no higher than 45ºC. If larger Petri dishes are used, the
The amount of agar medium is increased accordingly. For each microorganism.
listed in Table 2.6.12.-2, at least 2 Petri dishes are used. Incubate the plates as indicated in the
Table 2.6.12.-2. Obtain the arithmetic mean of the counts by culture method and calculate the
number of UFC in the original inoculum.

4-5-4-2-2. Dispersion method on the surface

For 9 cm diameter Petri dishes, add 15-20 mL of casein digested soybean agar or
if Sabouraud-dextrose is poured at approximately 45ºC into each Petri dish, and left to solidify. If used
Larger Petri dishes, the volume of the agar is increased accordingly.

Dry the plates, for example in a laminar flow hood or in an incubator. For each
Microorganism listed in Table 2.6.12.-2, at least 2 Petri dishes are used. Dispense a volume
measured no less than 0.1 mL of the prepared sample as described in 4-5-1 to 4-5-3 on the
surface of the medium. Incubate and count as indicated in 4-5-4-2-1.

4-5-4-3. Most Probable Number (MPN) Method. The accuracy and precision of the MPN method is
smaller than that of the membrane filtration method or the plate count method. They are obtained
unreliable results particularly for the counting of fungi. For these reasons, the method
NMP is reserved for the numbering of TAMC in situations where no other is available.
method. If the use of the method is justified, proceed as follows:

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Prepare a series of at least 3 decimal serial dilutions of the product, as described.

from 4-5-1 to 4-5-3. From each dilution level, 3 aliquots of 1 g or 1 mL are used to inoculate 3 tubes.
with 9 - 10 mL of digested soy casein broth. If necessary, it can be added in the
through a surfactant like polysorbate 80, or an inactivator of antimicrobial agents. From
In this way, if 3 levels of dilution are prepared, 9 tubes are inoculated.

Incubate all tubes at 30 – 35ºC for no more than 3 days. If the reading of the results is
difficult or uncertain due to the nature of the product that is going to be examined, subcultivar in the
same broth, or in agar of digested casein from soy, for 1 - 2 days at the same temperature, and
use these results. Using Table 2.6.12:4, determine the most probable number of
microorganisms per gram or milliliter of the examined product.

4-6. RESULTS AND INTERPRETATION

When the suitability of the membrane filtration method or the counting method is verified in
plate, an average count of the test microorganisms must be obtained that does not differ in a
factor greater than 2 with respect to the control value defined in 4-5-2 in the absence of the product.
When verifying the suitability of the NMP method, the value calculated from the inoculum must be
within the 95 percent confidence limits of the results obtained with the control.

If the criteria indicated above cannot be met for one or more of the
microorganisms tested with any of the described methods, to test the product is used
the method and testing conditions that most closely meet the criteria.

5. TESTING OF THE PRODUCTS

5-1. AMOUNT USED FOR TESTING

Unless otherwise specified, use 10 g or 10 mL of the product to be tested.

taken with the precautions mentioned above. For aerosol fluids or solids,
sample 10 containers. For the transdermal patches, sample 10 patches.

The amount to be tested can be reduced for active substances that will be formulated in
the following conditions: the quantity per dosage unit (for example, tablet, capsule, injection)
is less than or equal to 1 mg, or the amount per gram or milliliter (for preparations that are not presented
in dosage units) is less than 1 mg. In these cases, the amount to be tested is not
less than the amount present in 10 units of dosage or 10 g or 10 mL of the product.

For materials used as active substances, where the sample quantity is limited or the
the batch size is extremely small (i.e., less than 1000 mL or 1000 g), the amount
the sample must be 1 percent of the lot, unless prescribed or justified and authorized.
smaller amount.

For products where the total number of entities in a batch is less than 200 (for example,
samples used in clinical studies), the sample size can 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 amount, mix the contents of a sufficient number of
containers to obtain the sample.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

Table 2.6.12.-4. –Values of the most probable number of microorganisms

Observed combinations of tube numbers that NMP for

Limits of
they show growth in each set grams or by
trust in
Number of grams or milliliters of product per tube milliliter of
95%
0.1 0.01 0.001 producto
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 - 3.5
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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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

5-2. PRODUCT REVIEW

[Link] filtration

Use a filtration device designed to allow the transfer of the filter to the medium. Prepare the
sample using a method that has proven to be 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 that has proven to be appropriate.

For the determination of TAMC, transfer one of the membrane filters to the surface of the agar.
from soy casein digested. For the determination of TYMC, transfer the other membrane to the
Sabouraud-dextrose agar surface. Incubate the casein digest soy agar plate at 30
-35ºC for 3-5 days, and the Sabouraud-dextrose agar plate at 20-25ºC for 5-7 days.
Calculate the number of CFU per gram or per milliliter of product.

When examining transdermal patches, filter 10 percent of the volume of the preparation.
described in 4-5-1, separately through each of 2 sterile membrane filters. Transfer
one membrane to the digested casein soy agar for TAMC, and the other membrane to the agar of
Sabouraud dextrose for TYMC.

5-2-2. Plate counting methods

5-2-2-1. Plate pouring method

Prepare the sample using the method that has proven to be suitable, as described in the
section 4. Prepare at least 2 Petri dishes for each dilution level for each medium. Incubate the
casein digested soy agar plates at 30-35ºC for 3-5 days, and the agar plates
Sabouraud dextrose at 20-25 °C for 5-7 days. Select the plates that correspond to a
dilution given and showing the highest number of colonies less than 250 for TAMC and 50 for
TYMC. Obtain the arithmetic average of the counts by culturing method, and calculate the
UFC numbers per gram or per milliliter of product.

[Link] Dispersion Method

Prepare the sample using a method that has proven to be suitable, as described in the
section 4. Prepare at least 2 Petri dishes for each medium and each dilution level. For the
incubation and calculation of the number of CFU proceed as described for the counting method in
plate.

5.2-3. Most Probable Number Method

Prepare and dilute the sample using a method that has proven to be suitable, as described.
In section 4. Incubate all tubes at 30-35ºC for 3-5 days. If necessary, carry out a
subculture using the procedure that has proven to be suitable. Record for each level of
dilution the number of tubes that show microbial growth. Determine the most
probable microorganisms per gram or milliliter of examined product, based on the
Table 2.6.12.-4.

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European Pharmacopoeia 6.0 2.6.12 Microbiological examination of non-sterile products

5-3. INTERPRETATION OF THE RESULTS

The total count of aerobic microbes (TAMC) is considered to be equal to the number of CFU.
found using casein digested soy agar; if fungal colonies are detected in this
half, these colonies are counted as part of the TAMC. The total combined count of
Yeasts/fungi (TYMC) is considered to be equal to the number of CFU found using agar.
Sabouraud dextrose; if bacterial colonies are detected in this medium, they are counted as
part of the TYMC. When is it expected that the TYMC will exceed the acceptance criterion due to the
bacterial growth, Sabouraud-dextrose agar containing antibiotics can be used. If the
count is carried out using the NMP method, the calculated value is the TAMC.

When an acceptance criterion for microbial quality is prescribed, it is interpreted as

continue
- 101UFC: maximum acceptable recount = 20;
- 102UFC: maximum acceptable count = 200;
- 103UFC: maximum acceptable count = 2000, and so on.

The recommended solutions and means are described in chapter general 2.6.13 (in the section
B, harmonized method).

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

01/2008:20613
corrected 6.0

2.6.13. MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS (TESTS

FOR SPECIFIC MICROORGANISMS)

In this general chapter, two sets of tests are presented. The first set presents the
reference methods to determine compliance with the monographs. Therefore, the
Reference to this chapter in a monograph implies compliance with the first set of
tests, unless the use of the second set of tests has been authorized. The tests in
the second set also constitutes official methods of the European Pharmacopoeia, and can
they are referred to as such, especially in the requests for authorization for sale. It is intended
replace the first set with the second set once the relevant monographs
have been reviewed. The second set presents the tests developed in cooperation with
the Japanese Pharmacopoeia and the United States Pharmacopoeia to meet the requirements
harmonized.

A. METHOD OF THE EUROPEAN PHARMACOPOEIA

This general method proposes the use of certain selective means. A

A common characteristic of all selective media is that damaged microorganisms are not detected.
sublethally. Since these sublethally damaged microorganisms are important for quality
of the product, a revitalization of the examination procedures that are based on must be included
selective means.
If the product to be tested has antimicrobial activity, it must be properly neutralized.

Enterobacteria and other Gram-negative bacteria

Although the test has been designed to detect bacteria that belong to the family
Enterobacteriaceae, it is recognized that other types of organisms can be recovered (for example
AeromonasPseudomonas.
Bacteria detection. Prepare the product to be examined as described in the general method.
2.6.12, but using liquid medium D instead of the peptone-sodium chloride solution
buffered to pH 7.0, homogenize and incubate at 35-37 °C for a sufficient time to
revive the bacteria but insufficient to promote their multiplication (normally 2 hours but not
more than 5 h). Shake the container, transfer a volume of the liquid (homogenized A),
corresponding to 1 g or 1 mL of the product to be examined, to 100 mL of enrichment medium E, and
incubate at 35-37 °C for 1848 h. Make subcultures on F agar medium plates. Incubate at
35-37 °C for 18-24 h. The product being examined passes the test if no colonies develop.
of Gram-negative bacteria in none of the plates.

Quantitative evaluation. Inoculate appropriate amounts of the enrichment liquid medium E.

with homogenate A and/or with dilutions of it containing respectively 0.1 g,
0.01 g and 0.001 g (or 0.1 mL, 0.01 mL, and 0.001 mL) of the product to be examined. Incubate at 35-37 °C
for 24-48 h. From each culture, carry out subcultures on an agar F medium in order to
selectively isolate the colonies that develop. Incubate at 35-37 °C for 18-24 h. One
growth of well-developed colonies of Gram-negative bacteria, usually red or
reddish, constitutes a positive result. Note the minimum amount of the product to be examined that gives
positive result and the maximum amount that gives a negative result. Based on Table 2.6.13.-1,
determine the probable number of bacteria.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Table 2.6.13.-1
Results for each quantity of product
Probable number of bacteria
0.1 g of 0.01 g of 0.001 g of
per gram of product
0.1 mL 0.01 mL 0.001 mL
+ + + More than 103
+ + – Less than 103and more than 102
+ – – Less than 102and more than 10
– – – Less than 10

For the transdermal patch test, filter 50 mL of preparation B as described in the

general method 2.6.12 through a sterile membrane filter, place the membrane in 100 mL
from the enrichment liquid medium E, and incubate at 35-37 °C for 18-24 h. After the
incubation, spread on agar medium F for the detection of enterobacteria and others
microorganismos Gram negativos.
Escherichia coli
Prepare the product to be examined as described in the general method 2.6.12 and use 10 mL or
the amount corresponding to 1 g or 1 mL to inoculate 100 mL of liquid medium A, homogenize
Incubate at 35-37 °C for 18-48 hours. Shake the container, transfer 1 mL to 100 mL of the liquid medium.
Incubate at 43-45 °C for 18-24 hours. Perform subcultures on agar medium plates at 35-
37 °C for 18-72 h. The growth of red, non-mucoid colonies of Gram-negative bacilli,
indicates the possible presence of E. coli. This can be confirmed by appropriate biochemical reactions.
like the production of indole. The product passes the test if such colonies are not observed or if they
confirmation biochemical reactions are negative.

Salmonella
Prepare the product to be examined as described in the general method 2.6.12, but using
liquid medium A instead of the peptone-sodium chloride solution buffered at pH 7.0,
homogenize and incubate at 35-37 °C for 18-24 h. Transfer 1 mL of the enrichment medium
a 10 mL of liquid medium I and incubate at 41-43 °C for 18-24 h. Make subcultures in at least
2 different solid media, chosen from the following: agar medium J, agar medium K and medium
Incubate at 35-37 °C for 18-72 hours. The appearance of cultures with the following
characteristics indicate the possible presence of bacteria of the genus Salmonella:
 in the middle of agar J: well-developed, colorless colonies,
 in the middle of agar K: well-developed, red colonies that may or may not present centers
blacks,
 in the middle of agar L: small colonies, transparent, colorless or with a coloration that can
vary from pink to opaque white, often surrounded by a pink area
or red.
Transfer a few suspicious colonies separately to the agar medium M in tubes, using
inoculation on the surface and in depth. The presence of salmonella is confirmed.
provisionally if in the inoculation in depth, but not in the one carried out on the surface, it
produces a color change from red to yellow, generally accompanied by gas formation,
with or without hydrogen sulfide production in the agar. A confirmation can be made
definitively through appropriate biochemical and serological reactions. The product to be examined goes
la prueba si no se observan colonias del tipo descrito o si las reacciones bioquímicas y serológicas
confirmation ones are negative.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Pseudomonas aeruginosa
Prepare the product to be examined as described in the general method 2.6.12 and use 10 mL or
the amount corresponding to 1 g or 1 mL to inoculate 100 mL of liquid medium A, homogenize
Incubate at 35-37 °C for 18-48 hours. Perform subcultures on an agar N medium plate.
incubate them at 35-37 °C for 18-72 h. The substance to be examined passes the test if it is not detected.
growth of microorganisms. If colonies formed by Gram-negative bacilli appear,
transfer part of the morphologically different isolated colonies to liquid medium A and incubate at
41-43 °C for 18-24 h. The sample passes the test if no growth occurs at 41-43 °C.
For the transdermal patch test, filter 50 mL of preparation A as described in the
general method 2.6.12 through a sterile membrane filter, place the membrane in 100 mL
from liquid medium A and incubate at 35-37 °C for 18-48 h. After incubation, spread onto
the N agar medium.
Staphylococcus aureus
Prepare the product to be examined as described in the general method 2.6.12 and use 10 mL or
the amount corresponding to 1 g or 1 mL to inoculate 100 mL of liquid medium A, homogenize
Incubate at 35-37 °C for 18-48 hours. Perform subcultures on an agar medium plate O and
incubate them at 35-37 °C for 18-72 h. The appearance of black colonies of Gram-positive cocci, at
small surrounded by a transparent area, constitutes an indication of the presence of S. aureus.
confirmation can be made through appropriate biochemical reactions such as tests of the
coagulase and deoxyribonuclease. The product being examined passes the test if no observations are made
colonies of the type described in the agar medium O, or if the confirmation biochemical reactions
they are negative.

For the transdermal patch test, filter 50 mL of preparation A as described in the

general method 2.6.12 through a sterile membrane filter, place the membrane in 100 mL
from liquid medium A and incubate at 35-37 °C for 18-48 h. After incubation, spread onto
the agar medium O.
Nutritional and selective properties of the media and validity of the test
The tests described below must be carried out at least on each batch of dehydrated medium.
Proceed as follows: Cultivate each of the following reference strains separately in tubes.
that contain adequate means as indicated, at 30-35 °C for 18-24 hours:
Staphylococcus aureus for example ATCC 6538 (NCIMB 9518, CIP 4.83):
liquid medium A,
Pseudomonas aeruginosa for example ATCC 9027 (NCIMB 8626, CIP 82.118):
liquid medium A,
Escherichia coli for example ATCC 8739 (NCIMB 8545, CIP 53.126):
liquid medium A,
Salmonella typhimurium no reference strain is recommended (it can be used
also a non-pathogenic salmonella for humans, such as
Salmonella abony (NCTC 6017, CIP 80.39): liquid medium A.

Prepare dilutions of each culture using buffered peptone-sodium chloride solution at

pH 7.0, in order to obtain suspensions that contain about 1000 viable microorganisms per
milliliter. Mix equal volumes of each suspension and use 0.4 mL (which corresponds
approximately 100 microorganisms from each strain) as inoculum in the tests for the

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

detection of S. aureus, P. aeruginosa, E. coli, and Salmonella, in the presence and absence of the product
to be examined. A positive result must be obtained for the respective microorganisms.
Clostridia
The tests described below have various purposes. The first method is used
for products from which it is essential to exclude contamination by clostridia, what it does
necessary to demonstrate their absence. Generally in such products, the total count of
the microorganisms is low. The second method is a semi-quantitative test for Clostridium
perfringensy is intended for products in which the level of contamination with this species
constitutes a quality criterion.
Clostridia test
Prepare the product to be examined as described in the general method 2.6.12. Take 2
equal portions of the preparation, corresponding to 1 g or 1 mL of the product to be examined.
Heat 1 portion to 80 °C for 10 minutes and cool it quickly. Do not heat the other portion.
Transfer 10 mL of each of the homogenized portions to 2 containers (38 mm × 200 mm) or to
other suitable containers that hold 100 mL of medium P. Incubate under anaerobic conditions at
35-37 °C for 48 h. After this incubation, perform subcultures from each tube in Q medium.
with gentamicin, incubating under anaerobic conditions at 35-37 °C for 48 h. If not detected
microorganism growth, the product passes the test.
If there were growth, to re-cultivate each of the types of colonies formed.
in medium Q, without gentamicin, and incubate both under aerobic and anaerobic conditions. The
observation of growth exclusively anaerobic of Gram-positive bacilli (with or without formation of
endospores), which give a negative catalase reaction, indicates that it is Clostridium spp. If
It is necessary to compare the appearance of the colonies growing on both plates, using the
catalase test to eliminate Bacillus spp, aerobic or facultatively anaerobic, which give
positive said reaction. This test can be applied to isolated colonies on agar, or also
indirectly prior to transferring the bacteria to glass slides, depositing on the
cells a drop of diluted hydrogen peroxide solution R. The formation of gas bubbles
indicates that the catalase reaction is positive.
2. Clostridium perfringens count
Prepare the product to be examined as described in the general method 2.6.12 and prepare dilutions.
1:100 and 1:1000 in peptone-sodium chloride solution buffered at pH 7.0. Determine the
most probable number of bacteria as described for the count of aerobic microorganisms
total viable in 2.6.12, using culture medium R in tubes or in other suitable containers that
have a small Durham bell. Mix with the minimal possible agitation and incubate at
45.5-46.5 °C for 24-48 h. Containers that show blackening due to the formation
of iron(II) sulfide, as well as abundant gas in the Durham bell (at least 1/10 of the
Volume) indicate the presence of Cl. perfringens. Estimate the most probable number of Cl.
perfringens using Table 2.6.13.-2.

Controls
Use the following strains:
For method 1: Clostridium sporogenes, for example ATCC 19404 (NCTC 532) or CIP 79.3,
For method 2: Clostridium perfringens, for example ATCC 13124 (NCIMB 6125,
NCTC 8237, CIP 103 409.
If necessary, combine with Cl. sporogenes to verify selectivity and the conditions.
anaerobes.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Table 2.6.13.-2.–Values of the most probable number (MPN) of bacteria

Three tubes at each dilution level

Number of positive tubes NMP by Category* Confidence limits
0.1 g 0.01 g 0.001 g gram 1 2 at 95 percent
0 0 0 <3 - -
0 1 0 3 x <1 17
1 0 0 3 x 1 21
1 0 1 7 x 2 27
1 1 0 7 x 2 28
1 2 0 11 x 4 35
2 0 0 9 x 2 38
2 0 1 14 x 5 48
2 1 0 15 x 5 50
2 1 1 20 x 8 61
2 2 0 21 x 8 63
3 0 0 23 x 7 129
3 0 1 38 x 10 180
3 1 0 43 x 20 210
3 1 1 75 x 20 280
3 2 0 93 x 30 390
3 2 1 150 x 50 510
3 2 2 210 x 80 640
3 3 0 240 x 100 1400
3 3 1 460 x 200 2400
3 3 2 1100 x 300 4800
3 3 3 1100 - -
Category 1: Normal results obtained in 95 percent of cases.
Category 2: Less likely results obtained in only 4 percent of cases. These do not
used for important decisions. The results that are even less likely than
Those in category 2 are not mentioned and are always unacceptable.

The following section is included for information.

RECOMMENDED SOLUTIONS AND CULTURE MEDIA

The following solutions and culture media have proven satisfactory for applying them in the
microbial contamination tests indicated in the Pharmacopoeia. It is possible to use other methods,
as long as they have similar nutritional and selective properties for microorganisms for
those who take the test.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Peptone-sodium chloride solution buffered to pH 7.0

Potassium dihydrogen phosphate 3.6 g
Disodium hydrogen phosphate dihydrate 7.2 g, equivalent to phosphate 0.067 M
Sodium chloride 4.3 g
Peptone (from meat or casein) 1.0 g
Purified water 1000 mL
A esta solución pueden añadirse agentes tensioactivos o inactivadores de
antimicrobial agents such as:
Polysorbate 80 1 to 10 g/L.
Sterilize by heating in an autoclave at 121 °C for 15 min.

Liquid medium A (broth with casein hydrolysate and soy)

Pancreatic hydrolyzate of casein 17.0 g
Papainic hydrolyzed soy 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Monohydrate glucose 2.5 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 min.

Medium of agar B (agar with casein and soy hydrolysate)

Pancreatic hydrolysate of casein 15.0 g
Papainic hydrolyzed soy 5.0 g
Sodium chloride 5.0 g
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 min.

Medium C agar (Sabouraud-glucose agar with antibiotics)

Peptonas (from meat and casein) 10.0 g

Monohydrate glucose 40.0 g
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 5.6 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 min. Immediately before its
Use, add 0.10 g of sodium benzylpenicillin and 0.10 g of tetracycline per liter of medium, in the form of
sterile solutions. Alternatively, it is possible to add 50 mg of chloramphenicol per liter of medium,
before sterilization.

Liquid medium D (lactose monohydrate broth)

Beef extract 3.0 g
Pancreatic gelatin hydrolysate 5.0 g
Lactose monohydrate 5.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 6.9 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 min
and cool immediately.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Enriched liquid medium E (Mossel broth for Enterobacteriaceae enrichment)

Pancreatic gelatin hydrolysate 10.0 g
Monohydrate glucose 5.0 g
Dehydrated bull liver 20.0 g
Potassium dihydrogen phosphate 2.0 g
Dihydrate sodium hydrogen phosphate 8.0 g
Bright green 15 mg
Purified water 1000 mL
Adjust the pH so that after heating it is 7.2 ± 0.2.
Heat to 100 °C for 30 min and cool immediately.

Medium F agar (agar with crystal violet, neutral red and bile, with glucose)
Yeast extract 3.0 g
Pancreatic hydrolyzed gelatin 7.0 g
Biliary sales 1.5 g
Monohydrate lactose 10.0 g
Sodium chloride 5.0 g
Monohydrate glucose 10.0 g
If 15.0 g
Neutral red 30 mg
Crystal violet 2 mg
Purified water 1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2.
Heat to boiling; a autoclave should not be used.

Liquid medium G (MacConkey broth)

Pancreatic hydrolyzate of gelatin 20.0 g
Lactose monohydrate 10.0 g
Dehydrated bull's penis 5.0 g
Bromcresol purple 10 mg
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 minutes.

Medium H agar (MacConkey agar)

Pancreatic hydrolyzed gelatin 17.0 g
Peptones (from meat and casein) 3.0 g
Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Biliary sales 1.5 g
If 13.5 g
Neutral red 30.0 mg
Crystal violet 1 mg
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.1 ± 0.2.
Heat to boiling for 1 minute with constant stirring;
then sterilize by heating in an autoclave at 121 °C for 15 min.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Liquid Medium I (broth with tetrathionate, bile, and brilliant green)

Peptone 8.6 g
Dried beef tripe 8.0 g
Sodium chloride 6.4 g
Calcium carbonate 20.0 g
Potassium tetraborate 20.0 g
Bright green 70 mg
Purified water 1000 mL
Adjust the pH so that after heating it is 7.0 ± 0.2.
Calentar hasta inicio de la ebullición. No repetir el calentamiento.

Medium J agar (agar with deoxycholate and citrate)

Beef extract 10.0 g
Meat peptone 10.0 g
Monohydrate lactose 10.0 g
Sodium citrate 20.0 g
Iron(III) citrate 1.0 g
Sodium deoxycholate 5.0 g
If 13.5 g
Neutral red 20 mg
Purified water 1000 mL
Adjust the pH so that after heating it is 7.3 ± 0.2.
Gently heat until boiling, boil for 1 minute,
cool to 50 °C and transfer to Petri dishes. Do not heat in autoclave.

K agar medium (agar with desoxycholate, lysine, and xylose)

Xylose 3.5 g
L-Lysine 5.0 g
Monohydrate lactose 7.5 g
Sucrose 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol red 80 mg
Agar 13.5 g
Sodium deoxycholate 2.5 g
Sodium thiosulfate 6.8 g
Ammonium and iron(III) citrate 0.8 g
Purified water 1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2.
Calentar hasta el inicio de la ebullición, enfriar hasta 50 °C
and transfer to Petri dishes. Do not autoclave.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Medium of L agar (agar with sucrose, monohydrate lactose, phenol red, and brilliant green)
Peptones (from meat and casein) 10.0 g
Yeast extract 3.0 g
Sodium chloride 5.0 g
Monohydrate lactose 10.0 g
Sucrose 10.0 g
If 20.0 g
Phenol red 80 mg
Bright green 12.5 mg
Purified water 1000 mL
Heat to boiling for 1 minute.
Adjust the pH so that after sterilization it is 6.9 ± 0.2.
Immediately before use, sterilize by heating.
in an autoclave at 121 °C for 15 min, cool to 50 °C
and transfer to Petri dishes.

Medium M (agar with triple sugar and iron)

Beef extract 3.0 g
Yeast extract 3.0 g
Peptones (from casein and meat) 20.0 g
Sodium chloride 5.0 g
Lactose monohydrate 10.0 g
Sucrose 10.0 g
Monohydrate glucose 1.0 g
Ammonium and iron(III) citrate 0.3 g
Sodium thiosulfate 0.3 g
Phenol red 25 mg
If 12.0 g
Purified water 1000 mL
Heat to boiling for 1 minute, with stirring.
Adjust the pH so that after sterilization it is 7.4 ± 0.2.
Fill tubes to one third of their height, sterilize by heating
in autoclave at 121 °C for 15 minutes and let it cool in
inclined position, so that a deep part is obtained
and a sloping surface.

N medium agar (agar with cetyltrimethylammonium bromide)

Pancreatic hydrolyzed gelatin 20.0 g

Magnesium chloride 1.4 g
Potassium sulfate 10.0 g
Cetrimide 0.3 g
If 13.6 g
Purified water 1000 mL
Glycerol 10.0 mL
Heat to boiling for 1 minute with stirring.
Adjust the pH so that after sterilization it is 7.2 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 min.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Agar medium O (Baird-Parker agar)

Pancreatic casein hydrolysate 10.0 g

Beef extract 5.0 g
Yeast extract 1.0 g
Lithium chloride 5.0 g
If 20.0 g
Glycine 12.0 g
Sodium pyruvate 10.0 g
Purified water 950 mL
Heat to boiling for 1 minute with stirring. Adjust the pH so that after the
Sterilization should be 6.8 ± 0.2. Sterilize by heating in an autoclave at 121 °C for 15 minutes.
cool to 45-50 °C and add 10 mL of a sterile solution of 10 g/L of potassium tellurite and 50 mL
egg yolk emulsion.

Medium P (reinforced medium for clostridia)

Beef extract 10.0 g

Peptone 10.0 g
Yeast extract 3.0 g
Soluble starch 1.0 g
Monohydrate glucose 5.0 g
Cysteine hydrochloride 0.5 g
Sodium chloride 5.0 g
Sodium acetate 3.0 g
If 0.5 g
Purified water 1000 mL
Hydrate the agar and dissolve by heating until boiling on low.
continuous agitation. If necessary, adjust the pH so that
after sterilization it should be approximately 6.8.
Sterilize by heating in an autoclave at 121 °C for 15 minutes.

Half Q (like Columbia)

Pancreatic hydrolyzed casein 10.0 g
Peptic hydrolyzed meat 5.0 g
Pancreatic hydrolysate of heart 3.0 g
Yeast extract 5.0 g
Corn starch 1.0 g
Sodium chloride 5.0 g
If according to his ability from 10.0 g to 15.0 g
gelling agent
Purified water 1000 mL
Hydrate the agar and dissolve by heating to a low boil.
continuous agitation. If necessary, adjust the pH so that
after sterilization it is 7.3 ± 0.2.
Sterilize by heating in an autoclave at 121 °C for 15 minutes.
Let it cool to 45-50 °C; add, when appropriate,
gentamicin sulfate up to a concentration of 20 mg of
gentamicin base and pour into Petri dishes.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Medium R (medium with lactose monohydrate and sulfite)

Pancreatic casein hydrolysate 5.0 g
Yeast extract 2.5 g
Sodium chloride 2.5 g
Lactose monohydrate 10.0 g
Cysteine hydrochloride 0.3 g
Purified water 1000 mL
Dissolve, adjust to pH 7.1 ± 0.1 and package in amounts of up to
8 mL, in tubes of 16 mm × 160 mm that contain a small
Durham bell. Sterilize by heating in an autoclave.
at 121 °C for 15 min and store at 4 °C.
Before use, heat the medium in a water bath for 5 minutes and cool. Add to each tube.
0.5 mL de una solución de 12 g/L demetabisulfito de sodio Ry 0.5 mL de una solución de 10 g/L
of ammonium and iron(III) citrate, both freshly prepared and filtered through membranes
(pore size: 0.45 µm).

Agar medium S (R2A)

Yeast extract 0.5 g
Proteose peptone 0.5 g
Casein hydrolysate 0.5 g
Glucose 0.5 g
Starch 0.5 g
Potassium hydrogen phosphate 0.3 g
Anhydrous magnesium sulfate. 0.024 g
Sodium pyruvate 0.3 g
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.2 ± 0.2.
Sterilize by heating in an autoclave at 121 ºC for 15 minutes.

Neutralizing agents
Neutralizing agents can be used to neutralize the activity of agents.
antimicrobials. They can be added to the peptone-sodium chloride solution buffered at pH 7.0,
preferably before sterilization. If used, their efficacy and absence must be demonstrated.
of toxicity against microorganisms.
A typical neutralizing liquid has the following composition:
Polysorbate 80 30 g
Lecithin (egg) 3g
Histidine hydrochloride 1g
Peptone (from meat or casein) 1g
Sodium chloride 4.3 g
Potassium dihydrogen phosphate 3.6 g
Dihydrated sodium hydrogen phosphate 7.2 g
Purified water 1000 mL
Sterilize by heating in an autoclave at 121 °C for 15 minutes.
If the solution does not have enough neutralization capacity,
the concentration of Polysorbate 80 or lecithin can be increased.
Alternatively, the neutralizing agents mentioned in Table 2.6.13.-3 can be added.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Table 2.6.13.-3. –Inactivators of antimicrobial agents to add to the peptone solution

sodium chloride buffered to pH 7.0
Type of agent
Inactivator Concentration Comment
antimicrobial
Phenolic Lauryl sulfate of 4 g/L Add after the
sodium sterilization of the
30 g/L and 3 g/L
solution of
Polysorbate 80 and
5 mL/l-50 mL/L peptone chloride
lecithin
sodium buffered
Egg yolk a pH 7.0
Organo-mercurials Sodium thioglycolate 0.5 g/L-5 g/L
Thiosulfate
Halogens 5 g/L
sodium
Composed of Egg yolk 5 mL/L - 50 mL/L Add after the
quaternary ammonium sterilization of the
peptone solution-
sodium chloride
buffered to pH 7.0

B. HARMONIZED METHOD

1. INTRODUCTION

The tests described here will allow for the determination of the absence, or the limited presence, of
specific microorganisms that can be detected under the described conditions.

The tests have been designed primarily to determine whether a substance or preparation
meets an established specification for microbiological quality. If used for such
purposes, follow the instructions outlined below, including the number of
samples to be taken, and interpret the results as indicated later.

Alternative microbiological procedures can be used, including automated methods.

as long as its equivalence with the pharmacopoeial method has been demonstrated.

2. GENERAL PROCEDURES

La preparación de las muestras se realiza como se describe en el capítulo general 2.6.12(en la

section B, Harmonized Method).

If the product to be examined has antimicrobial activity, it must be removed or neutralized in

the measure of what is possible as described in chapter general 2.6.12 (in section B, Method
harmonized).

If surfactants are used in the sample preparation, their effectiveness must be demonstrated.
absence of toxicity for microorganisms and compatibility with the inactivators used,
as described in chapter general 2.6.12 (in section B, Harmonized method).

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

3. GROWTH PROMOTING AND INHIBITORY PROPERTIES OF THE

MEDIA, ADEQUACY OF THE TEST AND NEGATIVE CONTROLS

The test's ability to detect microorganisms in the presence of must be established.

product to be examined. The suitability of the test must be confirmed if a change is introduced in
the execution of the test or in the product, which could affect the results.

3-1. PREPARATION OF TEST STRAINS

Use standardized stable suspensions of test strains, or prepare them as indicated further.
forward. Crop maintenance techniques of the planting batch are used (batch systems
sowing), in such a way that the viable microorganisms used for inoculation correspond
no more than five passes from the original master planting lot.

3-1-1. Aerobic microorganisms. Cultivate each test bacterial strain separately in broth.
casein and soy digested agar or casein and soy digested agar at a temperature of 30-35ºC
for a period of 18 to 24 hours. Grow the test strain of Candida albicans separately.
in Sabouraud dextrose or in Sabouraud Dextrose Broth at a temperature of 20-25ºC for
a period of 2 to 3 days.

- Staphylococcus aureus, for example ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;
- Pseudomonas aeruginosa, for example ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC
13275
- Escherichia coli, for example ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972;
- Salmonella enterica subsp. enterica serotype Typhimurium, for example ATCC 14028 or, as
an alternative, Salmonella enterica subspecies enterica serotype Abony, for example NBRC
100797, NCTC 6017 or CIP 80.39;
- Candida albicans, for example ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.

To prepare the test suspensions, use buffered sodium chloride-peptone solution.

pH 7.0 or phosphate buffer solution of pH 7.2. Use the suspensions within 2 hours or
within 24 hours if stored at a temperature of 2-8ºC.

3-1-2. Clostridia. Use Clostridium sporogenes, such as ATCC 11437 (NBRC

14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.3) or NBRC 14293.
Cultivate the test clostridia strain under anaerobic conditions in reinforced medium for
clostridia at a temperature of 30-35ºC for a period of 24 to 48 hours. Alternatively
for the preparation and subsequent dilution of a fresh suspension of vegetative cells of Cl.
sporogenes, a stable suspension of spores is used for the test inoculation. The
The stable spore suspension can be maintained at a temperature of 2-8ºC for a period.
validated.

[Link] CONTROL

To verify the test conditions, perform a negative control using the diluent.
selected instead of test preparation. There should be no growth of microorganisms.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

3-3. GROWTH PROMOTING AND INHIBITORY PROPERTIES OF MEDIA

Test each batch of ready-to-use medium and each batch of medium prepared from medium.
dried or of the ingredients.

Verify the appropriate properties of the relevant media as indicated in Table 2.6.13.-1.

Testing the growth-promoting properties, liquid media: Inoculate a portion of the

appropriate medium with a small number (no more than 100 UFC) of the suitable microorganism.
Incubate at the specified temperature for a time not exceeding the shorter indicated period
in the test. A clearly visible growth of microorganisms comparable to the
obtained earlier with a previously analyzed and approved half batch.

Testing the properties of growth promotion, solid media: carry out the method of
surface area, inoculating each of the plates with a small number (no more than 100
UFC) of the appropriate microorganism. Incubate at the specified temperature for a time not
greater than the shorter period indicated in the test. There is a growth of microorganisms
comparable to that obtained previously with a batch of half analyzed and previously approved.

Test of inhibitory properties, liquid or solid media: Inoculate the appropriate medium with
at least 100 UFC of the appropriate microorganism. Incubate at the specified temperature for a
no less time than the greater period indicated in the test. No growth occurs of
test microorganism.

Test of indicator properties: using the surface extension method, inoculating

each of the plates with a small number (no more than 100 CFU) of the appropriate microorganism.
Incubate at the specified temperature for a period that falls within the indicated range.
in the test. The colonies are comparable, in appearance and indicator reactions, to the
previously obtained with a batch of medium that was analyzed and approved beforehand.

3-4. SUITABILITY OF THE TEST METHOD

For each product to be analyzed, prepare the sample as described in

the relevant paragraph in section 4. At the time of mixing, add each test strain in the
indicated growth medium. Inoculate the test strains individually. Use a number of
microorganisms equivalent to no more than 100 CFU in the inoculated test preparation.
Perform the test as indicated in the relevant paragraph in section 4, using the period of
shorter indicated incubation.

Specific microorganisms must be detected with the indicator reactions as described.

in section 4.

Any antimicrobial activity of the product requires a modification of the procedure.

the test (see 4-5-3 of general chapter 2.6.12) (in section B, Harmonized method)).

For a specific product, if the antimicrobial activity concerning the microorganism for
which indicates that the test cannot be neutralized, it is assumed that the inhibited microorganism does not
will be present in the product.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Table 2.6.13.-1 - Indicator properties of growth-promoting and inhibitory media

Medium Property Test strains
Gram test for bacteria Mussel broth for Promotion of growth E. coli
bile-tolerant negatives enrichment of [Link]
enterobacteria Inhibitory S. aureus
If violet red bile glucose Promotion of growth + E. coli
indicator [Link]
Test for Escherichia coli MacConkey broth Promotion of growth E. coli
Inhibitory S. aureus
If MacConkey Promotion of growth + E. coli
indicator
Salmonella test Caldo Rappaport Vassiliadis Promotion of growth Salmonella enterica subspecies
for the enrichment of enteric serotype Typhimurium or
Salmonella Salmonella enterica subspecies.
enteric serotype Abony
Inhibitory S. aureus
If chitosan, lysine, deoxycholate Promotion of growth + Salmonella enterica subspecies
indicator serotype Typhimurium or
Salmonella enterica subspecies.
serotype abony
Indicator [Link]
Test for If cetrimide Promotion of growth [Link]
Pseudomonas aeruginosa Inhibitory E. coli
Test for If mannitol salt Promotion of growth + S. aureus
Staphylococcus aureus indicator
Inhibitory E. coli
Test for clostridia Reinforced medium Promotion of growth Cl. sporogenes
for clostridia
If Columbia Promotion of growth Cl. sporogenes
Test for Sabouraud dextrose broth Promotion of growth C. albicans
Candida albicans If Sabouraud dextrose Promotion of growth + C. albicans
indicator

4. PRODUCT TESTING

4-1. BILE TOLERANT GRAM-NEGATIVE BACTERIA

[Link] preparation and [Link] a sample using a

dilution 1 in 10 of no less than 1 g of the product to be analyzed, as described in the chapter
general2.6.12 (in section B, Harmonized method), except that digested broth must be used
casein and soy as the diluent of choice, mix and incubate at a temperature of 20-25ºC for
a sufficient period to revive the bacteria but that does not stimulate their multiplication
organisms (usually 2 hours but no more than 5 hours).

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

4-1-2. Test of absence. Unless otherwise stated, use the volume

corresponding to 1 g of the product, prepared as indicated in 4-1-1, to inoculate Mossel Broth
for enrichment of enterobacteria. Incubate at a temperature of 30-35ºC for a period
from 24 to 48 hours. Subculture in red violet bile glucose agar plates. Incubate at a temperature
from 30-35ºC for a period of 18 to 24 hours.

The product passes the test if no colonies develop.

[Link] test
4-1-3-1. Selection and subculturing. Inoculate appropriate amounts of Mossel broth for
enrichment of enterobacteria with the preparation as indicated in 4-1-1 and/or dilutions of the
same that contain respectively 0.1 g; 0.01 g and 0.001 g (or 0.1 mL; 0.01 mL and 0.001 mL) of the
product to be examined. Incubate at a temperature of 30-35ºC for a period of 24 to 48 hours.
Subculture each of the crops on a red violet bile glucose agar plate. Incubate at a
temperature of 30-35ºC for a period of 18 to 24 hours.

4-1-3-2. Interpretation. The growth of colonies constitutes a positive result. Indicate the
the smallest amount of the product that produces a positive result and the largest amount that produces a
negative result. Based on Table 2.6.13.-2, determine the probable number of bacteria.

Table 2.6.13.-2 - Interpretation of the results

Results for each quantity of product Probable number of

0.1 g 0.01 g of 0.001 g o bacteria per gram or
0.1 mL 0.01 mL 0.001 mL milliliter of product
+ + + 103
+ + − < 103y > 102
+ − − < 102y > 10
− − − < 10

[Link] COLI

4-2-1. Sample preparation and pre-incubation. Prepare a sample using a dilution.

1 in 10 of no less than 1 g of the product to be examined as described in the general chapter
2.6.12 (in section B, Harmonized method), and use 10 mL or the corresponding amount to 1 g or
1 mL to inoculate an adequate amount (determined as described in 3-4) of broth
Casein and soy digested, mix and incubate at 30-35ºC for 18-24 hours.

4-2-2. Selection and subculture. Shake the container, transfer 1 mL of digested casein and soy broth.
100 mL of MacConkey Broth and incubate at 42 - 44ºC for 24 - 48 hours. Subculture on a plate of
Incubate MacConkey at 30-35ºC for 18 - 72 h.

4-2-3. Interpretation. The growth of colonies indicates the possible presence of E. coli. This is
confirm through identification tests.

The product passes the test if there are no colonies present or if the identification tests are
negatives.

16
European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

[Link]

4-3-1. Sample preparation and pre-incubation. Prepare the product to be examined as

is described in chapter general 2.6.12 (in section B, Harmonized method), and use the amount
corresponding to no less than 10 g or 10 mL to inoculate an adequate amount (determined
as described in 3-4) of digested casein and soybean broth, mix and incubate at 30-35ºC for
18-24 hours.

4-3-2. Selection and subculturing. Transfer 0.1 mL of digested casein and soy broth to 10 mL of
Rappaport Vassiliadis broth for Salmonella enrichment, and incubate at 30-35ºC for 18-
24 h. Subculture on xylose agar plates, lysine, deoxycholate. Incubate at 30-35ºC for 18-48 h.

4-3-3. Interpretation. The possible presence of Salmonella is indicated by the growth of

well-developed colonies of red color, with or without black centers. This is confirmed by
identification tests.

The product passes the tests if colonies of the described types are not present, or if they are
confirmatory identification tests are negative.

4-4. PSEUDOMONAS AERUGINOSA

4-4-1. Sample preparation and prior incubation. Prepare a sample using a dilution.
1 in 10 of no less than 1 g of the product to be examined as described in the general chapter
2.6.12 (in section B, Harmonized method), and use 10 mL or the amount corresponding to 1 g or
1 mL to inoculate an adequate amount (determined as described in 3-4) of broth
casein and soy digerido and mix. When analyzing transdermal patches, filter the volume
corresponding sample of a patch of the preparation described in 4-5-1 in the general chapter
2.6.12 through a sterile membrane filter, and place in 100 mL of digested casein broth and
soy. Incubate at a temperature of 30-35ºC for a period of 18 to 24 hours.

4-4-2. Selection and subculturing. Subculture on a cetrimide agar plate and incubate at 30-35ºC.
during 18-72 h.

4-4-3. Interpretation. The growth of colonies indicates the possible presence of P. aeruginosa. This
it is confirmed through identification tests.

The product passes the test if there are no colonies present or if the confirmatory tests of
Identification is negative.

[Link] AUREUS

4-5-1. Sample preparation and pre-incubation. Prepare a sample using a dilution.

1 in 10 of no less than 1 g of the product to be examined as described in the general chapter
2.6.12 (in section B, Harmonized method), and use 10 mL or the corresponding amount to 1 g or
1 mL to inoculate an adequate amount (determined as described in 3-4) of broth
Casein and soy digested and mixed. When testing transdermal patches, filter the volume.
sample corresponding to 1 patch of the preparation described in 4-5-1 in the general chapter
2.6.12 through a sterile membrane filter, and place in 100 mL of digested casein broth
soy. Incubate at 30-35ºC for 18-24 h.

17
European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

4-5-2. Selection and subculturing. Subculture on a mannitol salt agar plate and incubate at 30-35ºC.
during 18-72 h.

[Link]. The possible presence of S. aureus is indicated by the growth of

yellow/white colonies surrounded by a yellow area. This is confirmed by
identification tests.

The product passes the tests if colonies of the described types are not present, or if the
Confirmatory identification tests are negative.

[Link]

[Link] preparation and heat [Link] the product to be examined

using as described in the general chapter 2.6.12 (in section B, Harmonized method).
Take 2 equal portions that correspond to no less than 1 g or 1 mL of the product to be examined.
Heat one portion at 80ºC for 10 minutes, and cool quickly. Do not heat the other portion.

4-6-2. Selection and subculture. Transfer 10 mL of each mixed portion to 2 containers (38 mm
x 20 mmm or other suitable containers) that contain 100 mL of reinforced medium for
clostridia. Incubate under anaerobic conditions at a temperature of 30-35ºC for 48 hours.
After incubation, perform subcultures from each tube onto Columbia agar, and incubate in
anaerobic conditions at a temperature of 30-35ºC for 48 hours.

[Link] growth of anaerobic bacilli (with or without endospores) that produce a

Negative catalase reaction indicates the presence of clostridia. This is confirmed through tests.
of identification.

The product passes the tests if there are no colonies of the described types present, or if the
Confirmatory identification tests are negative.

4-7. CANDIDA ALBICANS

4-7-1. Sample preparation and prior incubation. Prepare the product to be examined as
is described in chapter general2.6.12 (in section B, Harmonized method), and use 10 mL or the
an amount corresponding to no less than 1 g or 1 mL to inoculate 100 mL of Sabouraud broth
Dextrose, and mix. Incubate at 30-35ºC for 3 - 5 days.

4-7-2. Selection and subculturing. Subculture on a Sabouraud dextrose agar plate and incubate at
30-35ºC for 24 - 48 h.

4-7-3. Interpretation. The growth of colonies may indicate the presence of C. albicans. This is
confirm through identification tests.

The product passes the tests if such colonies are not present or if the tests
Identification confirmatory tests are negative.

The following section is presented for information.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

5. RECOMMENDED SOLUTIONS AND CULTURE MEDIA

It has been found that the following solutions and culture media are satisfactory for the
purposes for which they are prescribed in the microbial contamination test in the
Pharmacopoeia. Other means can be used if they have growth-promoting properties and
similar inhibitors.

Buffer solution stock. Place 34 g of monobasic potassium phosphate in a flask.

Volumetric of 1000 mL, dissolve in 500 mL of purified water, adjust the pH to 7.2 ± 0.2 with
sodium hydroxide, dilute to 1000.0 mL with purified water, and mix. Distribute into containers, and
sterilize. Store at 2-8ºC.

Phosphate buffer solution pH 7.2. Prepare a mixture of stock buffer solution and
purified water (1:800 V/V), and sterilize.

Buffered solution of sodium chloride-peptone pH 7.0

Monobasic potassium phosphate 3.6 g

Dibasic sodium phosphate dihydrate 7.2 g, equivalent to phosphate 0.067 M
Sodium chloride 4.3 g
Peptone (from meat or casein) 1.0 g
Purified water 1000 mL
Sterilize in an autoclave using a validated cycle.

Digested casein and soy broth

Pancreatic casein digested 17.0 g
Soy papain digested 3.0 g
Sodium chloride 5.0 g
Dibasic potassium phosphate 2.5 g
Monohydrate glucose 2.5 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25ºC.
Sterilize in an autoclave using a validated cycle.

Casein and soy digested

Pancreatic casein digested 15.0 g
Digested soybean papain 5.0 g
Sodium chloride 5.0 g
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25ºC.
Sterilize in an autoclave using a validated cycle.

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Farmacopea Europea 6.0 2.6.13 Microbiological examination of non-sterile products

If Sabouraud dextrose
Dextrose 40.0 g
Mixture of peptic digested animal tissue and 10.0 g
pancreatic digest of casein (1:1)
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25ºC. Sterilize in a
autoclave using a validated cycle.

Agar dextrose
Potato infusion 200 g
Dextrose 20.0 g
If 15.0 g
Purified water 1000 mL
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25ºC.
Esterilizar en un autoclave usando un ciclo validado.

Sabouraud's Dextrose Broth

Dextrose 20.0 g
Mix of digested peptide from animal tissue and 10.0 g
pancreatic digested casein (1:1)
Purified water 1000 mL
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25 °C.
Sterilize in an autoclave using a validated cycle.

Caldo Mossel para enriquecimiento de enterobacterias

Pancreatic gelatin digested 10.0 g
Monohydrate glucose 5.0 g
Dehydrated ox tripe 20.0 g
Monobasic potassium phosphate 2.0 g
Dihydrate sodium dibasic phosphate 8.0 g
Bright green 15 mg
Purified water 1000 mL
Adjust the pH to be 7.2 ± 0.2 at 25ºC after heating.
Heat to 100ºC for 30 minutes and cool immediately.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

If violet red bile glucose

Yeast extract 3.0 g
Pancreatic gelatin digested 7.0 g
Biliary sales 1.5 g
Sodium chloride 5.0 g
Monohydrate glucose 10.0 g
If 15.0 g
Neutral red 30 mg
Violet crystal 2 mg
Purified water 1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25ºC.
Calentar a ebullición; no calentar en un autoclave.

MacConkey broth
Pancreatic gelatin digested 20.0 g
Lactose monohydrate 10.0 g
Dehydrated ox tongue 5.0 g
Bromcresol purple 10 mg
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25ºC.
Sterilize in an autoclave using a validated cycle.

If MacConkey
Pancreatic gel digestion 17.0 g
Peptones (from meat and casein) 3.0 g
Lactose monohydrate 10.0 g
Sodium chloride 5.0 g
Biliary sales 1.5 g
If 13.5 g
Neutral red 30.0 mg
Violet crystal 1 mg
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.1 ± 0.2 at 25ºC.
Boil for 1 minute with constant stirring, and sterilize in an autoclave using a validated cycle.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

Rappaport Vassiliadis broth for enrichment of Salmonella

Soy peptone 4.5 g
Hexahydrate magnesium chloride 29.0 g
Sodium chloride 8.0 g
Dipotassium phosphate 0.4 g
Monobasic potassium phosphate 0.6 g
Malachite green 0.036 g
Purified water 1000 mL
Dissolve, gently heated. Sterilize in an autoclave using a validated cycle, at a
temperature not exceeding 115ºC. The pH should be 5.2 ± 0.2 at 25ºC after heating and
sterilization in autoclave.

If you are looking for, lysine, deoxycholate

Xylose 3.5 g
L-Lysine 5.0 g
Lactose monohydrate 7.5 g
Sucrose 7.5 g
Cloruro de sodio 5.0 g
Yeast extract 3.0 g
Phenol red 80 mg
If 13.5 g
Sodium deoxycholate 2.5 g
Sodium thiosulfate 6.8 g
Ammonium ferric citrate 0.8 g
Purified water 1000 mL
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25ºC. Heat to boiling,
cool to 50ºC and pour into Petri dishes. Do not heat in an autoclave.

If cetrimide
Pancreatic gel digested 20.0 g
Magnesium chloride 1.4 g
Potassium sulfate 10.0 g
Cetrimide 0.3 g
If 13.6 g
Purified water 1000 mL
Glycerol 10.0 mL
Heat to boiling for 1 minute with stirring. Adjust the pH so that after the
sterilization should be at 7.2 ± 0.2 at 25ºC. Sterilize in an autoclave using a validated cycle.

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European Pharmacopoeia 6.0 2.6.13 Microbiological examination of non-sterile products

If mannitol salt
Pancreatic casein digested 5.0 g
Digested peptic of animal tissue 5.0 g
Meat extract 1.0 g
DMannitol 10.0 g
Sodium chloride 75.0 g
If 15.0 g
Phenol red 0.025 g
Purified water 1000 mL
Heat to boiling for 1 minute while stirring. Adjust the pH so that after the
sterilization should be at 7.4 ± 0.2 at 25ºC. Sterilize in an autoclave using a validated cycle.

Reinforced medium for clostridia

meat extract 10.0 g
Peptone 10.0 g
Yeast extract 3.0 g
Soluble starch 1.0 g
Monohydrate glucose 5.0 g
Cysteine hydrochloride 0.5 g
Sodium chloride 5.0 g
Sodium acetate 3.0 g
If 0.5 g
Purified water 1000 mL
Hydrate the agar and dissolve by heating to boiling, stirring constantly. If it were
necessary, adjust the pH so that after sterilization it is 6.8 ± 0.2 at 25ºC. Sterilize in
a sterilizer using a validated cycle.

If Columbia
Pancreatic casein digested 10.0 g
Peptic meat digest 5.0 g
Pancreatic heart digested 3.0 g
Yeast extract 5.0 g
Corn starch 1.0 g
Sodium chloride 5.0 g
If, according to the gelling capacity 10.0 - 15.0 g
Agua purificada 1000 mL

Hydrate the agar and dissolve by heating until boiling and stirring constantly. If it were
necessary, adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25ºC. Sterilize in
an autoclave using a validated cycle. Allow to cool to 45-50ºC; add sulfate if necessary
of gentamicin corresponding to 20 mg of base gentamicin, and pour into Petri dishes.

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