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JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products 121
4.05 Microbiological Examination
of Non-sterile Products
This chapter includes microbial enumeration tests and
tests for specified micro-organisms. For the test, use a mixture of several portions selected at random from the bulk or
from the contents of a sufficient number of containers. If
test specimens are diluted with fluid medium, the test should
be performed quickly. In performing the test, precautions
must be taken to prevent biohazard.
I. Microbiological Examination of Non-sterile Products:
Microbial Enumeration Tests
These tests are harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia.
The tests described hereafter will allow quantitative
enumeration of mesophilic bacteria and fungi which may
grow under aerobic conditions.
The tests are designed primarily to determine whether a
substance or preparation complies with an established specification for microbiological quality. When used for such
purposes follow the instructions given below, including the
number of samples to be taken and interpret the results as
stated below.
The methods are not applicable to products containing
viable micro-organisms as active ingredients.
Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeial method has been demonstrated.
1. General Procedures
Carry out the determination under conditions designed to
avoid extrinsic microbial contamination of the product to be
examined. The precautions taken to avoid contamination
must be such that they do not affect any micro-organisms
which are to be revealed in the test.
If the product to be examined has antimicrobial activity,
this is insofar as possible removed or neutralized. If inactivators are used for this purpose their efficacy and their absence
of toxicity for micro-organisms must be demonstrated.
If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated.
2. Enumeration Methods
Use the membrane filtration method, or the plate-count
methods, as prescribed. The most probable number (MPN)
method is generally the least accurate method for microbial
counts, however, for certain product groups with very low
bioburden, it may be the most appropriate method.
The choice of a method is based on factors such as the nature of the product and the required limit of micro-organisms. The method chosen must allow testing of a sufficient
sample size to judge compliance with the specification. The
suitability of the chosen method must be established.
3. Growth Promotion Test, Suitability of the Counting
Method and Negative Controls
The ability of the test to detect micro-organisms in the
presence of product to be tested must be established.
Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of
the test is introduced.
3.1. Preparation of test strains
Use standardised stable suspensions of test strains or prepare as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages
removed from the original master seed-lot. Grow each of the
bacterial and fungal test strains separately as described in
Table 4.05-I-1.
Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions;
to suspend Aspergillus brasiliensis spores, 0.05 per cent of
polysorbate 80 may be added to the buffer. Use the suspensions within 2 hours or within 24 hours if stored at 2 – 89C.
As an alternative to preparing and then diluting a fresh
suspension of vegetative cells of Aspergillus brasiliensis or
Bacillus subtilis, a stable spore suspension is prepared and
then an appropriate volume of the spore suspension is used
for test inoculation. The stable spore suspension may be
maintained at 2 – 89C for a validated period of time.
3.2. Negative control
To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as
described under 4. Testing of Products. A failed negative
control requires an investigation.
3.3. Growth promotion of the media
Test each batch of ready-prepared medium and each batch
of medium, prepared either from dehydrated medium or
from the ingredients described.
Inoculate portions/plates of casein soya bean digest broth
and casein soya bean digest agar with a small number (not
more than 100 CFU) of the micro-organisms indicated in
Table 4.05-I-1, using a separate portion/plate of medium for
each. Inoculate plates of Sabouraud-dextrose agar with a
small number (not more than 100 CFU) of the micro-organisms indicated in Table 4.05-I-1, using a separate plate of
medium for each. Incubate in the conditions described in
Table 4.05-I-1.
For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum. For a freshly prepared inoculum, growth
of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible
growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch
of medium occurs.
3.4. Suitability of the counting method in the presence of
product
3.4.1. Preparation of the sample
The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the
procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed.
(i) 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 casein soya bean digest
broth. If necessary adjust to pH 6 – 8. Further dilutions,
where necessary, are prepared with the same diluent.
(ii) Non-fatty products insoluble in water: Suspend the
product to be examined (usually a 1 in 10 dilution is prepared) in buffered sodium chloride-peptone solution pH 7.0,
phosphate buffer solution pH 7.2 or casein soya bean digest
broth. A surface-active agent such as 1 g/L of polysorbate
80 may be added to assist the suspension of poorly wettable
substances. If necessary adjust to pH 6 – 8. Further dilutions, where necessary, are prepared with the same diluent.
(iii) Fatty products: Dissolve in isopropyl myristate,
sterilised by filtration or mix the product to be examined
122
Table 4.05-I-1 Preparation and use of test micro-organisms
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
with the minimum necessary quantity of sterile polysorbate
80 or another non-inhibitory sterile surface-active reagent,
heated if necessary to not more than 409C, or in exceptional
cases to not more than 459C. Mix carefully and if necessary
maintain the temperature in a water-bath. Add sufficient of
the pre-warmed chosen diluent to make a 1 in 10 dilution of
the original product. Mix carefully whilst maintaining the
temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be
prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active reagent.
(iv) Fluids or solids in aerosol form: Aseptically transfer
the product into a membrane filter apparatus or a sterile
container for further sampling. Use either the total contents
or a defined number of metered doses from each of the containers tested.
(v) Transdermal patches: Remove the protective cover
sheets (``release liner'') of the transdermal patches and place
them, adhesive side upwards, on sterile glass or plastic trays.
Cover the adhesive surface with sterile porous material, for
example sterile gauze, to prevent the patches from sticking
together, and transfer the patches to a suitable volume of the
chosen diluent containing inactivators such as polysorbate 80
and/or lecithin. Shake the preparation vigorously for at least
30 minutes.
3.4.2. Inoculation and dilution
Add to the sample prepared as described above (3.4.1.)
and to a control (with no test material included) a sufficient
volume of the microbial suspension to obtain an inoculum of
not more than 100 CFU. The volume of the suspension of
the inoculum should not exceed 1 per cent of the volume of
diluted product.
To demonstrate acceptable microbial recovery from the
product, the lowest possible dilution factor of the prepared
sample must be used for the test. Where this is not possible
due to antimicrobial activity or poor solubility, further
appropriate protocols must be developed. If inhibition of
123
Table 4.05-I-2 Common neutralizing agents for interfering substances
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products
growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension may be added after neutralization, dilution or filtration.
3.4.3. Neutralization/removal of antimicrobial activity
The number of micro-organisms recovered from the prepared sample diluted as described in 3.4.2. and incubated
following the procedure described in 3.4.4., is compared to
the number of micro-organisms recovered from the control
preparation.
If growth is inhibited (reduction by a factor greater than
2), then modify the procedure for the particular enumeration
test to ensure the validity of the results. Modification of the
procedure may include, for example, (1) an increase in the
volume of the diluent or culture medium, (2) incorporation
of a specific or general neutralizing agents into the diluent,
(3) membrane filtration or (4) a combination of the above
measures.
Neutralizing agents. Neutralizing agents may be used to
neutralize the activity of antimicrobial agents (Table
4.05-I-2). They may be added to the chosen diluent or the
medium preferably before sterilization. If used, their efficacy and their absence of toxicity for micro-organisms must
be demonstrated by carrying out a blank with neutralizer
and without product.
If no suitable neutralizing method can be found, it can be
assumed that the failure to isolate the inoculated organism is
attributable to the microbicidal activity of the product. This
information serves to indicate that the article is not likely to
be contaminated with the given species of the micro-organism. However, it is possible that the product only inhibits
some of the micro-organisms specified herein, but does not
inhibit others not included amongst the test strains or for
which the latter are not representative. Then, perform the
test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion.
3.4.4. Recovery of micro-organism in the presence of
product
For each of the micro-organisms listed in Table 4.05-I-1,
separate tests are performed. Only micro-organisms of the
added test strain are counted.
3.4.4.1. Membrane filtration
Use membrane filters having a nominal pore size not
greater than 0.45 mm. The type of filter material is chosen in
such a way that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For
each of the micro-organisms listed in Table 4.05-I-1, one
membrane filter is used.
Transfer a suitable amount of the sample prepared as
described under 3.4.1. to 3.4.3. (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 an appropriate volume of diluent.
For the determination of total aerobic microbial count
(TAMC), transfer the membrane filter to the surface of
casein soya bean digest agar. For the determination of total
combined yeasts/moulds count (TYMC) transfer the membrane to the surface of Sabouraud-dextrose agar. Incubate
the plates as indicated in Table 4.05-I-1. Perform the counting.
3.4.4.2. Plate-count methods
Perform plate-count methods at least in duplicate for each
medium and use the mean count of the result.
(i) Pour-plate method: For Petri dishes 9 cm in diameter,
add to the dish 1 mL of the sample prepared as described
under 3.4.1. to 3.4.3. and 15 – 20 mL of casein soya bean
digest agar or Sabouraud-dextrose agar, both media being at
not more than 459C. If larger Petri dishes are used, the
amount of agar medium is increased accordingly. For each
of the micro-organisms listed in Table 4.05-I-1, at least 2
Petri dishes are used.
Incubate the plates as indicated in Table 4.05-I-1. Take the
arithmetic mean of the counts per medium and calculate the
number of CFU in the original inoculum.
(ii) Surface-spread method: For Petri dishes 9 cm in diameter, add 15 – 20 mL of casein soya bean digest agar or
Sabouraud-dextrose agar at about 459C to each Petri dish
and allow to solidify. If larger Petri dishes are used, the
volume of the agar is increased accordingly. Dry the plates,
for example in a laminar-air-flow cabinet or in an incubator.
For each of the micro-organisms listed in Table 4.05-I-1, at
least 2 Petri dishes are used. Spread a measured volume of
not less than 0.1 mL of the sample prepared as described
under 3.4.1. to 3.4.3. over the surface of the medium. Incubate and count as prescribed under 3.4.4.2. (i).
3.4.4.3. Most-probable-number (MPN) method
The precision and accuracy of the MPN method is less
than that of the membrane filtration method or the platecount method. Unreliable results are obtained particularly
for the enumeration of moulds. For these reasons the MPN
method is reserved for the enumeration of TAMC in situations where no other method is available. If the use of the
method is justified, proceed as follows.
124
Table 4.05-I-3 Most-probable-number values of micro-organisms
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
Prepare a series of at least 3 serial tenfold dilutions of the
product as described under 3.4.1. to 3.4.3.. From each level
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3
tubes with 9 – 10 mL of casein soya bean digest broth. If
necessary a surface-active agent such as polysorbate 80, or
an inactivator of antimicrobial agents may be added to the
medium. Thus, if 3 levels of dilution are prepared 9 tubes
are inoculated.
Incubate all tubes at 30 – 359C for not more than 3 days.
If reading of the results is difficult or uncertain owing to the
nature of the product to be examined, subculture in the same
broth, or casein soya bean digest agar, for 1 – 2 days at the
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products 125
same temperature and use these results. Determine the most
probable number of micro-organisms per gram or millilitre
of the product to be examined from Table 4.05-I-3.
3.5. Results and interpretation
When verifying the suitability of the membrane filtration
method or the plate-count method, a mean count of any of
the test organisms not differing by a factor greater than 2
from the value of the control defined in 3.4.2. in the absence
of the product must be obtained. When verifying the suitability of the MPN method the calculated value from the inoculum must be within 95 per cent confidence limits of the
results obtained with the control.
If the above criteria cannot be met for one or more of the
organisms tested with any of the described methods, the
method and test conditions that come closest to the criteria
are used to test the product.
4. Testing of Products
4.1. Amount used for the test
Unless otherwise prescribed, use 10 g or 10 mL of the
product to be examined taken with the precautions referred
to above. For fluids or solids in aerosol form, sample 10
containers. For transdermal patches, sample 10 patches.
The amount to be tested may be reduced for active substances that will be formulated in the following conditions:
the amount per dosage unit (e.g. tablet, capsule, injection) is
less than or equal to 1 mg or the amount per gram or millilitre (for preparations not presented in dose units) is less
than 1 mg. In these cases, the amount of sample to be tested
is not less than the amount present in 10 dosage units or 10 g
or 10 mL of the product.
For materials used as active substances where sample
quantity is limited or batch size is extremely small (i.e. less
than 1000 mL or 1000 g), the amount tested shall be 1 per
cent of the batch unless a lesser amount is prescribed or
justified and authorised.
For products where the total number of entities in a batch
is less than 200 (e.g. samples used in clinical trials), the sample size may be reduced to 2 units, or 1 unit if the size is less
than 100.
Select the sample(s) at random from the bulk material or
from the available containers of the preparation. To obtain
the required quantity, mix the contents of a sufficient number of containers to provide the sample.
4.2. Examination of the product
4.2.1. Membrane filtration
Use a filtration apparatus designed to allow the transfer of
the filter to the medium. Prepare the sample using a method
that has been shown suitable as described in section 3 and
transfer the appropriate amount to each of 2 membrane
filters and filter immediately. Wash each filter following the
procedure shown to be suitable.
For the determination of TAMC, transfer one of the
membrane filters to the surface of casein soya bean digest
agar. For the determination of TYMC, transfer the other
membrane to the surface of Sabouraud-dextrose agar. Incubate the plate of casein soya bean digest agar at 30 – 359C
for 3 – 5 days and the plate of Sabouraud-dextrose agar at
20 – 259C for 5 – 7 days. Calculate the number of CFU per
gram or per millilitre of product.
When examining transdermal patches, filter 10 per cent of
the volume of the preparation described under 3.4.1. separately through each of 2 sterile filter membranes. Transfer
one membrane to casein soya bean digest agar for TAMC
and the other membrane to Sabouraud-dextrose agar for
TYMC.
4.2.2. Plate-count methods
(i) Pour-plate method: Prepare the sample using a
method that has been shown to be suitable as described in
section 3. Prepare for each medium at least 2 Petri dishes for
each level of dilution. Incubate the plates of casein soya bean
digest agar at 30 – 359C for 3 – 5 days and the plates of
Sabouraud-dextrose agar at 20 – 259C for 5 – 7 days. Select
the plates corresponding to a given dilution and showing the
highest number of colonies less than 250 for TAMC and 50
for TYMC. Take the arithmetic mean per culture medium of
the counts and calculate the number of CFU per gram or per
millilitre of product.
(ii) Surface-spread method: Prepare the sample using a
method that has been shown to be suitable as described in
section 3. Prepare at least 2 Petri dishes for each medium
and each level of dilution. For incubation and calculation of
the number of CFU proceed as described for the pour-plate
method.
4.2.3. Most-probable-number method
Prepare and dilute the sample using a method that has
been shown to be suitable as described in section 3. Incubate
all tubes for 3 – 5 days at 30 – 359C. Subculture if necessary,
using the procedure shown to be suitable. Record for each
level of dilution the number of tubes showing microbial
growth. Determine the most probable number of microorganisms per gram or millilitre of the product to be examined from Table 4.05-I-3.
4.3. Interpretation of the results
The total aerobic microbial count (TAMC) is considered
to be equal to the number of CFU found using casein soya
bean digest agar; if colonies of fungi are detected on this medium, they are counted as part of TAMC. The total combined yeasts/mould count (TYMC) is considered to be equal
to the number of CFU found using Sabouraud-dextrose
agar; if colonies of bacteria are detected on this medium,
they are counted as part of TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial
growth, Sabouraud-dextrose agar containing antibiotics may
be used. If the count is carried out by the MPN method the
calculated value is the TAMC.
When an acceptance criterion for microbiological quality
is prescribed it is interpreted as follows:
－ 101 CFU: maximum acceptable count ＝ 20,
－ 102 CFU: maximum acceptable count ＝ 200,
－ 103 CFU: maximum acceptable count ＝ 2000,
and so forth.
The recommended solutions and media are described in II.
Tests for specified micro-organisms.
II. Microbiological Examination of Non-sterile Products:
Tests for Specified Micro-organisms
These tests are harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia.
The tests described hereafter will allow determination of
the absence or limited occurrence of specified microorganisms which may be detected under the conditions
described.
The tests are designed primarily to determine whether a
substance or preparation complies with an established specification for microbiological quality. When used for such
purposes follow the instructions given below, including the
number of samples to be taken and interpret the results as
stated below.
Alternative microbiological procedures, including automated methods may be used, provided that their equivalence
to the Pharmacopoeial method has been demonstrated.
126 4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
1. General Procedures
The preparation of samples is carried out as described in I.
Microbial enumeration tests.
If the product to be examined has antimicrobial activity,
this is insofar as possible removed or neutralized as
described in I. Microbial enumeration tests.
If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated
as described in I. Microbial enumeration tests.
2. Growth Promoting and Inhibitory Properties of the
Media, Suitability of the Test and Negative Controls
The ability of the test to detect micro-organisms in the
presence of the product to be tested must be established.
Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of
the test is introduced.
2.1. Preparation of test strains
Use standardized stable suspensions of test strains or prepare as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages
removed from the original master seed-lot.
2.1.1. Aerobic micro-organisms
Grow each of the bacterial test strains separately in containers containing casein soya bean digest broth or on casein
soya bean digest agar at 30 – 359C for 18 – 24 hours. Grow
the test strain for Candida albicans separately on
Sabouraud-dextrose agar or in Sabouraud-dextrose broth at
20 – 259C for 2–3 days.
Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83 or NBRC 13276,
Pseudomonas aeruginosa such as ATCC 9027, NCIMB
8626, CIP 82.118 or NBRC 13275,
Escherichia coli such as ATCC 8739, NCIMB 8545, CIP
53.126 or NBRC 3972,
Salmonella enterica subsp. enterica serovar Typhimurium
such as ATCC 14028
or, as an alternative,
Salmonella enterica subsp. enterica serovar Abony such as
NBRC 100797, NCTC 6017 or CIP 80.39,
Candida albicans such as ATCC 10231, NCPF 3179, IP
48.72 or NBRC 1594.
Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions.
Use the suspensions within 2 hours or within 24 hours if
stored at 2 – 89C.
2.1.2. Clostridia
Use Clostridium sporogenes such as ATCC 11437 (NBRC
14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC
532 or CIP 79.3). Grow the clostridial test strain under
anaerobic conditions in reinforced medium for Clostridia at
30 – 359C for 24 – 48 hours. As an alternative to preparing
and then diluting down a fresh suspension of vegetative cells
of Cl. sporogenes, a stable spore suspension is used for test
inoculation. The stable spore suspension may be maintained
at 2 – 89C for a validated period.
2.2. Negative control
To verify testing conditions a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as
described under 3. A failed negative control required an investigation.
2.3. Growth promotion and inhibitory properties of the
media
Test each batch of ready-prepared medium and each batch
of medium prepared either from dehydrated medium or
from ingredients.
Verify suitable properties of relevant media as described in
Table 4.05-II-1.
(i) Test for growth promoting properties, liquid media:
inoculate a portion of the appropriate medium with a small
number (not more than 100 CFU) of the appropriate microorganism. Incubate at the specified temperature for not
more than the shortest period of time specified in the test.
Clearly visible growth of the micro-organism comparable to
that previously obtained with a previously tested and approved batch of medium occurs.
(ii) Test for growth promoting properties, solid media:
perform surface-spread method, inoculating each plate with
a small number (not more than 100 CFU) of the appropriate
micro-organism. Incubate at the specified temperature for
not more than the shortest period of time specified in the
test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch
of medium occurs.
(iii) Test for inhibitory properties, liquid or solid media:
inoculate the appropriate medium with at least 100 CFU of
the appropriate micro-organism. Incubate at the specified
temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.
(iv) Test for indicative properties: perform surfacespread method, inoculating each plate with a small number
(not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of
time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch
of medium.
2.4. Suitability of the test method
For each product to be tested perform sample preparation
as described in the relevant paragraph in section 3. Add each
test strain at the time of mixing, in the prescribed growth
medium. Inoculate the test strains individually. Use a number of micro-organisms equivalent to not more than 100
CFU in the inoculated test preparation.
Perform the test as described in the relevant paragraph in
section 3 using the shortest incubation period prescribed.
The specified micro-organisms must be detected with the
indication reactions as described in section 3.
Any antimicrobial activity of the product necessitates a
modification of the test procedure (see 3.4.3. of I. Microbial
enumeration tests).
If for a given product the antimicrobial activity with
respect to a micro-organism for which testing is prescribed
cannot be neutralized, then it is to be assumed that the inhibited micro-organism will not be present in the product.
3. Testing of Products
3.1. Bile-tolerant gram-negative bacteria
3.1.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests, but using casein soya bean digest broth
as the chosen diluent, mix and incubate at 20 – 259C for a
time sufficient to resuscitate the bacteria but not sufficient to
encourage multiplication of the organisms (usually 2 hours
but not more than 5 hours).
127
Table 4.05-II-1 Growth promoting, inhibitory and indicative properties of media
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products
3.1.2. Test for absence
Unless otherwise prescribed use the volume corresponding
to 1 g of the product, as prepared in 3.1.1. to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30 – 359C
for 24 – 48 hours. Subculture on plates of violet red bile glucose agar. Incubate at 30 – 359C for 18 – 24 hours.
The product complies with the test if there is no growth of
colonies.
3.1.3. Quantitative test
3.1.3.1. Selection and subculture
Inoculate suitable quantities of enterobacteria enrichment
broth-Mossel with the preparation as described under 3.1.1.
and/or 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 30 – 359C for 24 – 48
hours. Subculture each of the cultures on a plate of violet
red bile glucose agar. Incubate at 30 – 359C for 18 – 24
128
Table 4.05-II-2 Interpretation of results
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
hours.
3.1.3.2. Interpretation
Growth of colonies constitutes a positive result. Note the
smallest quantity of the product that gives a positive result
and the largest quantity that gives a negative result. Determine from Table 4.05-II-2 the probable number of bacteria.
3.2. Escherichia coli
3.2.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth, mix and incubate at 30 – 359C for 18 – 24
hours.
3.2.2. Selection and subculture
Shake the container, transfer 1 mL of casein soya bean
digest broth to 100 mL of MacConkey broth and incubate at
42 – 449C for 24 – 48 hours. Subculture on a plate of MacConkey agar at 30 – 359C for 18 – 72 hours.
3.2.3. Interpretation
Growth of colonies indicates the possible presence of
E. coli. This is confirmed by identification tests.
The product complies with the test if no colonies are
present or if the identification tests are negative.
3.3. Salmonella
3.3.1. Sample preparation and pre-incubation
Prepare the product to be examined as described in I.
Microbial enumeration tests and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 2.4.) of casein
soya bean digest broth, mix and incubate at 30 – 359C for
18 – 24 hours.
3.3.2. Selection and subculture
Transfer 0.1 mL of casein soya bean digest broth to 10 mL
of Rappaport Vassiliadis Salmonella enrichment broth and
incubate at 30 – 359C for 18 – 24 hours. Subculture on plates
of xylose, lysine, deoxycholate agar. Incubate at 30 – 359C
for 18 – 48 hours.
3.3.3. Interpretation
The possible presence of Salmonella is indicated by the
growth of well-developed, red colonies, with or without
black centres. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.4. Pseudomonas aeruginosa
3.4.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth and mix. When testing transdermal patches,
filter the volume of sample corresponding to 1 patch of the
preparation described in I. Microbial enumeration tests
(3.4.1.) through a sterile filter membrane and place in 100
mL of casein soya bean digest broth. Incubate at 30 – 359C
for 18 – 24 hours.
3.4.2. Selection and subculture
Subculture on a plate of cetrimide agar and incubate at
30 – 359C for 18 – 72 hours.
3.4.3. Interpretation
Growth of colonies indicates the possible presence of
P. aeruginosa. This is confirmed by identification tests.
The product complies with the test if colonies are not
present or if the confirmatory identification tests are negative.
3.5. Staphylococcus aureus
3.5.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth and homogenise. When testing transdermal
patches, filter the volume of sample corresponding to 1
patch of the preparation described in I. Microbial enumeration tests (3.4.1.) through a sterile filter membrane and place
in 100 mL of casein soya bean digest broth. Incubate at 30 –
359C for 18 – 24 hours.
3.5.2. Selection and subculture
Subculture on a plate of mannitol salt agar and incubate at
30 – 359C for 18 – 72 hours.
3.5.3. Interpretation
The possible presence of S. aureus is indicated by the
growth of yellow/white colonies surrounded by a yellow
zone. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.6. Clostridia
3.6.1. Sample preparation and heat treatment
Prepare a sample using a 1 in 10 dilution (with a minimum
total volume of 20 mL) of not less than 2 g or 2 mL of the
product to be examined as described in I. Microbial enumeration tests. Divide the sample into two portions of at least 10
mL. Heat 1 portion at 809C for 10 minutes and cool rapidly.
Do not heat the other portion.
3.6.2. Selection and subculture
Use 10 mL or the quantity corresponding to 1 g or 1 mL of
the product to be examined of both portions to inoculate
suitable amounts (determined as described under 2.4.) of
reinforced medium for Clostridia. Incubate under anaerobic
conditions at 30 – 359C for 48 hours. After incubation,
make subcultures from each tube on Columbia agar and
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products 129
incubate under anaerobic conditions at 30 – 359C for 48 – 72
hours.
3.6.3. Interpretation
The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates
the presence of Clostridia. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.7. Candida albicans
3.7.1. Sample preparation and pre-incubation
Prepare the product to be examined as described in I.
Microbial enumeration tests and use 10 mL or the quantity
corresponding to not less than 1 g or 1 mL to inoculate 100
mL of Sabouraud-dextrose broth and mix. Incubate at 30 –
359C for 3-5 days.
3.7.2. Selection and subculture
Subculture on a plate of Sabouraud-dextrose agar and incubate at 30 – 359C for 24 – 48 hours.
3.7.3. Interpretation
Growth of white colonies may indicate the presence of C.
albicans. This is confirmed by identification tests.
The product complies with the test if such colonies are not
present or if the confirmatory identification tests are negative.
The following section is given for information.
4. Recommended Solutions and Culture Media
The following solutions and culture media have been
found satisfactory for the purposes for which they are
prescribed in the test for microbial contamination in the
Pharmacopoeia. Other media may be used provided that
their suitability can be demonstrated.
(i) Phosphate buffer solution pH 7.2
Prepare a mixture of water and stock buffer solution (800:1
V/V) and sterilize.
Stock buffer solution. Transfer 34 g of potassium dihydrogen phosphate to a 1000 mL volumetric flask, dissolve in 500
mL of purified water, adjust to pH 7.2 to ± 0.2 with sodium
hydroxide, add purified water to volume and mix. Dispense
in containers and sterilize. Store at a temperature of 2 – 89C.
(equivalent to 0.067 mol phosphate)
Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after heating it is 7.2 ± 0.2 at 259C.
Heat at 1009C for 30 minutes and cool immediately.
Adjust the pH so that after heating it is 7.4 ± 0.2 at 259C.
Heat to boiling; do not heat in an autoclave.
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
130 4.06 Sterility Test / General Tests JP XVII
Adjust the pH so that after sterilization it is 7.1 ± 0.2 at
259C. Boil for 1 minute with constant shaking then sterilize
in an autoclave using a validated cycle.
Dissolve, warming slightly. Sterilize in an autoclave using
a validated cycle, at a temperature not exceeding 1159C. The
pH is to be 5.2 ± 0.2 at 259C after heating and autoclaving.
Adjust the pH so that after heating it is 7.4 ± 0.2 at 259C.
Heat to boiling, cool to 509C and pour into Petri dishes. Do
not heat in an autoclave.
Heat to boiling for 1 minute with shaking. Adjust the pH
so that after sterilization it is 7.2 ± 0.2 at 259C. Sterilize in
an autoclave using a validated cycle.
Heat to boiling for 1 minute with shaking. Adjust the pH
so that after sterilization it is 7.4 ± 0.2 at 259C. Sterilize in
an autoclave using a validated cycle.
(xvi) Reinforced medium for Clostridia
Beef extract 10.0 g
Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after
sterilization it is about 6.8 ± 0.2 at 259C. Sterilize in an autoclave using a validated cycle.
Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after
sterilization it is 7.3 ± 0.2 at 259C. Sterilize in an autoclave
using a validated cycle. Allow to cool to 45 – 509C; add,
where necessary, gentamicin sulfate corresponding to 20 mg
of gentamicin base and pour into Petri dishes.
4.06 Sterility Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( ).
The test is applied to substances, preparations or articles
which, according to the Pharmacopoeia, are required to be
sterile. However, a satisfactory result only indicates that no
contaminating micro-organism has been found in the sample
examined in the conditions of the test.
1. Precautions against microbial contamination
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test
is performed. The precautions taken to avoid contamination
are such that they do not affect any micro-organisms which
are to be revealed in the test. The working conditions in
which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out
appropriate controls.
2. Culture media and incubation temperatures
Media for the test may be prepared as described below, or
equivalent commercial media may be used provided that they
comply with the growth promotion test.
The following culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium is
primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soya-bean
casein digest medium is suitable for the culture of both fungi
and aerobic bacteria.
4.05 Microbiological Examination
of Non-sterile Products
This chapter includes microbial enumeration tests and
tests for specified micro-organisms. For the test, use a mixture of several portions selected at random from the bulk or
from the contents of a sufficient number of containers. If
test specimens are diluted with fluid medium, the test should
be performed quickly. In performing the test, precautions
must be taken to prevent biohazard.
I. Microbiological Examination of Non-sterile Products:
Microbial Enumeration Tests
These tests are harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia.
The tests described hereafter will allow quantitative
enumeration of mesophilic bacteria and fungi which may
grow under aerobic conditions.
The tests are designed primarily to determine whether a
substance or preparation complies with an established specification for microbiological quality. When used for such
purposes follow the instructions given below, including the
number of samples to be taken and interpret the results as
stated below.
The methods are not applicable to products containing
viable micro-organisms as active ingredients.
Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeial method has been demonstrated.
1. General Procedures
Carry out the determination under conditions designed to
avoid extrinsic microbial contamination of the product to be
examined. The precautions taken to avoid contamination
must be such that they do not affect any micro-organisms
which are to be revealed in the test.
If the product to be examined has antimicrobial activity,
this is insofar as possible removed or neutralized. If inactivators are used for this purpose their efficacy and their absence
of toxicity for micro-organisms must be demonstrated.
If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated.
2. Enumeration Methods
Use the membrane filtration method, or the plate-count
methods, as prescribed. The most probable number (MPN)
method is generally the least accurate method for microbial
counts, however, for certain product groups with very low
bioburden, it may be the most appropriate method.
The choice of a method is based on factors such as the nature of the product and the required limit of micro-organisms. The method chosen must allow testing of a sufficient
sample size to judge compliance with the specification. The
suitability of the chosen method must be established.
3. Growth Promotion Test, Suitability of the Counting
Method and Negative Controls
The ability of the test to detect micro-organisms in the
presence of product to be tested must be established.
Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of
the test is introduced.
3.1. Preparation of test strains
Use standardised stable suspensions of test strains or prepare as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages
removed from the original master seed-lot. Grow each of the
bacterial and fungal test strains separately as described in
Table 4.05-I-1.
Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions;
to suspend Aspergillus brasiliensis spores, 0.05 per cent of
polysorbate 80 may be added to the buffer. Use the suspensions within 2 hours or within 24 hours if stored at 2 – 89C.
As an alternative to preparing and then diluting a fresh
suspension of vegetative cells of Aspergillus brasiliensis or
Bacillus subtilis, a stable spore suspension is prepared and
then an appropriate volume of the spore suspension is used
for test inoculation. The stable spore suspension may be
maintained at 2 – 89C for a validated period of time.
3.2. Negative control
To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as
described under 4. Testing of Products. A failed negative
control requires an investigation.
3.3. Growth promotion of the media
Test each batch of ready-prepared medium and each batch
of medium, prepared either from dehydrated medium or
from the ingredients described.
Inoculate portions/plates of casein soya bean digest broth
and casein soya bean digest agar with a small number (not
more than 100 CFU) of the micro-organisms indicated in
Table 4.05-I-1, using a separate portion/plate of medium for
each. Inoculate plates of Sabouraud-dextrose agar with a
small number (not more than 100 CFU) of the micro-organisms indicated in Table 4.05-I-1, using a separate plate of
medium for each. Incubate in the conditions described in
Table 4.05-I-1.
For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum. For a freshly prepared inoculum, growth
of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible
growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch
of medium occurs.
3.4. Suitability of the counting method in the presence of
product
3.4.1. Preparation of the sample
The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the
procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed.
(i) 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 casein soya bean digest
broth. If necessary adjust to pH 6 – 8. Further dilutions,
where necessary, are prepared with the same diluent.
(ii) Non-fatty products insoluble in water: Suspend the
product to be examined (usually a 1 in 10 dilution is prepared) in buffered sodium chloride-peptone solution pH 7.0,
phosphate buffer solution pH 7.2 or casein soya bean digest
broth. A surface-active agent such as 1 g/L of polysorbate
80 may be added to assist the suspension of poorly wettable
substances. If necessary adjust to pH 6 – 8. Further dilutions, where necessary, are prepared with the same diluent.
(iii) Fatty products: Dissolve in isopropyl myristate,
sterilised by filtration or mix the product to be examined
122
Table 4.05-I-1 Preparation and use of test micro-organisms
| Micro-organism | Preparation of test strain | Growth promotion |
Suitability of counting method in the presence of the product |
||
|
Total aerobic microbial count |
Total yeasts and moulds count |
Total aerobic microbial count |
Total yeasts and moulds count |
||
|
Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276 |
Casein soya bean digest agar or casein soya bean digest broth 30 – 359C 18 – 24 hours |
Casein soya bean digest agar and casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
Casein soya bean digest agar/MPN casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
||
|
Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275 |
Casein soya bean digest agar or casein soya bean digest broth 30 – 359C 18 – 24 hours |
Casein soya bean digest agar and casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
Casein soya bean digest agar/MPN casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
||
|
Bacillus subtilis such as ATCC 6633, NCIMB 8054, CIP 52.62 or NBRC 3134 |
Casein soya bean digest agar or casein soya bean digest broth 30 – 359C 18 – 24 hours |
Casein soya bean digest agar and casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
Casein soya bean digest agar/MPN casein soya bean digest broth ≦ 100 CFU 30 – 359C ≦ 3 days |
||
|
Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594 |
Sabouraud-dextrose agar or Sabouraud dextrose broth 20 – 259C 2 – 3 days |
Casein soya bean digest agar ≦ 100 CFU 30 – 359C ≦ 5 days |
Sabouraud-dex trose agar ≦ 100 CFU 20 – 259C ≦ 5 days |
Casein soya bean digest agar ≦ 100 CFU 30 – 359C ≦ 5 days MPN: not applicable |
Sabouraud-dex trose agar ≦ 100 CFU 20 – 259C ≦ 5 days |
|
Aspergillus brasiliensis such as ATCC 16404, IMI 149007, IP 1431.83 or NBRC 9455 |
Sabouraud-dextrose agar or potato-dex trose agar 20 – 259C 5 – 7 days, or until good sporula tion is achieved |
Casein soya bean digest agar ≦ 100 CFU 30 – 359C ≦ 5 days |
Sabouraud-dex trose agar ≦ 100 CFU 20 – 259C ≦ 5 days |
Casein soya bean digest agar ≦ 100 CFU 30 – 359C ≦ 5 days MPN: not applicable |
Sabouraud-dex trose agar ≦ 100 CFU 20 – 259C ≦ 5 days |
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
with the minimum necessary quantity of sterile polysorbate
80 or another non-inhibitory sterile surface-active reagent,
heated if necessary to not more than 409C, or in exceptional
cases to not more than 459C. Mix carefully and if necessary
maintain the temperature in a water-bath. Add sufficient of
the pre-warmed chosen diluent to make a 1 in 10 dilution of
the original product. Mix carefully whilst maintaining the
temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be
prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active reagent.
(iv) Fluids or solids in aerosol form: Aseptically transfer
the product into a membrane filter apparatus or a sterile
container for further sampling. Use either the total contents
or a defined number of metered doses from each of the containers tested.
(v) Transdermal patches: Remove the protective cover
sheets (``release liner'') of the transdermal patches and place
them, adhesive side upwards, on sterile glass or plastic trays.
Cover the adhesive surface with sterile porous material, for
example sterile gauze, to prevent the patches from sticking
together, and transfer the patches to a suitable volume of the
chosen diluent containing inactivators such as polysorbate 80
and/or lecithin. Shake the preparation vigorously for at least
30 minutes.
3.4.2. Inoculation and dilution
Add to the sample prepared as described above (3.4.1.)
and to a control (with no test material included) a sufficient
volume of the microbial suspension to obtain an inoculum of
not more than 100 CFU. The volume of the suspension of
the inoculum should not exceed 1 per cent of the volume of
diluted product.
To demonstrate acceptable microbial recovery from the
product, the lowest possible dilution factor of the prepared
sample must be used for the test. Where this is not possible
due to antimicrobial activity or poor solubility, further
appropriate protocols must be developed. If inhibition of
123
Table 4.05-I-2 Common neutralizing agents for interfering substances
| Interfering substance | Potential neutralizing method |
| Glutaraldehyde, Mercurials | Sodium hydrogen sulfite (Sodium bisulfite) |
| Phenolics, Alcohol, Aldehydes, Sorbate | Dilution |
| Aldehydes | Glycine |
|
Quaternary Ammonium Compounds (QACs), Parahydroxybenzoates (Parabens), Bis-biguanides |
Lecithin |
| QAC, Iodine, Parabens | Polysorbate |
| Mercurials | Thioglycollate |
| Mercurials, Halogens, Aldehydes | Thiosulfate |
| EDTA (edetate) | Mg or Ca ions |
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products
growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension may be added after neutralization, dilution or filtration.
3.4.3. Neutralization/removal of antimicrobial activity
The number of micro-organisms recovered from the prepared sample diluted as described in 3.4.2. and incubated
following the procedure described in 3.4.4., is compared to
the number of micro-organisms recovered from the control
preparation.
If growth is inhibited (reduction by a factor greater than
2), then modify the procedure for the particular enumeration
test to ensure the validity of the results. Modification of the
procedure may include, for example, (1) an increase in the
volume of the diluent or culture medium, (2) incorporation
of a specific or general neutralizing agents into the diluent,
(3) membrane filtration or (4) a combination of the above
measures.
Neutralizing agents. Neutralizing agents may be used to
neutralize the activity of antimicrobial agents (Table
4.05-I-2). They may be added to the chosen diluent or the
medium preferably before sterilization. If used, their efficacy and their absence of toxicity for micro-organisms must
be demonstrated by carrying out a blank with neutralizer
and without product.
If no suitable neutralizing method can be found, it can be
assumed that the failure to isolate the inoculated organism is
attributable to the microbicidal activity of the product. This
information serves to indicate that the article is not likely to
be contaminated with the given species of the micro-organism. However, it is possible that the product only inhibits
some of the micro-organisms specified herein, but does not
inhibit others not included amongst the test strains or for
which the latter are not representative. Then, perform the
test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion.
3.4.4. Recovery of micro-organism in the presence of
product
For each of the micro-organisms listed in Table 4.05-I-1,
separate tests are performed. Only micro-organisms of the
added test strain are counted.
3.4.4.1. Membrane filtration
Use membrane filters having a nominal pore size not
greater than 0.45 mm. The type of filter material is chosen in
such a way that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For
each of the micro-organisms listed in Table 4.05-I-1, one
membrane filter is used.
Transfer a suitable amount of the sample prepared as
described under 3.4.1. to 3.4.3. (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 an appropriate volume of diluent.
For the determination of total aerobic microbial count
(TAMC), transfer the membrane filter to the surface of
casein soya bean digest agar. For the determination of total
combined yeasts/moulds count (TYMC) transfer the membrane to the surface of Sabouraud-dextrose agar. Incubate
the plates as indicated in Table 4.05-I-1. Perform the counting.
3.4.4.2. Plate-count methods
Perform plate-count methods at least in duplicate for each
medium and use the mean count of the result.
(i) Pour-plate method: For Petri dishes 9 cm in diameter,
add to the dish 1 mL of the sample prepared as described
under 3.4.1. to 3.4.3. and 15 – 20 mL of casein soya bean
digest agar or Sabouraud-dextrose agar, both media being at
not more than 459C. If larger Petri dishes are used, the
amount of agar medium is increased accordingly. For each
of the micro-organisms listed in Table 4.05-I-1, at least 2
Petri dishes are used.
Incubate the plates as indicated in Table 4.05-I-1. Take the
arithmetic mean of the counts per medium and calculate the
number of CFU in the original inoculum.
(ii) Surface-spread method: For Petri dishes 9 cm in diameter, add 15 – 20 mL of casein soya bean digest agar or
Sabouraud-dextrose agar at about 459C to each Petri dish
and allow to solidify. If larger Petri dishes are used, the
volume of the agar is increased accordingly. Dry the plates,
for example in a laminar-air-flow cabinet or in an incubator.
For each of the micro-organisms listed in Table 4.05-I-1, at
least 2 Petri dishes are used. Spread a measured volume of
not less than 0.1 mL of the sample prepared as described
under 3.4.1. to 3.4.3. over the surface of the medium. Incubate and count as prescribed under 3.4.4.2. (i).
3.4.4.3. Most-probable-number (MPN) method
The precision and accuracy of the MPN method is less
than that of the membrane filtration method or the platecount method. Unreliable results are obtained particularly
for the enumeration of moulds. For these reasons the MPN
method is reserved for the enumeration of TAMC in situations where no other method is available. If the use of the
method is justified, proceed as follows.
124
Table 4.05-I-3 Most-probable-number values of micro-organisms
|
Observed combinations of numbers of tubes showing growth in each set |
MPN per g or per mL of product |
95 per cent confidence limits |
||
| Number of g or mL of product per tube | ||||
| 0.1 | 0.01 | 0.001 | ||
| 0 | 0 | 0 | Less than 3 | 0 – 9.4 |
| 0 | 0 | 1 | 3 | 0.1 – 9.5 |
| 0 | 1 | 0 | 3 | 0.1 – 10 |
| 0 | 1 | 1 | 6.1 | 1.2 – 17 |
| 0 | 2 | 0 | 6.2 | 1.2 – 17 |
| 0 | 3 | 0 | 9.4 | 3.5 – 35 |
| 1 | 0 | 0 | 3.6 | 0.2 – 17 |
| 1 | 0 | 1 | 7.2 | 1.2 – 17 |
| 1 | 0 | 2 | 11 | 4 – 35 |
| 1 | 1 | 0 | 7.4 | 1.3 – 20 |
| 1 | 1 | 1 | 11 | 4 – 35 |
| 1 | 2 | 0 | 11 | 4 – 35 |
| 1 | 2 | 1 | 15 | 5 – 38 |
| 1 | 3 | 0 | 16 | 5 – 38 |
| 2 | 0 | 0 | 9.2 | 1.5 – 35 |
| 2 | 0 | 1 | 14 | 4 – 35 |
| 2 | 0 | 2 | 20 | 5 – 38 |
| 2 | 1 | 0 | 15 | 4 – 38 |
| 2 | 1 | 1 | 20 | 5 – 38 |
| 2 | 1 | 2 | 27 | 9 – 94 |
| 2 | 2 | 0 | 21 | 5 – 40 |
| 2 | 2 | 1 | 28 | 9 – 94 |
| 2 | 2 | 2 | 35 | 9 – 94 |
| 2 | 3 | 0 | 29 | 9 – 94 |
| 2 | 3 | 1 | 36 | 9 – 94 |
| 3 | 0 | 0 | 23 | 5 – 94 |
| 3 | 0 | 1 | 38 | 9 – 104 |
| 3 | 0 | 2 | 64 | 16 – 181 |
| 3 | 1 | 0 | 43 | 9 – 181 |
| 3 | 1 | 1 | 75 | 17 – 199 |
| 3 | 1 | 2 | 120 | 30 – 360 |
| 3 | 1 | 3 | 160 | 30 – 380 |
| 3 | 2 | 0 | 93 | 18 – 360 |
| 3 | 2 | 1 | 150 | 30 – 380 |
| 3 | 2 | 2 | 210 | 30 – 400 |
| 3 | 2 | 3 | 290 | 90 – 990 |
| 3 | 3 | 0 | 240 | 40 – 990 |
| 3 | 3 | 1 | 460 | 90 – 1980 |
| 3 | 3 | 2 | 1100 | 200 – 4000 |
| 3 | 3 | 3 | More than 1100 |
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
Prepare a series of at least 3 serial tenfold dilutions of the
product as described under 3.4.1. to 3.4.3.. From each level
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3
tubes with 9 – 10 mL of casein soya bean digest broth. If
necessary a surface-active agent such as polysorbate 80, or
an inactivator of antimicrobial agents may be added to the
medium. Thus, if 3 levels of dilution are prepared 9 tubes
are inoculated.
Incubate all tubes at 30 – 359C for not more than 3 days.
If reading of the results is difficult or uncertain owing to the
nature of the product to be examined, subculture in the same
broth, or casein soya bean digest agar, for 1 – 2 days at the
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products 125
same temperature and use these results. Determine the most
probable number of micro-organisms per gram or millilitre
of the product to be examined from Table 4.05-I-3.
3.5. Results and interpretation
When verifying the suitability of the membrane filtration
method or the plate-count method, a mean count of any of
the test organisms not differing by a factor greater than 2
from the value of the control defined in 3.4.2. in the absence
of the product must be obtained. When verifying the suitability of the MPN method the calculated value from the inoculum must be within 95 per cent confidence limits of the
results obtained with the control.
If the above criteria cannot be met for one or more of the
organisms tested with any of the described methods, the
method and test conditions that come closest to the criteria
are used to test the product.
4. Testing of Products
4.1. Amount used for the test
Unless otherwise prescribed, use 10 g or 10 mL of the
product to be examined taken with the precautions referred
to above. For fluids or solids in aerosol form, sample 10
containers. For transdermal patches, sample 10 patches.
The amount to be tested may be reduced for active substances that will be formulated in the following conditions:
the amount per dosage unit (e.g. tablet, capsule, injection) is
less than or equal to 1 mg or the amount per gram or millilitre (for preparations not presented in dose units) is less
than 1 mg. In these cases, the amount of sample to be tested
is not less than the amount present in 10 dosage units or 10 g
or 10 mL of the product.
For materials used as active substances where sample
quantity is limited or batch size is extremely small (i.e. less
than 1000 mL or 1000 g), the amount tested shall be 1 per
cent of the batch unless a lesser amount is prescribed or
justified and authorised.
For products where the total number of entities in a batch
is less than 200 (e.g. samples used in clinical trials), the sample size may be reduced to 2 units, or 1 unit if the size is less
than 100.
Select the sample(s) at random from the bulk material or
from the available containers of the preparation. To obtain
the required quantity, mix the contents of a sufficient number of containers to provide the sample.
4.2. Examination of the product
4.2.1. Membrane filtration
Use a filtration apparatus designed to allow the transfer of
the filter to the medium. Prepare the sample using a method
that has been shown suitable as described in section 3 and
transfer the appropriate amount to each of 2 membrane
filters and filter immediately. Wash each filter following the
procedure shown to be suitable.
For the determination of TAMC, transfer one of the
membrane filters to the surface of casein soya bean digest
agar. For the determination of TYMC, transfer the other
membrane to the surface of Sabouraud-dextrose agar. Incubate the plate of casein soya bean digest agar at 30 – 359C
for 3 – 5 days and the plate of Sabouraud-dextrose agar at
20 – 259C for 5 – 7 days. Calculate the number of CFU per
gram or per millilitre of product.
When examining transdermal patches, filter 10 per cent of
the volume of the preparation described under 3.4.1. separately through each of 2 sterile filter membranes. Transfer
one membrane to casein soya bean digest agar for TAMC
and the other membrane to Sabouraud-dextrose agar for
TYMC.
4.2.2. Plate-count methods
(i) Pour-plate method: Prepare the sample using a
method that has been shown to be suitable as described in
section 3. Prepare for each medium at least 2 Petri dishes for
each level of dilution. Incubate the plates of casein soya bean
digest agar at 30 – 359C for 3 – 5 days and the plates of
Sabouraud-dextrose agar at 20 – 259C for 5 – 7 days. Select
the plates corresponding to a given dilution and showing the
highest number of colonies less than 250 for TAMC and 50
for TYMC. Take the arithmetic mean per culture medium of
the counts and calculate the number of CFU per gram or per
millilitre of product.
(ii) Surface-spread method: Prepare the sample using a
method that has been shown to be suitable as described in
section 3. Prepare at least 2 Petri dishes for each medium
and each level of dilution. For incubation and calculation of
the number of CFU proceed as described for the pour-plate
method.
4.2.3. Most-probable-number method
Prepare and dilute the sample using a method that has
been shown to be suitable as described in section 3. Incubate
all tubes for 3 – 5 days at 30 – 359C. Subculture if necessary,
using the procedure shown to be suitable. Record for each
level of dilution the number of tubes showing microbial
growth. Determine the most probable number of microorganisms per gram or millilitre of the product to be examined from Table 4.05-I-3.
4.3. Interpretation of the results
The total aerobic microbial count (TAMC) is considered
to be equal to the number of CFU found using casein soya
bean digest agar; if colonies of fungi are detected on this medium, they are counted as part of TAMC. The total combined yeasts/mould count (TYMC) is considered to be equal
to the number of CFU found using Sabouraud-dextrose
agar; if colonies of bacteria are detected on this medium,
they are counted as part of TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial
growth, Sabouraud-dextrose agar containing antibiotics may
be used. If the count is carried out by the MPN method the
calculated value is the TAMC.
When an acceptance criterion for microbiological quality
is prescribed it is interpreted as follows:
－ 101 CFU: maximum acceptable count ＝ 20,
－ 102 CFU: maximum acceptable count ＝ 200,
－ 103 CFU: maximum acceptable count ＝ 2000,
and so forth.
The recommended solutions and media are described in II.
Tests for specified micro-organisms.
II. Microbiological Examination of Non-sterile Products:
Tests for Specified Micro-organisms
These tests are harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia.
The tests described hereafter will allow determination of
the absence or limited occurrence of specified microorganisms which may be detected under the conditions
described.
The tests are designed primarily to determine whether a
substance or preparation complies with an established specification for microbiological quality. When used for such
purposes follow the instructions given below, including the
number of samples to be taken and interpret the results as
stated below.
Alternative microbiological procedures, including automated methods may be used, provided that their equivalence
to the Pharmacopoeial method has been demonstrated.
126 4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
1. General Procedures
The preparation of samples is carried out as described in I.
Microbial enumeration tests.
If the product to be examined has antimicrobial activity,
this is insofar as possible removed or neutralized as
described in I. Microbial enumeration tests.
If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated
as described in I. Microbial enumeration tests.
2. Growth Promoting and Inhibitory Properties of the
Media, Suitability of the Test and Negative Controls
The ability of the test to detect micro-organisms in the
presence of the product to be tested must be established.
Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of
the test is introduced.
2.1. Preparation of test strains
Use standardized stable suspensions of test strains or prepare as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages
removed from the original master seed-lot.
2.1.1. Aerobic micro-organisms
Grow each of the bacterial test strains separately in containers containing casein soya bean digest broth or on casein
soya bean digest agar at 30 – 359C for 18 – 24 hours. Grow
the test strain for Candida albicans separately on
Sabouraud-dextrose agar or in Sabouraud-dextrose broth at
20 – 259C for 2–3 days.
Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83 or NBRC 13276,
Pseudomonas aeruginosa such as ATCC 9027, NCIMB
8626, CIP 82.118 or NBRC 13275,
Escherichia coli such as ATCC 8739, NCIMB 8545, CIP
53.126 or NBRC 3972,
Salmonella enterica subsp. enterica serovar Typhimurium
such as ATCC 14028
or, as an alternative,
Salmonella enterica subsp. enterica serovar Abony such as
NBRC 100797, NCTC 6017 or CIP 80.39,
Candida albicans such as ATCC 10231, NCPF 3179, IP
48.72 or NBRC 1594.
Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions.
Use the suspensions within 2 hours or within 24 hours if
stored at 2 – 89C.
2.1.2. Clostridia
Use Clostridium sporogenes such as ATCC 11437 (NBRC
14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC
532 or CIP 79.3). Grow the clostridial test strain under
anaerobic conditions in reinforced medium for Clostridia at
30 – 359C for 24 – 48 hours. As an alternative to preparing
and then diluting down a fresh suspension of vegetative cells
of Cl. sporogenes, a stable spore suspension is used for test
inoculation. The stable spore suspension may be maintained
at 2 – 89C for a validated period.
2.2. Negative control
To verify testing conditions a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as
described under 3. A failed negative control required an investigation.
2.3. Growth promotion and inhibitory properties of the
media
Test each batch of ready-prepared medium and each batch
of medium prepared either from dehydrated medium or
from ingredients.
Verify suitable properties of relevant media as described in
Table 4.05-II-1.
(i) Test for growth promoting properties, liquid media:
inoculate a portion of the appropriate medium with a small
number (not more than 100 CFU) of the appropriate microorganism. Incubate at the specified temperature for not
more than the shortest period of time specified in the test.
Clearly visible growth of the micro-organism comparable to
that previously obtained with a previously tested and approved batch of medium occurs.
(ii) Test for growth promoting properties, solid media:
perform surface-spread method, inoculating each plate with
a small number (not more than 100 CFU) of the appropriate
micro-organism. Incubate at the specified temperature for
not more than the shortest period of time specified in the
test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch
of medium occurs.
(iii) Test for inhibitory properties, liquid or solid media:
inoculate the appropriate medium with at least 100 CFU of
the appropriate micro-organism. Incubate at the specified
temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.
(iv) Test for indicative properties: perform surfacespread method, inoculating each plate with a small number
(not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of
time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch
of medium.
2.4. Suitability of the test method
For each product to be tested perform sample preparation
as described in the relevant paragraph in section 3. Add each
test strain at the time of mixing, in the prescribed growth
medium. Inoculate the test strains individually. Use a number of micro-organisms equivalent to not more than 100
CFU in the inoculated test preparation.
Perform the test as described in the relevant paragraph in
section 3 using the shortest incubation period prescribed.
The specified micro-organisms must be detected with the
indication reactions as described in section 3.
Any antimicrobial activity of the product necessitates a
modification of the test procedure (see 3.4.3. of I. Microbial
enumeration tests).
If for a given product the antimicrobial activity with
respect to a micro-organism for which testing is prescribed
cannot be neutralized, then it is to be assumed that the inhibited micro-organism will not be present in the product.
3. Testing of Products
3.1. Bile-tolerant gram-negative bacteria
3.1.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests, but using casein soya bean digest broth
as the chosen diluent, mix and incubate at 20 – 259C for a
time sufficient to resuscitate the bacteria but not sufficient to
encourage multiplication of the organisms (usually 2 hours
but not more than 5 hours).
127
Table 4.05-II-1 Growth promoting, inhibitory and indicative properties of media
| Medium | Property | Test strains |
| Test for bile-tolerant gram-negative bacteria | ||
|
Enterobacteria enrichment broth Mossel |
Growth promoting |
E. coli P. aeruginosa |
| Inhibitory | S. aureus | |
| Violet red bile glucose agar |
Growth promoting＋ Indicative |
E. coli P. aeruginosa |
| Test for Escherichia coli | ||
| MacConkey broth | Growth promoting | E. coli |
| Inhibitory | S. aureus | |
| MacConkey agar |
Growth promoting＋ Indicative |
E. coli |
| Test for Salmonella | ||
|
Rappaport Vassiliadis Salmonella enrichment broth |
Growth promoting |
Salmonella enterica subsp. enterica serovar Typhimurium or Salmonella enterica subsp. enterica serovar Abony |
| Inhibitory | S. aureus | |
| Xylose, lysine, deoxycholate agar |
Growth promoting＋ Indicative |
Salmonella enterica subsp. enterica serovar Typhimurium or Salmonella enterica subsp. enterica serovar Abony |
| Indicative | E. coli | |
| Test for Pseudomonas aeruginosa | ||
| Cetrimide agar | Growth promoting | P. aeruginosa |
| Inhibitory | E. coli | |
| Test for Staphylococcus aureus | ||
| Mannitol salt agar |
Growth promoting＋ Indicative |
S. aureus |
| Inhibitory | E. coli | |
| Test for Clostridia | ||
| Reinforced medium for Clostridia | Growth promoting | Cl. sporogenes |
| Columbia agar | Growth promoting | Cl. sporogenes |
| Test for Candida albicans | ||
| Sabouraud-dextrose broth | Growth promoting | C. albicans |
| Sabouraud-dextrose agar |
Growth promoting＋ Indicative |
C. albicans |
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products
3.1.2. Test for absence
Unless otherwise prescribed use the volume corresponding
to 1 g of the product, as prepared in 3.1.1. to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30 – 359C
for 24 – 48 hours. Subculture on plates of violet red bile glucose agar. Incubate at 30 – 359C for 18 – 24 hours.
The product complies with the test if there is no growth of
colonies.
3.1.3. Quantitative test
3.1.3.1. Selection and subculture
Inoculate suitable quantities of enterobacteria enrichment
broth-Mossel with the preparation as described under 3.1.1.
and/or 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 30 – 359C for 24 – 48
hours. Subculture each of the cultures on a plate of violet
red bile glucose agar. Incubate at 30 – 359C for 18 – 24
128
Table 4.05-II-2 Interpretation of results
| Results for each quantity of product |
Probable number of bacteria per gram or mL of product |
||
| 0.1 g or 0.1 mL | 0.01 g or 0.01 mL | 0.001 g or 0.001 mL | |
| ＋ | ＋ | ＋ | more than 103 |
| ＋ | ＋ | － | less than 103 and more than 102 |
| ＋ | － | － | less than 102 and more than 10 |
| － | － | － | less than 10 |
4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
hours.
3.1.3.2. Interpretation
Growth of colonies constitutes a positive result. Note the
smallest quantity of the product that gives a positive result
and the largest quantity that gives a negative result. Determine from Table 4.05-II-2 the probable number of bacteria.
3.2. Escherichia coli
3.2.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth, mix and incubate at 30 – 359C for 18 – 24
hours.
3.2.2. Selection and subculture
Shake the container, transfer 1 mL of casein soya bean
digest broth to 100 mL of MacConkey broth and incubate at
42 – 449C for 24 – 48 hours. Subculture on a plate of MacConkey agar at 30 – 359C for 18 – 72 hours.
3.2.3. Interpretation
Growth of colonies indicates the possible presence of
E. coli. This is confirmed by identification tests.
The product complies with the test if no colonies are
present or if the identification tests are negative.
3.3. Salmonella
3.3.1. Sample preparation and pre-incubation
Prepare the product to be examined as described in I.
Microbial enumeration tests and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 2.4.) of casein
soya bean digest broth, mix and incubate at 30 – 359C for
18 – 24 hours.
3.3.2. Selection and subculture
Transfer 0.1 mL of casein soya bean digest broth to 10 mL
of Rappaport Vassiliadis Salmonella enrichment broth and
incubate at 30 – 359C for 18 – 24 hours. Subculture on plates
of xylose, lysine, deoxycholate agar. Incubate at 30 – 359C
for 18 – 48 hours.
3.3.3. Interpretation
The possible presence of Salmonella is indicated by the
growth of well-developed, red colonies, with or without
black centres. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.4. Pseudomonas aeruginosa
3.4.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth and mix. When testing transdermal patches,
filter the volume of sample corresponding to 1 patch of the
preparation described in I. Microbial enumeration tests
(3.4.1.) through a sterile filter membrane and place in 100
mL of casein soya bean digest broth. Incubate at 30 – 359C
for 18 – 24 hours.
3.4.2. Selection and subculture
Subculture on a plate of cetrimide agar and incubate at
30 – 359C for 18 – 72 hours.
3.4.3. Interpretation
Growth of colonies indicates the possible presence of
P. aeruginosa. This is confirmed by identification tests.
The product complies with the test if colonies are not
present or if the confirmatory identification tests are negative.
3.5. Staphylococcus aureus
3.5.1. Sample preparation and pre-incubation
Prepare a sample using a 1 in 10 dilution of not less than
1 g of the product to be examined as described in I. Microbial enumeration tests and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount
(determined as described under 2.4.) of casein soya bean
digest broth and homogenise. When testing transdermal
patches, filter the volume of sample corresponding to 1
patch of the preparation described in I. Microbial enumeration tests (3.4.1.) through a sterile filter membrane and place
in 100 mL of casein soya bean digest broth. Incubate at 30 –
359C for 18 – 24 hours.
3.5.2. Selection and subculture
Subculture on a plate of mannitol salt agar and incubate at
30 – 359C for 18 – 72 hours.
3.5.3. Interpretation
The possible presence of S. aureus is indicated by the
growth of yellow/white colonies surrounded by a yellow
zone. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.6. Clostridia
3.6.1. Sample preparation and heat treatment
Prepare a sample using a 1 in 10 dilution (with a minimum
total volume of 20 mL) of not less than 2 g or 2 mL of the
product to be examined as described in I. Microbial enumeration tests. Divide the sample into two portions of at least 10
mL. Heat 1 portion at 809C for 10 minutes and cool rapidly.
Do not heat the other portion.
3.6.2. Selection and subculture
Use 10 mL or the quantity corresponding to 1 g or 1 mL of
the product to be examined of both portions to inoculate
suitable amounts (determined as described under 2.4.) of
reinforced medium for Clostridia. Incubate under anaerobic
conditions at 30 – 359C for 48 hours. After incubation,
make subcultures from each tube on Columbia agar and
JP XVII General Tests / 4.05 Microbiological Examination of Non-sterile Products 129
incubate under anaerobic conditions at 30 – 359C for 48 – 72
hours.
3.6.3. Interpretation
The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates
the presence of Clostridia. This is confirmed by identification tests.
The product complies with the test if colonies of the types
described are not present or if the confirmatory identification tests are negative.
3.7. Candida albicans
3.7.1. Sample preparation and pre-incubation
Prepare the product to be examined as described in I.
Microbial enumeration tests and use 10 mL or the quantity
corresponding to not less than 1 g or 1 mL to inoculate 100
mL of Sabouraud-dextrose broth and mix. Incubate at 30 –
359C for 3-5 days.
3.7.2. Selection and subculture
Subculture on a plate of Sabouraud-dextrose agar and incubate at 30 – 359C for 24 – 48 hours.
3.7.3. Interpretation
Growth of white colonies may indicate the presence of C.
albicans. This is confirmed by identification tests.
The product complies with the test if such colonies are not
present or if the confirmatory identification tests are negative.
The following section is given for information.
4. Recommended Solutions and Culture Media
The following solutions and culture media have been
found satisfactory for the purposes for which they are
prescribed in the test for microbial contamination in the
Pharmacopoeia. Other media may be used provided that
their suitability can be demonstrated.
(i) Phosphate buffer solution pH 7.2
Prepare a mixture of water and stock buffer solution (800:1
V/V) and sterilize.
Stock buffer solution. Transfer 34 g of potassium dihydrogen phosphate to a 1000 mL volumetric flask, dissolve in 500
mL of purified water, adjust to pH 7.2 to ± 0.2 with sodium
hydroxide, add purified water to volume and mix. Dispense
in containers and sterilize. Store at a temperature of 2 – 89C.
| (ii) | Buffered sodium chloride-peptone solution pH 7.0 |
| Potassium dihydrogen phosphate | 3.6 g |
| Disodium hydrogen phosphate dihydrate | 7.2 g |
(equivalent to 0.067 mol phosphate)
| Sodium chloride | 4.3 g |
| Peptone (meat or casein) | 1.0 g |
| Water | 1000 mL |
Sterilize in an autoclave using a validated cycle.
| (iii) | Casein soya bean digest broth |
| Pancreatic digest of casein | 17.0 g |
| Papaic digest of soya bean | 3.0 g |
| Sodium chloride | 5.0 g |
| Dipotassium hydrogen phosphate | 2.5 g |
| Glucose monohydrate | 2.5 g |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (iv) | Casein soya bean digest agar |
| Panceratic digest of casein | 15.0 g |
| Papaic digest of soya bean | 5.0 g |
| Sodium chloride | 5.0 g |
| Agar | 15.0 g |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (v) | Sabouraud-dextrose agar |
| Dextrose | 40.0 g |
|
Mixture of peptic digest of animal tissue and pan creatic digest of casein (1:1) |
10.0 g |
| Agar | 15.0 g |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (vi) | Potato dextrose agar |
| Infusion from potatoes | 200 g |
| Dextrose | 20.0 g |
| Agar | 15.0 g |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (vii) | Sabouraud-dextrose broth |
| Dextrose | 20.0 g |
|
Mixture of peptic digest of animal tissue and pan creatic digest of casein (1:1) |
10.0 g |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (viii) | Enterobacteria enrichment broth-Mossel |
| Pancreatic digest of gelatin | 10.0 g |
| Glucose monohydrate | 5.0 g |
| Dehydrated ox bile | 20.0 g |
| Potassium dihydrogen phosphate | 2.0 g |
| Disodium hydrogen phosphate dihydrate | 8.0 g |
| Brilliant green | 15 mg |
| Purified water | 1000 mL |
Adjust the pH so that after heating it is 7.2 ± 0.2 at 259C.
Heat at 1009C for 30 minutes and cool immediately.
| (ix) | Violet red bile glucose agar |
| Yeast extract | 3.0 g |
| Pancreatic digest of gelatin | 7.0 g |
| Bile salts | 1.5 g |
| Sodium chloride | 5.0 g |
| Glucose monohydrate | 10.0 g |
| Agar | 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 at 259C.
Heat to boiling; do not heat in an autoclave.
| (x) | MacConkey broth |
| Pancreatic digest of gelatin | 20.0 g |
| Lactose monohydrate | 10.0 g |
| Dehydrated ox bile | 5.0 g |
| Bromocresol purple | 10 mg |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259C. Sterilize in an autoclave using a validated cycle.
| (xi) | MacConkey agar |
| Pancreatic digest of gelatin | 17.0 g |
| Peptones (meat and casein) | 3.0 g |
130 4.06 Sterility Test / General Tests JP XVII
| Lactose monohydrate | 10.0 g |
| Sodium chloride | 5.0 g |
| Bile salts | 1.5 g |
| Agar | 13.5 g |
| Neutral red | 30 mg |
| Crystal violet | 1 mg |
| Purified water | 1000 mL |
Adjust the pH so that after sterilization it is 7.1 ± 0.2 at
259C. Boil for 1 minute with constant shaking then sterilize
in an autoclave using a validated cycle.
| (xii) | Rappaport Vassiliadis Salmonella Enrichment broth |
| Soya peptone | 4.5 g |
| Magnesium chloride hexahydrate | 29.0 g |
| Sodium chloride | 8.0 g |
| Dipotassium hydrogen phosphate | 0.4 g |
| Potassium dihydrogen phosphate | 0.6 g |
| Malachite green | 36 mg |
| Purified water | 1000 mL |
Dissolve, warming slightly. Sterilize in an autoclave using
a validated cycle, at a temperature not exceeding 1159C. The
pH is to be 5.2 ± 0.2 at 259C after heating and autoclaving.
| (xiii) | Xylose, lysine, deoxycholate agar |
| Xylose | 3.5 g |
| L-Lysine | 5.0 g |
| Lactose monohydrate | 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 desoxycholate | 2.5 g |
| Sodium thiosulfate | 6.8 g |
| Ammonium iron (III) citrate | 0.8 g |
| Purified water | 1000 mL |
Adjust the pH so that after heating it is 7.4 ± 0.2 at 259C.
Heat to boiling, cool to 509C and pour into Petri dishes. Do
not heat in an autoclave.
| (xiv) | Cetrimide agar |
| Pancreatic digest of gelatin | 20.0 g |
| Magnesium chloride | 1.4 g |
| Dipotassium sulfate | 10.0 g |
| Cetrimide | 0.3 g |
| Agar | 13.6 g |
| Purified water | 1000 mL |
| Glycerol | 10.0 mL |
Heat to boiling for 1 minute with shaking. Adjust the pH
so that after sterilization it is 7.2 ± 0.2 at 259C. Sterilize in
an autoclave using a validated cycle.
| (xv) | Mannitol salt agar |
| Pancreatic digest of casein | 5.0 g |
| Peptic digest of animal tissue | 5.0 g |
| Beef extract | 1.0 g |
| D-Mannitol | 10.0 g |
| Sodium chloride | 75.0 g |
| Agar | 15.0 g |
| Phenol red | 25 mg |
| Purified water | 1000 mL |
Heat to boiling for 1 minute with shaking. Adjust the pH
so that after sterilization it is 7.4 ± 0.2 at 259C. Sterilize in
an autoclave using a validated cycle.
(xvi) Reinforced medium for Clostridia
Beef extract 10.0 g
| Peptone | 10.0 g |
| Yeast extract | 3.0 g |
| Soluble starch | 1.0 g |
| Glucose monohydrate | 5.0 g |
| Cysteine hydrochloride | 0.5 g |
| Sodium chloride | 5.0 g |
| Sodium acetate | 3.0 g |
| Agar | 0.5 g |
| Purified water | 1000 mL |
Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after
sterilization it is about 6.8 ± 0.2 at 259C. Sterilize in an autoclave using a validated cycle.
| (xvii) | Columbia agar |
| Pancreatic digest of casein | 10.0 g |
| Meat peptic digest | 5.0 g |
| Heart pancreatic digest | 3.0 g |
| Yeast extract | 5.0 g |
| Maize starch | 1.0 g |
| Sodium chloride | 5.0 g |
| Agar, according to gelling power | 10.0 g to 15.0 g |
| Purified water | 1000 mL |
Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after
sterilization it is 7.3 ± 0.2 at 259C. Sterilize in an autoclave
using a validated cycle. Allow to cool to 45 – 509C; add,
where necessary, gentamicin sulfate corresponding to 20 mg
of gentamicin base and pour into Petri dishes.
4.06 Sterility Test
This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( ).
The test is applied to substances, preparations or articles
which, according to the Pharmacopoeia, are required to be
sterile. However, a satisfactory result only indicates that no
contaminating micro-organism has been found in the sample
examined in the conditions of the test.
1. Precautions against microbial contamination
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test
is performed. The precautions taken to avoid contamination
are such that they do not affect any micro-organisms which
are to be revealed in the test. The working conditions in
which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out
appropriate controls.
2. Culture media and incubation temperatures
Media for the test may be prepared as described below, or
equivalent commercial media may be used provided that they
comply with the growth promotion test.
The following culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium is
primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soya-bean
casein digest medium is suitable for the culture of both fungi
and aerobic bacteria.
| (i) | Fluid thioglycollate medium |
| L-Cystine | 0.5 g |
| Agar | 0.75 g |
| Sodium chloride | 2.5 |
