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MLT

Equipments Required
LAF
Filtration Assembly
Sterile 0.45 micron membrane filter
Sterile Pipettes 1 ml and 10 ml
2.0 Material Required
Sterile 0.1% w/v peptone 3 X 100 ml
70% IPA solution
Sample for testing
SCDA and SCDA plates
SDA and SDA plates
SCDM 3 X 100 ml
3.0 Procedure:
3.1 Total Aerobic Microbial Count:
3.1.1 Collect the sample to be tested for microbial limit test as per sampling plan.
3.1.2 Use specified quantity of sample for each of the test specified in the individual
monograph and pre-treat the sample as following method-
3.1.3 Water Soluble Product: Dissolve 10 gm or dilute 10 ml of sample, unless otherwise
specified, in sterile 90 ml peptone water or buffered sodium chloride-peptone solution pH 7.0
3.1.4 Product Insoluble In Water: Suspend 10 gm or 10 ml sample, unless otherwise
specified, in sterile 90 ml peptone water with 0.1% polysorbate 80 or buffered sodium
chloride-peptone solution pH 7.0
3.1.5 Fatty Products: Homogenize 10 gm or 10 ml of the sample, unless otherwise
specified, with 5 g of polysorbate 80. If necessary heat to not more than 40C. Mix carefully
while maintaining temperature on water bath. Add 85 ml of sterile peptone water or buffered
sodium chloride-peptone solution pH 7.0
3.2 Examination of sample by Membrane Filtration Method:
3.2.1 Aseptically connect the rubber tube of sterile manifold to receiver tank and receiver
tank rubber pipe to vacuum pump.
3.2.2 Using sterile smooth tip forceps, place a 47 mm diameter 0.45m sterile membrane
filter on the center of the filter support screen. Without disturbing the filter, place the funnel
on top of the filter holder base.
3.2.3 Separately transfer 10 ml of pretreated sample from step no. 3.1.3 to each two 90 ml of
sterile 0.1% peptone water. Mix well and transfer the whole quantity of dilution to each of
two membrane filters and filter immediately.
3.2.4 Wash both the membrane filter with each 3 x 100 ml of sterile 0.1% peptone water into
the filtration funnel and filter under partial vacuum
3.2.5 After completion of filtration process, shut off the vacuum with the help of vacuum
control key.
3.2.6 Transfer one of the membrane filters, intended for the enumeration of bacteria, to the
surface of the plate containing SCDA and other, intended for the enumeration of fungi, to the
surface of the plate of SDA.
3.2.7 For positive control carry out the same procedure in duplicate except for sample use
100 ml fluid A inoculated with 100 bacterial cells and another 100 ml fluid A is inoculated
with c.albicans intended for identification of total aerobic and fungal count respectively.
3.2.8 For negative control carry out the same procedure except for sample use 100 ml sterile
0.1% peptone water.
5.3.2.9 Incubate the plates for 5 days, unless a more reliable count is obtained in shorter
time, at 30 to 35C in the test of bacteria and 20 to 25C in the test for fungi.
3.2.10 Count the number of colonies that are formed. Calculate the number of cfu
per gram or per ml of the sample being examined.
3.3 Examination of sample Plate Count Method:
3.3.1 Use this method for fatty products and product insoluble in water.
3.3.2 Use 90 mm sterile petri plate. Take a four-petri plate and label two plates for bacteria
and remaining two for fungi count. Transfer 1 ml quantity of each pretreated dilution sample
solution to each of four petri plates.
3.3.3 Add 15 ml of sterile liquefied SCDA at not more than 45C, in to two plates labeled
for bacterial count.
3.3.4 Then add 15 ml of sterile liquefied SDA at not more than 45C, into two plates labeled
for fungal count.
3.3.5 Allow to solidify the plates at room temperature, invert and incubate at 30 to 35C for
5 days and 20 to 25C for 5 days respectively.
3.3.6 Count the number of colonies that are formed. Calculate the number of cfu
per gram or per ml of the sample being examined.
3.4 Test For Specified Microorganisms:
3.5 For Membrane Filtration Method:
3.5.1 Follow the same procedure described under 3.2.1 to 3.2.5, transfer one of the
membrane filter, intended for enrichment of E. coli and Salmonella to a tube containing 100
ml of sterile nutrient broth, and other membrane intended for enrichment of Pseudomonas
aeruginosa and Staphylococcus aureus, to a tube containing 100 ml of sterile Soybean casein
digest medium.
3.5.2 For positive control carry out the same filtration procedure in duplicate except for
sample use 100 of peptone water inoculated with approx 100 cells of E. coli or Salmonella
and another 100 ml of peptone water inoculate with Staph. aureus or Ps. aeruginosa and
transfer the membrane to 100 ml of sterile nutrient broth and soyabean casein digest medium
respectively.
3.5.3 For negative control carry out the same procedure except for sample use 100 ml of
sterile peptone water for both the tubes.
3.6 For Plate count method (Direct Inoculation)
3.6.1 Use this method for Fatty products and Product insoluble in water.
3.6.2 Transfer separately 1 ml quantity of pretreated sample from step no. 3.1.4 and 3.1.5 to
a tube containing 100 ml of sterile nutrient broth and soybean casein digest medium.
3.6.3 For positive control inoculate approx 10 to 100 cells of E. coli or salmonella into
nutrient broth and Staph. aureus or Ps. aeruginosa in Soybean casein digest medium.
3.6.4 For Negative control inoculate 1 ml of sterile peptone water in both the medium.
3.6.5 Incubate all the tubes at 35 37C for 18 to 24 hours.
3.6.6 Observe the tubes for growth, by means of turbidity. If the growth is present in sample
tube and positive control tube and absent in negative control tube, proceed for further
identification of specific microorganisms i.e. E. coli, Salmonella, Ps
aeruginosa and Staphylococcus aureus.
3.6.7 If growth is not observed in sample tube and negative control tube and observed in
positive control tube, need not proceed for further identification of specific microorganisms
i.e. E. coli, Salmonella, Ps aeruginosa and Staphylococcus aureus.
3.7 Escherichia coli:
3.7.1 By means of inoculating loop, streak a portion from enrichment culture (obtained from
nutrient broth of previous test) on the surface ofMacConkeys agar plate.
3.7.2 Simultaneously carry out the positive control by streaking a growth of E. coli on the
surface of MacConkeys agar plate. For negative control incubate the plate as it is without
inoculation.
3.7.3 Invert and incubate all the plates at 35 to 37C for 24 hours.
3.7.4 Upon examination, if none of the colonies are brick red in colour and have a
surrounding zone of precipitated bile, the sample meets the requirements of the test for the
absence of Escherichia coli.
3.7.5 If the colonies described above are found, transfer the suspect colonies individually to
the surface of Levine eosin-methylene blue agarmedium.
3.7.6 Simultaneously carry out the positive control by streaking a growth of E. coli on the
surface of MacConkeys agar plate. For negative control incubate the plate as it is without
inoculation
3.7.7 Invert and incubate all the plates at 35 to 37C for 24 hours
3.7.8 Upon examination, if none of the colonies exhibits both a characteristic metallic sheen
under reflected light, the sample meets the requirements of the test for the absence
of Escherichia coli
3.8 Salmonella:
3.8.1 Primary Test: Aseptically add 1.0 ml of the enrichment culture (obtained from nutrient
broth of previous test) to each of two tubes containing (a) 10 ml of sterile Selenite F
broth and (b) tetrathionate-bile-brilliant green broth and incubate at 35 to 37C for 24 to 48
hours.
3.8.2 From each of these two cultures subculture on at least two of the following four agar
media: Bismuth sulphite agar, Brilliant green agar, Deoxycholate-citrate agar and Xylose-
lysine-deoxycholate agar.
3.8.3 Simultaneously carry out the positive control by streaking a loop full growth
of Salmonella on surface of one of the above media, which is used for testing. For negative
control incubate the agar plate without streaking or inoculation.
3.8.4 Invert and incubate all the plates at 35 to 37C for 18 to 24 hours.
3.8.5 Upon examination, if none of the colonies confirms to the description given in Table-
1, the sample meets the requirements of the test for the absence of the genus Salmonella.
3.8.6 If any colonies confirming to the description in Table-1, carry out the secondary test.
Table-1
Sr. No Medium Description of colony
1 Bismuth sulphite agar Black or green
2 Brilliant Green Agar Small, transparent and colorless, or opaque,
pinkish or white (frequently surrounded by a
pink or red zone)
3 Deoxycholate-citrate agar Colorless, and opaque, with or without black
center.
4 Xylose-lysine-deoxycholate Red with or without black centers.
agar
3.8.7 Secondary Test: Subculture any colonies showing the characteristics given in Table
1, in triple sugar-iron agar by first inoculating the surface of the slope and then making a stab
culture with the same inoculating needle.
3.8.8 At the same time inoculate a tube of urea broth. Incubate at 36 to 38C for 18 to 24
hours.
3.8.9 Upon examination, no evidence of tubes having alkaline (red) slant and acid (yellow)
butt (with or without concomitant blackening of the butt from hydrogen sulfide production),
the sample meets the requirements of the test for absence of genus salmonella.
3.9 Pseudomonas aeruginosa:
3.9.1 Streak a portion of the medium from soyabean casein digest medium (obtained from
nutrient broth of previous test) on the surface of cetrimide agar medium,
3.9.2 Simultaneously carry out the positive control by streaking a loop full growth of Ps.
aeruginosa on the surface of cetrimide agar. For negative control incubate the cetrimide agar
plate without inoculation. Invert and incubate all the plates at 35 to 37C for 18 to 24 hours.
3.9.3 If, upon examination, none of the plate contains colonies having the characteristic
listed in Table-2 for the media used, the sample meets the requirements for freedom from Ps.
aeruginosa.
3.9.4 If any colonies confirming to the description in table 3 are produced, carry out the
Oxidase and pigment test.
Table-2
Medium Colony Fluorescence Oxidase Gram stain
characteristic in UV light
Cetrimide Generally Greenish Positive Negative rods
Agar greenish
Pseudomonas Generally Yellowish Positive Negative rods
agar for colorless to
detection yellowish
fluorescein
Pseudomonas Generally Blue Positive Negative rods.
agar for greenish
detection
Pyocyanin

3.9.5 Pigment Test: Streak representative suspect colonies from the agar surface of cetrimide
agar on the surface of pseudomonas agar medium for detection of fluorescein and
pseudomonas agar medium for detection of Pyocyanin.
3.9.6 Cover and invert the inoculated plates and incubate at 33 to 37C for not less than 3
days.
3.9.7 Examine the streaked surface area under UV light and determine whether colonies
confirming to the description in Table-2.
3.9.8 Oxidase Test: If growth of suspect colonies occurs, place 2 or 3 drops of a freshly
prepared 1% w/v solution of N, N, N1, N1-tetramethyl-4-phenylenediamine dihydrochloride
on filter paper and smear with the suspected colony. If there is no development of a pink
color, changing to purple, the sample meets the requirements of the test for absence
of Pseudomonas aeruginosa.
3.10 Staphylococcus aureus:
3.10.1 Streak a portion of the medium from soyabean casein digest medium (obtained from
nutrient broth of previous test) on the surface of one of the agar medium listed in Table-3
3.10.2 Simultaneously carry out the positive control by streaking a loop full growth of
Staphylococcus aureus on the surface of agar medium. For negative control incubate the agar
plate without inoculation.
3.10.3 Invert and incubate all the plates at 35 to 37C for 18 to 24 hours
3.10.4 If, upon examination, none of the plate contains colonies having the characteristic
listed in Table-3 for the media used, the sample meets the requirements for freedom from
Staphylococcus aureus.
3.10.5 If any colonies confirming to the description in table 3 are produced, carry out the
coagulase test.
Table 3
Sr. No Selective Medium Colony characteristic Gram Stain
1 Vogel-Johnson agar Black surrounded by yellow Positive cocci in
zones clusters
2 Mannitol-salt agar Yellow colonies with yellow Positive cocci in
zones clusters
3 Baird-Parker agar Black, shiny, surrounded by Positive cocci in
clear zone of 2 to 5 mm clusters
3.10.6 Coagulase Test: Transfer representative suspect colonies from the agar surface or any
of the media listed in Table-3 to individual tubes, each containing 0.5 ml of mammalian,
preferably rabbit or horse plasma with or without additives.
3.10.7 Incubate at 37C and examine the tubes at 3 hours and subsequently at suitable
intervals up to 24 hours.
3.10.8 If no coagulation in any degree is observed, the sample meets the requirements of the
test for the absence of Staphylococcus aureus.
4.0 Precaution
4.1 Keep the hands clean and used strictly IPA rinsed hand gloves throughout the
operations.
4.2 Run positive and negative control along with each test.
4.3 The microbial limit test must be carried out under LAF.
4.4 For pour plate method, if necessary dilute the sample in the sample solution to obtain
100 to 300 cfu.
5.0 Frequencies
Batch wise

6.0 Abbreviation
SOP : Standard Operating Procedure
IPA : Isopropyl Alcohol
LAF : Laminar Air Flow
cfu : Colony forming unit
SCDA : Soybean casein digest Agar
SCDM : Soybean casein digest medium
SDA : Sabouraud Dextrose Agar

MLT (Microbial Limit Test) Validation

The validation test is done by following methods

A. Preparation of culture suspension


B. Recovery of Viable Microorganism
A. Preparation of the Test Suspensions
1. Remove following culture slant from the refrigerator and allow it to attain room
temperature.

Bacillus subtilis

Candida albicans

2. Inoculate loop full of the culture from each slant separately into 10 ml of sterile saline
solution (0.9% sodium chloride solution).

3. Transfer 1.0 ml of the solution into 9.0 ml of sterile saline solution (0.9% sodium chloride
solution) to obtain a test dilution of 10-1.

4. Transfer 1.0 ml of the 10-1 dilution into 9.0 ml of sterile saline solution to give 10-
2
dilution.
5. Similarly serially dilute the culture suspension to obtain dilution of 10-3, 10-4, 10-5, 10-6,
10-7 and 10-8

6. Plate 1.0 ml of the culture suspension from dilution 10-3 to 10-8 in duplicate into sterile
petri dishes.

7. Pour approximately 15-20 ml of sterile Soybean Casein Digest Agar cooled to about
45C in each plate. Incubate at 32.5 2.5C for 24 to 48 hours.

8. Count the number of colonies on each plate and select the dilution, which gives a count of
10 to 100 cfu for recovery of viable microorganisms.
B. Recovery of Viable
Microorganisms
For Total Bacterial Count:
Step Negative control Positive control Product Control Positive Product control
No

1 Add 1.0 ml of sterile Add 1.0 ml of the culture Prepare sample by Prepare sample by dissolving
water into the 100ml of suspension (containing 10 dissolving 10g of product 10g of product under test in
Soyabean Casein digest to 100 cells of B. subtilis) under test in 100 ml 100 ml
Medium. in 100 ml of of Soybean Casein digest of Soybean Casein digest
sterile Soybean Casein medium. medium and add 1 ml of
digest medium. culture suspension
(containing 10 to 100 cells o
B.Subtilis).

2 Aseptically pipette out 1 Aseptically pipette out 1 Aseptically pipette out 1 Aseptically pipette out 1 ml
ml of the pretreated ml of the pretreated ml of the pretreated of the pretreated specimen in
specimen in duplicate in specimen in duplicate in specimen in duplicate in duplicate in sterile
sterile petridishes. sterile petridishes. sterile petridishes. petridishes.

3 To each of the To each of the petridishes To each of the petridishes To each of the petridishes
petridishes add about 20 add about 20 ml add about 20 ml add about 20 ml
ml of Soybean Casein of Soybean Casein of Soybean Casein of Soybean Casein Digest
Digest Agar Digest Agar Digest Agar Agar (HiMedia Code No.
(HiMedia Code No. (HiMedia Code No. (HiMedia Code No. M290), mix and allow to
M290), mix and allow M290), mix and allow to M290), mix and allow to solidify.
to solidify solidify solidify.

4 Invert the petridishes Invert the petridishes and Invert the petridishes and Invert the petridishes and
and incubate at 30c to incubate at 30c to 35c incubate at 30c to 35c incubate at 30c to 35c for
35c for five days. for five days. for five days. five days.
5 Count the number of Count the number of Count the number of Count the number of Colony
Colony Forming Units Colony Forming Units Colony Forming Units Forming Units developed at
developed at the end of developed at the end of developed at the end of the end of incubation and
incubation and record incubation and record the incubation and report that report that highest number o
the results. results. highest number of Colony Colony Forming Units
Forming Units obtained obtained in the plates.
in the plates. Multiply the Multiply the highest number
highest number of Colony of Colony Forming Units by
Forming Units by 10 and 10 and report this value as
report this value as Total Total bacterial Count per 1
bacterial Count per 1 gm gm of the sample.
of the sample.

For Total Fungal Count:

Step Negative control Positive control Product Control Positive Product control
No

1 Add 1.0 ml of sterile Add 1.0 ml of the culture Prepare sample by Prepare sample by dissolving
water into the 100ml of suspension (containing 10 dissolving 10g of product 10g of product under test in
Soybean Casein digest to 100 cells of C. albicans) under test in 100 ml 100 ml
Medium. in 100 ml of of Soybean Casein digest of Soybean Casein digest
sterile Soybean Casein medium. medium and add 1 ml of
digest medium. culture suspension
(containing 10 to 100 cells o
C. albicans).

2 Aseptically pipette out 1 Aseptically pipette out 1 Aseptically pipette out 1 Aseptically pipette out 1 ml
ml of the pretreated ml of the pretreated ml of the pretreated of the pretreated specimen in
specimen in duplicate in specimen in duplicate in specimen in duplicate in duplicate in sterile
sterile petridishes. sterile petridishes. sterile petridishes. petridishes.

3 To each of the To each of the petridishes To each of the petridishes To each of the petridishes
petridishes add about 20 add about 20 ml of add about 20 ml of add about 20 ml of
ml of Sabouraud Sabouraud Dextrose Agar Sabouraud Sabouraud Dextrose Agar
Dextrose Agar (HiMedia Code No. Dextrose Agar (HiMedia (HiMedia Code No. M063),
(HiMedia Code No. M063), mix and allow to Code No. M063), mix and mix and allow to solidify
M063), mix and allow solidify allow to solidify.
to solidify .

4 Invert the petridishes Invert the petridishes and Invert the petridishes and Invert the petridishes and
and incubate at 20c to incubate at 20c to 25c incubate at 20c to 25c incubate at 20c to 25c for
25c for five days. for five days. for five days. five days.

5 Count the number of Count the number of Count the number of Count the number of Colony
Colony Forming Units Colony Forming Units Colony Forming Units Forming Units developed at
developed at the end of developed at the end of developed at the end of the end of incubation and
incubation and record incubation and record the incubation and report that report that highest number o
the results. results. highest number of Colony Colony Forming Units
Forming Units obtained obtained in the plates.
in the plates. Multiply the Multiply the highest number
highest number of Colony of Colony Forming Units by
Forming Units by 10 and 10 and report this value as
report this value as Total Total Fungal Count per 1 gm
Fungal Count per 1 gm of of the sample.
the sample.

Acceptance Criteria
The product under test is considered non inhibitory to microorganism under the defined test
condition if the following condition are met.

The test is valid, i.e. negative control shows no growth.


The recovery of organism from positive product control is not less than 75% when
compared with the recovery of organisms from positive control.

Validation of Microbiological Tests


The variety of microbiological tests makes it difficult, if not impossible, to prescribe a single,
comprehensive method for validating all types of tests. By their very nature, microbiological
tests possess properties that make them different from chemical tests. Consequently, the
well-known procedures for validating chemical tests are not appropriate for many
microbiological tests. Yet, it is necessary to validate microbiological tests if they are to be
useful for controlling the quality of drug products and devices. Test-method validation
provides assurance that a method is suitable for its intended use. Given this definition, any
rational company would want to be sure that its methods are validated.

Some tests, such as bioburden or viral titer tests, are quantitative in nature while other tests, such as
those for the presence of objectionable organisms, are qualitative. As with chemical tests, these
differences necessitate different validation approaches. The purpose of a test also may change the
procedures for running and validating it. As an example, consider a drug that will be orally
administered. Normally, sterility is not a major issue, and the specification allows for a considerable
number of organisms. However, if the drug will be administered to immunocompromised cancer or
AIDS patients, the bioburden level must be reduced considerably, increasing the test sensitivity
required in the validation study.

The nature of the test material itself changes how a test is run and the validation protocol.
Consequently, testing for objectionable organisms is different when testing a diuretic for
hypertension or an antibiotic for treating pneumonia. Also, a procedure that works perfectly well for
checking the bioburden of granulated sugars may fail with sodium chloride. These differences make
full coverage of the topic impossible within the context of this primer. This article will present the
general considerations that apply to most microbiological tests. However, three excellent
publications are available to analysts preparing validation study protocols for microbiological
methods (see Suggested Reading).

Note also that certain microbiological tests are already associated with well defined validation
procedures. For example, the endotoxin test and USP bacterial enumeration tests have clearly
defined validation procedures. In addition, individual countries may have specific requirements that
modify or change standard procedures. If a test is associated with a compendial or regulatory
validation procedure, workers are advised to follow that procedure unless there are clear reasons for
not doing so. In such cases, the reasons should be documented and filed with the test procedure.

Media The suitability of the medium used for cultivating organisms or cells obviously can have a
major impact on the test results. Some organisms are extremely fastidious and require a precisely
defined medium with several complex nutrients, while others grow in the presence of inorganic salt
mixtures and simple carbon sources. It is commonly argued that delicate, fastidious organisms
cannot survive manufacturing processes and should not be of concern, but organisms as delicate
and fastidious as mycoplasmas can appear in final preparations of biologics.

In addition to the nutrient composition of the media, more general factors such as pH and ionic
strength must be validated. While it is commonly believed that media in the range of pH 6.0 8.0 are
suitable for sterility and bioburden studies, individual organisms may require a more restricted range.
The same holds true for ionic strengths and osmolalities outside of the human physiological range.
Shifting the pH range from 6.0 7.0 to 7.0 8.0 and raising the ionic strength to 300 mOsm may
select for a different set of organisms than those that would be present in the lower pH range at 150
mOsm.

Most validation schemes require the use of five or more "indicator organisms" to demonstrate the
medium's ability to support growth. In addition to aerobic bacteria, anaerobic organisms, yeasts, and
molds are usually included. This is an important step since a finding of "no growth detected" is
meaningless if the medium was incapable of growing any organisms. This leads to two important
points.

First, the indicator organisms are supposedly representative of the types of organisms that will be
encountered during the testing, but this is not necessarily true. The indicator organisms are a subset
of organisms that are known to grow on properly prepared media, but the organisms contaminating a
manufacturing process may not belong to that subset. As a result the quality control laboratory may
repeatedly face what appears to be a microbial contamination event despite monitoring cultures that
show no growth. It is very important to know whatorganisms are normally present in the working
environment and to include these environmental isolates in a validation program. There is little value
in proving that a medium will support the growth of indicator organisms if the environment is full of
organisms with very different cultivation requirements.

The second issue involves media handling. The qualification or validation study may require
autoclaving the medium and then pouring culture plates as the autoclaved material cools. In
laboratories with a low testing load, the excess material is often poured into large tubes or culture
flasks to cool and solidify and then stored for future use, usually in a refrigerator. However, when
future testing is done, the second heating of the medium may not be captured in the qualification or
validation check and may not even be mentioned in the test procedure. If the agar is melted under
gentle conditions and quickly poured, there may be no problem, but in some cases, technicians have
placed the flasks in microwave ovens to heat the medium while taking a short break. With a powerful
microwave oven it is easy to boil the medium for an unknown period of time. This can destroy
nutrients or produce toxic or inhibitory substances. Consequently, in laboratories where this second
heating is a common practice, this procedure must be captured in the validation and described
exactly in the test procedures.

When preparing the validation protocol, the analyst should specify the recovery level expected for
each of the indicator organisms. Generally, recovery of at least 80% of the inoculum or control is
desirable. Recovery of less than 50% is usually unacceptable and should raise questions about the
presence of inhibitory substances, especially when the testing is taking place in the presence of a
raw material or product intermediate. It may be necessary to introduce and validatethe
performance of an agent that inactivates the inhibitor. It is important to set the specifications
before the study is conducted and to hold to these specifications. If specifications are not pre-set and
the test system cannot meet general acceptance specifications, it is very easy to set "acceptable"
specifications that would otherwise have been unacceptable. The other problem is the "specification
creep" that occurs when a recovery of 78% is found and the specification is 80%. A quality
assurance or quality control worker who allows the 78% to pass will soon face the expectation that
75% should pass because it is "only slightly different from the other one." Over the course of a few
years, an 80% specification can gradually turn into a 70%, then 65%, specification.

Environment The incubation temperature can have a major effect on the ability of an organism to
grow in a given medium. It is well known that yeasts and molds require a different incubation
temperature than bacteria in a sterility test. Similarly,cells in tissue culture are often extremely
sensitive to small changes in temperature, not only for their growth but also in their susceptibility to
being infected or lysed by viruses. The analyst may need to develop temperature curves to justify
the incubation temperatures used for the test. It is also important to verify the incubator's ability to
maintain the set temperature within the specified range. If a four-degree temperature variation can
cause a significant change in the test results, the incubator's ability to hold a 1 C range at all
internal locations is critical. This may not be covered in a validation study, but it should be included
in the incubator's qualification studies.

In addition to the usual range from 20 40 C, it may be necessary to demonstrate the ability to
grow organisms at extreme temperatures. If it is necessary to monitor the presence of microbes in a
hot or cold room, it will be necessary to demonstrate an ability to cultivate thermophiles or
psychrophiles in addition to organisms that grow under more normal conditions. While the
significance of these extremophiles may be open to question, their presence and the possibility that
they may leave residues such as endotoxins must be considered.

The atmosphere in which the test system is immersed can have a major effect. Anaerobic organisms
cannot grow in the presence of oxygen, and tissue cultures may require the presence of 5% CO2 to
grow well. Certain facultative organisms will adjust their metabolic paths to cope with reduced levels
of oxygen. This, in turn, can affect their growth rates. When media for general purposes, such as
sterility tests, are being considered, it is normal to include one medium that provides anaerobic
conditions. The detection of anaerobes is important as they include toxin-producing and other
pathogenic bacteria.

Quantitative Issues One of the problems with quantitative microbiological tests is that as microbe
counts become smaller, straight-forward linear behavior is less common than that which follows the
Poisson distribution. This is because random distribution is not even distribution. Most quantitative
tests for microorganisms require the plating of dilute liquid samples, and it is normal to prepare
samples to ensure the dispersion of microbes and a random distribution of bacteria or viruses. When
concentrations are high, the lack of even distribution is not a problem; simple linear averaging
methods can compensate for the uneven distribution. Problems arise with smaller numbers of
microbes.

Consider an example where there are exactly 100,000 organisms per mL. If 0.1 mL is taken and
mixed with 0.9 mL of a diluent, it is highly unlikely that the new suspension will contain exactly
10,000 organisms; it would not be surprising to have anywhere from 9,800 10,200 organisms.
Back-calculating the result produces a range from 98,000 102,000 organisms in the original
sample, and, if there were enough replicates, the results could be averaged to obtain a number
indistinguishable from 100,000. This is the result that would be expected based on linear thinking.

However, if there were only 10 organisms per mL, it is quite possible that a 0.1 mL aliquot would not
contain any organisms at all. In fact, in this situation about one third of the aliquots will not contain a
single organism. This could lead to the conclusion, on averaging, that the sample only contained 6.7
organisms per mL, which is a significant deviation from the true value.

A transition occurred from a high density that produces a fairly smooth, homogeneous distribution of
organisms to a low density that results in organisms that are distributed with significant distances
between them. Under these conditions, the suspension behaves according to the Poisson
distribution and assumptions related to a normal distribution no longer hold. The Poisson distribution
is an exponential function. The problem is that parameters such as the standard deviations may be
logarithmic in nature, and when attempts are made to make these numbers "real" by taking the
antilogarithms, the results may actually have no "real" meaning. This can cause great difficulties
when attempting to validate quantitative microbial test procedures.

When it is necessary to deal with the Poisson distribution, it is wise to consult a statistician who is
versed in the use of this distribution. It appears that the transition to the Poisson distribution occurs
when approximately 100 colonies or plaques are counted. This is unfortunate because at this level
many analysts will declare a colony or plaque count to be "too numerous to count" (TNTC) to avoid
the tedium of these measurements. Therefore, most colony or plaque counting procedures actually
operate under the Poisson distribution and calculations based on the normal distribution will be
incorrect.

Revalidation The frequency of revalidation is a contentious question. There are many tests, such as
the growth promotion test on culture media, that are essentially self-validating and are run
frequently. It could be argued that if performance parameters (for example, percent recovery of
indicator organisms) are monitored via control charting and no significant changes are seen,
revalidation is unnecessary. However, control charting usually does not measure all the parameters
included in validation studies. Consequently, it is wise to revalidate tests after any major change in
constituents or procedures; in fact, revalidation may be needed to justify the changes. Changes in
suppliers (especially of media components) and changes in the composition of test samples have
resulted in major changes in microbiological tests. Finally, it is probably wise to revalidate
procedures approximately every second year to protect against unseen or unreported changes. A
media supplier may change its own suppliers or change its processing procedures without notifying
customers. The supplier may have no idea of the impact these changes could have on the end use
of their product. In addition, personnel changes in the laboratory and the maturing of analysts'
techniques can have an effect.

Suggested Reading Carroll MC. A multifaceted look at the microbial limits test. In: R Prince,
editor. Microbiology in Pharmaceutical Manufacturing. Baltimore, MD: PDA; 2001. pp. 519535.

PDA. Evaluation, validation and implementation of new microbiological testing methods: PDA
Technical Report No. 33. PDA Journal of Pharmaceutical Science and Technology 2000; 54(Suppl.
TR33).

Petitti DM. Practical considerations for the development, validation, and transfer of analytical test
methods. In: R Prince, editor. Microbiology in Pharmaceutical Manufacturing. Baltimore, MD: PDA;
2001. pp. 723746.

Steven S. Kuwahara, Ph.D., is the principal consultant and founder of GXP BioTechnology LLC,
PMB 506, 1669-2 Hollenbeck Avenue, Sunnyvale, CA 94087-5042, 408.530.9338

Obtaining successful validations of certain raw materials and finished products can be tricky. Researching the
materials beforehand is important. By working closely with development scientists and chemists, setting
appropriate specifications, and performing research, method development trials can be streamlined to produce
an adequate method. Key factors in developing a proper method include some experimentation as well as
knowledge of the pH of the material, the water activity of the material, the water solubility of the material, and
any antimicrobial properties of the material, to name a few. Risk assessments can be used to determine what to
test, how frequently to test, the stability program, and objectionable microorganism identification. Clear
documentation and adequate scientifically sound justifications are necessary so that the any future questions
can be easily answered.
Introduction
The Harmonized Microbial Limit Test (HMLT) was made effective in May 2009. The international
harmonization included United States Pharmacopeia (USP), Japanese Pharmacopeia (JP), and European
Pharmacopeia (EP). Companies were encouraged to revalidate their raw materials and finished products in
order to obtain compliance to the enhanced guidance documents prior to the implementation date. This paper
provides insight to the test and strategies for obtaining successful validations of the test.

Application of the Harmonized Microbial Limits


Test
The HMLT is used to evaluate raw materials and non-sterile products for acceptable microbial quality. It
involves the examination of non-sterile products that will determine whether a substance or preparation
complies with established specifications for microbiological quality (1).

The HMLT was not originally designed as a quality control test. It was designed as a referee test to demonstrate
compliance with monograph requirements (2). However, many companies use the test as a means to determine
the quality of their finished products or raw materials. Microbial limit testing is seen as an attribute of good
manufacturing practice (GMP) and of quality assurance (3).

The tests allow quantitative enumeration of mesophilic bacteria and fungi that grow under aerobic conditions
(1). Certain microorganisms can adversely impact (reduce or inactivate) the activity of certain products (4). It is
important to understand the microbial load of the raw materials or finished products in order to determine if
the product will react the way it was intended.

Microorganisms can also affect the health of the patients (4). Knowing the amount and types of
microorganisms in a product or device is important to patient safety.

The guidance documents listed in Table I govern the Microbial Limits Test. This may not be an all-inclusive list.

Table I: Governing Documents for the Microbial Limits Test

Document
Document Title
Number
USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
Microbiological Examination of Nonsterile Products: Acceptance Criteria for
USP <1111>
Pharmaceutical Preparations and Substances for Pharmaceutical Use
EP 2.6.12. Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
EP 2.6.13. Microbiological Examination of Non-Sterile Products: Test for Specified Micro-Organisms
JP 4.05 Microbial Limit Test
ICHGuidance Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on
QB4 Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
General Chapter
In regards to the documents listed in Table I, USP <61>, EP 2.6.12, and JP 4.05 are harmonized with one
another, and the assays described in the documents are equivalent. In addition, USP <62>, EP 2.6.13,
and JP 4.05 are harmonized with one another, and the assays described in those documents are equivalent.

Even though the tests are internationally harmonized with one another, individual monographs for specific raw
materials may not be harmonized in the various regions.

It is essential to learn the product and the excipients so that important testing decisions can be made. Effective
strategies to learn about the product include collaborating with the research and development (R&D) team,
joining the product team, and listening to the chemist working on the project. Discover how the product is used,
the target audience, the maximum dose, the delivery routes, how the product reacts in the body or with other
chemicals, the water solubility, the pH, the water activity, and the antimicrobial properties, to name a few.

Knowing the product and the excipients can help in making risk-based decisions on which items to test and
how often to conduct the test. Some guidance documents give instruction as to the regulatory expectations for
testing. Table II lists some of these documents (4-8).

Table II: Guidance Documents Listing Regulatory Expectations.

Document Number Document Title


Each lot of a component, drug product container, or closure with potential for
21 CFR 211.84 (d) microbial contamination that is objectionable in view of its intended use shall be
subjected to microbiological tests before use.
Appropriate written procedures, designed to prevent objectionable microorganisms in
21 CFR 211.113 (a)
drug products not required to be sterile, shall be established and followed.
There shall be appropriate laboratory testing, as necessary, of each batch of drug
21 CFR 211.165 (b)
product required to be free of objectionable microorganisms.
The significance of microorganisms in nonsterile pharmaceutical products should be
USP Chapter <1111> evaluated in terms of the use of the product, the nature of the product, and the
potential hazard to the user.
ICH Guideline Q6A
NA
Decision Trees 6 and 8
Individual Monographs NA
Based on Code of Federal Regulations Title 21 Part 211.165, it is wise to test every batch of non-sterile finished
products that are required to be free of objectionable microorganisms. If the tests are not conducted, not
validated properly, or the microorganisms recovered are not identified; there is a risk that objectionable
microorganisms may go undetected.

In order to determine if any excipients require specific tests, start by researching the monographs. If a
monograph exists for a particular raw material, test per that monograph. Use the more stringent monograph
(or a mix of the international monographs) to be able to use one test for global markets. If a monograph doesnt
exist, use a risk-based approach to determine if testing needs to be conducted.
During the risk-based approach, ask the following questions.

How much of the material is used in the finished product?


What is the potential the amount of the excipient added will negatively impact the bioburden of the finished
product?
What is the nature of the excipient (e.g., is it plant-based?)?
Is the manufacturing process going to reduce the microbial load?
What is the water activity?
Does the excipient have natural anti-microbial properties?

Use the gathered information to aide in writing the specifications. The specifications of the material will enable
the method development and validation process to be more streamlined. In order to set an appropriate
specification for the material, start by researching the product or excipient. If guidance or monographs do not
exist, reference USP<1111>.

USP <1111> lists acceptance criteria for non-sterile products. It lists the recommended total aerobic microbial
count limits, the total combined yeast/mold limits, and the recommended specified microorganism to screen
for. Depending on the product and application, a company may want to screen for additional specified
microorganisms that are not listed in USP <1111>. It is highly recommended to identify every microorganism
recovered in order to evaluate the presence or absence of objectionable microorganisms.

There are three commonly used enumeration methods listed in the compendial chapters to choose from. The
methods include the membrane filtration method, the plate count method, and the most probable number
(MPN) method. Whichever method is utilized depends on factors such as the nature of the product and the
required limit of microorganisms.

The compendial methods are already validated by scientists at USP; however, the suitability of the method to
recover microorganisms if they are present must be established. This is not a complete validation but rather a
verification of the suitability of the method (2).

Method development trials are the appropriate time-to-experiment for new products and will inform a facility
of the reaction to diluents, heat, filtration, sonication, shaking, etc. Method development trials are typically
recorded in notebooks and kept separate from the validation exercises.

Start method development by utilizing information about the product that has been gathered from project
teams, scientists, or chemists. Utilizing known product information will decrease the amount of manipulation
during the method development trials. For example, if a product is not soluble in water, try adding polysorbate
to the diluents or using isopropyl myristate instead. If the pH is acidic, it will need to be adjusted to a neutral
range (i.e., pH of 6-8) to recover microorganisms. If a product has antimicrobial activities, try adding
neutralizers to the media or utilizing the membrane filtration method. Sample preparation depends on the
physical characteristics of the product to be tested (1).

The compendial chapters outline useful information for the development process as well:

If the product contains antimicrobial activity, this should be neutralized.


If inactivators are used, their efficacy and their absence of toxicity for microorganisms must be demonstrated.
Common neutralizing agents and methods include the addition of polysorbate, the addition of lecithin, and/or
dilution methods (1).

USP <61> states that if no suitable neutralizing method can be found, it can be assumed that the failure to
isolate the inoculated microorganisms is attributable to the microbial activity of the product (1).Proceed by
performing the test with the highest dilution factor compatible with microbial growth and the specific
acceptance criterion in case other microorganisms are not inhibited by the product (1). USP <62> continues on
to say that for a given product, if the antimicrobial activity with respect to a microorganism for which testing is
prescribed cannot be neutralized, then it is to be assumed that the inhibited microorganism will not be present
in the product (10). Most companies continue in the method development trials until a suitable method is
identified.

Depending on the nature of the product and the required limit of microorganisms allowed, choose the
appropriate method to use in the method development trials. Again, the methods include the membrane
filtration method, the plate count method, and the MPN method.

For the membrane filtration method, filtration must be performed with filters that have a pore size not greater
than 0.45m (1).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 (1). Common filter materials include cellulose, nylon, and
Polyvinylidene fluoride (PVDF).

Membrane Filtration Example


1. Transfer a suitable quantity of the sample prepared (preferably representing 1 gram [g] of the product) to the
membrane and filter immediately. Rinse the filter with an appropriate diluent (1).
2. For Total Aerobic Microbial Counts (TAMC), transfer the filter to soybean-casein digest agar.
3. For Total Yeast Microbial Counts (TYMC), transfer the filter to sabouraud dextrose agar.
4. Incubate all of the plates according to the compendial guidance (1).

There are two methods listed in the compendial chapters for the plate count methods. They are the pour plate
method and the surface-spread method.

Pour Plate Method Example:

1. Add 1 milliliter (mL) of the sample preparation to duplicate petri dishes.


2. Cover the sample with 15-20 mL of molten media (cooled to ~45C).
3. Allow the plates to solidify at room temperature.
4. Invert the plates and incubate according to the compendial guidance (1).

Spread Plate Method Example:

1. Add media to sterile petri dishes and allow the media to solidify.
2. Add a measured amount of not less than 0.1 mL of the prepared sample to duplicate solidified media plates.
Spread the sample over the media surface.
3. Invert the plates and incubate according to the compendial guidance (1).
Another method described in the compendial chapters is the MPN method. The precision and accuracy of the
MPN method is less than that of the membrane filtration method or the plate-count method. Unreliable results
are obtained particularly for the enumeration of mold (1). The MPN method may be appropriate with products
with very low bioburden if no other method is available (1).

Example of MPN Method:

1. A series of at least three serial 10-fold dilutions of the product/raw material are prepared. From each level of
dilution, three aliquots of 1 g or 1 mL are used to inoculate three tubes with 9 to 10 mL of soybean-casein digest
broth. Neutralizers may be added to the media if needed.
2. Incubate all tubes at 30-35 C for no more than three days.
3. If reading the results is difficult, subculture to soybean-casein digest agar and incubate at the same temperature
for one to two days.
4. Use Table 3 from USP <61> to determine the MPN of microorganisms per g or mL (1).

Whichever method is chosen, it is imperative to prove the suitability of the test to recover microorganisms. To
do this appropriately, the media utilized must be properly growth promoted as described in the compendial
chapters (USPand USP). Proper positive and negative controls along with titer plates must also be utilized
throughout the process. Fresh microorganism dilutions must be used (no more than five passages from the
original master seed-lot) (1, 10). Microorganisms may be purchased from vendors in a ready-to-use format or
prepared as described in the compendial chapters.

USP <62> covers the testing of the following specified organisms:

Bile-Tolerant Gram-Negative bacteria


Escherichia coli
Salmonella
Pseudomonas aeruginosa
Staphylococcus aureus
Clostridia
Candida albicans.

Under each section, USP states how much of the product or excipient is to be examined and how to incubate
with each type of media with product in order to isolate any of the potential specified microorganisms within
that product. These specified microorganism challenges must be validated to recover microbial growth as well.
This portion of the microbial limits test is a presence/absence test. Depending on the product or excipient, one
may choose to validate any number of the specified microorganisms from USP <62>.

It is important to state which microorganisms are validated in the specifications appropriately. If a company
says, Comply with current USP <62>, without delineating which of the specified microorganisms were
validated, one could assume that the company validated all of the specified microorganisms from that chapter.

To save time and money during the initial method development trials, use a subset of microorganisms to test
the product reactions. For example:

An antibiotic that targets gram-negative bacteria needs to be tested. The product is readily soluble in water.
The product is diluted in Phosphate Buffer pH 7.2 and the membrane filtration method is chosen.
Because the product targets gram negative bacteria, varying dilutions and rinsing agents are utilized with gram
negative bacteria for the method development trials.
After an acceptable method has been identified, the full compendial panel of microorganisms are performed to
assure that all of the required microorganisms can be recovered accurately utilizing the employed method.

Remember, during method development, be creative! This is the right time to learn the product or excipient:

How does the material react to varying scenarios?


How does the material dissolve?
How easily/accurately are inoculated microorganisms recovered?
Does the pH need to be adjusted?
Which method (membrane filtration, pour plate, or MPN) is the right method?
Which neutralizers are needed, if any?
Creative Example: One may need to filter the specified microorganism challenges and then put the filter into
the media.

Once data has been collected and a reasonable method has been identified, go back over the data to make sure
the best method is chosen and can be repeated during the validation exercises. If a method is validated in the
United States, it should be repeatable in Japan.

By this point, one should have identified a method, a diluent, a dilution factor, a sample preparation, any
neutralizers, how to adjust the pH, and, possibly, the water activity of the material.

Knowing the water activity is not a requirement for the validation, but it is a useful tool. Water activity is
discussed inUSP <1112> Application of Water Activity Determination To Nonsterile Pharmaceutical Products.
Microorganisms may still be present at levels of <0.6 aw, but they will not proliferate. The test aids in the
decisions relating to the following:

Optimizing product formulations to improve antimicrobial effectiveness of preservative systems


Reducing the degradation of active pharmaceutical ingredients within product formulation susceptible to
chemical hydrolysis
Reducing the susceptibility of formulations (especially liquids, ointments, lotions, and creams) to microbial
contamination
Providing a tool for the rationale for reducing the frequency of microbial limit testing and screening for
objectionable microorganisms for product release and stability testing using methods contained in the general
chapters <61> and <62>
Reducing water activity to greatly assist in the prevention of microbial proliferation (11).

After the method development data has been evaluated, choose the method in which the compendial
microorganisms can be recovered by at least 50% of the positive controls.

Other points to consider:

Is the method one that any analyst can easily perform without easily contaminating the sample?
Are the recovery counts at least 50% of the positive controls?
Growth should not be inhibited (reduction by a factor greater than two)(1).
Does the pH need to be adjusted for the validation? If so, the pH will need to be adjusted for routine testing.

The validation exercises should be performed under method validation protocols that are controlled like other
cGMP documents. The validation is more of a verificationof the method suitability to recover microorganisms
from the testing material (2). Typical analytical validations consider the following:

Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range.

Alternative microbiological procedures, including automated methods, may be used as long as these methods
have been demonstrated to be equivalent to the Pharmacopeia method (1). One may need to perform side-by-
side comparisons with the traditional methods and alternative method to show equivalency to the compendial
methods.

Prepare the sample to be tested according to the protocol (which should match the method development
preparation). Unless otherwise directed (e.g., in a monograph), use 10 g or 10 mL of the product to be examined
to prepare the sample preparation (1).

The amount to be tested may be reduced for active substances if:

The amount per dosage (tablet, capsule, etc.) is less than or equal to 1 mg.
The amount per g or mL (for preparations not presented in dose units) is less than 1 mg.

Therefore, the amount of sample to be tested is not less than the amount present in 10 dosage units or 10 g/10
mL of the product

For materials used as active substances where the sample quantity is limited or batch size is extremely small
(less than 1000 mL or 1000 g), the amount tested shall be 1% of the batch unless a smaller amount is prescribed
or justified and authorized. For products where the total number of entities in a batch is less than 200, the
sample size may be reduce to two units or one unit if the sample size is less than 100. . Samples should be at
random from the bulk material or from available containers of the preparation. To obtain the required quantity,
mix the contents of a sufficient number of containers to provide the sample(1).

Method validation is typically performed on three lots of material to demonstrate the robustness of the method.
The first step of the validation is the total aerobic/yeast and mold count portion of the validation (1). Inoculate
separate portions of the sample preparation that are equivalent to 1 g or 1 mL of the material being examined
(1).The inoculums are made with less than 100 cfu of the microorganisms specified in USP <61>. The
inoculums must not exceed 1% of the volume of the diluted product (1).
If the pour plate method is used, inoculate the sample so that 1 mL of the dilution will contain <100 cfu of the
challenged microorganism. Otherwise, the plated sample may not contain growth.

For the membrane filtration method, transfer the filters onto tryptic soy agar (TSA) plates or sabouraud
dextrose agar (SDA) plates after filtering. Incubate the TSA plates for no more than three days at 3035C. This
is below the incubation range of three to five days; thereby, this proves that recovery can be obtained by the
minimum of three days. Un-inoculated product plates should be incubated for the three to five day incubation
range (1).

Incubate the SDA plates for not more than five days at 2025C. This is below the incubation range of five to
seven days, proving that recovery can be obtained by the minimum of five days. Un-inoculated product plates
should be incubated for the three to five-day incubation range (1).

After incubation, calculate the number of cfu per g or per mL of the tested material.

For the plate count method, prepare sample as stated in the protocol. Inoculate duplicate sterile petri dishes
with the inoculated product sample. Cover the dishes with media (or spread the sample onto the media for the
spread plate method). Incubate the TSA plates for no more than three days at 3035C. This is below the
incubation range of three to five days, proving that recovery can be obtained by the minimum of three days. Un-
inoculated product plates should be incubated for the three to five-day incubation range.

Incubate the SDA plates for no more than five days at 2025C. This is below the incubation range of five to
seven days, thus proving that recovery can be obtained by the minimum of five days. Un-inoculated product
plates should be incubated for the three to five-day incubation range.

Select the plates corresponding to a given dilution and show the highest number of colonies less than 250 for
TAMC and 50 for TYMC (1).Take the mean per culture medium of the counts and calculate the number of cfu
per g or per mL of product (1).

For the MPN method, incubate all tubes for not more than three days at 3035 C. Subculture, if necessary,
using the procedure shown to be suitable. For each level of dilution, record the number of tubes showing
microbial growth. Determine the MPN of microorganisms per g or mL of the product to be examined from
Table 3 in USP <61>.

The specified microorganism portion of the validation is second step of the validation (9). Inoculate separate
portions of the sample preparation that are equivalent to 1 g or 1 mL of the material being examined (this may
be a 1 in 10 dilution) (10).. The inoculums are made with less than 100 cfu of the specified microorganisms being
examined in USP<62>.

Incubate each step of the specified microorganism challenges using the lesser time of the incubation range. For
example, if the incubation range is 1824 hours, remove the validation sample at 18 hours.

The first step in USP <62> is dilution of the product in an enrichment media to encourage low-levels of
microbial growth (2).The second step is to subculture the microbial growth suspensions on selective agar to
depress general microbial growth and allow for the specified microorganisms to grow (2). The differential
media was designed to distinguish the colony morphology of the specified microorganisms thereby allowing
visual identification (2).
For many companies, the question of how to treat clinical products verses commercial products arises in
meeting rooms. For commercial products, most companies agree that the industry practice is to validate three
lots at a minimum for method validations.

What about clinical products? Clinical products are a different story. It is difficult sometimes to have three lots
of product in order to perform a complete validation. Small amounts of product are often made for use in
clinics. Formulations change frequently depending on field studies and developments discovered throughout
the early phases of a project. It is acceptable to perform method validation on one lot for clinical products.
Chances are the company will be redeveloping and revalidating with each formulation change of the product. It
may take years for a product to reach commercial launch.

Keep an eye on the product, however. The moment the product moves to commercial manufacturing, a three-
lot validation needs to occur.

Validation acceptance criteria is essential when determining if a method was properly validated. When verifying
the suitability of the membrane filtration 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 in the absence of product must
be obtained (1). In other words, the percent recovery between the inoculated product dilutions and the positive
controls must be at least 50%. Some companies use an internal 70% recovery criterion that is acceptable
because it is more stringent.

The compendial chapters give additional detail to the validation criteria for the MPN method. When verifying
the suitability of the MPN Method, the calculated value from the inoculums must be within 95% confidence
limits of the results obtained with the control (1). If the criterion 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 (1).

Specified microorganisms must be recovered during the validation for the assay to be valid. The titer plates
must demonstrate not more than 100 cfu was utilized to achieve the positive results (10).

After validations are complete, routine testing may begin. Following validation activities, reports are usually
written to approve the studies and methods (or standard operating procedures [SOPs]) are written to lock down
the way the tests are routinely performed.

Routine testing for the HMLT will contain the TAMC, the TYMC, and any specified microorganism challenges.
All representative colonies of growth obtained from any portion of the test needs to be identified to the species
level if possible. The microorganisms need to be researched and determined if they are objectionable
microorganisms.

There is a regulatory expectation that recovered microorganisms are identified from nonsterile products. There
have been warning letters issued to companies failing to comply with this expectation. Product recalls have also
occurred.

In order to determine if a microorganism is objectionable, USP <1111> provides information to aide in the risk
assessment. The microorganisms significance should be evaluated by researching the following:

Number of microorganisms (1 cfu or 1X106 cfu)


Microorganisms characteristics
Use of the product/route of administration
The nature of the product: Does it support growth? Does it have antimicrobial preservatives? What is the water
activity?
The method of application
The intended recipient (e.g., elderly, infants, etc.)
Potential impact to patients
Use of immunosuppressive agents
The presence of disease, wounds, or organ damage
Does the microorganism have a history of causing infections?

There are many sources to research microorganisms on the Internet. A good source of reference for researching
potential objectionable microorganisms is the FDA Bad Bug Book. There is not an all-inclusive list of
objectionable microorganisms. Microorganisms must be identified and evaluated to determine the potential
impact to patient health.

To determine the frequency of testing, one can utilize a risk-based approach. Like the objectionable
microorganisms, this is going to be a product-by-product risk assessment.

ICH QA6, Harmonised Tripartite Guideline: Specifications: Test Procedures and Acceptance Criteria for New
Drug Substances and New Drug Products: Chemical Substances provides some guidance on determining the
appropriate testing intervals:

In general, it is advisable to test the drug product unless its components are tested before manufacture and the
manufacturing process is known, through validation studies, not to carry a significant risk of microbial
contamination or proliferation (3).

With acceptable scientific justification, it may be possible to propose no microbial limit testing for solid oral
dosage forms or skip lot testing.

ICH Q6A Decision Tree Number 6: Microbiological Quality Attributes of Drug Substances and Excipients walks
through a series of questions about the drug substance and excipients to aid in the risk-based decisions about
the frequency of routine testing.

ICH Q6A Decision Tree Number 8: Microbiological Attributes of Non-Sterile Drug Products is another decision
tree. It walks through a series of questions about the drug product to aide in the risk-based decisions about the
frequency of routine testing.

Stability testing is performed under various temperatures and humidity set-points. Microbial limits testing
performed for stability testing is typically performed at initial, six months, 12 months, 24 months, etc. When
designing the program, take into account the product formulations, strengths, and packaging configurations. It
may be appropriate to leverage bracketing or matrixing stability testing designs as allowed under ICH Q1D.
Bracketing or matrixing can greatly reduce the test samples needed for the stability program.

Conclusion
Learn about the material before beginning. Set appropriate specifications so that the method can be
appropriately targeted. For international compliance, use the most stringent international method or a mixture
of the methods.

When developing methods, be creative! Learn as much as possible from development scientists, chemists,
research, and project teams to aide in the method development trials. Utilize the information to target the
method development trials in order to save time and money. Once a suitable method has been identified,
perform a trial as one would do during the validation with all of the microorganisms to demonstrate the method
will work properly. Perform these trials in a notebook and keep excellent notes about the trials. Be sure to test
the pH and choose the best suitable method for the application. Dont underestimate the value of knowing the
water activity.

During validation, write a method validation protocol utilizing the method developed in the trial runs. Be
certain that the method is performed the same way each time and that it is repeatable. For clinical products, a
one-lot validation is sufficient due to various clinical constraints. For commercial products, a minimum of a
three-lot validation should be performed.

Create a clean validation package for regulatory audits. During routine testing, lock down the method so that it
is performed the same way every time. Bracketing or matrixing can be utilized in decreasing the volume of
stability testing (12).

Risk-based approaches should be utilized in investigating objectionable microorganisms and in determining the
testing frequencies. Clear documentation and justifications should be maintained so that decisions can be
clearly understood in the future.

References
1. USP Chapter <61>, Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.
2. S. Sutton "Does International Harmonization of the USP Microbial Limits Tests Require Re-Validation of
Finished Product Tests?", Journal of Validation Technology 16 (3), 2009.
3. ICH Q6A, Specifications: Test Procedures for New Drug Substances and New Drug Products: Chemical
Substances.
4. USP Chapter <1111>, Microbiological Examination of Nonsterile Products: Acceptance Criteria for
Pharmaceutical Preparations and Substances for Pharmaceutical Use.
5. Code of Federal Regulations, Title 21, Food and Drugs (Government Printing Office, Washington, DC), Part
211.84(d).
6. Code of Federal Regulations, Title 21, Food and Drugs (Government Printing Office, Washington, DC), Part
211.113(a).
7. Code of Federal Regulations, Title 21, Food and Drugs (Government Printing Office, Washington, DC), Part
211.165(b).
8. ICH Guideline Q6A, Decision Trees 6 and 8.
9. USP Chapter <62>, Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms.
10. USP Chapter <1112>, Application of Water Activity Determination To Nonsterile Pharmaceutical Products.
11. ICH Q2(R1), Validation of Analytical Procedures: Text and Methodology.
12. ICH Q1D, Bracketing and Matrixing Designs for Stability Testing of New Drug Substances and Products.
Microbial limit test 112070804013

1. 1. MICROBIAL LIMIT TEST


2. 2. This test are designed to perform qualitative quantitative estimation of the no of
viable aerobic micro-organisms present or detecting the presence of designated
microbial species in pharmaceutical product. The term growth is used to designate the
presence & presumed proliferation of viable micro-organism. The most care must be
taken while performing microbial test so that contamination from outside can be avoided.
3. 3. Preliminary testing: The method given herein are invalid unless it is demonstrated
that the test specimen to which they are applied do not themselves inhibit the
multiplication of under the test condition of micro- organism that can be present.
Therefore, inoculate diluted specimen of substance being examined with separate viable
culture of (1)E.coli (2)S.aures (3)S.typhi (4)Psudomonas aeruginosa
4. 4. If organism fails to grow in medium the procedure should be modified by: a)
incresing the volume diluents with quantity of diluents remain same, or b) incresing a
sufficient quantity of inactivating agent in diluents ,or c) combining aforementioned
modification so as to permit growth of organisms in media.
5. 5. If inhibitory subtances are present in sample,0.5% soyalecithin & 4%of polysorbate
20 may be added to the culture medium. Repeat the same procedure using fluid casin
digest soyslecithin,-polysorbate20 medium to demonstrate neutralization of
preservative or other antimicrobial agent in test material. Where inhibitory substance
are contained in product & latter is soluble, the Membrane filtration method may be
used.
6. 6. If inspite of incorporation of suitable inactivating agent & a substantial increase in
volume of diluents it is still not possible to recover the viable culture described above &
where article is not suitable for applying the membrane filtration method it can be
assumed that the failure to isolate the inoculated organism may be due to the
bactericidal activity of product. This may be indicated that the article not likely to be
contaminated with the given species of organism. Monitoring should be continued to
establish the spectrum of inhibition & bactericidal activity of product.
7. 7. Media: culture media may be as per procedure & they have similar ingredient &/or
yield media comparable to those obtained from the formula. Where agar specified in
formula, use agar that has moisture content nmt15 %. Where water is mention in
formula, use purified water. Media should be sterilized by heating in autoclave at 115c
for 30 min.
8. 8. In preparing media as per formula, dissolve the soluble solid in water, using the heat
if necessary to effect complete solution & add HCL & NaOH in sufficient quantities to
yield require pH in medium. SAMPLING: USE 10ML OR 10G SPECIMEN FOR EACH
OF TEST.
9. 9. METHODS(1)TOTAL AEROBIC MICROBIAL COUNT MEMBRANE FILTRATION -
METHOD PLATE COUNT METHOD POURED PLATE SPREAD PLATE MUTIPLE OR
SERIAL DILUTION METHOD
10. 10. PREPARATION OF TEST FLUID: Water soluble product: Dissolve 10g or 10ml of
the sample in buffer or fluid medium( 1) TOTAL AEROBIC MICROBIALCOUNT &
adjust volume to 100ml. Product insoluble in water(non fatty): Take 10g of sample, grind
to fine powder & suspend it in buffer or fluid medium & adjust the volume to 100ml.
11. 11. A suitable surface-active agent such as 0.1%w/v of polysorbate 80 may be added to
assist the suspension of poorly wettable substance. Fatty product: Homogenise 10g or
10ml of sample with 5g ofpolysorbate20 or polysorbate80. -If necessary ,heat to nmt
40`c for 30min. -Add 85ml of buffer or fluid medium. -Adjust the PH to about 7.
Membrane filtration: Use membrane filter 50mm in diameter & having nominal pore size
NGT 0.45 um orless.
12. 12. Sterilized the filters,filteration apparatus,media & other apparatus used. Transfer
10ml or quantity of each dilution contain 1g of preparation being examined to each of
two membrane filter & filter immediately. If necessary dilute the pretreated preparation
so that 10-100 colony count may be expectd. After filtration wash the each filter three or
more time with appropriate fluid such as phosphate buffer,sodium chloride-peptone
buffer or fluid medium. For fatty susbtance add polysorbate20 or polysorbate80 to
washing.
13. 13. Transfer one of the membrane filter, intended for enumeration of bacteria to surface
of plate of casein soyabean digest agar & intend for enumeration of fungi to surface if
sabouraud dextrose agar with antibiotics. Incubate the plate for 5 days, unless more
reliable count is obtanied in shorter time,at 30 to 35c in test for bacteria & 20 to 25c in
test for fungi. Count the number of colonies that are formed. Calculate the no of
organism per g or ml of preparation being examined.
14. 14. POUR PLATE METHOD FOR BACTERIA: Use Petri dish 9 to 10 cm diameter, add
to each dish a mixture of 1ml of the pretreated preparation & about 15ml of liquefied
casein soyabean digest agar at NMT 45c If necessary dilute the preparation as
described above so that colony count NMT300 may be expected. Incubate the plate at
30 to 35 c for 5 days unless more reliable count is obtained in shorter time. Calculate
the result using plate with greatest no. of colonies but taking 300 colonies per plate as
maximum consistent with good evaluation.
15. 15. FOR FUNGI: Use saboraud dextrose agar with antibiotics & incubate the plate at
20 to 25 c for 5 days. Calculate the result using plate with nmt 100 colonies. SPREAD
PLATE METHOD Place 0.05-.2 ml of test fluid on solidified dried surface of agar
medium spread it uniformly using spreader. Proceed under same condition as for the
pour plate method.
16. 16. MULTIPLE TUBE METHOD Use 12 test tubes : 9 containing 9 ml of soybean-
casein digest medium each and 3 containing 10 ml of the same medium each for control.
Prepare dilutions using the 9 tubes. First, add 1 ml of the test fluid to each of three test
tubes and mix to make 10- times dilutions.(100ul) Second, add 1 ml of each of the 10-
times dilutions to each of another three test tubes and mix to make 100- times
dilutions.(10ul)
17. 17. Third, add 1 ml of each of the 100-times dilutions to each of the remaining three
test tubes and mix to make 1,000- times dilutions (1ul) Incubate all 12 test tubes for at
least 5 days at 30 - 35c. No microbial growth should be observed for the control test
tubes. If the determination of the result is difficult or if the result is not reliable, take a
0.1ml fluid from each of the9 test tubes and place it to an agar medium or fluid medium,
incubate all media for 24 72 hours at 30 35c, and check them for the absence or
presence of microbial growth. Calculate the most probable number of microorganisms
per ml or gram of the sample.
18. 18. Test for specified micro organism - E.COLI: Place the prescribed quantity in sterile
screw-capped container, add 50ml of nutrient broth, shake allow to stand for 1hr
&incubate at 37 for 18 to 24hr. Primary test: Add 1ml of enrichment culture to tube
contain 5ml MacConkey broth&incubate in water bath at 36 to 38 for 48hr.
19. 19. If content show acid &gas carry out secondary test. Secondary test: Add 0.1ml of
content of tube containing (a)5 ml of MacConkey broth (b)5ml of peptone water incubate
in a water bath at 43.5 to 44.5 for 24hr &examine tube for (a) acid &gas (b) indole For
indole: Add 0.5 ml of kovacs reagent, if red colour is produced ,indole is present.
20. 20. That indicates presence of e.coli. For control: Repeat primary &secondary test
adding 1.0ml of enrichment culture &volume of broth containing 10 to 50 e.coli
organism,prepared from 24hr culture in nutient broth,to 5ml MacConkey broth. The test
is not valid unless the result indicate that the control contain e.coli.
21. 21. OTHER TEST: streak a portion from enrichment culture on surface of
MacConkey agar medium.cover the dish &incubate. -If none of the colonies are brick-
red in colour, sample meet the requirement of test for absence of e.coli. If colony
described above are found, transfer the suspect colony to surface of Levine eosin
ethylene blue agar medium. cover &incubate.
22. 22. Upon examination, none of colony exhibit both metallic sheen under reflected light
& blue-black under transmitted light ,sample meet requirement test for absence of
E.coli. SALMONELLA: Transfer a quantity of pretreaed prepration being
examined containing 1g or 1ml of product to 100ml of nutrient broth in sterile screw
capped jar ,shake & incubate at 35 to 37 for 24hr.
23. 23. Preliminary test; Add 1.0 ml of enrichment culture to each of two tubes
containing(A)10ml of selenite F broth & (B)tetrathionet -bile- briliant green broth
&incubate at 36 to38 for 48 hr. From each of this two cultures subculture on following
four agar medium & incubate at 36 to 38for24 hr if none of colonies conform to
description given in table, sample meet requirment for absence of salmonella.
24. 24. Characteristic Colonial MorphologySelective Medium Brilliant Green Small,
transparent, colorless or pink to white Agar Medium opaque (frequently surrounded by
pink to red zone) Xylose-Lysine- Red, with or without black centers Desoxycholate Agar
Medium Bismuth Sulfite Black or green Agar Medium
25. 25. Secondary test: subculture any colonies showing characteristics given in table
in triple sugar-iron agar by first inoculating the surface of slope & at the same time
inoculate a tube of urea broth &incubate both at 36 to 38 for 18 to 24 hr. The absence
of acidity from the surface growth in triple sugar iron agar & together with absence of red
colour in urea broth, indicates the presence of salmonella.
26. 26. FOR CONTORL: Repeat the primary &secondary test using 1.0ml of
enrichment culture& volume of broth containing 10 to 50 salmonella organism, prepare
from 24hr broth culture in nutrient broth, for inoculation of tubes (a) &(b). The result is
not valid unless the result indicate that the control contains salmonella.
27. 27. PSUDOMONAS AERUGINOSA: Inoculate 100ml of fluid soyabean casein digest
medium with quantity of solution ,suspension or emulsion thus obtained containing 1g or
1ml of preparation being examined &incubate at 35 to 37for 24 to 48hr
28. 28. Characteristic Fluorescence Selective Colonial in Medium Morphology UV Light
Oxidase Test Gram Stain Centrimide Generally Greenish Positive Negative rodsAgar
Medium greenishPseudomonas Generally Yellowish Positive Negative rodsAgar
Medium colorless to for yellowishDetection of FluorescinPseudomonas Generally Blue
Positive Negative rodsAgar Medium greenish forDetection of Pyocyanin
29. 29. If upon examination none of colonies having characteristics listed in table for media
used, sample meet requirement for absence of micro- organisms. If colony conform to
description in table ,carry out oxidase & pigment test. OXIDASE &PIGMENT TEST:
Streak representative suspect colony from cetrimide agar medium on surface of
pseudomonas agar medium for
30. 30. detection of florescein &pseudomonas agar medium for detection of pyocyanin
contained in Petri dish & incubate at 33 to 37 for NLT 3 days. Examine the streak
surface under u.v light. Examine plate to determine whether colonies conforming to
description in table are present.
31. 31. If, growth of suspect colonies occur ,place 2 or 3 drops freshly prepared 1%w/v
solution of N,N- tetramethyl-4-phenylenediamine dihydrochloride on filter paper &smear
with colony. If there is no development of pink colour changing to purple, the sample
meet requirements of test for absence of pseudomonas aeruginosa.
32. 32. STAPHAYLOCOCCUS AURES: Proceed as described under psudomonas
aeruginosa ,if upon examination of incubate plate. none of them contain colony having
characteristic listed table sample meet requirment for absence of S.aures. If, growth
occurs ,carry out COAGULASE test. Transfer repsentative suspect colony from agar
surface to individual tubes, each contain 0.5ml of mammalian,
33. 33. preferably horse or rabbit plasma with or without additive. Incubate in water bath
at 37 examine tubes at 3hr & subsequently at suitable interval up to 24hr. If no
coaggulation is observed sample meet requirment of test for absence of S.aures
34. 34. CharacteristicSelective Medium Colonial Morphology Gram Stain Vogel-Johnson
Black Surrounded by yellow Positive cocci Agar Medium zone (in clusters) Mannital-Salt
Yellow colonies with yellow Positive cocci Agar Medium zones (in clusters) Baird-Parker
Black, shiny, surrounded by Positive coccid Agar Medium clear zones 2 to 5 mm (in
clusters)
35. 35. REFERENCES: (1)INDIAN PHARMACOPIEA (2)U .S. P (3) BY: JAMES
SWARBRICK ENCYCLOPEDIA OF PHARMACEUTICAL TECHNOLOGY,THIRD
EDITION ,VOL-1 (4) BY: GILBERT S. BANKER MARTIN M . RIGER
PHARMACEUTICAL DOSAGE FORM DISPERSE SYSTEM, VOL-2