The document outlines antimicrobial effectiveness testing for pharmaceutical products, emphasizing the need for preservatives in both sterile and non-sterile formulations to inhibit microbial growth. It details testing procedures, required microorganisms, and acceptance criteria for determining the efficacy of antimicrobial agents in various product categories, including injections, topical applications, and oral preparations. The information presented is crucial for ensuring product safety and compliance with pharmacopoeial standards.

1. National Institute of Pharmacy and Education
Research, Ahmedabad
Presented By –
Rushikesh Tamhane (PA25)
Sachin Patel (PA23)
ANTIMICROBIAL EFFECTIVENESS TESTING
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2. REFERENCES
1. THE UNITED STATES PHARMACOPEIA 41 NF 36 Page No. 5959
2. INDIAN PHARMACOPOEIA 2022 Volume I Page No. 29
3. BRITISH PHARMACOPOEIA Volume 5 2018 Appendix XVI C V-A519
4. EUROPEAN PHARMACOPOEIA 11.0 Volume 1 Page No. 205
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3. WHAT ARE ANTIMICROBIALS?
• Antimicrobials are the substances added to aqueous products.
• Nonsterile product contains preservatives to protect them from inadvertent
introduction during manufacturing process.
• Sterile products contains preservatives to inhibit growth of microorganism
introduced from repeated withdrawal of individual dose.(1,2)
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4. WHAT ARE ANTIMICROBIALS ?
• All Antimicrobial agents are toxic substances so for protection of patients concentration should be
maintained. (1)
• Concentration can be kept minimum if active ingredients of formulation possess intrinsic antimicrobial
activity.(1)
• However one or more antimicrobial preservatives are expected in sterile multidose unit. (1)
• Antimicrobial should not be used solely for Good Manufacturing Practices. (1,2,3)
• Challenge organisms are generally based on contaminants to drug product considering its physical attributes,
formulation ,intended use .(1,2)
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5. • Antimicrobial effectiveness whether formerly present in product due to intrinsic activity of active
ingredient or produced because of addition of antimicrobial preservatives must be demonstrated for all
sterile & non sterile formulations. (1,2)
• Antimicrobial Effectiveness must be demonstrated for aqueous-based, multiple-dose topical and oral
dosage forms and other such as ophthalmic, otic, nasal, irrigation and dialysis fluids.(1,2) (Aqueous is
defined as water activity more than 0.6.)
• The Efficacy of antimicrobial preservative may be enhanced or diminished by active constituent of
formulation in which it is incorporated or by container and closure used.(3)
PARTICULARS OF ANTIMICROBIAL EFFECTIVENESS TESTING
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6. PARTICULARS OF ANTIMICROBIAL EFFECTIVENESS TESTING
• The test for effectiveness of antimicrobial preservatives shall be demonstrated during development of
pharmaceutical preparation and during the commercial manufacturing.(1,2,3)
• Test is not intended to be used for routine control purpose. (1,2,3)
• It should be recognized that the presence of dead microorganisms or their metabolic by-products may
cause adverse reactions in sensitized persons.(2)
• The antimicrobial activity of preparation in its final container is investigated over period of validity to
ensure that such activity has not been impaired by storage and done immediately prior to testing.(3)
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7. GENERAL PROCEDURES (As per USP)
 General Considerations:-
 Ability of procedure to detect challenge microorganisms in the presence of suitably neutralized product to
be tested must be established.
 Suitability of the procedure must be reconfirmed if change is made in material , method, product or direct
product contact materials that may affect outcome of test.
 Preparation of Test Strains:-
 Use standardized suspensions of test strains or prepare as stated below.
• Seed-lot culture maintenance techniques are used so that the viable microorganisms used for inoculation
are NMT five passages removed from original master seed lot.
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8. USE CULTURES OF FOLLOWING MICROORGANISMS
• Viable microorganisms used should be part of freshly growing culture with exception of A. brasiliensis
spores.
• Culture conditions are described in next table from which suitable media are Soyabean casein digest or
Sabouraud Dextrose Agar medium.
• To Harvest:-
• C. albicans:- use sterile saline TS to wash surface growth and collect.
• A. brasiliensis:- use sterile saline TS with 0.05% of polysorbate 80.
Candida Albicans (ATCC No. 10231) Escherichia coli (ATCC No. 8739)
Aspergillus Braslliensis (ATCC No. 16404) Pseudomonas aeruginosa (ATCC No. 9027)
Staphylococcus aureus (ATCC No. 6538)
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9. • The spore suspension should be aseptically treated (filtration through sterile glass wool) to remove hyphae.
• All microbial suspensions should be prepared to ensure there is no carry over of residual growth medium
from the inoculum (e.g. centrifugation followed by resuspension in appropriate sterile suspending fluid.)
• Stock culture organisms may be grown in suitable liquid medium (i.e. Soyabean-Casein Digest Broth or
Sabouraud Dextrose Broth) and the cells harvested by centrifugation washed and resuspended in
appropriated sterile suspending fluid.
• Microbial suspension adjusted to obtain microbial count of 1*108 cfu/ml.
• Use bacterial and yeast suspension within 2 h , or within 24 h if stored between 20 – 80 for up to 7 days.
 PREPARATION OF TEST STRAINS CONTD
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10.  GROWTH PROMOTION OF MEDIA
• Media used must be capable of supporting microbial growth.
• Test Each batch of ready-prepared medium and each batch of medium prepared either from dehydrated
medium or from ingredients described.
• Solid media counts must be at least 50% of calculated value for standardized inoculum.
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11. SUITABILITY OF THE COUNTING METHOD IN
THE PRESENCE OF THE PRODUCT
1. Prepare a 10-1 dilution by adding 1 mL of product in 9 mL of saline or other neutralizing diluent and
continue dilution scheme to 10-2 and 10-3 dilution levels.
2. Add appropriate no. of challenging organisms to each tube of diluted product, mix, and place suitable
volume from each dilution, to yield less than 250 cfu/plate for bacteria and yeast (ideally 25-250 cfu) or
less than 80 cfu/plate for A.brasiliensis (8-80 cfu) plating should be done in duplicate.
3. A positive control for this procedure is to introduce same inocula into saline and transfer similar volumes of
saline to agar plates. 50% of saline control count will be a suitable recovery.
4. If the diluted product exhibits antimicrobial properties specific neutralizers may need to be incorporated
into the diluents
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12. • The ability of the procedure to measure preservative efficacy may be compromised if the method suitability
requires significant dilution (10-2 or 10-3) as this will affect measured recovery.(It may be difficult to measure
a 3 log unit reduction for a 105-106 inoculum.)
• If no suitable neutralizing agent or method is found and method suitability requires significant dilution a
higher level of inoculation (107-108) may be used so that 3 log unit reduction can be measured.
• Membrane filtration may be used to filter larger volumes of dilutions to overcome this difficulty or to assist
in the neutralization of antimicrobial properties.
• Reported recovery cannot be less than 1 cfu/plate on average (or 100 cfu/ml if 1 mL is plated in duplicate at
the 10-2 dilution).
SUITABILITY OF THE COUNTING METHOD IN
THE PRESENCE OF THE PRODUCT
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13. ANTIMICROBIAL EFFECTIVENESS TESTING
(AS PER IP)(2)
• Challenging the preparation in its final container with a prescribed inoculum of suitable microorganism.
• Storing the inoculated product at a prescribed temperature.
• Withdrawing samples from container at specified intervals of time and counting organisms in samples
removed.
• The preservative properties are considered adequate if there is significant fall or no increase in the number
of microorganisms in inoculated preparation after storage at specified conditions
• The organism specified for use in the tests are intended to be representative of those that might be expected
to be found in the environment in which preparation is manufactured, stored and used.
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14. • However they should be supplemented by other strains or species especially those likely to be found in
conditions under particular product is made or used or offer a particular challenge to the product being tested.
• Precautions:-
• Challenge test should be conducted under conditions that prevent accidental contamination of the product
during test.
• It should not affect survival of organisms in the product being examined . (2)
ANTIMICROBIAL EFFECTIVENESS TESTING
(AS PER IP)
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15. TEST ORGANISMS
Candida albicans ATCC 10231 Aspergillus brasiliensis ATCC 16404
Escherichia coli ATCC 8739 Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
• To prevent any phenotypic changes in the strains used, the organisms used in test should not be more than
5 passages made from the original culture.
• One passage is defined as inoculation and growth of organism from existing culture to fresh medium.
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16. PREPARATION OF INOCULUM (AS PER IP)
• Grow each bacterial species separately in casein soyabean digest agar and incubate them at 300 to 350 for
18 to 24 hours.
• Grow Candida albicans on Sabouraud dextrose agar and incubate at 200 to 250.
• Then Harvest the growth and resuspend each of the microorganism separately in sterile saline to obtain a
microbial count of 1 * 108 CFU per ml.
• To suspend spores of Aspergillus brasiliensis 0.05% polysorbate 80 may be added to saline.
• Use this suspension within 2-4 hours and can be stored at 40 to 80 for a validated period of time.
• Remove immediately a suitable sample from each suspension and determine CFU/ml by pour plate or
filtration method to determine inoculum concentration and baseline
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17. PROCEDURE (AS PER IP)
• If sufficient volume (at least 20 ml) of product is available in each container and the product container can be
inoculated aseptically then test can be conducted in five original containers of product.
• If filled volume is less or container cannot be inoculated aseptically then transfer (at least 20 ml) the product
in each of 5 suitable sterile containers.
• Inoculate each container with one of the prepared and standardized inoculum like that after inoculation the
final concentration of organisms remains between 1 * 105 and 1 * 106 CFU/ml and volume don’t exceed
1% of volume of product.
• Initial concentration estimated based on concentration of microorganisms in each standardized inoculum by
pour plate or membrane filtration method
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18. PROCEDURE (AS PER IP)
• Incubate the inoculated containers at room temperature.
• Determine the viable count by plate-count method at 7, 14, and 28 days subsequent to the inoculation.
• Record any changes observed in the appearance at these intervals.
• Calculate % of reduction in CFU/ml by concentration of CFU/ml at start of the test.
• Express changes in terms of % of initial concentration.
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19. INTERPRETATION (AS PER IP)
• For parenteral, ophthalmic, sterile nasal and otic preparations:-
a) Concentration of the viable bacteria NMT 10% of initial concentration at 7 day. NMT 0.1 % of the
initial concentration at 14 day and there is a further decrease in count at 28 day.
b) There is no increase in yeast and mold count at 7, 14, and 28 day from initial count.
• For topical preparations made with aqueous base, non-sterile nasal preparation and emulsions
including those applied to mucous membrane:
a) The concentration of the viable bacteria are NMT 1 % of initial concentration at 14 days and further
decrease in count at 28 days.
b) There is no increase in yeast and mold count at 14 and 28 day from initial count.
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20. INTERPRETATION (AS PER IP)
• For oral Preparations:
a) The concentration of the viable bacteria are NMT 10% of the initial concentration at 14 days and there
is a further decrease in count at 28 day.
b) There is no increase in yeast and mold count at 14 and 28 days from initial count.
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21.  For the purpose of testing, compendial articles have been divided into four categories .The criteria of
antimicrobial effectiveness for these products are a function of the route of administration. It is expected that
formulations containing preservatives will meet minimal efficacy standards, whether packaged as multidose
or unit dose.
PRODUCT CATEGORIES
Category Product Description
1 Injections; other parenteral including emulsions , otic products, sterile nasal products and
ophthalmic products made with aqueous bases or vehicles
2 Topically used products made with aqueous bases or vehicles; nonsterlie nasal products
and emulsions, including those applied to mucous membranes
3 Oral products other than antacids, made with aqueous bases or vehicles
4 Antacids made with an aqueous base
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22. PROCEDURE (AS PER USP)
 The procedure can be conducted either in five original containers if sufficient volume of product is available in
each container and the product container can be entered aseptically (i.e., needle and syringe through an elastomeric
rubber stopper),or in five sterile caped bacteriological containers[inert relative to antimicrobial agents ] of suitable
size into which a sufficient volume of product has been transferred.
 Inoculate each container with one of the prepared and standardized inocula, and mix. The volume of suspension
inoculum used is between 0.5% and 1.0% of the volume of the product to minimize potential effects on the
product.
 The concentration of viable microorganisms that is added to the product (Category 1,2,3) is such that the final
concentration of test preparation after incubation is between 1x105and 1x106 cfu/ml of product.
 For category 4 products (antacids) final concentration of test preparation is 1x103 and 1x104 cfu/ml of product.
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23.  The initial concentration of viable microorganism in each test preparation is estimated based on the
concentration of microorganism in each of the standardized inocula as determined by the plate-count method.
 Incubate the inoculated containers at 22.5 ± 2.5º. Sample each container at the appropriate intervals specified .
Record any changes observed in appearance at these intervals. Determine by the plate-count procedure the
number of cfu present in each test preparation for the applicable intervals .
 Using the calculated concentrations of cfu/ml present at the start of the test, calculate the change in log10 values
of the concentration of cfu/ml for each microorganism at the applicable test intervals, and express the changes
in concentration in terms of log reductions.
 The log reduction is defined as the difference between the log10 unit value of the starting concentration of
cfu/ml in the suspension and the log10 unit value of cfu/ml of the survivors at that time point.
PROCEDURE
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24. Organisms Suitable
Medium
Incubation
Temperature
Inoculum
Incubation
time
Microbial
Recovery Incubation
Time
Escherichia Coli
(ATCC No.8739)
Soybean-Casein Digest Broth;
Soybean-Casein Digest Agar
32.5 ± 2.5 18-24 h 3-5 days
Pseudomonas
aeruginosa
(ATCC No.9027)
Soybean-Casein Digest Broth;
Soybean-Casein Digest Agar
32.5 ± 2.5 18-24 h 3-5 days
Staphylococcus aureus
(ATCC No.6583)
Soybean-Casein Digest Broth;
Soybean-Casein Digest Agar
32.5 ± 2.5 18-24 h 3-5 days
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25. 25
Candida albicans
(ATCC No.10231)
Sabouraud Dextrose Agar;
Sabouraud Dextrose Broth
22.5 ± 2.5 44-52 h 3-5 days
Aspergillus brasiliensis
(ATCC No.16404)
Sabouraud Dextrose Agar;
Sabouraud Dextrose Broth
22.5 ± 2.5 6-10 days 3-7 days

26. Bacteria NLT 1.0 log reduction from the initial calculated count at 7 days,
not less than 3.0 log reduction from the initial count at 14 days, and
no increase from the 14 day’s count at 28 days.
Yeast and Molds No increase from the initial calculated count at 7, 14 and 28 days.
The requirements for antimicrobial effectiveness are met if the criteria specified in given table. No increase in
counts is counts is defined as NMT 0.5 log10 unit more than the value to which it is compared.
ACCEPTANCE CRITERIA (USP)
For Category 1 Products
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27. For Category 2 Products
Bacteria NLT 2.0 log reduction from the initial count at 14 days, and no
increase from the 14 day‘s count at 28 days.
Yeast and Molds No increase from the initial calculated count at 7, 14 and 28 days.
For Category 3 Products
Bacteria NLT 2.0 log reduction from the initial count at 14 days, and no
increase from the 14 day‘s count at 28 days.
Yeast and Molds No increase from the initial calculated count at 7, 14 and 28 days.
Bacteria, Yeast, Molds No increase from the initial calculated count at 14
and 28 days.
For Category 4 Products
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28. The criteria for evaluation of antimicrobial activity are given below in terms Of the log10 reduction in the
number of viable micro-organisms against the value obtained for the inoculum.
6h 24h 7d 14d 28d
Bacteria A
B
2
―
3
1
—
3
—
―
NR
NI
Fungi A
B
―
―
―
―
2
—
―
1
NI
NI
 Parenteral preparations, eye preparations , intrauterine preparations
ACCEPTANCE CRITERIA BP
A: recommended
efficacy to
achieved.
B: if criteria A not
attained B must be
satisfied.
NR : no recovery
NI : no increase
in number
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29. 6h 7d 14d 28d
Bacteria A
B
2
―
3
―
―
3
NI
NI
Fungi A
B
—
―
―
—
2
1
NI
NI
 Ear preparations, nasal preparation, preparations for cutaneous application and Preparations for inhalation
14d 28d
Bacteria 3 NI
Fungi 1 NI
 Oral preparations, oromucosal peparations and rectal preparations
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30. THANK YOU