PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-VPart-3
Preservation of pharmaceutical products using antimicrobial agents.
Introduction. Ideal Properties of Preservatives:
Antimicrobial Chemical Preservatives
Development of a Preservative System.
Factors affecting efficacy of a preservative: 1. Interaction With components of the formulation
2. Properties of the Preservatives:
3) Effect of Containers.
4) Type of microbes:
5) Influence of pH:
Challenge Test: Efficacy Test of Preservative : Medium used, Choice of test organism:
Preparation of the inoculum:
Procedure:
Interpretation of Results:
2. Introduction.
• A preservative is an antimicrobial substance added in the
pharmaceutical formulations with the view to avoid contamination in
the formulation.
3. Ideal Properties of Preservatives:
• Should not cause irritation.
• Should be non toxic.
• Stable.
• Should be effective during shelf life of the formulation.
• Should kill all contaminants effectively and rapidly.
• Should not react with ingredients of the formulation.
• Should not interact with the container and closure system of the dosage form.
4. • Should not produce allergic reactions to the patients.
• Should not increase the cost of the end product.
• Should have a good spectrum of activity at lower dose.
• However not a single preservative is found to possess all the requirements.
• Even some microbes like Pseudomonas have already started showing
resistance to many of the present preservatives and hence a combination of
preservatives is used.
• Sometimes an action potentiator is added to increase the efficacy of a
preservative.
5. Antimicrobial Chemical Preservatives:
• The main purpose of these preservatives is to inhibit growth of microbes in the
pharmaceutical product.
• They can be classified into four major groups as,
• Acidic,
• Mercuric,
• Neutral,
• Quaternary Ammonium Compounds.
• Commonly used antimicrobial chemical preservatives are given in table 1.1
7. • Being chemicals these preservatives are known to react with the
contents of formulation and container closure system hence must
be selected carefully.
• e.g. Plastic containers may absorb some preservatives making
formulation susceptible to microbial attack.
• Some glass containers may release alkali in the formulation which in turn
may make your preservative ineffective.
8. • Some other ingredients or vehicles themself act as preservatives
sometimes like,
• Syrup contains high concentrations of sugar which inhibit growth of microbes.
• Chloroform water, alcohol are known preservatives.
• While using preservative in pharmaceutical emulsions the partition
coefficient of the preservative must be considered as microbes may grow
in lipids as well as water layers.
9. Development of a Preservative System.
•A single preservative is not found to possess all the ideal
requirements of a preservative.
•Even some microbes like Pseudomonas have already started
showing resistance to many of the present preservatives.
10. • Hence a combination of preservatives is preferred instead of using a single preservative.
• By combining two preservatives Synergy can be obtained means increased inhibitory effect, e.g.
Methyl para hydroxybenzoic acid is combined with propyl para hydroxybenzoic acid in ratio
of 10:1.
• By combining the preservatives spectrum of activity of the system gets widened e.g. eye drops and
contact lens cleaners combine “Phenoxetol with Phenyl ethyl alcohol and Benzalkonium
Chloride.
• Preservative Germall 115 is an antibacterial when combined with parabens the combination
becomes antibacterial as well as antifungal.
• However, the combination system when used should have enough half lifes with
reference to the life of the formulation.
11. Factors affecting efficacy of a preservative:
1. Interaction With components of the formulation:
• A chemical antimicrobial preservative may interact with the contents of the
formulation and lose its activity e.g.
• Tablets contain many additives which may be incompatible with the preservatives.
• Some drugs can interact with preservatives making them ineffective e.g. Kaolin, Sulfadimidine,
can absorb preservatives from the formulation.
• When used in an emulsion a preservative with good lipid solubility gets concentrated in oil
phase leaving the water phase of the formulation vulnerable.
• Hydrocolloids such as tragacanth, alginates pvp can interact with preservatives and make them
ineffective.
12. 2. Properties of the Preservatives:
• Plastic containers may absorb some preservatives making formulation
susceptible to microbial attack.
• Some glass containers may release alkali in the formulation which inturn
may make your preservative ineffective.
• While using preservative in pharmaceutical emulsions the partition
coefficient of the preservative must be considered as microbes may grow
in lipids as well as water layers
13. 3) Effect of Containers.
• Plastic containers may absorb some preservatives making formulation
susceptible to microbial attack.
• Some glass containers may release alkali in the formulation which inturn
may make your preservative ineffective.
• Screw-capped containers and corks are a common source of
contamination.
• Rubber used in closures may interact with the preservatives.
14. 4) Type of microbes:
• Many products of biological origin contains contamination from specific
genuses of organisms and hence include preservative effective against such
microbes e.g.
• Plant products are contaminated with dust which contains microbes from Clostridia,
Bacillus, Staphylococcus species.
• Animal products like gelatin are usually found contaminated with salmonella,
clostridium etc.
15. 5) Influence of pH:
• Quaternary ammonium compounds need higher pH for their
increased efficacy.
• Acidic preservatives usually demand and acidic pH for better
action e.g. Benzoic acid.
• However, many preservatives are known to show action at a
wider range of pH.
16. Challenge Test: Efficacy Test of Preservative.
• This test is performed on a formulation in its final container to determine
efficacy of the preservative added against the microbial spoilage.
• This test is applicable to, multi dose parenterals, otic, nasal, oral, topical
and ophthalmic products made with aqueous base or vehicles.
• Medium used:
• Soybean Casein Digest Medium
17. • Choice of test organism:
• The microbes which are likely to arise in the formulation through raw
material are usually selected.
• However to check effectiveness of the preservative against a wide range
of microbes Gram +ve, Gram -ve bacterias, yeasts and moulds are
selected in the IP test.
Microorganism ATCC No.
Staphylococcus aureus ATCC 6538
Pseudomonas aeruginosa ATCC 9027
Escherichia coli ATCC 8739
Candida albicans ATCC 10231
Aspergillus brasiliensis ATCC 16404
Microbes used as test organisms for Challenge Test.
18. • Preparation of the inoculum:
• Fresh stock cultures of test microorganisms are subcultured on the surface of
Soyabean Casein Digest Medium.
• Incubate,
• bacterial cultures @ 30-35 ℃ for 18-24 hours.
• Candida albicans @ 20-25 ℃ for 48 hours.
• Aspergillus brasiliensis @ 20-25 ℃ for 7 days.
• Using sterile solution harvest the microorganisms to get a count of 1 x 108
CFU/ ml (Colony FOrming Unit).
19. • Procedure:
• Inoculate each final container or product test tube with one of the standardized
microbial suspension in a ratio of 0.1 ml : 20 ml and mix well.
• The final concentration of microorganisms in the product should be between 1 x 105
to 1 x 106 microorganisms per ml.
• Determine final concentration of microorganisms in final product by plate count
method.
• Incubate inoculated containers at room temperature.
• Determine viable count after 7, 14 and 28 days of inoculation.
• Calculate the percentage reduction in CFU / ml of each microorganism at stated test
intervals and get the changes in terms of percentage of initial concentration.
20. Interpretation of Results:
• For parenteral, ophthalmic, sterile nasal and otic preparations:
• Viable bacterial concentration is,
• NMT 10 % on after 7days,
• NMT 0.1 % after 14 days.
• There is no further decrease in count at 28 day.
• No increase in yeast and mould count at 7, 14 & 28 days test from the initial
count.
21. • For topical preparations made with aq. base , non sterile nasal preparations and
emulsions:
• Viable bacterial concentration is NMT 1% of initial concentration at 14 days and there is
further decrease in count at 28 days.
• No increase in yeast and mould count at 14 & 28 days test from the initial count.
• For oral preparations:
• Viable bacterial concentration is NMT 10% of initial concentration at 14 days and there
is further decrease in count at 28 days.
• No increase in yeast and mould count at 14 & 28 days test from the initial count.
