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Assessment of Microbial Contamination and Spoilage

The evaluation of a medicinal product's microbiological composition is critical. A sterile product should be completely devoid of microorganisms.

Assessment of Microbial Contamination and Spoilage

The evaluation of a medicinal product's microbiological composition is critical. A sterile product should be completely devoid of microorganisms, as determined by a sterility test. Non-sterile items, on the other hand, may include microorganisms. These microorganisms have the potential to be both pathogenic and non-pathogenic. Microorganisms like these can cause deterioration, which can pose health risks. The overall number of microorganisms present in a product must be minimal and below the allowable limit. To detect the existence of certain bacteria, the types and characteristics of the microbes should also be examined.

Tests to determine the total amount of microorganisms and types of bacteria are called microbial limit tests. The following tests are carried out to evaluate microbial contamination and consequent deterioration.

Test of sterility - Some pharmaceutical items should be tested for sterility, according to the Indian pharmacopoeia, the British pharmacopoeia, and the United States pharmacopoeia. The procedures are comparable with minor differences. The tests prescribed by the Indian Pharmacopoeia are briefly reviewed. There are two methods: direct inoculation and membrane filtering.

1. Direct inoculation - In this procedure, a little amount of material is immediately introduced to the pharmacopeia-specified culture medium. This inoculation medium is then incubated for a certain amount of time. The existence of growth implies the presence of a microorganism derived from the sample. As a result, it is possible to determine that the sample is not sterile. In the lack of any growth, a sample is said to be sterile.

2. Membrane filtration - The material is filtered through a membrane filter and rinsed with diluting solution in this procedure. If microbes are present, they will be near the top of the filter paper. This filter paper is now injected into the appropriate culture medium. If growth is detected, it shows that the product is not sterile.

In both the above-mentioned methods, a positive and negative control test must be performed.

Microbial limit test - The European Pharmacopoeia suggests assessing microorganisms both qualitatively and quantitatively. The microbiological Limit test is recommended by the United States Pharmacopoeia. It is divided into two sections: (i) Total Aerobic Microbial Count and (ii) Test for Specific Microorganisms.

i) Total Aerobic Microbial Count
In this approach, a defined amount of test sample (10gm) is combined with a specific amount of peptone water (90ml).

This is called sample dilution. Dilutions of water-insoluble and fatty compounds must follow precise procedures. The total microbial count is determined using the following procedure: To begin with, the sample is filtered through a membrane filter by mixing 10 ml of distilled water with 90 ml of peptone water. After that, it is rinsed three times with sterile peptone water. One filter paper is incubated for 5 days at 30-35oC in Petri dish with Soyabean Caesin Digest Agar medium. Colonies are counted to ascertain bacterial count.

Another filter paper is immersed in Sabouraud Dextrose Agar Media and incubated at 20-25oC for 5 days. The fungal count is then calculated.

Second, the sample plate count technique is examined. In this approach, created dilution is directly transferred to four Petri dishes. There are two for bacteria and two for fungus. 15 ml of Soyabean Caesin Digest Agar medium is added to the first two Petri dishes. After 5 days of incubation at 30-35oC, colonies are counted. 15 ml of Sabouraud Dextrose Agar Media was transferred to the remaining two Petri dishes and incubated at 20-25oC. Colonies are tallied.

ii) Test for Specific Microorganisms
Escherichia coli, Salmonella, Pseudomonas aeruginea, and Staphylococcus aureus are identified using specific assays.

Mac-Conkey agar medium is used to culture E. coli. Colonies are distinguished by their metallic gleam.

Salmonella colonies are detected as black or green on bismuth sulphite agar medium.

Pseudomonas aeruginosa is recognized by growing it on cetrimide agar medium. Colonies gradually turn a greenish colour.

Staphylococcus aureus is grown on Vogel Johnson agar media and is distinguished by black colonies surrounded by yellow zones.

Periodic test
To assess the product's continuous efficacy, a total microbiological count must be performed on a regular basis.

There are a number of microorganism identification and determination tests available. They include Luciferase tests, Epifluorescence tests, electrical impedance tests, etc.
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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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