Assessment of microbial contamination and spoilage
Assessment of microbial contamination and spoilage is done as following,
Physical and chemical changes:
The changes of different pharmaceutical formulations indicate microbial contamination and spoilage.
Change in viscosity, pH, emulsion stability and loss of surface activity of formulation indicates microbial spoilage.
Measurement of oxygen consumption of the product can teel us the degree of oxidative attack and microbial growth
Assessment of viable microorganisms in non-sterile products:
Non-sterile products are tested for viable microorganisms for detection of pathogens and total viable counts.
In microbiological terms, pharmaceutical products can be divided into two groups: sterile and nonsterile.
Non-sterile drugs must satisfy the appropriate microbiological purity criteria by passing Microbial Limit Tests which are included in pharmacopoeial monographs.
Pharmacopoeial studies are prepared specifically with a view to ensuring that the medicinal product is therapeutically effective and safe for the patient.
Sterility test: (Click here for detailed process of sterility tests)
Sterility Testing confirms that products are free from the presence of viable microorganisms.
Test is conducted by competent and experienced personnel in an adequately clean room with laminar flow cabinet facilities.
All injectables and ophthalmic preparations are sterile hence, these preparations are tested by the sterility test.
Estimation of pyrogens: (Click here for detailed process of pyrogen tests)
Pyrogen is a substance, typically produced by a bacterium, which produces fever when introduced or released into the systemic circulation.
The lipopolysaccharides and lipoproteins which comprises a major part of the cell wall of Gram –ve bacteria are called endotoxins which are the most commonly called pyrogens.
To test the pyrogens presence, two tests are used
1. RP Test (Rabbit Pyrogen Test)
2. LAL Test (Limulus Amoebocyte Lysate Test).
Microbial Limit Tests:
The microbial content of non sterile dosage forms, herbal products, herbs and processed herbals are controlled by using following methods,
Estimation of total viable aerobic microbes in a given sample (Total Viable Count).
Detection of specific species of microbe in the sample.
These microbial limit tests are mainly applicable to the raw materials of natural or biological origin (e.g. Starch, Gelatin etc) and some finished products like Calamine lotion, Dried Aluminium Hydroxide Gel, Betamethasone valerate ointments etc.
Detection of specific microbes includes Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Shigella, Clostridia and Candida albicans as per Indian Pharmacopoeia 2014.
Total Aerobic Microbial Count:
The total count of viable aerobic microbes per unit weight or volume of the sample is a very commonly used technique in microbiology.
However, it is necessary first to determine the presence of antimicrobial substances in the sample.
For determination of total aerobic microbial count, it is necessary to pretreat the samples as per specifications given in I.P..
Total aerobic count of the test samples is determined by the following methods.
Membrane Filtration.
Plate Count Methods.
Pour Plate Method.
Surface spread Method.
Most Probable Number(MPN)
Membrane Filtration:
For this use: filter 50 mm in diameter and pore size not greater than 0.45µm.
Procedure:
Filter 10 ml or quantity of each dilution containing 1 gm of the preparation through each two membrane filters immediately.
If necessary the pretreated solution to be diluted to make up the volume.
Wash each membrane by filtering it through three quantities of buffered sodium chloride peptone solution (For fatty substances add Polysorbate 20/80 in the solution.)
Transfer the membranes each to the surface of Casein Soybean Digest Agar plate and Sabouraud Dextrose Agar plate with antibiotics.
Incubate the plates @ 30 to 35 ℃ for 3-5 days for testing bacteria and 5-7 days for fungi.
Calculate no. of microbes per gm or per ml of the sample.
2. Plate Count Methods:
Pour Plate Method:
Mix 1 ml of pretreated sample and about 15 ml of liquified Casein Soybean Digest Agar plate or Sabouraud Dextrose Agar in petri dishes (9 cm diameter) with antibiotics @ 45 ℃.
If necessary dilute the sample so that colony count of not more than 300 is obtained.
Incubate the Casein Soybean Digest Agar plate @ 30 to 35 ℃ for 3-5 days.
Incubate the Sabouraud Dextrose Agar plate @ 20 to 25 ℃ for 2-7 days.
Select the plates containing not less than 30 and not more than 250 colonies of bacteria and 30-50 colonies of fungi.
Calculate no. of microbes per gm or per ml of the sample.
Surface spread Method:
Add 15 ml of liquified Casein Soybean Digest Agar plate or Sabouraud Dextrose Agar in petri dishes (9 cm diameter) with antibiotics @ 45 ℃ and allow to solidify them.
Dry the plates under laminar air flow or in an incubator.
Spread a measured volume of not less than 0.1 ml of pretreated sample over the plates.
Use at least two plates for each sample and each strain of test microbe.
Incubate the Casein Soybean Digest Agar plate @ 30 to 35 ℃ for 3-5 days.
Incubate the Sabouraud Dextrose Agar plate @ 20 to 25 ℃ for 2-7 days.
Select the plates containing not less than 30 and not more than 250 colonies of bacteria and 30-50 colonies of fungi.
Calculate no. of microbes per gm or per ml of the sample.
3) Most Probable Number (MPN):
Also known as “Multiple Tube method” or “Serial Dilution Method”.
The accuracy is less than filtration and plate count methods and hence is carried out only when they are not suitable.
Procedure:
Prepare a series of at least three subsequent ten fold dilutions of the product as described in the pretreatment of the sample.
From each level of dilution, thee aliquots of 1 gm or 1ml are used to inoculate three tubes with 9 ml of Sterile Casein Soybean Digest Broth.
Hence if three levels of dilutions are prepares then 9 test tubes will be used and likewise.
Incubate all the test tubes @ 30 to 35 ℃ for 3-5 days.
Record for each level of the dilution the number of tubes are showing microbial growth.
Determine the most probable number of bacteria per gm or per ml of the product as per image bellow.
