Sterility & Pyrogen testing: Sterility Test; Culture Media.

 

Introduction:

• Sterilization is the process of complete removal of microorganisms from any surface or product.

• Sterility hence can be defined as “Complete freedom from microorganisms”.

• The tests for sterility are conducted to make sure there is complete absence of viable forms of microorganisms on a pharmaceutical product.

• The products which are to be strictly supervised for absence of the microorganisms are those who come directly in contact with the systemic circulation e.g. ophthalmic preparations, parenteral injections, implants, bandages, surgical dressings, needles, surgical instruments etc.

• The test for sterility has to be conducted in an aseptic environment so as to avoid contamination of the product.

Test for Sterility.

Principle:

• A viable microorganism when provided with favorable conditions and nutrients shows growth indicating its presence in the product.

• The probability of detecting the microbes in a product increases with the number of the testings done.

• The external surfaces of the ampoules and vials or the containers should be cleaned with the suitable antimicrobial substance and test be carried out in an aseptic environment in order to avoid any contamination of microbes in the products under testing.

Culture Media.

• Following culture media are used for conducting the sterility tests,

• Fluid Thioglycollate Medium (FTM).

• Alternative Thioglycollate Medium (ATM).

• Soybean Casein Digest Medium (SCDM).

1. Fluid Thioglycollate Medium (FTM):

• For use with Clear fluid products.

• Mainly intended for “Anaerobic Bacteria” but can also detect Aerobic Bacterias.

• It is an indicator culture medium as it contains “Resazurin” a dye which becomes pink on contact with oxygen.

• While using not more than one tenth of the upper portion of the medium should have pink color.

• If more than one third of the medium is looking pink (Showing presence of oxygen) it can be simply restored by reheating in the water bath until color disappears.

• pH: 7.1 ± 0.2.

• Sterilization: Autoclaving @ 121℃ for 20 mins.

2) Alternative Thioglycollate Medium (ATM):

• For use with turbid or viscous products and devices with small layers.

• It is an anaerobic medium i.e. made for cultivation of anaerobes and hence anaerobic conditions must be followed while preparing and using.

• pH: 7.1 ± 0.2.

• Sterilization: Autoclaving @ 121℃ for 20 mins.

3) Soybean Casein Digest Medium (SCDM):

• Suitable for cultivation of both fungi and aerobic bacterias.

• pH: 7.3 ± 0.2.

• Sterilization: Autoclaving @ 121℃ for 20 mins.

Table 2.1: Composition of the Culture Medias used for Sterility Test.

Sr No.	Component	FTM	ATM	SCDM
1	L-Cysteine	0.5 gm	0.5 gm	--
2	Sodium CHloride	2.5 gm	5.5 gm	5. gm
3	Dextrose (Monohydrate or Anhydrous)	5.5 gm / 5.0 gm	5.5 gm / 5.0 gm	2.5 gm / 2.3 gm
4	Yeast Extract (Water Soluble)	5.0 gm	5.0 gm	--
5	Pancreatic Digest of Casein	15.0 gm	15.0 gm	17.0 gm
6	Sodium Thioglycollate / Thioglycolic Acid	0.5 gm / 0. ml	0.5 gm / 0.3 ml	--
7	Papic digest of Soybean meal	--	--	3.0 gm
8	Dipotassium Hydrogen Phosphate	--	--	2.5 gm
9	Resazurin Sodium	1.0 ml	--	--
10	Distilled water upto	1000 ml	1000 ml	1000 ml
11	pH after Sterilization by Autoclave @ 121℃ for 20 mins.	7.1 ± 0.2	7.1 ± 0.2	7.3 ± 0.2

Tests for Culture Media:

• The media to be used in sterility testings should pass the following tests,

• Sterility of Media.

• Growth Promotion Test.

• Test for Bacteriostatic and Fungistatic.

1. Sterility of Media:

• Incubate the portions of FTM and ATM @ 30-35℃ and SCDM at 20-25℃ and incubate them for not less than 14 days.

• The presence of microbial growth fails the test, while absence of growth passes the test.

2. Growth Promotion Test:

• This test is used to check the capability of the medium to promote the bacterial growth.

• Medium sample from each autoclaved medium container is taken and inoculated with about 100 test organisms of each “Test Strain” and incubated as per the requirements of the given strain.

• The sample passess the test if it shows satisfactory growth of the organisms.

• The test is considered failed on inadequate growth of the microbes in the sample.

Table 2.2: List of Test Microorganisms for the Growth Promotion Test of Media.

Medium	Test Microorganisms	Incubation Parameters
		Temperature	Duration in Days	Conditions
FTM	Clostridium Sporogenes	30-35℃	3	Anaerobic
	Staphylococcus aureus	30-35℃	3	Aerobic
	Pseudomonas aeruginosa.	30-35℃	3	Aerobic
ATM	Bacteroides vulgatus	30-35℃	3	Anaerobic
	Clostridium Sporogenes.	30-35℃	3	Anaerobic
	Bacillus subtilis.	30-35℃	3	Aerobic
SCDM	Aspergillus brasiliensis	20-25℃	5	Aerobic
	Candida albicans	20-25℃	5	Aerobic
	Bacillus subtilis.	30-35℃	3	Aerobic

3) Test for Bacteriostatic and Fungistatic:

• Prepare cultures of the test bacterias and fungi.

• Inoculate the required volume of culture media with 100 viable microorganisms.

• Add the required amount of the preparation under test to the half of the containers containing medium and microbes.

• The half of the containers are kept as control.

• After specified time the growth of microbes is visually compared in test and control containers.

• If the test preparation is found to be bacteriostatic or fungistatic it can be neutralized using “Neutralising agents” as specified.

Table 2.2: List of Neutralizing substances for antimicrobial compounds.

Sr No.	Antimicrobial agent	Neutralizing Agent.
1	Penicillin	Penicillinase
2	Streptomycin	Streptomycin phosphotransferase
3	Cephalosporins	Cephalosporinase.
4	Sulfonamides	P Amino benzoic Acid