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.

Sterility Test Methods.

• Sterility tests can be carried out using one of the following methods,

• Methods A: Membrane Filtration.

• Method B: Direct Inoculation.

Method A: Membrane Filtration.

• This method is preferred when substance to be tested is,

• a) an oil,

•  b) an ointment that can be put into solution,

•  c) a non bacteriostatic solid not readily soluble in the culture medium and

•  d) a soluble powder or a liquid that possesses inherent bacteriostatic and fungistatic properties.

• For liquid products where the volume > 100ml or more

• Precautions: 

• A laminar sterile airflow cabinet – to avoid accidental contamination.

• Working conditions monitored regularly by sampling the air and surfaces of the working area.

• Apparatus: 

• A suitable unit consists of a closed reservoir and a receptacle between which a properly supported membrane of appropriate porosity is placed. 

• Nominal pore size not > 0.45µm and diameter of approx. 47mm.

• Flow rate is adjusted to 55-75 ml of water / minute.

• Pressure 70 mm of Hg.

• Cellulose nitrate filter for aqueous, oily and weakly alcoholic solutions.

• Cellulose acetate solution for strong alcoholic solutions.

• Dilution Fluids:

• Fluid A: Dissolve 1g of peptic digest of animal tissue in water to make 1 liter, filter / centrifuge, adjust to pH 7.1±0.2, dispense into flasks in 100-ml quantities and sterilise at 121º for 20 minutes.

• Fluid B: If the test sample contains lecithin or oil, use fluid A to each liter of which has been added 1 ml of polysorbate 80, adjust to pH 7.1±0.2, dispense into flasks in 100-ml quantities and sterilise at 121º for 20 minutes.

• Quantities of sample to be used:

Table 3.1 Quantities of sample to be used:

Preparation	Quantity in each container of the preparation	Minimum quantity to be used for each culture medium
Liquids	Less than 1 ml.	Total contents of container.
	1-40 ml	Half of the container but not less than 1 ml.
	40 ml to 100 ml.	20 ml
	More than 100 ml	10% of the container but not less than 20 ml.
Antibiotic liquids		1 ml
Other soluble preparations		The whole contents of each container to provide not less than 200mg.
Insoluble preparations ointments & creams.		The whole contents of each container to provide not less than 200mg.
Solids	Less than 50mg	Total container.
	50 mg - 300 mg	Half of the container but not less than 50 mg.
	300 mg - 5g	150 mg
	More than 5 gm	500 mg
Catgut and other surgical sutures for veterinary use.		Three sections of a strand each 30 cm long
Surgical dressings/ cotton/ gauze (In packages)		100 mg per package
Sutures and other individually packed single use material.		Whole device
Other medical devices		Whole device or material cut into pieces or diassembled

Method of test:

• 1) For aqueous solution: - 

• Prepare each membrane by aseptically transferring fluid A + media + preparation being examined. -

• Alternatively, first, combined quantities of preparation + prescribed in the two media. 

• Draw the liquid rapidly through a filter with aid of vacuum.

• If the solution being examined has antimicrobial properties, wash the membrane by filtering 100ml of sterile fluid A, three times, or quantities should be sufficient to allow growth of small inoculum of organisms.

• After filtration, aseptically remove membrane from holder, cut the membrane in half, immerse one half of membrane in 100ml of soyabean-casein digest medium and incubate at 20º to 25º for not < 7days. 

• Similar, other membranes in FTM (30º to 35º).

• 2)  For liquids immiscible with aqueous vehicles and suspensions: - 

• Same as above but add sufficient quantity of fluid A to achieve rapid filtration.

• Sometimes use sterile enzyme preparations such as penicillinase or cellulase to aid in dissolving insoluble substances. 

• If lecithin is there, use fluid B for diluting.

• 3)  For oils and oily solutions: 

• Low viscous, filter without dilution through dry memb.

• Viscous oils – dilute as per needed using sterile diluent like isopropyl myristate – filter by applying pressure or suction gradually. 

• Wash the membrane at least 3 times sterile fluid B (100 ml) or to heat not > than 45º and use warm solutions for washing membrane. 

• 4)  For ointments and creams: 

• Dilute ointments in a fatty base and emulsions of the w/o type to give a fluid concentration of 1%w/v, by heating (if necessary), not > than 40º with a suitable sterile diluent.

• 5)  For soluble solids: 

• Dissolve substance in a suitable sterile solvent for each medium and carry out test described under aqueous solutions. 

• 6)  For sterile devices:

• Aseptically pass a sufficient volume of fluid B through each of not less than 20 devices so that not less than 100ml is recovered from each device. 

• Collect fluids in sterile containers and filter the entire volume through a membrane filter funnel, and follow the test as under for aqueous solutions.

Method B Direct Inoculation

Method of test:

• 1) For aqueous solutions and suspensions: 

• Remove liquid from test containers with a sterile pipette or with a sterile syringe or a needle 

• Aseptically transfer specified volume of the material from each container to a vessel of the culture medium 

• Mix the liquid with the medium but do not aerate excessively 

• Incubate the inoculated media for not less than 14 days, unless otherwise specified in the monograph.

• Incubate at 30º to 35º in case of fluid thioglycollate medium and at 20º to 25º in case of soyabean casein digest medium. 

• If a test sample renders the medium turbid, it is difficult to examine presence or absence of microbial growth by visual examination. 

• Transfer suitable portions of the same medium to fresh vessels between the third and seventh days after the test is started. 

• Continue incubation of transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days

• 2) For oils and oily solutions:

• In media, add 0.1% w/v of (4 –tert -octylphenoxy) polyethoxy ethanol, 1% w/v of polysorbate 80 or other suitable emulsifying agent, in an appropriate conc. 

• Oil containing cultures should be shaken gently each day. 

• 3) For ointments: - 

• Preparation is diluted tenfold in a sterile diluent such as fluid B or any other aqueous vehicle capable of dispersing test material homogeneously throughout the fluid mixture. 

• Mix 10ml of fluid mixture with 80 ml of the medium and proceed the same as aqueous solutions & suspension.

• 4) For solids: - 

• Mix, and incubate same as 1)

• 5) For sterile devices: - 

• For articles of such size and shape as permit complete immersion in not more than 1000ml of culture medium test the intact article, using the appropriate media and incubate same as 1).

• For transfusion or infusion assemblies or where the size of an item makes immersion impracticable, flush the lumen of each of 20 units with a sufficient quantity of fluid thioglycollate medium and soyabean casein medium to yield a recovery of not less than 15ml of each medium, and incubate with not less than 100 ml of each of two media.

• Where the presence of the specimen being tested interferes with the test because of bacteriostatic action, rinse the article thoroughly with minimum amount of fluid A. Recover the rinsed fluid and test as described for sterile devices under method A.

Observation and Interpretation of Results.

• At interval during incubation period, and at its conclusion, examine media for macroscopic evidence of microbial growth. 

• If no evidence of growth is found, the preparation being examined passes the test for sterility. 

• If evidence of microbial growth is found, reserve the containers, and it is demonstrated that it is not due to preparation, hence the tests for sterility are invalid and may be recommenced. 

• Perform a retest using the same number of samples, volumes to be tested and the media 

• if no evidence of microbial growth is then found, the preparation being examined passes the test for sterility.

• If evidence of microbial growth is found, isolate and identify the organisms.

• If they are not readily distinguishable from those growing in the containers reserved in the first test, the preparation being examined fails the test for sterility.  

• If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second retest using twice the number of samples. 

• If no evidence of microbial growth is found in the second retest, the preparation being examined passes the test for sterility. 

• If evidence of growth of any micro-organisms is found in second retest, the preparation being examined fails the test for sterility.

Advantages of Filtration Technique

1. Wide application – can be used for solutions, soluble solid, insoluble solids, oils, ointments, syringes like articles 

2. Very large volume can be tested with one pad 

3. Small volume of broth is required than direct inoculation into culture media 

4. Apply to substances for which no satisfactory inactivators are known 

5. Some strong antibacterial agents like mercurials and quaternary ammonium compounds, inactivated by treatment with appropriate neutralising solution. 

6. Subculturing is often eliminated

Disadvantages of Filtration Technique 

1. The possibility of adsorption of sufficient medicament 

2. Highly skilled staff and exceptionally good aseptic techniques are necessary. 

3. A virtually sterile environment is essential 

4. A room with laminar air flow and conventional asepsis laboratory is needed.