7.1 Total Viable Spore Count of Biological indicator, Paper carriers: 
7.1.1 Aseptically remove three specimens of the relevant biological indicators from their original individual containers.
7.1.2 Disperse the paper into component fibers by placing the test specimens in a sterile 250 ml screw-capped bottle of a suitable mixer containing 100 mL of chilled, sterilized purified water and mix for a adequate time to achieve a homogeneous suspension.
Note: 15 minutes or more to be required for optimal recovery.
7.1.3 Transfer a 10 ml aliquot of the suspension to a sterile 100 ml screw-capped bottle.
7.1.4 Heat the bottle containing the suspension in a water bath at 95°C to 100°C for 15 minutes (Heat Shock), starting the timing when the temperature reaches 95°C.
7.1.5 Cool rapidly in an ice-water bath at 0°C to 4°C.
7.1.6 Transfer 1ml aliquots in duplicate to tubes containing 9 ml of sterile purified water and make 10 fold appropriate serial dilutions in sterilized purified water.
7.1.7 The dilutions being selected as calculated to yield preferably 30 to 300 colonies, but not less than 6, on each of a pair of plates.
Note: Where the biological indicator has a low spore concentration, it may be necessary to modify the dilution series and to use more plates at each dilution.
7.1.8 Prepare a separate series of plates for each aliquot.
7.1.9 Place 1 ml of each selected dilution in each of two sterile Petri dishes.
7.1.10 Within 20 minutes, add to each plate15 to 20 ml of Soybean Casein Digest Agar media that has been melted and cooled to 40°C to 45°C. Swirl to attain a homogeneous suspension, and let it to solidify.
7.1.11 Incubate the plates in an inverted position at 55°C to 60°C or at the optimal recovery temperature specified by the manufacturer.
7.1.12 Examine the plates after 24 and 48 hours, recording for each plate the number of colonies, and use the number of colonies observed after 48 hours to calculate the results.
7.1.13 Calculate the average number of spores per specimen from the results, using the appropriate dilution factor.
Note: The test is valid if the log number of spores per carrier at 48 hours is equal to or greater than the log number after 24 hours in each case.

7.2 Total Viable Spore Count of Biological indicator, Non paper Carriers:
7.2.1 Aseptically remove the three carriers from their original packaging or container. Place each carrier in a suitable sterile container containing 100 ml of chilled sterile Purified water, and mix for an appropriate time.
Note: 15 minutes or more may be required for optimal recovery.
7.2.2 Transfer a 10 ml aliquot of the suspension to a sterile 100 ml screw-capped bottle.
7.2.3 Heat the bottle containing a suspension of Geobacillus stearothermophilus (ATCC 7953) at 95°C to 100°C for 15 minutes. Start the timing when the lowest temperature reached the specified range.
7.2.4 Cool rapidly in an ice-water bath at 0°C to 4°C.
7.2.5 Transfer 1ml aliquots in duplicate to tubes containing 9 ml of sterile purified water and make 10 fold appropriate serial dilutions in sterilized purified water.
7.2.6 The dilutions being selected as calculated to yield preferably 30 to 300 colonies, but not less than 6, on each of a pair of plates.
Note: Where the biological indicator has a low spore concentration, it may be necessary to modify the dilution series and to use more plates at each dilution.
7.2.7 Prepare a separate series of plates for each aliquot.
7.2.8 Place 1 ml of each selected dilution in each of two sterile Petri dishes.
7.2.9 Within 20 minutes add the aliquot to each plate containing 15 to 20 ml of Soybean Casein Digest agar media that has been melted and cooled to between 40°C to 45°C. Swirl to attain a homogeneous suspension and, allow it to solidify.
7.2.10 Incubate the plates in an inverted position aerobically at the following respective temperatures at 55°C to 60°C or at the optimum temperature specified by the biological indicator manufacturer.
7.2.11 Examine the plates after 24 and 48 hours. Number of colonies observed in each plate should be recorded. Average number of spores per carrier shall be calculated from the results, using the appropriate dilution factor.
Note: The test is valid if the log number of spores per carrier at 48 hours is equal to or greater than the log number after 24 hours in each case.
7.3 Total Viable Spore Count of Biological indicator, Liquid Spore Suspensions:
Prepare an appropriate serial dilution of the original spore suspension in chilled sterile purified water contained in a sterile 100 ml screw-capped bottle, and proceed with the viable spore count procedures specified under Total Viable Spore Count of Biological indicator for steam sterilization, Non paper Carriers.
7.4 Acceptance criteria:
Population count of Biological indicator must be 50% to 300% of the label claim.