Biological Indicators Conformation of viable spore count and resistance performance test

• Use the biological Indicator as per manufacture recommendation.
• Randomly pick 3 no's of biological indicators.
• Take 250 ml beaker of 100 ml of chilled sterilized water.
• Dip the 3 no's of biological indicators.
• With the help of sterilized forceps crush the ampules / paper carrior/ spore disc  in to pieces.
• Blending or mixing more than 15 minutes.
• Achieve the homogeneous suspension.
• Allow the bottle to stand for at least 5 minutes without disturbing to allow air bubbles to dissipate prior to sonication.
• Sonicate the sample for 3 to 5 minutes at 45-60 kHz.
• Transfer 10 ml of aliquot of the suspension to a sterile scrow- capped bottle (16 x 125 mm).
• Next Heat shocking treatment.
• After heat shocking treatment rapidly cool 0-4 (ice bath).
• Plate within 20 minutes after heat shocking treatment.
• Transfer two 1 ml of aliquots to suitable tube contains 9 ml of sterile purified water.
• Ana vortex the tube.
• From each tube prepare the ten fold serial dilution up to a suitable dilution to get countable colonies.
• For example if the labeled spore population is 10⁶  then prepare dilution up to 10⁶. Vortex the tubes for at least 10 seconds before proceeding for next dilution.
• Yield preferably 30-300 cfu but not less than 6.
• Place 1.0 ml of each selected dilution in each of 15 x 100 mm petri dishes within the 20 minutes.
•  Add each plate molted SCDA media.
• After solidification invert the plate and incubate at 55-60°C for biological indicators for steam sterilization or Invert the plate incubated at 30-35°C for Biological indicator for Ethyl oxide sterilization.
• After Incubation observe the plate and record the colony forming unit (CFU).

       Name of the Organism: Geo bacillus stereo thermophilus.

       Spore population : 3.6 x 10⁶ spores/ vial.

       Lot No: 

Dilution	24 hours			48 hours			Average
	P1	P2	AVG	P1	P2	AVG	24 hr	48 hr
10-1	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC
10-2	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC
10-3	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC
10-4	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC	TNTC
10-5	82	88	85	83	88	85.5	85	85.5
10-6	4	6	5	4	6	5	5	5
10-7	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil

Examine the plates after 24 and 48 hours. Record the number of colonies observed on each plate.

Calculate the average number of spores per carrier from the results, using the appropriate dilution factor. 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.

  

Lower Limit (LL)= 3.6 x 10⁶ spores/ vial = 3600000 X 50/100  =  1800000 = 6.25

Upper Limit (UL)= 3.6 x 10⁶ spores/ vial = 3600000 X 300/100 = 10800000 = 7.03

No. of Spores for specimen = Avg no.of cfu   x  Dilution factor  / No's of Bi's

After 24 hrs Spore count   =  85 x 10⁵   / 3  = 28.3 x 10⁵ = 28.3 x 10^5,  2.83 x 10⁶ = 6.45

After 48 hrs Spore count =85.75 x 10⁵ / 3  = 28.3 x 10⁵ = 28.6 x 10^5,  2.86 x 10⁶ = 6.46

Acceptance Criteria : 50 % to 300 % recovery of labelled spore count is acceptable.

As per USP 39 & 40  General chapter 55 revision details:

1. Total viable spore count conformation at least four samples from their individual container ( previous 3 samples).
2. Total viable spore count confirmation 100 mL of sterilized purified water temperature also specified at 2-8 degree C .
3. Conformation test after incubation examine the plates at least 48 h ( previous 24 h and 48 h incubation) recording for each plate the number of colonies.