1.0 Objective
To validate autoclave with a biological indicator for sterilization efficiency.2.0 Scope
This procedure is applicable for Autoclave installed in microbiology area of quality control laboratory.3.0 Responsibility
3.1 Doing: Tech.Assistant (Microbiologist)/Executive3.2 Checking: Executive/Manager
4.0 Accountability
Head of the Department5.0 Procedure
5.1 Frequency: Twice in a year5.2 Organism: Bacillus steriothermophilus spores. (Certified Strength 106)
5.3 Perform the initial count of one strip of B.steriothermophilus spores from the lot/batch which are to be used for validation purpose.
5.4 Initial count of spores:
5.4.1 Transfer the strip aseptically into 100 ml sterile Normal Saline, keep it for 1/2 hr and give heat shock, keep the tube in water bath at 70°C-80°C for 15 minutes.
5.4.2 Shake the tube by palming or vortex it to homogenise suspension of the spores
5.4.3 Depending upon the initial count, dilute further in sterile Normal Saline
5.4.4 Take each aliquot in duplicate from different dilutions in such a way that the colony count should not come more than 300 cfu/plate.
5.4.5 Pour the SCD agar medium which has been melted & cooled to 45°C to 50°C into the allocated plates, swirl it & let it be solidified.
5.4.6 Incubate the plates in an inverted position at 55 ± 2°C for 48-72 hours.
5.4.7 Count the colonies per plate and calculate the average number/strip using dilution factor.
5.4.8 Use the strip for validation purpose if the initial count is not more than 150 % of the label claim and not less than 75% of the label claim.
5.4.9 Record the result in Annexure-I
5.5 Autoclave validation
5.5.1 Load the autoclave chamber with accessories and glassware.
5.5.2 Take requires nos. of strip, label it and put inside the autoclave as describe below.
5.5.3 Place the strip in Petri plates, arrange it in the autoclave as per location chart is given in Annexure III & IV for double door & vertical autoclave respectively.
5.5.4 Keep one strip unexposed for POSITIVE CONTROL.
5.5.5 Run the autoclave cycle at 121°C and 15 lb pressure for 20 minutes as per S.O.P. for vertical Autoclave and as per S.O.P. for double door autoclave.
5.5.6 After cycle is over, withdraw all the strips.
5.5.7 Mark the sterile SCD (Soybean Casein Digest) medium tubes (10ml) with location no., date and Equipment I.D. No. Transfer the strips aseptically to respective labelled SCD medium tubes.
5.5.8 Transfer one unexposed strip to a SCD medium tube and label it as POSITIVE CONTROL.
5.5.9 Keep one SCD medium tube uninoculated for NEGATIVE CONTROL.
5.5.10 Incubate all the tubes at 55°C ± 2°C for 14 days.
5.5.11 Observe the tubes daily up to 14 days for turbidity /growth in the SCD medium tubes having strips.
5.5.12 POSITIVE CONTROL should show growth within 72 hrs.
Related: New Genetically Engineered Biological Indicators
5.6 Interpretation
5.6.1 If no growth observed in any of the SCD medium tubes, indicates satisfactory performing of equipment.
5.6.2 If growth observed in any one of the SCD medium tubes, identify the growth of B.stearothermophilus performing gram staining, simultaneously compare it with POSITIVE CONTROL. In case of failure, investigate for non-compliance of validation, stop the use of equipment. Meanwhile, rectify the cause and perform the re-validation. On compliance, equipment can be used for routine use. and repeat the cycle.
5.6.3 Maintain the validation record and affix calibration card having following criteria.
Name of equipment :
Identification No :
Validation frequency :
Validation On :
Due date of Validation :
Validation done by :
5.7 Record the result in Annexure-II Attach the sterilization cycle record chart along with the report.
N.saline = Normal saline
hr.= Hour
cfu= Colony forming unit
B.= Bacillus
ml= millilitre
%= Percentage
°C = Degree centigrade
U.S.P.= The United States Pharmacopoeia
&= and
S.O.P.= Standard operating procedure
5.6 Interpretation
5.6.1 If no growth observed in any of the SCD medium tubes, indicates satisfactory performing of equipment.
5.6.2 If growth observed in any one of the SCD medium tubes, identify the growth of B.stearothermophilus performing gram staining, simultaneously compare it with POSITIVE CONTROL. In case of failure, investigate for non-compliance of validation, stop the use of equipment. Meanwhile, rectify the cause and perform the re-validation. On compliance, equipment can be used for routine use. and repeat the cycle.
5.6.3 Maintain the validation record and affix calibration card having following criteria.
Name of equipment :
Identification No :
Validation frequency :
Validation On :
Due date of Validation :
Validation done by :
5.7 Record the result in Annexure-II Attach the sterilization cycle record chart along with the report.
6.0 Abbreviations
SCD= Soyabean Casein digestN.saline = Normal saline
hr.= Hour
cfu= Colony forming unit
B.= Bacillus
ml= millilitre
%= Percentage
°C = Degree centigrade
U.S.P.= The United States Pharmacopoeia
&= and
S.O.P.= Standard operating procedure
ANNEXURE -I
MICRO BIOLOGICAL DEPARTMENT
INITIAL COUNT OF BIOLOGICAL INDICATOR
Name of Indicator Organism :
Claimed Initial count :
Form of Indicator :
Batch /Lot No. : Carrier material :
Mfg./Exp. Date :
Diluting fluid : 0.9% Sod.Chloride (N.Saline)
Growth medium : Soyabean casein digest agar PH : _________(7.3+/-0.2) Lot No:
SCD broth PH : _________(7.3+/-0.2) Lot No:
Incubation temperature :
Incubation Period :
1ml 1ml 1ml
Dilution step : Spore'strip ---------->100 ml ----> 10ml----> 10 ml-----> 10 ml
(Dil.1) (Dil 2) (Dil 3) (Dil 4 )
Results:
Dil No.
|
Dil.factor
|
Aliquotes
plated
|
Cfu/plate
|
Mean cfu/plate
|
Cfu/ml
|
Cfu/strip
|
Mean cfu/strip
|
Actual initial count :
Comment :
Sign of Microbiologist :
Date of Report :
Sign. of Q.C. Incharge :
ANNEXURE -II
MICRO BIOLOGICAL DEPARTMENT
VALIDATION OF AUTOCLAVE
Organism : Bacillus stearothermophilus spore strip (106 cfu) Mfg.: Exp:
Label claim :
Location and No. of strip :
Batch No.of strip :
Medium : Soyabean Casein Digest Medium (SCD) Lot No:
pH : __________ ( 7.3 +/- 0.2)
Incubation Temperature : 55 +/- 2oC
Sterilization :Temp: _______ oC
Cycle parameters : Hold Time :________ min.
Observation :
Date
|
1st
|
2nd
|
3rd
|
4th
|
5th
|
6th
|
7th
|
8th
|
9th
|
10th
|
11th
|
12th
|
13th
|
14th
|
Strip No:
| ||||||||||||||
1
| ||||||||||||||
2
| ||||||||||||||
3
| ||||||||||||||
4
| ||||||||||||||
5
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6
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7
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8
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9
| ||||||||||||||
10
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Sign
|
-ve= No growth Negative control :_______
+ve= Growth Positive control :_______
Comment :
Sign. Microbiologist :
Date of Report :
Sign. of Q.C. Incharge
ANNEXURE-I
QUALITY CONTROL LABORATORY
VALIDATION OF AUTOCLAVE
INITIAL COUNT OF BIOLOGICAL INDICATOR
Date of Testing
|
: _________________________
|
Date of Completion
|
: _________
| ||||||||
Name of Indicator Org.
Batch / Lot No.
Mfg. / Exp. Date
Growth medium
Incubation Temp & period
|
:_________________
:_________________
:_________________
: Soyabean casein digest agar
PH:____________(7.3+/-0.2)
: 53°C to 57°C for 2 to 3 Days
|
Claimed Initial Count :___________
Carrier Material :__________
Diluting fluid : 0.9% Sodium Chloride
| |||||||||
Dilution Steps:
1ml 1ml 1ml
(dil. 1) (dil. 2) (dil. 3) (dil. 4)
| |||||||||||
Dilution
No.
|
Dilution
Factor
|
Aliquots
Plated
|
Cfu/plate
|
Mean cfu/Plate
|
cfu/ml
|
cfu/strip
| |||||
1
|
2
| ||||||||||
Mean cfu / strip : ______________ Actual initial count : _________________
Acceptance criteria : Initial count should NMT 150% of the label claimed & NLT 75% of the label claimed.
Analyst : Checked By :
Date : Date :
ANNEXURE-II
QUALITY CONTROL LABORATORY
VALIDATION OF AUTOCLAVE WITH BIOLOGICAL INDICATOR
Name of Organism
B.No. / Lot No.
Mfg. / Exp. Date
Label Claim
Sterilization Temp
|
:
:
:
:
:
|
Medium Used
Medium Lot No.
PH of Medium
Incubation Temp.
|
: Soyabean Casein Digest medium
:
:
:
: 53oc to 57oc
| |||||||||||||||
Ster-hold time
Loading Time
|
:
|
Incubation Period
|
: 14 Days
| |||||||||||||||
GPT of Medium
|
:
| |||||||||||||||||
Position of Spores strips : As per attached location chart
| ||||||||||||||||||
Location
Date --->
|
Observation (Days)
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1
|
2
|
3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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13
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14
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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13
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14
| ||||||||||||||||||
15
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Sign.
| ||||||||||||||||||
+ Ve = Growth observed Negative Control : _____________
- Ve = No Growth observed Positive Control : ______________
Conclusion :
Analyst : Checked By :
Date : Date :
ANNEXURE -II
MICRO BIOLOGICAL DEPARTMENT
VALIDATION OF AUTOCLAVE
Organism : Bacillus stearothermophilus spore strip (106 cfu) Mfg.: Exp:
Label claim :
Location and No. of strip :
Batch No.of strip :
Medium : Soyabean Casein Digest Medium (SCD) Lot No:
pH : __________ ( 7.3 +/- 0.2)
Incubation Temperature : 55 +/- 2oC
Sterilization :Temp: _______ oC
Cycle parameters : Hold Time :________ min.
Observation :
Date
|
1st
|
2nd
|
3rd
|
4th
|
5th
|
6th
|
7th
|
8th
|
9th
|
10th
|
11th
|
12th
|
13th
|
14th
|
Strip No:
| ||||||||||||||
1
| ||||||||||||||
2
| ||||||||||||||
3
| ||||||||||||||
4
| ||||||||||||||
5
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6
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7
| ||||||||||||||
8
| ||||||||||||||
9
| ||||||||||||||
10
| ||||||||||||||
Sign
|
-ve= No growth Negative control :_______
+ve= Growth Positive control :_______
Comment :
Sign. Microbiologist :
Date of Report :
Sign. of Q.C. Incharge
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