SOP for Validation of DHS with Biological Indicator : Pharmaceutical Guidelines

SOP for Validation of DHS with Biological Indicator

Standard operating procedure to validate DHS with biological indicator for sterilization efficiency.

1.0 Objective

To validate DHS with the biological indicator for sterilization efficiency.

2.0 Scope

This procedure is applicable for DHS, installed in microbiology area of quality control laboratory.

3.0 Responsibility

3.1 Doing: Tech.Assistant ( Microbiologist)
3.2 Checking: Executive/Manager

4.0 Accountability

Head of the Department

5.0 Procedure

5.1 Frequency: At every six month
5.2 Organism: Bacillus subtillis spore.
5.3 Perform the initial count of one strip of B. subtillis spores from the lot/batch which is 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 and keep it for 1/2 hr and give heat shock, keep the tube in water bath at 70-80°C for 15 minutes.
5.4.2 Shake the tube by palming or vortex it to homogenise the 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 so as to get not more than 50 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 30°-35° 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 results in Annexure-I

5.5 DHS validation

5.5.1 Load the DHS chamber with perforated SS container with closer..
5.5.2 Take nine strips cover with aluminium foil properly marked with location and put inside the DHS covering the coldest point as per location chart attached.
5.5.3 Keep one strip unexposed for POSITIVE CONTROL.
5.5.4 Run the DHS cycle as per S.O.P.
5.5.5 After completion of cycle, withdraw all the strips.
5.5.6 Mark the sterile SCD (Soyabean Casein Digest) medium tubes (10ml) with location no., date and transfer the strips aseptically to respective labelled SCD medium tubes.
5.5.7 Transfer one unexposed strip to a SCD medium tube and label it as POSITIVE CONTROL.
5.5.8 Keep one SCD medium tube uninoculated for NEGATIVE CONTROL.
5.5.9 Incubate all the tubes at 30°C to 35°C for 14 days.
5.5.10 Observe the tubes daily up to 14 days for turbidity /growth in the SCD medium tubes having spores strips.
5.5.11 POSITIVE CONTROL should show growth within 72 hrs.

5.6 Interpretation

5.6.1 If no growth observed in any one of the SCD medium tube ,test complies and DHS stands validated.
5.6.2 If growth observed in any one of the SCD medium tube, identify the growth for B.subtillis
performing gram staining, simultaneously compare it with POSITIVE CONTROL. In case of failure, rectify the DHS problem and repeat the cycle.
5.6.3 Maintain the validation record and affix calibration card having following criteria.
Name of equipment :
Identification No :
Calibration frequency :
Calibration on :
Due date of calibration:
Calibration done by
Calibration checked by :
5.7 Record the results as per Annexure-II

6.0 Abbreviations

6.1 SCD= Soyabean Casein digest
6.2 N.saline = Normal saline
6.3 hr.= Hour
6.4 cfu= Colony forming unit
6.5 B.= Bacillus
6.6 ml= millilitre
6.7 %= Percentage
6.8 °C = Degree centigrade
6.9 U.S.P.= The United States Pharmacopoeia
6.10 &= and
6.11 S.O.P.No.= Standard operating procedure number
6.12 S.S = Stainless steel
6.13 DHS = Dry Heat sterilisation


ANNEXURE-I
QUALITY CONTROL LABORATORY
S.O.P.NO.___________
VALIDATION OF D.H.S
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
  Lot No.:________________
  PH:____________(7.3+/-0.2)

: 30°C to 35°C for 2 to 3 Days
Claimed Initial Count   :
Carrier Material            :
Diluting fluid  : 0.9% Sodium Chloride
                             


Dilution Steps:
                                                                                         1ml                1ml                1ml                                 
                                      Carrier Material              100ml             10ml               10ml              10ml
                                                                              (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
S.O.P.NO.____________
VALIDATION OF D.H.S WITH BIOLOGICAL INDICATOR
Name of Org.
B.No. / Lot No.
Mfg. / Exp. Date
Label Claimed
Sterilization Temp
:
:
:
:
:
Medium Used

Medium Lot No.
PH of Medium
Incubation Temp.
: Soyabean Casein Digest
  medium
:
:
: 30°C  to  35°C
Ster-hold time
Loading Time
:
Incubation Period
: 14 Days
:
GPT of Medium
:
Position of Spores strips : As per attached location chart

Sr. No.
Location


        Date à
Observation (Days)
1
2
3
4
5
6
7
8
9
10
11
12
13
14














1














2














3















4















5















6















7















8















9















10















11















12















13















14















15















Sign.

















+ Ve = Growth observed                                 Negative Control : _____________

- Ve = No Growth observed                            Positive Control   : ____________

Conclusion :



Analyst :                                                                                      Checked By :
Date      :                                                                                      Date            :





Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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