SOP for Qualification of Biological Indicators

This Document describes (Standard Operating procedure) SOP for Qualification of Biological Indicators

SOP for Qualification of Biological Indicators

I. Purpose & Scope:

  • The purpose of this SOP is to lay down the procedure for Qualification of Biological Indicators
  • This Standard operating procedure shall be applicable for Qualification of Biological Indicators

II. Responsibilities:

  • All Quality Control and Quality Assurance personnel’s shall be responsible to follow and implement this SOP.

III : Introduction and Procedural Part :

Introduction :

  • Steam heat sterilization depends upon the exposure of microorganisms to the saturated steam at temperatures sufficient to destroy it.
  • The Biological indicators are used for qualification of Steam Heat Sterilizer. To evaluate its performance, for providing greater than 6 Log reduction in challenged microbial population of resistant strain, to achieve Sterility Assurance Level (SAL) of 10-6.
  • Achieving greater than 6 Log reduction in biological population of resistant strain thereby resulting in sterile load.
  • The spore population determination of the Biological Indicator (BI) strips (Geobacillus stearothermophilus ATCC 7953) shall be performed prior to the bacterial challenge activities.
  • Confirm the D value of biological indicators in certificate of analysis (COA) of supplier. Proceed only if D value of biological indicator is Not more than 2.5 minutes. The BI strips shall be tested for spore population determination to ensure that each BI is having a minimum spore population of 1×106
  • The Biological Indicators to be challenged must have minimum 1.0 x 106 spores/unit population of viable spores of Geobacillus stearothermophilus ATCC 7953.
  • Biological Indicators must meet the In-house standards, so determine the Inhouse spore population of Biological indicator strips/ampoules.

Procurement :

  • Procure the required biological indicator strips/ampoules of Geobacillus stearothermophillus (ATCC 7953) from any of the sources.
  • Procured biological indicator strips/ampoules should be checked for its Spore population details, Date of manufacturing, Date of Expiry, D-Value, Storage condition.
  • After Procurement, perform the Qualification of biological indicator strips/ampoules. At first perform Growth promotion characteristics and identification of strain and then determine the spore population of given biological indicator strips.

Requirement :

  • Sterile Soybean casein digest medium (SCDM).
  • Sterile Soybean casein digest Agar (SCDA).
  • Sterile Purified water/sterile saline
  • Vortex mixer
  • Heating block
  • Sterile pipette tips and sterile test tubes.

Growth promotion characteristics and Identification:

  • Perform the Growth promotion test of biological indicator strips in Preincubated Soybean casein digest medium.
  • Clean the LAF work station with 70% IPA.
  • Take the one strip and inoculate in Preincubated Soybean caseine digest medium(10ml or 100ml) aseptically.
  • Incubate this inoculated medium at 55-60°C for minimum 24-48 hours. Keep negative control of that medium along with test.
  • After incubation, observe the medium for any growth in the form of turbidity.
  • For identification purpose, streak from inoculated medium on the preincubated Soybean casein digest agar plate. Incubate the SCDA plate at 55-60°C for minimum 48 hours.
  • After observing the growth characteristics perform the Gram staining of the observed colonies.
  • Record the observation and results in Annexure-I.
  • Acceptance criteria: Luxuriant growth within 48 hours.

Procedure for determination of spore population : 

  • After Growth promotion characteristics and identification of strain, perform the spore population determination.
  • Take 03 number of biological indicator strips/ampoules and inoculated into 100ml of sterilized purified water/saline.
  • Vortex whole contents on vortex mixer for 15-20minutes for dislodge and disperse spores.
  • After mixing, take 10ml stock solution in sterile test tube aseptically.
  • Give the heat shock to this stock solution at 95-100°C in heating block/water bath for minimum 15 minutes.
  • Perform the serial dilution of this stock solution for enumeration of spore population.
  • Aseptically pipette out 1ml of stock solution and inoculate in 9ml sterile saline in test tube, vortex the tube to get homogenized suspension. This shall be treated as 10-1
  • Serially dilute the microbial suspension by taking 1ml into another tube containing 9ml of sterile saline solution .
  • Continue the dilution process by following the below mentioned steps up to 10-6 Vortex each dilution for approximately 30 seconds.

1 ml   stock solution +  9 ml  saline  →  10-1    

1 ml  10-1     +  9 ml  saline  →  10-2

1 ml  10-2     +  9 ml  saline  →  10-3

1 ml  10-3     +  9 ml  saline  →  10-4

 1 ml  10-4     +  9 ml  saline  →  10-5

1 ml  10-5     +  9 ml  saline  →  10-6                      

  • Pour 1ml each in two sterile petri plates from 10-2 to 10-5
  • Add 20-25 ml sterile soybean casein digest agar medium in each plate (at temperature NMT 45°C) and swirled for uniform mixing. Allow the media to solidify. Keep the negative control plate of SCDA media.
  • After solidification, Incubate the plates at 55° – 60°C for 24-48 hrs.
  • After incubation period count the number of colonies on each dilution. Record the mean results of two plates for each dilution in Annexure-I.
  • Obtain the spore population of stock solution per 10ml, per 100ml and finally per strip or per ampoule.
  • Acceptance criteria: Spore population should be ±1 log of the label claim.

Precautions :

  • Maintain the aseptic condition through the process to avoid contamination.
  • After completion of qualification, destruct the plates and suspensions in media destruction autoclave.

Annexures :

Annexure-I : Report for Qualification of Biological Indicator         


Annexure I

                      Report for Qualification of biological indicator 

                                                                                              Page No.: 1 of 2

  1. I) Details of Biological Indicator:
Date of Receipt Name of Biological Indicator/Strain No.
Date of Mfg. Type of Biological Indicator
Date of Expiry Lot No/D Value

Storage condition

Spore Population

(As per COA)


  1. II) Growth Promotion Characteristics and Identification:

      (Acceptance Criteria : Luxuriant growth within 48 hours)

Purpose and Date

of Inoculation

Inhouse Media

Lot No.

Date of Observation after incubation at


Growth Promotion:


III) Spore Population Determination :

Date of Testing : Done By :
No. of indicators/ampoules used : Volume of Sterile Purified water/saline used and sterilization Inhouse lot No.:
Blending Time : Volume of Stock Solution :
Heat Shock Temperature : Heat Shock Time :
Inhouse lot No. of used Media : Incubation Condition :
Incubator I.D : Observed On/By :

Annexure I

                    Report for Qualification of Biological Indicator    

                                                                                            Page No.: 2 of 2


Dilution Used 10-2 10-3 10-4 10-5
Observation after 24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs
Plate-1 (cfu/ml)                


Average of Obtained Population of Stock suspension:————————–

= ——————— (X)

Obtained Spore Population in Stock solution (10ml)  = ———————-

= ———————-

= ——————–  (Y)

Obtained Spore Population in Stock solution (100ml) = ———————

= ——————— (Z)

Obtained Spore Population/Strip                                  = ————————

=  ————————

Acceptance Criteria for spore Population Determination:
Spore Suspension : ±1 log value of the label claim.
Obtained Spore Population : Meeting the Acceptance Criteria/Not Meeting the Acceptance Criteria.


Remark: The Biological Indicator lot No.——————— to be used for Steam sterilization    Qualification stands qualified/disqualified for its intended use.


Analysed By:                                                                                          Checked By:

Date :                                                                                                       Date :                

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