Growth Promotion Test Procedure of Culture Media & its SOP

Growth Promotion Test Purpose: Growth Promotion Test, To verify that the culture media is capable to growth in order to use in Microbiology Test specified in Pharmacopoeia. Scope of Growth Promotion Test: This SOP applies for verification of the effectiveness of culture media which is used in Microbiology Laboratory at XX Pharmaceuticals Limited. Definitions/Abbreviation : […]


Growth Promotion Test Purpose:

Growth Promotion Test, To verify that the culture media is capable to growth in order to use in Microbiology Test specified in Pharmacopoeia.

Scope of Growth Promotion Test:

This SOP applies for verification of the effectiveness of culture media which is used in Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/Abbreviation :

CFU      : Colony Forming Unit

CSDA   : Casein Soyabean Digest Agar

CSDB   : Casein Soyabean Digest Broth

GPT      : Growth Promotion Test

SDA     : Sabouraud Dextrose Agar

SDB     : Sabouraud Dextrose Broth

TAMC   : Total Aerobic Microbial Count

TYMC   : Total Yeast & Mould Count

Responsibilities:

The roles and responsibility is summarized as follows:

Executive, Microbiology

To execute Inoculation, incubation of Culture media and plate count of Growth Promotion Test.

Assistant Manager/Manager, Microbiology

To ensure Growth Promotion Test, documentation and application of sound technical information.

Head of Quality Assurance

Approval of this SOP

Procedure:

Note:

  • Don’t move forcefully into the test area. Move always gently.
  • Disinfect all apparatus using 70% IPA before transfer into the Laminar Air Flow.
  • When enter into test area, wear sterile latex free gloves, lab coat/apron and eye protection (when required).
  • To prevent the unauthorized contamination, make sure that all personal ornaments, all type of cellular phone are left before enter into the test room.

General Requirement :

Glass Apparatus :

  • Pipette 2 ml, 10 ml
  • Screw Capped Test Tube
  • Sterilized 90 mm Glass Petridish
  • Screw capped Conical Flask 100 ml
  • Volumetric Flask 500 ml
  • Volumetric Flask 1000 ml

Media and Reagents:

  • Meat peptone
  • Neutralized Peptone
  • Selected Media

Others Requirements:

  • 70% IPA or ethanol
  • 0.45 µm Membrane Filter
  • Forceps
  • Filtration Unit( sterilized filter disk and filtering funnel)
  • Glass spreader
  • Scissors
  • Surgical Gloves
  • Surgical Cotton

Test Conditions:

  • Monitoring the testing area using by Microbial Air Sampler during working day.
  • Perform test under LAF to avoid any type contamination.

Culture Media Preparation:

  • Prepare different culture media  as per specific requirement
  • Weigh the amount declared in the manufacturer label into the appropriate flask.
  • Bring to boil completely to dissolve media.
  • Sterilize at 1210C for 15 minutes or as per Manufacturer declaration mention on the label.
  • Store the prepared culture media in air tight flask controlled room temperature at controlled environment
  • Store prepared agar media at (2-8)0
  • Preserve the dehydrated culture media up to its expiry date.
  • Never use the expired culture media.
  • Use agar media when the temperature reduce near at 450C and cool in case of broth media.

Stock Buffer Solution:

  • Place 34 g of Potassium Dihydrogen Phosphate[KH2PO4] in the 1000 ml volumetric flask
  • Dissolve in 500 ml of Purified Water then adjust to pH (7.2 ± 0.2) and dilute to make 1000ml with Purified Water.
  • Dispense 90 ml into each screw capped flask [5 or more flask required]. 
  • Sterilize at 1210C for 15 minutes.
  • Keep the prepared buffer solution at (2-80C) for a validated period.

Glassware Cleaning & Sterilization:

  • First of all clean all glassware with 1% detergent and then rinse with sufficient tap water.
  • Finally Rinse with sufficient Purified Water to remove the residual content of detergent.
  • Sterilize all glassware with Dry Heat Sterilizer at 2000C for 1 hour.

General Procedures

Growth Promotion Test (GPT) of General Media

  • Carry out Growth Promotion Test for each prepared culture media.
  • Prepare standardized suspension for the different test strains as mentioned in Table 1
  • Use sterile Buffer Sodium Chloride [NaCl]-Peptone solution where pH 7.0 or Phosphate Buffer which pH 7.2 as respective diluents.
  • Use the standard test strains are not more than 5 passages from original master seed lot.
  • During preparation of Aspergillus brasiliensis suspension, add 0.5% Polysorbate 80 to the buffer solution.
  • Use the suspension within 24 hours when preserve at (2-8)0C otherwise use within 2 hours.
  • Freshly prepare the Bacillus subtilis and A. brasiliensis suspension and  store at (2-8)0C for validated period, then dilute it for use.
  • Inoculate distinctly 100 cfu of each microorganism to 10 ml of each liquid media & follow spread plate method for solid media as mention in Table 1.
  • Incubate the medium for fungi at (20-25)0C for 5 days and for bacteria at (30-35)0C for 3 days.
Growth Promotion Test

Interpretation of Results:

  • Liquid media is suitable for use, if growth found, then it is clearly on each media.
  • Solid media is suitable when count is not greater than 2 from the calculated value of the standardized value.

Inoculation and Dilution:

  • Add the sufficient volume of suspension of inoculums to the sample to maintain not more than 100 cfu
  • Add inoculums suspension not more than 1% of the diluted product.
  • Prepare lowest possible dilution for tolerable microbial recovery of sample
  • When sample contains any antimicrobial properties add the neutralizer to remove Interfering factor.
  • When growth is inhibited then increase the use of diluents or membrane filtration or combination of all   above.
  • Add (20-25) ml of SCDA[Soyabean Casein Digest Agar] & SDA[Sabouraud Dextrose Agar] to 90 mm diameter petridish at least duplicate.
  • Incubate the medium for bacteria at 30-350C for 3 days and the medium for fungi at 20-250C      for 5 days.

Interpretation of  Results :

  • Liquid media is suitable for use, if growth found, then it is clearly on each media.
  • Solid media is suitable when count is not greater than 2 from the calculated value of the standardized value.
  • Growth obtained must not be differing by a factor greater than 2 from the calculated value for standardized inoculums for Solid Media, For freshly prepared inoculums, growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.

Growth Promotion Test of Selective Media:

Growth Promotion & Inhibitory Test :

  • Perform this test for each prepared media.
  • Inoculate the each media not more than 100 cfu microorganisms as per Table 2
  • Perform surface-spread method for solid media
  • Incubate the media as per specified in that microorganisms.

Interpretation of Result :

  • The media is suitable for use if growth found clearly in liquid media and found the specific  colony characteristics on solid media.
  • Inhibitory test is failed when no growth found occurs the specific colony.
growth promotion test

Test for E. coli :

  • Add the specific microorganism to 100 ml of CSDM. Incubate at 30-350C for 18-24 hours.
  • Shake the container, transfer 1 ml of SCDA[Soyabean Casein Digest Agar] to 100 ml of MacConkey Broth. Incubate at [42-44]0C for 24 hours.
  • Subculture on MacConkey Agar plate from MacConkey broth. Incubate at 30-350C for 18-72 hours.
  • GPT of that culture media complies with the test for E. coli if the red colonies are present with precipitated zone & the biochemical tests are negative[-ve].

Test for Salmonella

  • Add the specific microorganism in 100 ml of CSDM. Incubate at 30-350C for [18-24] hours.
  • Shake the container, transfer 0.1 ml of CSDM to 10 ml of Rappaport Vassiliadis Salmonella (RVS) Broth. Incubate at 30-350C hours for 18-24 hours.
  • Subculture on Xylose Lysine Deoxycholate (XLD) Agar plate from Rappaport Vassiliadis Salmonella (RVS) Broth. Incubate at [30-35]0C for [18-48] hours.
  • GPT of that culture media complies for Salmonella if no red colonies are present with or without black centres and the biochemical tests are negative.

Test for Pseudomonas aeruginosa

  • Add the specific microorganism to 100 ml of CSDM. Incubate at 30-350C for 18-24 hours.
  • Subculture on the Cetrimide Agar plate from CSDM. Incubate it at 30-350C for 18-72 hours.
  • GPT of that culture media complies with the test for Ps. aeruginosa if no bluish green  colonies are present & the biochemical tests are negative[-ve].

Test for staphylococcus aureus

  • Add the specific microorganism into 100 ml of CSDM. Incubate it at (30-35)0C for 18-24 hours.
  • Sub-culture on Mannitol Salt Agar plate from CSDM. Incubate at (30-35)0C for 18-72 hours.
  • GPT of that culture media complies with the test for St. aureus if no yellow/white colonies        
  • Surrounded yellow zone are present and the biochemical tests are negative.

Test for Candida albicans

  • Add specific microorganism to 100 ml of Soubaurad Dextrose Broth(SDB). Incubate at (30-35)0C for (3-5) days.
  • Subculture on Soubaurad Dextrose Agar[SDA] plate from SDB[Soubaurad Dextrose Broth]. Incubate at [30-35]0C for [24-48] hours.
  • GPT of that culture media complies with the test for C. albicans if no white colonies are present and the biochemical tests are negative[-ve].

Growth Promotion Test Report Preparation:

  • Prepare Report in Growth Promotion Test Report of General Culture Media, Annexure-I & Growth Promotion Test Report of Selective Culture Media, Annexure-II.

Download: All Annexure

Annexure-I Growth Promotion Test of General Culture Media

Annexure-II Growth Promotion Test of Selective Culture Media


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