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 :

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)⁰C 
• 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.

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.

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