Growth Promotion Test and Inhibition Test of Media

Growth promotion test (GPT): Also referred to as fertility or nutritive properties test, which is performed on the media used during different tests like sterility test, microbial limit test, preservative efficacy test to demonstrate that it is capable of supporting the growth of micro-organisms

SOP for Growth Promotion Test (USP)

1.0   PURPOSE:

    • To lay down the procedure for evaluating the growth promotion test and Inhibition property of the sterilized media used for microbiological testing.

2.0   SCOPE:

    • This Standard Operating Procedure is applicable to the Microbiology Department for the Growth Promotion Test and Inhibition property.

3.0   REFERENCES:

    • Guideline for the receipt, storage, preparation, growth promotion test, use and disposal of microbiological media

4.0   PROCEDURE – Growth Promotion Test and Inhibition Test of Media:

    • General Procedure for Growth Promotion Test (GTP) :

    • Growth promotion test shall be performed whenever a new medium is received / Batch No. or lot No. is changed / Make or Vendor is changed.
    • After procuring the Media, check the Media container for the following details i.e. manufacturing date, Expiry date, Code No., and lot. number.
    • Store all the Media in a cool dark place at NMT 25 ± 5°
    • Before taking the media for use, perform the growth promotion (i.e. nutritive properties) or growth inhibition studies specific to the media as per Annexure-4.
    • Whenever there is an approved and “in use” medium available, inoculate it with the same organism.
    • If the growth promotion qualities of the media are not the same as compared to the previously approved lot then discard that media.
    • Procedure to Check the Growth Promotion qualities of the media :

    • Check the growth promotion qualities of the media (Using the organisms as mentioned in Annexure-4) by anyone of the following methods.
    • For newly received agar media

    • containers transfer 1.0 ml of culture having a cell count of 10 to 100 cells in the dilution into two sterile Petri plates and pour the media of New Container [B].
    • Swirl the plates and allow solidifying.
    • Similarly, transfer 1.0 ml of the culture of having a cell count of 10 to 100 cells in the dilution into two sterile Petri plates and pour the prepared Previous Container media [A].
    • Swirl the plates and allow solidifying.
    • Incubate the plates at respective temperatures, after incubation counts the colonies and compare the count with the previous container results.
    • After incubation record the results as per Annexure-2
    • The recovery in the growth promotion test for the new container must be within factor 2 of the actual inoculum concentration obtained for the previous container.
    • For Example: If the standard culture inoculum used has count 100 cfu/ml then the recovery should be within 100/2= 50 cfu/ml and 100*2= 200 cfu/ml.
    • For agar media :

    • Prepare inoculum of known concentration (approximately 10 to 100 cells/ml) of the organism and then transfer it to the plate followed by pouring with the agar media.
    • After incubation record the results as per Annexure-1.
    • For selective agar media :

    • Carryout growth promotion test by streaking specified organisms on the plate and observe for the characteristics of colonies and record the result in Annexure-1.
    • In case of liquid media or broth inoculate 100cfu of inoculum, in 100 ml of media or 10 ml of media by using the organisms as mentioned in Annexure-4 incubate the media,
    • Observe the growth and record the result in Annexure-3.
    • Liquid media are suitable if should show clearly visible growth in media.
    • Put an uninoculated Bottle/Tube/plate of media as a negative control to confirm the sterility of the media.
    • Label or mark using by a marker the name of media, batch/lot no., the organism used tested by, and date on the Bottle/Tube/plate of media.
    • For the growth promotion test of daily prepared media, if more organisms are prescribed for the test, in that case, minimum of two bacteria and one yeast/mold shall be used for tests on a daily rotation basis.
    • For solid media, the recovery of inoculated organisms should not be factor 2 from the calculated value of inoculums added.

Note: Before the use of any batch of prepared media for testing if the growth promotion test is not possible to perform before testing, it can be performed simultaneously with testing.

    • The shelf life of the opened media bottle shall not be more than 12 months and for an unopened bottle, it is till the shelf life of the container.
    • Assign the batch Number :

    • To prepared media with respect to Growth Promotion Test as GPT/MMXXX/YY,
    • Where
      • GPT stands for Growth Promotion Test
      • MM stands for Microbiological Media
      • XXX – 001, 002, 003, ——— to 999
      • YY    –  Last 2 digits of the calendar year.
    • For example, the first batch number for the year 2020 shall stand as GPT/MM001/20.
    • Growth promotion test shall be performed every finished (Prepared) lots of dehydrated medium.
  • General Instructions:

    • All live cultures shall be segregated from areas used for sample testing and optimally,
    • Handle all live cultures in a different area of the laboratory within a Biosafety Cabinet.
    • Positive control dilutions and inoculation shall be performed in a biological safety hood/cabinet
    • Agar plates containing fungal cultures shall be sealed with parafilm to prevent the spread of spores.
    • Surfaces in areas where a was opened shall be sanitized immediately after use by using an approved sanitizer for the appropriate contact time like..
      • Live culture plate,Growth Promotion Test
      • Tube,
      • Bottle,
      • Pellet, etc.,
    • Gloves shall be used when working with live cultures.

5.0   RESPONSIBILITY:

    • Officer or Executive of the Microbiology Department shall be responsible for the preparation of new or revision of existing SOPs.
    • The Head of the department/designee of respective areas & QA shall be responsible for reviewing the SOPs.

6.0   ABBREVIATIONS USED IN SOP FOR GROWTH PROMOTION TEST:

    • ATCC:   American Type Culture Collection 
    • cfu:   Colony-forming unit
    • EHS:   Environment, Health, and Safety
    • MTCC: Microbial Type Culture Collection
    • NCTC: National Collection of Type Cultures

7.0   ANNEXURES – SOP FOR GROWTH PROMOTION TEST:

Annexure- 1: Media Growth Promotion and Inhibition Test For Agar Media.

Prepare the LOG by using the following table contents-

    • Sr. No.
    • Date
    • Name of Media
    • Media Lot No.
    • Sterilization Cycle No
    • GPT Ref. No.
    • Test for Growth Promoting and Inhibitory Property
      • Test Organism
      • Temp. of Incubation
      • Incubator No.
      • No. of Cells Inoculated (10-100 cfu)
      • Observation
        • Plate I
        • Plate II
        • Avg.
      • % Recovery
      • Test Performed (Pr / In)
      • Incubation Completed
      • Recorded by
    • Checked By

Annexure- 2: Newly Received Agar Media-Comparative Test Report.

Name of the Media : Date of Inoculation :
Batch No. / Lot No. :   Date of Report  :       
Batch No./ Lot No. (Previous container) : Expiry date               :
Incubator ID. : Acceptance Criteria :
GPT Ref. No. : Recovery                  :
S. No. Test Organism Method of Inoculation

 

No. of Cells Inoculated

(10-100 cfu)

Previous Container [A] New Container [B] %

Recovery

(Bx100/A)

I II Avg I II Avg
           

Annexure- 3: Growth Promotion/ Inhibition Test Record for Liquid Media.

Name of Medium  : Inoculation Date :   
Lot No. : Date of Incubation :
Date of Manufacturing : Incubator ID :
Date of Expiry : Date of Observation :
Received on : Date of Report :
GPT Ref. No. :
Name of Organism (s) No. of Cells Inoculated

(10-100 cfu)

Method of Inoculation Test Performed

(Pr / In)

Observation Results

Satisfactory/Not Satisfactory

Annexure- 4: Acceptance Criteria for the Growth Promotion Test for Culture Media.

Name of Media Microorganism Name Incubation Conditions Method of inoculation Growth characteristics on media/ Microscopic examination Organisms to be used for Growth Inhibitory Test
Temp. (°C) Time (Hrs.)

Soyabean Casein Digest Broth

Escherichia coli 30-35 24-48 Direct Turbidity, -Ve rods. NA
Pseudomonas aeruginosa 30-35 24-48 Direct Turbidity, -Ve rods.
Staphylococcus aureus 30-35 24-48 Direct Turbidity, +Ve Cocci.
Bacillus subtilis 30-35 24-48 Direct Turbidity, +Ve rods.
Micrococcus luteus 30-35 24-48 Direct Turbidity, +Ve Cocci.
Salmonella 30-35 24-48 Direct Turbidity, -Ve rods.
Candida albicans 20-25 48-72 Direct Turbidity, Yeast Budding Cells
Aspergillus  brasiliensis 20-25 48-72 Direct Turbidity, Cotton Like growth

Soyabean Casein Digest Agar

Pseudomonas aeruginosa 30-35 24-48 Pour Plate Colorless to greenish colonies, -Ve rods. NA
Staphylococcus. aureus 30-35 24-48 Pour Plate Yellow shining colonies, +Ve Cocci.
Bacillus subtilis 30-35 24-48 Pour Plate Irregular whitish colonies, +Ve rods.
Micrococcus luteus 30-35 24-48  Pour Plate Small yellowish colonies, +Ve Cocci.
EM Isolate 30-35 24-48 Pour plate Suitable growth observed.
Candida albicans 20-25 48-72 Pour Plate Cream/white colored small colonies,
Aspergillus brasiliensis 20-25 48-72 Pour Plate Formation of mycelia.

Mac Conkey broth

Escherichia coli /        E. coli Mutant 42-44 24-48 Direct Gas formation in durum tube, Color change,   -Ve rods. Staphylococcus aureus

Sabouraud. Dextrose agar

Candida albicans 20-25 24-48 Pour Plate Rounded shining whitish to creamish colonies, NA
Aspergillus brasiliensis 20-25 48-72 Pour Plate Cottony growth with mycelia, Spores appear as pale to bright greenish/black.

Sabouraud. broth

Candida albicans 30-35 24-48 Direct Turbidity, Yeast Budding Cells
EE broth Enterobacteria 35-37 18-24 Direct Turbidity, -Ve rods Staphylococcus aureus
VRBGA Enterobacteria 35-37 18-24 Streaking Red/reddish colonies, -Ve rods. Staphylococcus aureus
MacConkey. Agar Escherichia coli 30-35 18-24 Streaking Brick red colonies, -Ve rods. Staphylococcus aureus
Mannitol salt agar Staphylococcus aureus 30-35 18-24 Streaking Yellow colonies with yellow zones, +Ve Cocci. Escherichia coli
Xylose-Lysine-Deoxychola-te Agar medium Salmonella 30-35 18-24 Streaking Red-colored translucent colony, -ve rods. Staphylococcus aureus
Xylose-Lysine-Deoxycholate Agar medium Shigella 30-35 24-48 Streaking Red-colored translucent colony without black centers, -ve rods. Staphylococcus aureus
Cetrimide Agar Medium Pseudomonas aeruginosa 30-35 18-24 Streaking Generally Greenish -Ve rods Escherichia coli
GN Broth Shigella 30-35 24-48 Direct Turbidity, -Ve rods. Staphylococcus aureus
R2A Pseudomonas aeruginosa 30-35 24-48 Pour Plate Colorless to yellowish colonies, -Ve rods. NA
Staphylococcus. aureus 30-35 24-48 Pour Plate Yellow shining colonies, +Ve Cocci.
Bacillus subtilis 30-35 24-48 Pour Plate Irregular whitish colonies, +Ve Cocci.
Micrococcus luteus 30-35 24-48 Pour Plate Small yellowish colonies, +Ve Cocci.
Candida albicans 20-25 48-72 Pour Plate Small colonies, creamish to white color. +Ve, Yeast budding cells.
Aspergillus niger 20-25 48-72 Pour Plate Formation of mycelia, Spores appears as pale to bright red.
Rappaport Vassiliadis Salmonella Enrichment Broth Salmonella abony 30-35 24-48 Direct Turbidity, -Ve rods. Staphylococcus aureus
MRSA Lactobacillus planetarium 35-37 24-48 Pour Plate Red color colony,  +Ve rods. NA
Sabouraud chloramphenicol agar C. albicans 30-35 24-48 Streaking Small size, Creamish/White color colony NA
Reinforced Medium for Clostridia Clostridia 30-35 24-48 Direct Good-Luxuriant growth under Anaerobic Condition. NA
Columbia Agar Clostridia 30-35 24-48 Pour Plate The colony is slightly yellowish. Staphylococcus aureus

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pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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