SOP on Media growth Promotion, Inhibition and Sterility Check

SOP on Media growth Promotion, Inhibition, and Sterility Check

Objective

To lay down the procedure for Media Growth Promotion, Inhibition, and Sterility Check.

Scope

This SOP is applicable for Media Growth Promotion, Inhibition, and Sterility Checks in the Microbiology Laboratory of (Pharmaceutical Company Name).

Responsibility

Chemist or above of QC laboratory.

Head – Microbiology Section.

Accountability

Head – Quality Control.

Abbreviations and Definitions

QC: Quality Control

GPT:  Growth Promotion Test

SCDM: Soyabean Casein digest Medium

FTM: Fluid Thioglycollate medium.

Procedure

Check all the sterile lots of the media (Prepared as per the directions given ‘Preparation of Sterile media SOP No.: for its sterility, ability to promote the growth of specific organisms, and Growth Inhibitory property.

Prepare Culture suspension as per SOP No.:

Inoculate portions of the test media in plates/tubes/bottles with 0.1 ml of specified microbial culture suspension containing 10-100 CFUs.

For the Selection of Organisms and method of Inoculation, follow Table-1.

Use any one of the organisms for the Growth promotion Test for Media.

Use one or more environmental isolates if available, preferably in the media used for environmental monitoring, sterility test, media used for enumeration tests of water, products, etc.

Incubate the inoculated media at recommended temperatures specified for the organism as per Table 2 and Table 3.

Similarly, incubate the uninoculated media as Negative control for sterility check at a specified temperature for a time specified as per Table 2 and Table-3.

Retain the Negative controls meant for sterility test under incubation for 14 Days at 22.5+2.5oC for SCDM and at 32.5+2.5oC for Fluid Thioglycollate medium.

Use any one of the organisms for inoculating SCDM and one aerobic and one anaerobic organism for inoculating FTM used as a positive control in Sterility Test respectively from Table-1.

There should not be any microbial contamination present in the Sterility check portions of media by the completion of the incubation period.

Observe and note the observation in the record as per Annexure No.:

Write the interpretation by comparing the acceptance criteria mentioned in table 1.

Record the details of Media GPT, Sterility, and inhibition tested for each autoclaved media Lot as per Annexure No.:

Perform GPT on each received lot of dehydrated media and note down the results as per Annexure. No.

Growth should be clear and visible in the form of turbidity in the case of Liquid media and distinct colony formation in the case of Agar media and the results shall be comparable to the previously approved batch of the media.

Perform all inoculations aseptically underclass ‘A’ LAF cabinet in the subculturing area.

Perform Microbial recovery for solidified media to demonstrate the suitability of the test method.

Discard all the used media/tubes/bottles after completion of the test as per SOP No.: QC- (SOP on Decontamination and Disposal of Used Media).

Discard the used media plates/tubes/bottles even before the complete incubation period, if the acceptance criteria are met.

Table 1: Growth-promoting and inhibitory properties of media

1. Media: Soyabean-Casein

  • Digest Agar
  • Medium
  • Test Microbial culture: 
  • S.aureus  (ATCC 6538)
  • Property:  Growth promoting
  • Method of inoculation: Pour Plate
  • Acceptance criteria:  More than 70% of initial count of culture suspension used.
    Test Microbial culture: B. subtilis (ATCC 6633)
    Test Microbial culture: S. abony (ATCC 14028)
    Test Microbial culture: E. coli  ( ATCC 8739)
    Test Microbial culture: C.albicans(ATCC 10231)
    Test Microbial culture: A. niger (ATCC 16404)

2 Media :Potato  Dextrose Agar

  • Test Microbial culture: C.albicans( ATCC 10231)
  • Test Microbial culture: A.niger(ATCC 16404)
  • Property: Growth promoting
  • Method of inoculation: Pour Plate
  • Acceptance criteria: More than 70% of the initial count of culture suspension used.

3 Media: Sabouraud

  • Dextrose Agar
  • Media (or)
  • Potato Dextrose
  • Agar Medium
  • Test Microbial culture: C.albicans (ATCC 10231)
  • Test Microbial culture: A.niger( ATCC 16404)
  • Property: Growth promoting
  • Method of inoculation: Pour Plate
  • Acceptance criteria: More than 70% of the initial count of culture suspension used.

4 Media: Plate Count Agar  Medium

  • Test Microbial culture:  P. aeruginosa( ATCC 9027)
  • Property: Growth promoting
  • Method of inoculation: Pour Plate
  • Acceptance criteria: More than 70% of initial count of culture suspension used.
    Test Microbial culture: S. aureus (ATCC 6538)
    Test Microbial culture: Salmonella.abony (ATCC 14028)
    Test Microbial culture: E. coli( ATCC 8739)

5. Media: Soyabean –Casein Digest Medium (Other than sterility test)

  • Test Microbial culture: P. aeruginosa( ATCC 9027)
  • Property: Growth promoting
  • Method of inoculation: Addition
  • Acceptance criteria: Visible growth should be observed and compared to previously tested and approved Media.
  • Test Microbial culture: S. aureus  (ATCC 6538)
    Test Microbial culture: C. albicans (ATCC 10231)
    Test Microbial culture: A. niger( ATCC 16404)
    Test Microbial culture: B. subtilis( ATCC 6633)

6. Media:Soyabean –Casein Digest Medium (for sterility test)

  • Test Microbial culture: C. albicans (ATCC 10231)
  • Property: Growth promoting
  • Method of inoculation: Addition
  • Acceptance criteria: Visible growth should be observed and compare to previously tested and approved Media.
    Test Microbial culture: A. niger ( ATCC 16404)
    Test Microbial culture: B. subtilis ( ATCC 6633)

7. Media :Fluid  thio-glycollate Medium

  • Test Microbial culture: Cl.Sporogenes(ATCC 19404)
  • Property: Growth promoting
  • Method of inoculation: Addition
  • Acceptance criteria: Visible growth should be observed and compare to previously tested and approved Media.
    Test Microbial culture: S. aureus  (ATCC 6538)
  • Test Microbial culture: P. aeruginosa( ATCC 9027)
  • Test Microbial culture: M. luteus (ATCC 9341)

8. Media :Fluid Lactose Medium

  • Test Microbial culture: Salmonella. abony (ATCC 14028)
    Property: Growth promoting
    Method of inoculation: Addition
    Acceptance criteria: Visible growth should be observed and compare to previously tested and approved Media.
    Test Microbial culture: E. coli( ATCC 8739)

9 Media :Levine Eosin-Methylene Blue Agar Medium

  • Test Microbial culture: E. coli( ATCC 8739)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Metallic sheen under reflected light and a  blue-black appearance under transmitted light.

10 Media : MacConkey Agar

  • Test Microbial culture: E. coli ( ATCC 8739)
  • Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Brick-red may have a surrounding zone of precipitated bile.

11 Media :Fluid Selenite-Cystine Medium

  • Test Microbial culture: Salmonella. abony (ATCC 14028)
    Property: Growth promoting
  • Method of inoculation: Addition
  • Acceptance criteria: A characteristic growth on Selective agar Media*
  • Test Microbial culture: S. aureus (ATCC 6538)
    Property: Inhibitory
    Method of inoculation: Addition
    Acceptance criteria: No growth or No Characteristic growth*

12 Media :Fluid Tetrathionate Medium

  • Test Microbial culture: Salmonella.abony (ATCC 14028)
    Property: Growth promoting
    Method of inoculation: Addition
    Acceptance criteria: A characteristic growth on Selective agar medium*
  • Test Microbial culture: S. aureus  (ATCC 6538)
  • Property: Inhibitory
    Method of inoculation: Addition
    Acceptance criteria: No growth or No Characteristic growth*

13. Media :Bismuth Sulfite Agar Medium

  • Test Microbial culture: Salmonella. abony (ATCC 14028)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Black or green colonies
    Test Microbial culture: S. aureus  (ATCC 6538)
  • Property: Inhibitory
  • Method of inoculation: Pour plate/
  • Method of inoculation: Spreading/
  • Method of inoculation: Streaking
  • Acceptance criteria: No growth or No Characteristic growth

14. Media :Brilliant Green Agar Medium

  • Test Microbial culture: Salmonella abony (ATCC 14028)
    Property: Growth promoting
    Method of inoculation:  Pour plate/Spreading/Streaking
    Acceptance criteria: Small, transparent, colorless or pink to white opaque colonies (frequently surrounded by pink to red zone
    Test Microbial culture: Salmonella.aureus  (ATCC 6538)
    Property: Inhibitory
    Method of inoculation: Pour plate/Spreading/Streaking
    Acceptance criteria: No growth or No Characteristic growth

15.Media :Xylose-Lysine-Desoxycholate Agar Medium

  • Test Microbial culture: Salmonella. abony(ATCC 14028)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Red-colored colonies, with or without black centers
    Test Microbial culture: E.coli ( ATCC 8739)
    Property: Inhibitory
    Method of inoculation: Pour plate/Spreading/Streaking
    Acceptance criteria: No growth or No Characteristic growth

16. Media :Rappaport Vassiliadis

  • Test Microbial culture: Salmonella. abony  (ATCC 14028)
    Property: Growth promoting
    Method of inoculation: Addition
    Acceptance criteria: Visible growth should be observed and compare to previously tested and approved Media.
    Test Microbial culture: S. aureus  (ATCC 6538)
    Property: Inhibitory
    Method of inoculation: Addition
    Acceptance criteria: No growth or No Characteristic growth

17. Media: Cetrimide Agar Medium

  • Test Microbial culture: P. aeruginosa( ATCC 9027)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Generally greenish colonies
  • Test Microbial culture: E. coli ( ATCC 8739)
  • Property: Inhibitory
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: No growth or No Characteristic growth

18.Media :Pseudomonas Agar Medium for the Detection of Fluorescin

  • Test Microbial culture: P. aeruginosa( ATCC 9027)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Generally colorless to yellowish colonies.
  • 19 Media :Pseudomonas Agar Medium for detection of Pyocyanin
    Test Microbial culture: P. aeruginosa( ATCC 9027)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Generally greenish colonies

20. Media :Vogel-Johnson Agar Medium

  • Test Microbial culture: S. aureus (ATCC 6538)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Black colonies Surrounded by yellow zone
  • 21 Media :Baird-Parker Agar Medium
    Test Microbial culture: S. aureus (ATCC 6538)
    Property: Growth promoting
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: Green purple  colonies, surrounded by clear zones 2 or 5 mm

22. Media : Mannitol-Salt

  • Agar Medium
  • Test Microbial culture: S. aureus (ATCC 6538)
    Property: Growth promoting
  • Method of inoculation: Pour plate/ Spreading/Streaking
  • Acceptance criteria: Yellow colonies with yellow zones
    Test Microbial culture: E. coli( ATCC 8739)
    Property: Inhibitory
  • Method of inoculation: Pour plate/Spreading/Streaking
  • Acceptance criteria: No growth or No Characteristic growth

23. Media :Sabouraud Dextrose Broth

  • Test Microbial culture: C. albicans (ATCC 10231)
  • A. niger (ATCC 16404)
  • Property: Growth promoting
    Method of inoculation: Addition
  • Acceptance criteria: Visible growth should be observed and compared to previously tested and approved Media.

24.Media : Reinforced Agar medium 

  • Test Microbial culture: Cl. Sporogenes (ATCC 19404)
  • Property: Growth promoting
  • Method of inoculation: Pour plate
  • Acceptance criteria: More than 70% of the initial count of culture suspension used

25. Media : Reinforced broth

  • Test Microbial culture: Cl.sporogenes(ATCC 19404)
    Property: Growth promoting
  • Method of inoculation: Addition
  • Acceptance criteria: Visible growth should be observed and compared to previously tested and approved Media

26. Media : ** R 2A Medium

  • Test Microbial culture :
  • P. aeruginosa( ATCC 9027)
    S. aureus(ATCC 6538)
    Salmonella.abony(ATCC 14028)
    E. coli ( ATCC 8739)
  • Property: Growth promoting
  • Method of inoculation: Pour plate
  • Acceptance criteria: More than 70% of the initial count of culture suspension use.

*- To be confirmed by streaking on one of Salmonella selective media (i.e. Bismuth Sulfite Agar

Medium / Brilliant Green Agar Medium / Xylose-Lysine-Desoxycholate Agar Medium)

**-For R2A incubate the inoculated media at 22.5 + 2.5OC for maximum up to 5 days

Table 2: Culture conditions for Microbial recovery

Name of the Organism: A. niger (ATCC 16404)
Incubation Time: 5 days
Incubation Temperature: 22.5 + 2.5OC

Name of the Organism: C. albicans(ATCC 10231)
Incubation Time: 3 days
Incubation Temperature: 22.5 + 2.5OC

Name of the Organism: S. Typhimurium (ATCC 14028)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: P. aeruginosa ( ATCC 9027)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: E. coli ( ATCC 8739)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: S. aureus( ATCC 6538)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: B. subtilis (ATCC 6633)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: Cl. sporogenes (ATCC 19404)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: B. vulgatus (ATCC 19404)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: M. luteus(ATCC (ATCC 8482)
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: Environmental isolate-Bacteria
Incubation Time: 48 hours
Incubation Temperature: 32.5 + 2.5OC

Name of the Organism: Environmental isolate-Yeast
Incubation Time: 3 days
Incubation Temperature: 22.5 + 2.5OC

Name of the Organism: Environmental isolate-Mold
Incubation Time: 5 days
Incubation Temperature: 22.5 + 2.5OC

Table 3: Incubation conditions for Broth Medium

Medium: FTM

  • Name of Organism: Cl. sporogenes(ATCC 19404)
    Incubation Time: 3 days
    Incubation Temperature: 32.5 + 2.5OC
  • Name of Organism: P. aeruginosa ( ATCC 9027)
    Incubation Time: 3 days
    Incubation Temperature: 32.5 + 2.5OC
  • Name of Organism: S. aureus ( ATCC 6538)
  • Name of Organism: B. subtilis (ATCC 6633)
  • Incubation Time: 3 days
    Incubation Temperature: 32.5+ 2.5 OC

Medium: SCDM

  • Name of Organism: B. subtilis (ATCC 6633)
    Incubation Time: 3 days
    Incubation Temperature: 22.5 + 2.5OC
    Name of Organism: A. niger (ATCC 16404)
    Incubation Time: 5 days
    Incubation Temperature: 22.5 +2.5OC
    Name of Organism: C. albicans(ATCC 10231)
    Incubation Time: 5 days
    Incubation Temperature: 22.5 + 2.5OC

Medium: RAPPAPORT VASSILIADIS

  • Name of Organism: Salmonella abony(ATCC 14028)
    Incubation Time: 1 days
    Incubation Temperature: 32.5 + 2.5OC
    Name of Organism: Salmonella aureus(ATCC 6538)
    Incubation Time: 1 days
    Incubation Temperature: 32.5 + 2.5OC

Medium: FTM

  • Name of Organism: Environmental Isolate- Bacteria
    Incubation Time: 3 days
    Incubation Temperature: 32.5 + 2.5OC

Medium: SCDM

  • Name of Organism: Environmental Isolate- Bacteria
    Incubation Time: 3 days
    Incubation Temperature: 22.5 + 2.5OC

Medium: SCDM

  • Name of Organism: Environmental Isolate-Yeast
    Incubation Time: 5 days
    Incubation Temperature: 22.5 + 2.5OC

Medium: SCDM

  • Name of Organism: Environmental Isolate Mold
    Incubation Time: 5 days
    Incubation Temperature: 22.5 + 2.5OC

Forms and Records (Annexures)

  • Media growth Promotion, Inhibition and Sterility Check
  • Growth Promotion Test Report

Distribution

  • Master copy        –       Quality Assurance
  • Controlled copies –       Quality Assurance, Production, Quality Control, Stores, Engineering, and Human Resource

History:

Date Revision Number
Reason for Revision
00   NEW SOP

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About Pharmaceutical Guidanace

Ms. Abha Maurya is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 18 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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