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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 Check in 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 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 CFU’s.
    • For Selection of Organisms and method of Inoculation, follow the Table-1.
    • Use any one of the organisms for Growth promotion Test for a 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 specified temperature for 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 organism for inoculating SCDM and one aerobic and one anaerobic organism for inoculating FTM used as positive control in Sterility Test respectively from the Table-1.
    • There should not be any microbial contamination present in the Sterility check portions of media by the completion of 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 under class ‘A’ LAF cabinet in the sub culturing area.
    • Perform Microbial recovery for solidify media to demonstrate the suitability of 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 complete incubation period, if the acceptance criteria is met.

Table 1: Growth promoting and inhibitory properties of media

S.

No

Media Test Microbial culture Property Method of inoculation Acceptance criteria
1 Soyabean-Casein

Digest Agar

Medium

S.aureus  (ATCC 6538)  

 

Growth promoting

 

 

Pour Plate

 

 

 

More than 70% of initial count of culture suspension used.
B. subtilis (ATCC 6633)
S. abony (ATCC 14028)
E. coli  ( ATCC 8739)
C.albicans(ATCC 10231)
A. niger (ATCC 16404)
2 Potato  Dextrose

Agar

C.albicans( ATCC 10231)

A.niger(ATCC 16404)

 

 

Growth promoting

 

 

 

Pour Plate

 

 

 

More than 70% of initial count of culture suspension used.
3 Sabouraud

Dextrose Agar

Media (or)

Potato Dextrose

Agar Medium

C.albicans (ATCC 10231)

A.niger( ATCC 16404)

 

 

 

Growth promoting

 

 

 

Pour Plate

 

 

 

More than 70% of initial count of culture suspension used.
4 Plate Count Agar

Medium

P. aeruginosa( ATCC 9027)  

Growth promoting

 

Pour Plate

 

 

 

 

 

More than 70% of initial count of culture suspension used.
S. aureus (ATCC 6538)
Salmonella.abony (ATCC 14028)
E. coli( ATCC 8739)
5 Soyabean –Casein Digest Medium (Other than sterility test) P. aeruginosa( ATCC 9027)  

Growth promoting

 

Addition

 

Visible growth should be observed and compare to previously tested and approved Media.

 

S. aureus  (ATCC 6538)
C. albicans (ATCC 10231)
A. niger( ATCC 16404)
B. subtilis( ATCC 6633)
6 Soyabean –Casein Digest Medium (for sterility test) C. albicans (ATCC 10231)  

Growth promoting

 

Addition

Visible growth should be observed and compare to previously tested and approved Media.
A. niger ( ATCC 16404)
B. subtilis ( ATCC 6633)
7 Fluid  thio-glycollate Medium Cl.Sporogenes(ATCC 19404)  

Growth promoting

 

Addition

Visible growth should be observed and compare to previously tested and approved Media.
S. aureus  (ATCC 6538)
P. aeruginosa( ATCC 9027)

M. luteus (ATCC 9341)

8 Fluid Lactose Medium Salmonella. abony (ATCC 14028) Growth promoting Addition Visible growth should be observed and compare to previously tested and approved Media.
E. coli( ATCC 8739)
9 Levine Eosin-Methylene Blue Agar Medium E. coli( ATCC 8739) Growth promoting Pour plate/

Spreading/

Streaking

 

 

Metallic sheen under reflected light and a  blue-black appearance under transmitted light.
10 MacConkey Agar E. coli ( ATCC 8739) Growth promoting

 

Pour plate/

Spreading/

Streaking

 

 

 

Brick-red may have surrounding zone of precipitated bile.
11 Fluid Selenite-Cystine Medium Salmonella. abony (ATCC 14028) Growth promoting Addition

 

 

 

 

Characteristic growth on Selective agar Media*
S. aureus (ATCC 6538) Inhibitory Addition No growth or No Characteristic growth*
12 Fluid Tetrathionate Medium Salmonella.abony (ATCC 14028) Growth promoting Addition Characteristic growth on Selective agar medium*
S. aureus  (ATCC 6538)

 

 

 

Inhibitory Addition No growth or No Characteristic growth*
13 Bismuth Sulfite Agar Medium Salmonella. abony (ATCC 14028) Growth promoting Pourplate/

Spreading/

Streaking

Black or green colonies
S. aureus  (ATCC 6538)  

Inhibitory

Pourplate/

Spreading/

Streaking

No growth or No Characteristic growth
14 Brilliant Green Agar Medium Salmonella abony (ATCC 14028) Growth promoting Pourplate/Spreading/Streaking Small, transparent, colorless or pink to white opaque colonies (frequently surrounded by pink to red zone
Salmonella.aureus  (ATCC 6538) Inhibitory Pourplate/Spreading/Streaking No growth or No Characteristic growth
15 Xylose-Lysine-Desoxycholate Agar Medium Salmonella. abony(ATCC 14028) Growth promoting Pourplate/Spreading/Streaking

 

 

Red colored colonies, with or without black centers
E.coli ( ATCC 8739) Inhibitory Pourplate/Spreading/Streaking No growth or No Characteristic growth
16 Rappaport Vassiliadis Salmonella. abony  (ATCC 14028) Growth promoting Addition Visible growth should be observed and compare to previously tested and approved Media.
S. aureus  (ATCC 6538) Inhibitory Addition No growth or No Characteristic growth
17 Cetrimide Agar Medium P. aeruginosa( ATCC 9027) Growth promoting Pourplate/Spreading/

Streaking

Generally greenish

colonies

E. coli ( ATCC 8739)  

Inhibitory

Pourplate/Spreading/

Streaking

No growth or No Characteristic growth
18 Pseudomonas Agar Medium for the Detection of Fluorescin P. aeruginosa( ATCC 9027) Growth promoting Pourplate/

Spreading/

Streaking

Generally colorless to yellowish colonies.
19 Pseudomonas Agar Medium for detection of Pyocyanin P. aeruginosa( ATCC 9027) Growth promoting Pourplate/

Spreading/

Streaking

Generally greenish colonies
20 Vogel-Johnson Agar Medium S. aureus (ATCC 6538) Growth promoting Pourplate/

Spreading/

Streaking

Black colonies Surrounded by yellow zone
21 Baird-Parker Agar Medium S. aureus (ATCC 6538) Growth promoting Pourplate/

Spreading/

Streaking

 

 

Green purple  colonies, surrounded by clear zones 2 or 5 mm
22 Mannitol-Salt

Agar Medium

S. aureus (ATCC 6538) Growth promoting Pourplate/ Spreading/

Streaking

Yellow colonies with yellow zones
E. coli( ATCC 8739) Inhibitory Pourplate/

Spreading/

Streaking

No growth or No Characteristic growth
23 Sabouraud Dextrose Broth C. albicans (ATCC 10231)

A. niger (ATCC 16404)

Growth promoting Addition Visible growth should be observed and compare to previously tested and approved Media.

 

 

24

 

Reinforced Agar medium

 

Cl. Sporogenes (ATCC 19404)

 

 

Growth promoting

 

Pour plate

 

More than 70% of initial count of culture suspension used

25 Reinforced broth Cl.sporogenes(ATCC 19404) Growth promoting Addition

 

Visible growth should be observed and compare to previously tested and approved Media
 

26

 

** R 2A Medium

P. aeruginosa( ATCC 9027)  

Growth promoting

 

Pour plate

More than 70% of initial count of culture suspension use.
S. aureus(ATCC 6538)
Salmonella.abony(ATCC 14028)
E. coli ( ATCC 8739)

 

*- 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

Incubation
Time Temperature
A. niger (ATCC 16404) 5 days 22.5 + 2.5OC
C. albicans(ATCC 10231) 3 days 22.5 + 2.5OC
S. typhimurium (ATCC 14028) 48 hours 32.5 + 2.5OC
P. aeruginosa ( ATCC 9027) 48 hours 32.5 + 2.5OC
E. coli ( ATCC 8739) 48 hours 32.5 + 2.5OC
S. aureus( ATCC 6538) 48 hours 32.5 + 2.5OC
B. subtilis (ATCC 6633) 48 hours 32.5 + 2.5OC
Cl. Sporogenes (ATCC 19404) 48 hours 32.5 + 2.5OC
B. vulgatus (ATCC 19404) 48 hours 32.5 + 2.5OC
M. luteus(ATCC (ATCC 8482) 48 hours 32.5 + 2.5OC
Environmental lsolate-Bacteria 48 hours 32.5 + 2.5OC
Environmental lsolate-Yeast 3 days 22.5 + 2.5OC
Environmental lsolate-Mold 5 days 22.5 + 2.5OC

 

Table 3: Incubation conditions for Broth Medium

Medium Name of Organism Incubation
Time Temperature
FTM

 

Cl. Sporogenes(ATCC 19404) 3 days 32.5 + 2.5OC
P. aeruginosa ( ATCC 9027) 3 days 32.5 + 2.5OC
S. aureus ( ATCC 6538)

B. subtilis (ATCC 6633)

3 days 32.5+ 2.5 OC
SCDM

 

B. subtilis (ATCC 6633) 3 days 22.5 + 2.5OC
A. niger (ATCC 16404) 5 days 22.5 +2.5OC
C. albicans(ATCC 10231) 5 days 22.5 + 2.5OC
RAPPAPORT VASSILIADIS Salmonella abony(ATCC 14028) 1 days 32.5 + 2.5OC
Salmonella aureus(ATCC 6538) 1 days 32.5 + 2.5OC
FTM Environmental Isolate- Bacteria 3 days 32.5 + 2.5OC
SCDM Environmental Isolate- Bacteria 3 days 22.5 + 2.5OC
SCDM Environmental Isolate-Yeast 5 days 22.5 + 2.5OC
SCDM Environmental Isolate Mold 5 days 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

Mr. Shiv Kumar is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 14 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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