SOP for Growth Promotion Test of Microbiological media
1.0 Purpose:
To lay down a procedure to check growth promotion test of different media used for Microbiological analysis.
2.0 Scope:
3.0 Responsibilities:
Microbiologist : To perform and maintain the procedure as per SOP.
Head-QC : To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0 Procedure:
4.1 Prepare and sterilize the media as per SOP of Media preparation. Take prepared culture dilution of (10-100 CFU/ml) which is applicable for specific microorganisms for specific media as given in Annexure-I.
4.2 For enumeration of media, inoculate 1.0 ml of the inoculum in every plate by pour plate method and for liquid media directly inoculate 1.0 ml to the specific media.
4.3 For selective solid media streak loop full culture from dilution of selected organism & for liquid selective media inoculate loop full culture from dilution of selected organism.
4.4 For bacteria, incubate solid media for 24-72 hrs. & for liquid media, incubate for 18-48 hrs at specified temperature and for fungus, incubate solid media for 72-120 hrs & for liquid media, incubate for 24-72 hrs at specified temperature.
4.5 Simultaneously keep a positive control of the previously approved medium. Solid enriched media should be compare with positive control and test media recovery should be not less than 70 %.
4.6 Calculate the Microbial Recovery (in %) of media as given below.
% Recovery = Mean CFU observed X 100
Inoculated CFU/ml
4.7 If media recovery is not above 70% than reject the batch/Lot of the media or return to the supplier.
4.8 Observe for the characteristic growth as mentioned in the annexure-I.
5.0 Attachment:
Annexure-I : Growth characteristics:
Name of the Media
|
Growth promoting Test Strains
|
Growth inhibitory Test strains
|
Observed Growth
|
Incubation Period
|
Incubation temperature
|
MacConkey agar
|
E.coli
|
S.aureus
|
Luxuriant Growth, Brick red colonies may have surrounding zone of precipitated bile
|
18-72hrs
|
30-35 °C
|
MacConkey broth
|
E.coli
|
S.aureus
|
Turbidity
|
24-48 hrs
| 42-44 °C |
l salt agar
|
S.aureus
|
E.coli
|
Luxuriant Growth, Yellow/ White colonies surrounded by yellow zones
|
18-72 hrs.
|
30-35°C
|
Nutrient broth
|
E.coli
Salmonella abony
|
-------
|
Turbidity
|
24-48 hrs.
|
30-35°C
|
Pseudomonas agar for Fluorescein
|
P. aeruginosa
|
S. aureus
|
Luxuriant Growth
|
24-48 hrs.
|
30-35°C
|
Pseudomonas agar for Pyocyanin
|
P. aeruginosa
|
S. aureus
|
Luxuriant Growth
|
24-48 hrs.
|
30-35°C
|
Sabourauds dextrose agar
|
C. albicans
A. niger
|
E.coli
|
Recovery NLT 70 %
|
72-120 hrs.
|
20-20°C
|
Sabourauds dextrose broth
|
C. albicans
A. niger
|
E.coli
|
Turbidity
|
72-120 hrs.
|
30-35°C
|
Sabourauds Chloramphenicol agar
|
C. albicans
A. niger
|
E.coli
|
Recovery NLT 70 %
|
72-120 hrs.
|
20-20°C
|
Soyabean casein digest agar
|
B. subtilis
C. albicans
S. aureus
P.aeruginosa
A. niger
|
------
|
Recovery NLT 70 %
|
24-72hrs. for Bacteria & 72-120 hrs. for Fungi.
|
30-35°C for Bacteria &
20-20°C for fungi.
|
Reinforced Medium for Clostridia Broth
|
Clostridium sporogenes
|
------
|
Turbidity
|
48 hrs.
|
30-35°C
|
Colombia Agar
|
Clostridium sporogenes
|
-------
|
Luxuriant Growth
|
48-72 hrs.
|
30-35°C
|
Eosin methylene blue agar
|
E.coli
|
S. aureus
|
Blue–black with metallic sheen
|
24-48
|
30-35°C
|
Cetrimide Agar
|
P.aeruginosa
|
S. aureus
E.coli
Salmonella abony
|
Greenish colonies under UV light
|
18-72
|
30-35°C
|
Triple sugar iron agar
|
Salmonella abony
|
S. aureus
|
Luxuriant Growth, Pink slant, black butt with gas & H2S
|
24-48
|
30-35°C
|
Violet Red Bile Glucose Agar
|
E.coli
|
-------
|
Luxuriant Growth, red to reddish colonies
|
18-24
|
30-35°C
|
Soyabean casein digest medium
|
B. subtilis
C. albicans
S. aureus
P. aeruginosa
A. niger
|
------
|
Turbidity
|
24-72hrs. for Bacteria & 72-120 hrs. for Fungi.
|
30-35°C for Bacteria &
20-20°C for fungi.
|
Xylose Lysine Deoxycholate Agar
|
Salmonella abony
|
S.aureus
|
Luxuriant Growth, red colonies with or without black centered
|
18-48 hrs.
|
30-35°C
|
Nutrient agar
|
B. subtilis
C. albicans
S. aureus
P.aeruginosa
A. niger
|
-------
|
Recovery NLT 70 %
|
24-72hrs. for Bacteria & 72-120 hrs. for Fungi.
|
30-35°C for Bacteria &
20-20°C for fungi.
|
Enterobacteria Enrichment Broth Mossel
|
E.coli
P.aeruginosa
|
S.aureus
|
Yellow color form
|
24-48 hrs.
|
30-35°C
|
Violet Red Bile Glucose Agar
|
E.coli
Salmonella abony
|
S.aureus
|
Luxuriant Growth
|
18-24 hrs.
|
30-35°C
|
R2A agar
|
B. subtilis
C. albicans
S. aureus
P. aeruginosa
A. niger
|
------
|
Luxuriant Growth
|
24-72hrs. for Bacteria & 72-120 hrs. for Fungi.
|
30-35°C for Bacteria &
20-20°C for fungi.
|
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