1.0 OBJECTIVE
To lay down a standard procedure for growth promotion test of culture media.
2.0 SCOPE
This Standard Operating Procedure is applicable to the technical staff of Microbiology laboratory for growth promotion test of culture media.
3.0 RESPONSIBILITY
Microbiologist shall be responsible to follow the procedure mention in this SOP.
4.0 ACCOUNTABILITY
Department Head & QA Head shall be accountable for effective implementation of this SOP.
5.0 ATTACHMENTS
List of Micro-organisms used for growth promotion test – Attachment-I
Format for the record of growth promotion test of culture media – Attachment-II
6.0 PROCEDURE
6.1 Precautions
6.1.1 The dehydrated culture media as well as their ingredients are highly hygroscopic and must be stored
in a cool dry place away from bright light
6.1.2 Use fresh sterile pipette for each transfer.
6.1.3 If any spillage of cultures, immediately wash with 70% IPA Solution.
6.1.4 Always use dry spatula for weighing the dehydrated media. The weighing operation shall be
completed as quickly as possible to prevent absorption of moisture by the hygroscopic contents.
6.1.5 Always wear the face mask while weighing the dehydrated media to avoid inhalation of fine particles
of media.
6.2 Test for growth Promoting Properties of agar media
6.2.1 Label the plates with culture name, Media B.No., date of incubation on the base of petri plate.
6.2.2 Select the quantified microbial culture as per attachment-I for the growth promotion test.
6.2.3 Add 1.0ml suspension of specific culture containing 10 to 100 CFU/ml into two sterile petri plate.
6.2.4 Aseptically pour the cooled media at 40 to 45 ºC into both the labeled plates, mix the plates by gently
rotating clockwise and anticlockwise direction. Allow the plates to solidify at room temperature.
6.2.5 Simultaneously run a negative control to verify testing conditions, using the same procedure in place
of the test organism use diluents i.e. 1.0ml of saline solution.
6.2.6 Incubate the plates for 24 to 72 hours for the bacterial count 30-35ºC and 5 to 7 days for fungal count
at 20-25 ºC.
6.2.7 After incubation observe the plates for number of microbial colonies, and express the number of cfu
by following formula and record the average results in the attachment-II.
P1+P2
2
Where P1 and P2 are plate 1 and plate 2.
6.2.8 Calculate the microbial recovery in percentage by equation-
%Recovery = Mean cfu observed X100
Inoculated CFU/ml
6.2.9 Recovery should not less than 75%.
6.2.10 Frequency : for each batch of consignment.
6.3 Test for growth promoting properties of Broth Medium
6.3.1 Prepare required quantity of liquid culture media, dispense 100ml in test tube and sterilize as per
manufacturer’s instructions.
6.3.2 After sterilization transfer the media to LAF room and allow it to cool at room temperature.
6.3.3 Start the LAF as per SOP and proceed further work under LAF.
6.3.4 Add 1.0 ml of positive culture for growth promoting properties, containing 100 CFU cells into broth
tube and label with media B. No., Name of positive control and date of inoculation.
6.3.5 Incubate the tubes at 30-35ºC for 24-48 hours for bacterial count and at 20-25ºC 5-7 days for fungal
count.
6.3.6 Daily observe the tubes for growth for turbidity.
6.3.7 Satisfactory growth should be observed within 3 days of incubation in the test.
6.3.8 In case the media passes the growth promotion test, a approved label shall be affixed on the media
container, then the same should be used for analysis.
6.3.9 In case the media fails for growth promotion test then a rejected label shall be affixed on the
container and then the same shall be rejected and accordingly the rejection entry should be made in
the stock register.
6.3.10 The rejected media should be returned back to the supplier.
7.0 REFERENCES
In- house
8.0 ABBREVIATIONS
SOP: Standard operating procedure
°C : Degree Celsius
Psi : Pound per square inch
I.P.A.: Isopropyl Alcohol
CFU: Colony forming unit
LAF: Laminar air flow
QA: Quality Assurance
QC: Quality Control
9.0 DISTRIBUTION LIST
Quality Assurance Department
Quality Control Department
10.0 HISTORY OF REVISION
| Version Number | Effective Date | Reason for Revision |
List of Micro-organisms used for growth promotion test – Attachment-I
| S.No.
|
Media | Micro-organisms used for growth promotion test | ||||
| 01
|
Soyabean Casein Digest Agar | E.coli, p.aeruginosa | Bacillus subtilis | S.aureus, Salmonella spp. | A.brasiliensis | Candida albicans |
| 02
|
Soyabean Casein Digest Medium | E.coli | Bacillus subtilis | S.aureus | A.brasiliensis | Candida albicans |
| 03
|
Sabouraud Dextrose Agar | A.brasiliensis | Candida albicans | N.A. | N.A. | N.A. |
| 04
|
Cetrimide Agar | P.aeruginosa | N.A. | N.A. | N.A. | N.A. |
| 05
|
Mannitol Salt Agar | S.aureus | N.A. | N.A. | N.A. | N.A. |
| 06
|
MacConkey Agar | E.coli | N.A. | N.A. | N.A. | N.A. |
| 07
|
Xylose Lysine Deoxycolate Agar | Salmonella sp. | Shigella spp. | N.A. | N.A. | N.A. |
| 08
|
Baired Parker Agar | S.aureus | N.A. | N.A. | N.A. | N.A. |
| 09
|
MacConkey broth | E.coli | N.A. | N.A. | N.A. | N.A. |
| 10
|
Bismuth sulfite agar | Salmonella sp. | N.A. | N.A. | N.A. | N.A. |
| 11
|
Nutrient agar | E.coli | Bacillus subtilis | S.aureus | Salmonella | p.aeruginosa |
| 12
|
Nutrient broth | E.coli | Bacillus subtilis | S.aureus | A.brasiliensis | Candida albicans |
| 13
|
Peptone water | E.coli | Salmonella spp. | N.A. | N.A. | N.A. |
Format for the record of growth promotion test of culture media – Attachment-II
| S. No | Date of analysis | Name of Media | Batch No. | Use Before | Container No. issued | No. of colonies | Done By | Checked by | Remarks |
