STANDARD OPERATING PROCEDURE

TITLE : PREPARATION OF MEDIA

SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 1 of 10

1.0 OBJECTIVE
To lay down the procedure for the preparation of the media used by microbiology department for
microbiological testing.

2.0 SCOPE
This SOP is applicable for the preparation of all the media used in Microbiology lab.

3.0 RESPONSIBILITY
Officer/Executive - Microbiology

4.0 ACCOUNTABILITY
Head-QC

5.0 ABBREVIATIONS
SOP : Standard Operating Procedure
QC : Quality Control
QA : Quality Assurance
SCDA : Soyabean Casein Digest Agar

6.0 PRECAUTIONS
6.1 Read the instructions given on the Bottle label for - ingredients, directions for use, expiry date,
before use of commercial dehydrated media.
6.2 During weighing of media always wear gloves and mask so that the media is not inhaled.
6.3 Ensure that the dehydrated media powder has the following characteristics:-
a) Free flowing, uniform color, absence of lumps.
b) Do not use the media if not satisfied with reference to the above mentioned characteristics.

7.0 MATERIALS AND EQUIPMENTS

7.1 MATERIALS:
Dehydrated cultural media, dried spatula, Glass beaker, Glycerol, and Non absorbent cotton,
Aluminum foil, Butter paper.
7.2 EQUIPMENTS:
Autoclave, Analytical Balance, pH meter

8.0 PREPARATION OF MEDIA:

8.1 Always ensure that the Growth Promotion test of each lot of medium should be done before or
parallel of the test, for each autoclaved load or each lot of medium for its growth promoting
qualities by separately inoculating container of each medium with less than 100 viable
microorganisms of each of the strains and incubating according to the conditions specified as
per SOP.
8.2 Place a piece of butter paper on the weighing pan of the analytical balance large enough to
accommodate the quantity to be weighed.
8.3 Open the bottle containing the dehydrated media.
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 2 of 10

8.4 With the help of a clean and dried spatula/spoon add the dehydrated media powder on the butter
paper.
8.5 Weigh the media as given in Table I

TABLE I
SUPPLEMENT
TO ADD PER
pH AFTER
S.No. MEDIA LITRE OF SHORT NAME
STERILIZATION
STERILISED
MEDIA
Soyabean Casein
1. None SCDM 7.3 ± 0.2
Digest Medium
Fluid Thioglycollate
2. None FTM 7.1± 0.2
Medium
Enterobacteria
3. Enrichment Broth None EEBM 7.2 ± 0.2
Mossel
Bacteriological
4. None PW 7.1 ± 0.2
Peptone

5. MacConkey Broth None MCB 7.3 ± 0.2

Buffered Sodium
6. Chloride Peptone None BPW 7.2 ± 0.2
Water
7. GN Broth None GNB 7.0 ± 0.2
Rappaport
8. Vassiliadis None RVSEB 5.2 ± 0.2
Salmonella
Enrichment Broth
9. Columbia Agar None COA 7.0 ± 0.2
10. Cetrimide Agar None CA 7.2 ± 0.2
11. Mannitol Salt Agar None MSA 7.4± 0.2

12. Eosin Methylene

None EMB 7.1 ± 0.2
Blue Agar
Soyabean Casein
13. 1% Glycerol SCDA 7.3 ± 0.2
Digest Agar

14. Pseudomonas Agar

None PAF 7.2 ± 0.2
Medium for
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 3 of 10

SUPPLEMENT
TO ADD PER
pH AFTER
S.No. MEDIA LITRE OF SHORT NAME
STERILIZATION
STERILISED
MEDIA
Detection of
Fluorescin
Pseudomonas Agar
Medium
15. None PAP 7.2 ± 0.2
For Detection of
Pyocyanin
Sabouraud Dextrose
16. None SDA 5.6 ± 0.2
Agar
17. MacConkey Agar None MCA 7.1 ± 0.2
18. Reinforced Media None RFM 7.2 ± 0.2
19. R2 A Agar None R2 A 7.2 ± 0.2
Xylose Lysine De-
20. None XLDA 7.4 ± 0.2
oxycholate Agar
Violet Red Bile
21. None VRBGA 7.2 ± 0.2
Glucose Agar

8.6 After weighing the required amount take it slowly from the pan without spilling transfer it into a
Clean glass conical flask and cover it with aluminum foil.
8.7 SCDA media shall be supplemented with 1% glycerol media before sterilization to avoid
dehydration during exposure.
8.8 Add a required volume of Water for Injection and mix to dissolve, if necessary heat the medium
to dissolve completely by using a hot plate/ water bath at temp range of 80-90°C. Do not over
heat the medium.
Table II
TYPE OF
TEST TO BE QUANTITY OF TEMPERATURE OF MEDIA
GLASSWARE
PERFORMED THE MEDIA DURING DISPENSING
FOR DISPENSING

Swab Test Bottles At ambient temperature

(Peptone water) 500 ml / bottle

Sub culturing
(Slant Test Tubes 10 ml / test tube Approximately 40-50°C
preparation)
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 4 of 10

TYPE OF
TEST TO BE QUANTITY OF TEMPERATURE OF MEDIA
GLASSWARE
PERFORMED THE MEDIA DURING DISPENSING
FOR DISPENSING

Test Tubes, 10ml/test tube, At ambient temperature/40-

Microbial limit
Conical flask/ Roux 100ml/conical flask, 50°C
test
bottle, Plates 20ml/plates

8.9 Close the test tubes and conical flasks with autoclavable caps or cotton plug of non absorbent
cotton and wrap them with aluminum foil.
8.10 Before sterilization always check the pH of the media if the pH is not within the specified
limits, it should be adjusted by adding 1N or 0.1N Hydrochloric acid or Sodium Hydroxide
solution.
After that autoclave the media as per SOP No.
8.11 Check the pH of the media after sterilization if the pH of the media is not within the specified
limits as per the details, discard the media.
8.13 Frequency-Daily during the activity.

9.0 PRE-INCUBATION OF MEDIA:

9.1 Pre-incubation of agar media:
9.1.1 Pre incubate 100% of all agar media plates at 30-35oC for 24 hours for all media with
following exception:-
9.1.2 Soyabean casein digest agar shall be incubated at 20-25oC for 48 hours and then incubated at
30-35oC for 24 hours.
9.1.3 R2A agar shall be incubated at 30-35oC for 72 hours.
9.1.3 Fore pour plate media shall be prepared freshly and shall be used, in such case keep negative
control plate up to the test completion.

9.2 Pre incubation of liquid media:

9.2.1 Pre incubate 100% of all media tubes at 30-35oC for 24 hours for all media with following
exception:-
9.2.2 Soyabean casein digest medium shall be incubated at 20-25oC for 72 hours and Fluid
thioglycollate medium shall be incubated at 30-35oC for 72 hours.
9.2.3 Mac Conkey Broth shall be incubating at 42-44oC for 24 hours.
9.3 Checking of media plates/liquid media for the following after Pre-incubation
9.3.1 Breakage of plate or lids.
9.3.2 Less volume or unequal filling of plates/tubes.
9.3.3 Dehydration resulting in crakes or dimpled surfaces on solid medium.
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 5 of 10

9.3.4 Excessive number of bubbles and Microbial contamination.

9.3.5 Excessive darkening or colour change of the medium.
9.3.6 Microbial contamination of the liquid medium.
9.3.7 Raised surface in case of 55 mm contact plate.
9.3.8 Visually checks remove the all defected plates and discard.
9.4 Acceptance Criteria for Solid Media plates after Pre-Incubation:
9.4.1 There should not be any deficiency in media plates as given under point no. 9.3 and the
contaminated plates should not be more than 5% of the prepared plates. If more than 5% of
contaminated plates observed, discard the particular lot.
9.5 Acceptance Criteria for Liquid Media after Pre-Incubation:
9.5.1 No contamination should be observed in pre incubated containers representing the lot. If
contamination is observed then discard the particular.
10.0 ENCLOSURES:
Annexure I – Media Preparation and consumption record
Annexure II _ Media Receipt and Issue record
Annexure III _ Sterile Media Reconciliation Record
Annexure IV –Media Preparation Label
Annexure V _ Media Receipt Label

11.0 DISTRIBUTION

11.1 The SOP shall be distributed to respective department by QA. The QA department
Shall authorize for the distribution of SOP and shall sign for “Issued by: “
Follow the distribution of SOP as per following:
Master Copy - QA Department
Control Copy - User Department

12.0 HISTORY OF REVIEW:

PREVIOUS
PREVIOUS CCF No. IF
EFFECTIVE REASON FOR REVIEW
REVISION NO. ANY
DATE
00 NIL New SOP
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 6 of 10

Annexure I
MICROBIOLOGY DEPARTMENT MEDIA PREPARATION AND CONSUMPTION RECORD

Name of Media………………………………………..
pH pH Destructio
Preparation Prepara Media Chec
Media Stoc before after Use Used n
Date tion used ked
Qty Volum B. No k steriliz sterili before on
B. No for On By by
in g e ml ation zation
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 7 of 10

Annexure II
MICROBIOLOGYDEPARTMENT MEDIA RECEIPT AND ISSUE RECORD

Media Name: ………………………… Code No: ……………..

RECEIPT ISSUE
Balance Checked
Date
Batch Exp.D Received Batch Issued Quantity by
Qty Qty GPT No.
No. ate by No. by
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 8 of 10

Annexure III
MICROBIOLOGY DEPARTMENT STERILE MEDIA RECONCILATION RECORD

Name of Media: .....................

Date Prepa No. of No. of Used for Used Used Remain Destru Destru Chec
of ration Plates/Tubes Plates/Tubes (Purpose) on By ing ction ction ked
Prep Batch /Bottles /Bottles Stock On By by
arati no. prepared used
on
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 9 of 10

Annexure IV
MEDIA PREPARATION LABEL:

MEDIA PREPARATION
LABEL
Name of the Media
Autoclave lot no.
Date of preparation
Use before
Prepared by
 STANDARD OPERATING PROCEDURE
TITLE : PREPARATION OF MEDIA
SOP No.: Department : Microbiology
Effective Date :
Revision No.: 00
Revision Date :
Supersede SOP No.: NIL Page No.: 10 of 10

Annexure V
MEDIA RECEIPT LABEL
Name of the Media
Qty. Received
Bottle No.
Date of Opening
Date of Release
Done by