MPK | SOP FOR THE METHOD OF THE PREPARATION AND STORAGE OF DIFFERENT MICROBIOLOGICAL CULTURE MEDIA.

 

1.0.      Purpose

To describe the method of the preparation and storage of different Microbiological culture media.

 

Sterility culture broth

2.0.      Scope

This procedure is applicable to the preparation and storage of different culture media used in microbiological testing in Micro lab of Q.A. department.

 3.0.      Responsibilities

Action	Responsibility
Follow this procedure.	Microbiologist
Implementation of this procedure.	Lab Incharge

4.0        Requirements:

4.1 Dehydrated prepared media containers,

4.2  Glass Conical flasks,

4.3  Caped glass bottles,

4.4  Spatulas,

4.5  Balance (Q.C. – 000 ),

4.6  pH meter (Q.C. – 000 ),

4.7  Double distilled water,

4.8  Water bath, (Q.C – 000)

4.9  Refrigerator (Q.C – 000)

4.10  Refrigerator (Q.C – 000)

5.0  Procedure

5.1   Preparation of Culture Media

S. No	Action	Responsibility
5.1.1	Reconstitute media as per manufacturer’s instructions and as per the required volume in a clean glass conical flask and dissolve in double distilled water. Following are the methods of reconstitution of different Microbiological media:	Microbiologist                                                       Microbiologist                                                             Microbiologist                                                                 Microbiologist                                             Microbiologist
5.1.2	Tryptic Soy Agar (TSA)   Take 40 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.3 ± 0.2.
5.1.3	Sabouraud Dextrose Agar (SDA) Take 65 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 5.6 ± 0.2.
5.1.4	Brilliant Green Agar (BGA) Take 50 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 6.9 ± 0.2.
5.1.5	Mannitol Salt Agar Take 111 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.5 ± 0.2.
5.1.6	Cetrimide Agar Take 45.3 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.2 ± 0.2.
5.1.7	Eosine Methylene Blue (EMB) Take 36 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.1 ± 0.2.
5.1.8	MacConkey Agar (with crystal violet) Take 50 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.1 ± 0.2.
5.1.9	Salmonella Shigella Agar (SSA) Take 63 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.0 ± 0.2.
5.1.10	R2A Agar  Take 18.1 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.2 ± 0.2.
5.1.11	Bismuth Sulphite Agar (Modified) Take 40 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.6 ± 0.2.
5.1.12	Trypticase Soy Agar Take 45.7 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.3 ± 0.2.
5.1.13	Mannitol Salt Agar (Phenol-red) Take 108 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.4 ± 0.2.
5.1.14	Tryptic Soy Broth (TSB)   Take 30 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.3 ± 0.2.
5.1.15	Fluid Thioglycollate Medium (FTM)     Take 29.5 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.1 ± 0.2.
5.1.16	Lactose Broth Take 13 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 6.9 ± 0.2.
5.1.17	Tetrathionate Broth Take 77 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 8.0 ± 0.2.
5.1.18	Selenite Cystine Broth Base Take 19 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.0 ± 0.2.
5.1.19	1% Peptone water Take 1 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.2 ± 0.2.
5.1.20	Letheen Broth, Modified Take 43.8 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.2 ± 0.2.
5.1.21	Salmonella Enrichment Broth Take 41.8 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 5.2 ± 0.2.
5.1.22	MacConkeys Broth   Take 40 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.4 ± 0.2.
5.1.23	Enterobacteria Enrichment Broth Mossel (EEB) Take 43.5 gm powder and dissolve completely in 1 liter of double distilled water it is heat sensitive and use without autoclave.
5.1.24	Xylose Lysine Deoxycholate Agar (XLD) Take 53 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.4 ± 0.2.
5.1.25	MacConkey Agar Take 52 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.4 ± 0.2.
5.1.26	Standard Plate Count Agar Suspend 32.5 gm powder in 1 liter of double distilled water. Dissolve completely; maintain pH up to 7.0 ± 0.2.
5.1.27	VRBD Agar Suspend 39.5 gm powder in 1 liter of double distilled water. Dissolve completely; maintain pH up to 7.4 ± 0.2.
5.1.28	Antibiotic Medium 11 Take 30.5 gm powder and dissolve completely in 1 liter of double distilled water, maintain pH up to 7.9 ± 0.2
5.1.29	Antibiotic Medium 10 Add 42 gm of powder in 1 liter of double distilled water. Add 10 ml of polysorbate 80.Heat to boil and maintain pH up to 7.2 ± 0.2.
5.1.30	Dissolve the above media by heating in water bath by constant shaking till it turns to a clear transparent appearance.
5.1.31	At this stage, note down the pH of the media and record in the Annexure – I as before autoclave.
5.1.32	If require, dispense the media in tubes or caped bottles. Incase of flask or tube cotton plugs are use to close them.
5.1.33	Autoclave the media as per SOP # Q.C/0 /SOP/000.
5.1.34	After autoclave cycle, take out the media and allow cooling.
5.1.35	Note down the pH of the media and record in Annexure – I as after autoclave.
5.1.36	The pH of the media should comply the specification given in the manufacturer’s certificate of analysis.
5.1.37	Maintain the media consumption record Mentioning name of the media, batch/lot number, date of preparation and pH of each batch of finished medium in the attached format Annexure-I.
5.1.38	Criteria of Lab ID is Year / Media Code / Month / # of times preparation in 1 month
5.1.39	Media codes are allotted for each media bottle as 01, 02, and 03 … and it pasted on the bottles of media.
5.1.40	Month is identified by small alphabets as a, b, c, d, e, f, g, h, I, j, k, l.

 

5.2             Storage of Culture Media

 

storage of media

S.No	Action	Responsibility
5.2.1	Store dehydrated media as per manufacturer’s recommendations until expiration. Record the storage temperature and relative humidity on daily basis in annexure- II	Microbiologist
5.2.2	Stored prepared media at 2 - 8°C in the refrigerator for one month. Daily monitor the temperature of both refrigerators in their respective formats.

 

5.3          Precautions

 

S.No	Action	Responsibility
5.3.1	Avoid prolong heating of media flask in water bath.	Microbiologist
5.3.2	Constant stirring should be done to avoid any sediment at the bottom of the flask.

 

 6.0             Definition

 

Culture Media	A substance which promote the growth of microorganism under     laboratory condition
Sterilization	Refers to any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) from a surface, equipment, article of food or medication, or biological culture medium.

 

7.0.         References Used:

S. No	Description
I.	USP/ NF 2009, <62> “Microbiological Test”
II.	USP/ NF 2009, <71> “Microbiological Test”

 

8.0          List of Record Generated

 

S. No	Description of Annexure
I.	Preparation and Consumption Record of Culture Media
II.	Daily Monitoring of Storage temperature and Humidity of Dehydrated Media in Microbiology Lab

 

9.0          Referred Documents (s)

 

S. No	Description of Annexure
N/A	N/A

 10.0.      Annexure

 

S. No	Description of Annexure
I.	Preparation and Consumption Record of Culture Media
II	Daily Monitoring of Storage temperature and Humidity of Dehydrated Media in Microbiology Lab