Institution Laboratory name Location Head/Responsible person

Standard Operating Procedure (SOP) Preparation of blood agar

Code: Version: no. Date: of release Page: 1 of 4

1. Preparation of Blood Agar (BA) 2. Objectives and scope This SOP describes the preparation of Blood Agar (BA) media that is used in the laboratory to determine or rule out contamination of positive MGIT tube cultures. Mycobacterium tuberculosis does not grow on blood agar hence any growth on the blood agar plate after a period of 24 hours is considered as a contamination. This is growth due to other fastidious microorganisms and not necessarily Mycobacterium tuberculosis. This SOP applies to all laboratory staff who perform preparation of BA media as well as perform MGIT Culture and MGIT SIRE DST, and must be followed at all times while preparing BA. 3. Abbreviations, definitions and terms N.A. MGIT BA SIRE DST AFB SOP TSBA Not applicable Mycobacteria Growth Indicator Tube Blood Agar Streptomycin, Isoniazid, Rifampicin and Ethambutol Drug Susceptibility Testing Acid-Fast Bacilli Standard Operating Procedure Tryptic Soy Blood Agar Base

4. Tasks, responsibilities and accountabilities Task Preparation of Blood Agar Responsible Techs in MGIT culture and DST section Accountable Lab Manager

5. Safety and environment N.A. 6. Procedure 6.1 Reagents and Materials Weighing balance Tryptic Soy Blood agar base Distilled water One 1 litre capacity cornical flask Autoclave Safety cabinet

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Institution Laboratory name Location Head/Responsible person

Standard Operating Procedure (SOP) Preparation of blood agar

Code: Version: no. Date: of release Page: 2 of 4

25ml of sterile defibrinated blood Petri dishes Measuring cylinder Bunsen burner

6.2 Procedure 1. 2. 3. 4. 5. 6. 7. Weigh 500ml of distilled water using a measuring cylinder. Transfer the distilled water into a 1 litre cornical flask. Weigh 20g of Tryptic Soy Blood Agar Base (TSBA) using a weighing balance. Suspend the measured TSBA into the 500ml of distilled water. Mix thoroughly (dissolving occurs during autoclaving). Autoclave at 121C for 15 minutes. Allow the autoclaved TSBA to cool to 45-50C and then aseptically add 25ml of sterile defibrinated blood. Mix thoroughly. 8. Arrange the petri-dishes onto the clean safety hood and then gently pour the warm blood agar onto the plates. 9. Using a bunsen burner gently invert and pass the flame over the poured blood agar in the plate such that the air bubbles are removed. 10. Cover the petri-dishes and allow the blood agar to coagulate before storage in a refrigerator. 11. Label on the bottom top of the blood agar plates the batch number, date prepared and expiration date, and tech. initials. 12. Document/fill in he worksheet for BA preparation, Appendix 1. 6.3 Quality Control a. Perform Sterility Check 1. Randomly select 2 blood agar plates and incubate them at 37C for 24 hours. 2. If there is no visible growth or haemolysis of the media then the blood agar is sterile and ready for use. b. Test to support growth of bacterial contaminants 1. After sterility check, inoculate two BA plates with a strain of Staphylococcus aureus. 2. Incubate the plates at 37C for 24 hours. 3. Observe for a luxurious growth of S. aureus on both plates. 4. If only one plate shows growth, repeat QC with two other plates. 5. If there is no growth or only one plate shows growth, then QC fails. Complete Corrective Action Log and report to Laboratory Supervisor. 7. Related documents SOP MGIT Liquid Culture of Mycobaterium tuberculosis 8. Related forms

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Institution Laboratory name Location Head/Responsible person

Standard Operating Procedure (SOP) Preparation of blood agar

Code: Version: no. Date: of release Page: 3 of 4

N.A. 9. References N.A. 10. Attachments / Annexes Appendix 1, Worksheet for Preparation of Blood Agar (BA)

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Worksheet for Preparation of Blood Agar

Tech. Name:____________________________________
Procedure 1. 2. 3. 4. 5. 6. 7. 8. 9. Measure 500ml of distilled water into a 1 litre cornical flask. Weigh 20g of Tryptic Soy Blood Agar Base. Add and suspend the measured TSBA into the 500ml of distilled water. Mix thoroughly. Heat with frequent agitation and boil for one minute to completely dissolve the powder. Autoclave at 121C for 15 minutes. Cool to 45-50C and then aseptically add 25ml of sterile defibrinated blood. Mix thoroughly. Arrange the petri-dishes onto the clean safety hood and then gently pour the warm blood agar onto the plates. Invert and pass the Bunsen burner flame over the poured blood agar in the plate such that the air bubbles are removed. Cover the petri-dishes and allow the blood agar to coagulate before storage in a refrigerator.

Date: _______________________________

10. Label on the bottom top of the blood agar plates the batch number, date prepared and expiration date, and tech. initials.

Quality Control # Plates prepared: Sterility Check Sterile? [yes] or [no] Assigned Batch # S. Aureus Growth Support test Growth Present? [Yes] [No]

Plate 1 Plate 2

Plate 1 Plate 2

QC Passed [YES] [NO] ** Both plates should be sterile, no haemolysis visible and should support S. Aureus growth Reviews Tech. Sign: ________________________ Date:__________________________

Reviewer Sign:______________________ Date: __________________________

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