Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Preparation of blood agar
Code:
Version: no.
Date: of release
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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
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•
•
•
•
•
•
•
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
5. Safety and environment
N.A.
6. Procedure
6.1 Reagents and Materials
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Weighing balance
Tryptic Soy Blood agar base
Distilled water
One 1 litre capacity cornical flask
Autoclave
Safety cabinet
25ml of sterile defibrinated blood
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Accountable
Lab Manager
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
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 121°C for 15 minutes.
Allow the autoclaved TSBA to cool to 45-50°C 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 37°C 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 37°C 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
N.A.
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Preparation of blood agar
9. References
N.A.
10. Attachments / Annexes
Appendix 1, Worksheet for Preparation of Blood Agar (BA)
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Code:
Version: no.
Date: of release
Page: 3 of 4
Worksheet for Preparation of Blood Agar
Tech. Name:____________________________________
Date: _______________________________
Procedure
1.
Measure 500ml of distilled water into a 1 litre cornical flask.
2.
Weigh 20g of Tryptic Soy Blood Agar Base.
3.
Add and suspend the measured TSBA into the 500ml of distilled water. Mix thoroughly.
4.
Heat with frequent agitation and boil for one minute to completely dissolve the powder.
5.
Autoclave at 121°C for 15 minutes.
6.
Cool to 45-50°C and then aseptically add 25ml of sterile defibrinated blood. Mix thoroughly.
7.
Arrange the petri-dishes onto the clean safety hood and then gently pour the warm blood agar onto the plates.
8.
Invert and pass the Bunsen burner flame over the poured blood agar in the plate such that the air bubbles are removed.
9.
Cover the petri-dishes and allow the blood agar to coagulate before storage in a refrigerator.
10. Label on the bottom top of the blood agar plates the batch number, date prepared and expiration date, and tech. initials.
Quality Control
Assigned Batch
#
# Plates prepared:
Sterility Check
Sterile? [yes] or [no]
S. Aureus Growth Support
test
Plate 1
Plate 2
Growth Present? [Yes]
[No]
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: __________________________
Source: GLI Stepwise Process towards TB Laboratory Accreditation