Institution
Laboratory name
Location
Head/Responsible person / Standard Operating Procedure (SOP)
Preparation of Middlebrook / Code:
Version: no.
Date: of release
Page: 1of 5

1. Preparation of Middlebrook
2. Objectives and scope

The storage and maintenance of mycobacterial strains allows for future reference studies or investigations, etc. It is possible to maintain mycobacterial cultures by re-culturing on solid medium such as Löwenstein–Jensen medium but ongoing metabolic activity during in-vitro culturing may cause mutations in mycobacterial strains.

Lyophilization has been the most frequently used approach to preserve cultures while avoiding genetic rearrangements. However, because the viability of the lyophilized bacteria drops dramatically within a few years, storage of cultures in –70-80°C freezers is now preferred.

This SOP applies to all technicallaboratory personnel involved in the preparation of Reagents in the laboratory.

3. Abbreviations, definitions and terms

•N.A.Not applicable

•OADCOleic Albumin Dextrose Catalyse

4. Tasks, responsibilities and accountabilities

Task / Responsible / Accountable
Preparation of Middlebrook / Techs in Reagent Preparation section / Lab Manager
Quality control of Middlebrook / Techs in Reagent Preparation section and LJ section / Lab Manager

5. Safety and environment

Wear laboratory coat and gloves when performing this procedure.

6. Procedure

6.1 Reagents and Materials

•Middlebrook powder

•Glycerol

•OADC enrichment media

•Distilled water

•Duran bottles

•Cryovials (1.8 and 1.25ml)

6.2 Preparation of Middlebrook (7H9) media

  1. Stepwise Procedure 1000ml
  1. Suspend 4.7g of the Middlebrook powder in 900ml of distilled water.
  2. Add 2ml of glycerol.
  3. Autoclave the solution at 121oC for 10 minutes.
  4. Allow to cool to 45oC, then add 100ml of OADC enrichment media.
  5. Allow to cool.
  6. Aliquot into 250 ml sterile duran bottles.
  7. Incubate for 72 hours for sterility check (fill in appendix 1).
  8. Keep at 2-8oC.

b. Aliquoting media in cryovials

  1. Retrieve one duran 250ml containing sterile middlebrook from the refrigerator.
  2. Aliquout 1 ml into cryovials.
  3. Incubate for sterility checks for 48 hours.
  4. Inspect for cloudness. Discard cryovials that are cloudy.
  5. Fill out appendix 2.
  6. Keep at 2-8oC.

6.3Storage

Store properly labeled Middlebrook (7H9) bottles at 2-8ºC up to 3 months.

Store aliquouted cryovials at 2-8oC up to 3 months.

6.4 Quality Control

6.4.1 Sterility Check

Incubate prepared media for 72 hours. Discard the whole solution if it becomes cloudy. Record on appendix 1.

7. Related documents

N.A.

8. Related forms

N.A.

9. References

N.A.

10. Attachments / Annexes

SOP “Preparation of Middlebrook” Appendix 1.

SOP “Preparation of Middlebrook Aliquots” Appendix 2

GLI Stepwise Process towards TB Laboratory Accreditation

Appendix 1

Preparation of Middlebrook

Date Prepared: ______Prepared by: ______

Ingredients / Lot: Number / Expiration date / Amount Used
Middlebrook powder
Glycerol
OADC
Distilled Water

Quality Control Check:

Sterility Check:Incubate for 72 hours; should not be cloudy.

Pass/Fail:______

Middlebrook BATCH NO:______Expiration Date:______

NB: Storage at 4oC for 3 months.

Date Prepared: ______Prepared by: ______

Ingredients / Lot: Number / Expiration date / Amount Used
Middlebrook powder
Glycerol
OADC
Distilled Water

Quality Control Check:

Sterility Check: Incubate for 72 hours; should not be cloudy.

Pass/Fail:______

Middlebrook BATCH NO:______Expiration Date:______

NB: Storage at 4oC for 3 months.

Reviewed by/Date:______

GLI Stepwise Process towards TB Laboratory Accreditation

Appendix 2

Preparation of Middlebrook Aliquots

Date Prepared:______Prepared by:______

No: of cryovials prepared / No: Passed QC check / No: Failed QC check

Sterility check: Incubated for 48 hours, should not be cloudy. Discard all that are cloudy.

Middlebrook BATCH NO:______Expiration Date:______

(Batch No: written of the duran bottle)

NB: Storage at 4oC for 3 months.

Date Prepared:______Prepared by:______

Sterility check: Incubated for 48 hours, should not be cloudy. Discard all that are cloudy.

No: of cryovials prepared / No: Passed QC check / No: Failed QC check
No: of cryovials prepared / No: Passed QC check / No: Failed QC check
No: of cryovials prepared / No: Passed QC check / No: Failed QC check

Middlebrook BATCH NO:______Expiration Date:______

(Batch No: written of the duran bottle)

NB: Storage at 4oC for 3 months.

Date Prepared:______Prepared by:______

Sterility check: Incubated for 48 hours, should not be cloudy. Discard all that are cloudy.

No: of cryovials prepared / No: Passed QC check / No: Failed QC check

Middlebrook BATCH NO:______Expiration Date:______

(Batch No: written of the duran bottle)

NB: Storage at 4oC for 3 months.

Reviewed by/Date: ______

GLI Stepwise Process towards TB Laboratory Accreditation