Media Preparation_TB 02-09_V1.0.doc

TABLE OF CONTENTS

1.PURPOSE......

2.SCOPE......

3.RESPONSIBILITIES......

4.CROSS-REFERENCES......

5.PROCEDURES......

5.1. General......

5.2. Preparation of Middlebrook Media......

5.2.1. Middlebrook 7H9 medium

5.2.2. Middlebrook 7H11 medium (selective or non-selective)

5.3. Preparation of Blood Agar......

6.REFERENCES......

7.CHANGE HISTORY......

1.PURPOSE

This SOP describes preparation of several types of media that are commonly used for mycobacterial isolation or other procedures related to the TB diagnostics laboratory: Middlebrook 7H11 agar (selective and non-selective), Middlebrook 7H9 media and Blood Agar:

  • Middlebrook 7H11 agar base contains many inorganic salts which help the growth of mycobacteria. Citric acid from sodium citrate helps in retaining inorganic cations in solution. Glycerol supplies carbon and energy. The Middlebrook OADC supplement contains oleic acid, bovine albumin, sodium chloride, dextrose and catalase. Oleic acid and other long chain fatty acids are essential for mycobacteria metabolism. Dextrose is energy source. Catalase neutralizes toxic peroxides. Albumin protects tubercle bacilli from toxic agents. Malachite green inhibits other bacteria.
  • Middlebrook 7H9 Broth is used to subculture stock strains, prepare inoculum, and freeze culture isolates.
  • Blood agar is used for growth of contaminating organisms, most commonly in MGIT liquid cultures.

2.SCOPE

This SOP covers all procedures involving preparing media in the ______TB Laboratory.

3.RESPONSIBILITIES

All staff members working in the ______TB Laboratory are responsible for the implementation of this SOP. All users of this procedure who do not understand it or are unable to carry it out as described are responsible for seeking advice from their supervisor.

4.CROSS-REFERENCES

See: / Document Matrix_TB 01-01_V1.0.doc
Location:

Refer to SOPs listed under 07-01 (General Procedures), 07-02 (Specimen Handling), 07-04 (Culture and Drug Susceptibility Testing) and 07-06 (Equipment Use and Maintenance).

5.PROCEDURES

5.1. General

  • The media preparation is carried out in the ______, with autoclaving being done by ______.
  • Sterile technique is essential to prevent contamination.
  • All work surfaces, including the biological safety cabinet (BSC), must be cleaned with 5% Lysol.
  • Stock solutions, such as OADC and antibiotics, must be aseptically prepared and stored.
  • Excessive or prolonged heat affects the quality of the media.
  • Never autoclave 7H11 medium base and store it in the refrigerator for future use. The heat required to melt it produces a medium of poor quality.
  • Remove from the autoclave as soon as the pressure allows.
  • Dispense within an hour of sterilization. When the autoclaved media stands for more than one hour in the water bath, a precipitate begins to form.
  • Do not breathe dust from the dehydrated media (7H9).
  • Wear suitable protective clothing
  • Place antibiotics in the media immediately before pouring the plates to reduce their degradation.
  • Always check the medium when taking it out of the autoclave. It should be clear and transparent. If a precipitate is present, it may be due to 1). Sterilization temperature within the autoclave being reached to rapidly to dissolve the base in 10 minutes, 2) poor quality basal medium, and/or 3) dirty glassware.

5.2. Preparation of Middlebrook Media

5.2.1.Middlebrook 7H9 medium

Materials and equipment

  • Biological safety cabinet (BSC)
  • Autoclave
  • Waterbath
  • Middlebrook 7H9 broth base.
  • Glycerol
  • Oleic acid dextrose citrate (OADC) supplement
  • Petri dishes
  • Distilled water
  • 500 ml flasks
  • Sterile 10 ml pipettes
  • Pipet-aid
  • Sterile micropipette tips

Preparation

To prepare 450ml Middlebrook 7H9 medium:

  • Weigh out 2.35 grams of Middlebrook 7H9 broth base and dissolve in distilled water in a suitable autoclavable container. Once dissolved add up to 450ml with distilled water.
  • Add 2ml of glycerol or 0.5g of Tween 80.
  • Aliquot into smaller quantities for autoclaving, to reduce the risk of contamination during repeated use.
  • Aliquot two small volumes (5ml each) of medium for QC purposes.
  • Autoclave at 121°C for 15 minutes.
  • Remove from the autoclave and cool to at least 45°C in a water bath.
  • Aseptically add 50ml of oleic acid-albumin-dextrose-catalase (OADC).
  • Label with name and date of preparation.
  • Store at 2-8°C for up to three weeks. Check that screw caps are tight before storing.
  • Discard any prepared media that shows signs of contamination, discoloration, or evaporation.

Quality Control

Sterility check: Place one QC tube at 37°C for 3 days and record growth or no growth.

M. tuberculosisgrowth check:

  • Use frozen M.tuberculosis suspensions as described below.
  • Remove one aliquot of 10-4 dilution from the freezer.
  • Allow to equilibrate to room temperature.
  • Inoculate 2 QC broth tubes (5ml) with 200μl of the M.tuberculosissuspension and incubate at 37°C for 14 days.
  • Examine weekly for growth.
  • At 14 days, mix and prepare a ZN smear and examine according to ZN Microscopy.

See: / Ziehl Neelsen Microscopy_TB 04-01_V1.0.doc
Location:

5.2.2. Middlebrook 7H11 medium (selective or non-selective)

Materials and equipment

  • Biological safety cabinet (BSC)
  • Autoclave
  • Waterbath
  • Middlebrook 7H11 Agar base
  • Glycerol
  • Oleic acid dextrose catalase (OADC) supplement
  • Petri dishes
  • Distilled water
  • 500 ml flasks
  • Sterile 10 ml pipettes
  • Pipette-aid
  • Sterile micropipettetips

Preparation

This procedure makes about 25 plates:

  • Weigh out 10.25gm Middlebrook 7H11 agar base and dissolve together with 2.5 ml glycerol in sufficient distilled water in a suitable autoclavable container. Once dissolved add up to 450ml distilled water.
  • Cover flask and autoclave for 15 minutes at 121oC
  • As soon as possible remove from autoclave and place in a 50°C waterbath.
  • Turn on meticulously cleaned BSC five minutes before use.
  • Arrange sterile plates in the BSC and mark to indicate the type of media (7H11 or 7H11S) and the pour date.
  • Remove OADC from refrigerator and place in 50°C waterbath.
  • As soon as media has cooled to 50oC, remove one flask of medium and OADC and place in the BSC.
  • Using a sterile 50ml falcon tube add 50 ml of OADC to the flask of 7H11 and swirl to mix.
For non-selective 7H11
  • Working rapidly so that medium does not solidify, add 20 ml of medium to each plate until all the medium has been used.
  • Cover plates as soon as the media has been dispensed.
For Selective 7H11 (7H11S)
  • Selective 7H11 medium can be made by addition of various antibiotic supplements, e.g. PANTA (polymixin B, amphotericin, nystatin, trimethoprim, azlocillin), PACT (polymixin B, amphotericin, carbenicillin) or NOA (nystatin, oxacillin, aztreonam).
  • Once the media has cooled to 50oC, add recommended volume of antibiotic supplement according to manufacturer’s recommendation.
  • Swirl solution to mix well. Add approximately 20 ml medium to each plate.
  • Cover plates and leave at room temperature until well solidified(plates may be left at room temperature in the BSC overnight).
  • Place solidified plates in inverted position in the incubator at 37+1oC for 24 hours and check for contamination. Discard contaminated plates.
  • Remove plates from the incubator and place at 2-8oC until use. Plates should be covered (use a plastic bag) to prevent contamination by dripping condensation or other spillage inside the refrigerator.

Quality control

For each lot of 7H11 or 7H11S plates prepared:

  • Prepare serial 10 fold dilutions to 10-4 in 7H9 medium from a McFarland No.1 M.tuberculosisATCC 25177 and freeze 20-50 aliquots of the 10-3 and 10-4dilutions in labeled cryovials.
  • Remove one aliquot of each dilution (10-3 and 10-4) from the freezer.
  • Prepare two plates for inoculation.
  • Allow to equilibrate to room temperature.
  • Inoculate both plates with 500μl droplets of each dilution.
  • Wrap plates in a Ziplock bag and incubate at 37±1°C for 4 weeks.
  • Count number of colonies on plates and note colony size and number.
  • Record on Quality Control Middlebrook Media form.

Use: / QC Middlebrook Media_form.doc
Location:

5.3. Preparation of Blood Agar

Materials and equipment

  • Tryptic Soy Blood Agar Base
  • Defibrinated blood (Could be expired)
  • Distilled water
  • 500 mL flasks
  • Petri dishes

Preparation

  • Suspend 40g Tryptic Soy Blood Agar Base No. 2 in 1 litre of distilled water.
  • Autoclave at 121°C for 15 minutes.
  • Cool to 45 – 50oC.
  • Aseptically add 5% sterile defibrinated blood.
  • Mix thoroughly.
  • Pour about 20 ml into each whole plate.
  • Store plates in new plastic bags at 2-8°C for up to 1 month.
  • Store protected from any direct light.
  • Discard any media that shows signs of contamination, discoloration, or evaporation.

Quality control

  • Incubate 10% of the prepared number of plates at 37°C for at least 48hr to rule out contamination.
  • Inoculate 2 plates per batch with E.coli to ascertain the media supports growth of contaminants.
  • Prepare a stock concentration of E. coli and store aliquots at -80°C.
  • Remove one aliquot from freezer and allow to thaw.
  • Allow Blood Agar plates to equilibrate to room temperature.
  • Streak onto 2 Blood Agar plates and allow to dry.
  • Incubate at 37°C for 4 days.

Record results on Blood Agar QC form.

Use: / Blood Agar QC_form.doc
Location:

6.REFERENCES

MacFaddin,JF.,(1985).Media for isolation –Cultivation-Identification-Maintainance of medical bacteria.Vol. 1 . Williams and Wilkins.Baltimore, Maryland.

American Type Culture Collection,Manassas,Va.U.S.A.

Middlebrook and Cohn,AmJPublic Health, 48: 844 (1958)

7.CHANGE HISTORY

New version # / date / Old version # / date / No. of changes / Description of changes / Source of change request

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