VIII. METHODS FOR TYPING MYCOBACTERIA
371
F. The soft-agar overlay method for phage typing M . tuberculosis
1. 7H9 broth medium
Add 4.7 g of Middlebrook 7H9 broth base (Difco) and 0.5 ml of Tween80 to 900 ml of distilled water and stir until each is in solution. Sterilise
by autoclaving at 15 lb (121°C) pressure for 15 min. Allow to cool and add
aseptically, 100 ml of sterile Middlebrook ADC enrichment (BBL).
Dispense in 5 ml amounts into sterile 20 x 150 mm screw-cap tubes.
2. Soft agar overlay
1. Dubos broth base without Tween-80 (Difco) 6.5 g
2. Proteose peptone No. 3 (Difco)
10.0 g
3. Glycerol
10.0 g
4. Agar
7.5 g
5. Distilled water
1000 ml
Dissolve ingredients 1, 2, and 3 in water then add agar. Heat to dissolve
agar. Stir frequently while heating to dissolve the agar in order to prevent
scorching. Dispense in 3.5 ml amounts into sterile 16 x 125 screw-cap
tubes. Sterilise medium after tubing by autoclaving at 15 lb (121°C)
pressure for 15 min.
3. Hard basal medium (oleic acid-albumin agar)
1. Dubos Oleic Agar Base (Difco)
20.0 g
2. Bacto-Dubos Oleic Albumin Complex (Difco) 100 g
3. Distilled water
900 ml
Dissolve agar base in water with frequent stirring to prevent scorching.
Sterilise by autoclaving at 15 lb (121°C) for 15 min. Temper medium to
52°C then add aseptically, 100 ml of sterile Racto-Dubos oleic albumin
complex (Difco). Mix contents of flask by gentle swirling to prevent formation of bubbles and dispense into sterile Petri dishes.
All supplements are available commercially in sterile amounts of
20 ml/tube. Smaller quantities of each medium may be prepared by using
10 ml supplement for each 90 ml of basal medium.
Inoculate 7H9 broth with the isolates of M . tuberculosis that are to be
phage typed. Incubate the 7H9 broth cultures at 37°C for ten days. Shake
cultures daily to maintain dispersed growth. Just prior to use, shake
cultures and allow to stand undisturbed for 5 min to permit the larger
clumps to settle.
Melt the soft-agar overlays in a boiling water bath. After melting the
agar temper the soft-agar to 52°C. Pipette 0-5 ml of the 7H9 broth culture
into the tube with the tempered soft-agar, shake the tube gently to mix
371
F. The soft-agar overlay method for phage typing M . tuberculosis
1. 7H9 broth medium
Add 4.7 g of Middlebrook 7H9 broth base (Difco) and 0.5 ml of Tween80 to 900 ml of distilled water and stir until each is in solution. Sterilise
by autoclaving at 15 lb (121°C) pressure for 15 min. Allow to cool and add
aseptically, 100 ml of sterile Middlebrook ADC enrichment (BBL).
Dispense in 5 ml amounts into sterile 20 x 150 mm screw-cap tubes.
2. Soft agar overlay
1. Dubos broth base without Tween-80 (Difco) 6.5 g
2. Proteose peptone No. 3 (Difco)
10.0 g
3. Glycerol
10.0 g
4. Agar
7.5 g
5. Distilled water
1000 ml
Dissolve ingredients 1, 2, and 3 in water then add agar. Heat to dissolve
agar. Stir frequently while heating to dissolve the agar in order to prevent
scorching. Dispense in 3.5 ml amounts into sterile 16 x 125 screw-cap
tubes. Sterilise medium after tubing by autoclaving at 15 lb (121°C)
pressure for 15 min.
3. Hard basal medium (oleic acid-albumin agar)
1. Dubos Oleic Agar Base (Difco)
20.0 g
2. Bacto-Dubos Oleic Albumin Complex (Difco) 100 g
3. Distilled water
900 ml
Dissolve agar base in water with frequent stirring to prevent scorching.
Sterilise by autoclaving at 15 lb (121°C) for 15 min. Temper medium to
52°C then add aseptically, 100 ml of sterile Racto-Dubos oleic albumin
complex (Difco). Mix contents of flask by gentle swirling to prevent formation of bubbles and dispense into sterile Petri dishes.
All supplements are available commercially in sterile amounts of
20 ml/tube. Smaller quantities of each medium may be prepared by using
10 ml supplement for each 90 ml of basal medium.
Inoculate 7H9 broth with the isolates of M . tuberculosis that are to be
phage typed. Incubate the 7H9 broth cultures at 37°C for ten days. Shake
cultures daily to maintain dispersed growth. Just prior to use, shake
cultures and allow to stand undisturbed for 5 min to permit the larger
clumps to settle.
Melt the soft-agar overlays in a boiling water bath. After melting the
agar temper the soft-agar to 52°C. Pipette 0-5 ml of the 7H9 broth culture
into the tube with the tempered soft-agar, shake the tube gently to mix