Table 2
Most common differentiation tests used for mycobacterial characterization
Test
Inoculation
Incubation
Reagents
Positive reaction
Negative reaction
Nitrate
Emulsify a loopful of growth
into 3–4 drops of water,
add to 2 mL NaNO
3
2 h, 37
C, 5% CO
2
One drop of 1:2 HCl, two
drops AFB nitrate reagent
A and AFB nitrate
reagent B, if clear, add
nitrate reagent C (zinc)
Red/pink (without
zinc)
No color development
(with zinc)
No color
development
(without zinc)
Red/pink (with
zinc)
Niacin
Add 1.5 mL water or saline to
culture grown on an L-J
slant or Middlebrook agar.
Scrape off surface growth
with a pipette. Stab pipette
through the growth into
the medium to permit
extraction of the niacin.
Allow the fluid to remain in
contact with the culture by
slanting the L-J or plate so
the surface of the medium
is in the horizontal
position. Incubate ~30 min
in this position. Remove
0.6 mL of the extract with a
sterile pipette and transfer
to the bottom of a
13
 75 mm test tube
N/A
Add one niacin test strip to
each 13
 75 mm tube.
Read 12–15 min after
strips are added
Yellow
No color
development
Pyrazinamidase Streak two slants of Remel
pyrazinamidase medium
with a loopful of colonies
from solid media. The
inoculum should be visible
37
C, 4 days.
Tube 1 is checked at 4 days. If
negative, check tube 2 at
7 days
Prepare reagent: Add 5 mL
water to one tube of
granulated ferrous
ammonium sulfate. Add
1 mL ferrous ammonium
sulfate to slant.
Refrigerate for 1 h
Red/pink
No color
development
16
Elizabeth Wallace et al.