Protocol
For Motility, H2S Production & Indole Tests:
1. Motility Test:
Ø Prepare
semi-solid media (0.4% agar)
I.
R2A (add 0.4% agar)
II.
SIM (no need to add agar
Ø Dispense
in sterile glass tubes & autoclave
Ø To
test for motility, an isolated colony is stabbed to within 1 of the bottom of
the tube with sterile needle. But care should be taken not to touch to the
bottom completely.
Ø Incubate
the tube at 280C for 24 hrs.
Ø A
positive motility test is indicated by turbid area extending away from the line
of inoculation.
Ø A
negative test is indicated by growth along the inoculation line but no further.
 |
| Motility test |
2. H2S
Production & Indole Test:
Ø Using isolated colonies from an 18-24 hour culture on solid media,
inoculate the SIM Medium by stabbing the center of the medium to a depth of 1/2
inch.
Ø Incubate the inoculated
medium aerobically at 28 degrees C. for 18-24 hours.
Ø Observe for H 2 S production and motility.
Ø Once H 2 S and motility reaction have
been read and recorded, apply three drops of Kovacs Reagent to the surface of
the medium.
Ø
Observe for the development
of a pink to red color.
INTERPRETATION OF
RESULTS
·
A positive H 2 S
test is denoted by a blackening of the medium along the line of inoculation. A
negative H 2 S test is denoted by the absence of
blackening.
·
A positive motility test
is indicated by a diffuse zone of growth flaring from the line of inoculation.
·
A negative motility test
is indicated by growth confined to the stab line.
·
A positive test for
indole is denoted when a pink to red color band is formed at the top of the
medium after addition of Kovacs Reagent. A yellow color denotes a negative
indole test after addition of Kovacs Reagent.
Reagent:
Kovac’s Reagent: (For 10 ml)
1. Add 2.5ml of concentrated HCl to 7.5ml of Amyl alcohol
2. Dissolve 0.5g of paradimethylamino-benzaldehyde in the
solution from step 1
3. Aliquot to single use vials
4. Store at 4oC in closed vials
