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.
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| 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
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