Perfringens Agar Base

Biochemical Confirmation:

I: Confirmation technique using Lactose Sulphite Broth Base (M1287):

Inoculation and Incubation:

Inoculate each selected colony into the Fluid Thioglycollate Medium (M009). Incubate under anaerobic conditions at 37°C for 18 h to 24 h.

After incubation, tranfer with no delay 5 drops of the thioglycollate culture to the Lactose Sulphite Broth Base (M1287) by means of a sterile pipette. Incubate aerobically at 46°C for 18 h to 24 h in the water bath.

Interpretation of the result:

Examine the tubes of Lactose Sulphite Broth Base (M1287) for the production of gas and the presence of a black color (iron sulfite precipitate). Durham tubes more than one-quater full of gas and tubes having a black precipitate are considered positive.

In case of doubt, when the Durham tube in a blackened medium is less than one-quater full of gas, transfer with no delay, using a sterile pipette, 5 drops of the previous growth in Lactose Sulphite Broth Base (M1287) to another tube of Lactose Sulphite Broth Base (M1287). Incubate in the water bath at 46°C for 18 h to 24 h. Examine this tube as described above.

Bacteria which form characteristic colonies in the T.S.C. medium are considered as being C. perfringens. In all other cases, the tubes should be considered as negative.

II: Confirmation technique using Motility Nitrate Medium, Buffered (M6301):

Inoculation and reading of nitrate motility medium:

Stab-inoculate each selected colony into the freshly deaerated Motility Nitrate Medium, Buffered (M6301).

Incubate under anaerobic conditions at 37 °C for 24 h. Examine the tube of the nitrate motility medium for the type of growth along the stab line. Motility is evident from diffuse growth out into the medium away from the stab line.

Test for the presence of nitrite by adding, with the graduated pipette and the rubber bulb, 0.2 ml to 0.5 ml of the nitrite detection reagent (5.6) to each tube of nitrate motility medium.

The formation of a red colour confirms the reduction of nitrate to nitrite. If no red colour is formed within 15 min, add a small amount of zinc dust and allow to stand for 10 min. If a red colour is formed after the addition of zinc dust, no reduction of nitrate has taken place.

Inoculation and reading of Lactose Gelatin Medium, Modified (M9871):

Inoculate each selected colony into the freshly deaerated lactose-gelatin medium (5.8). Incubate under anaerobic conditions at 37 °C for 24 h.

Examine the tubes of the lactose-gelatin medium for the presence of gas and a yellow colour (due to acid formation) indicating fermentation of lactose. Chill the tubes for 1 h at 5°C and check for gelatin liquefaction. If the medium has solidified, re-incubate for an additional 24 h and again check for gelatin liquefaction.

Interpretation:

Bacteria that produce black colonies in T.S.C. media (M837I), are non-motile, usually reduce nitrate to nitrite, produce acid and gas from lactose, and liquefy gelatin in 48 h are considered to be C. perfringens. Cultures that show a faint reaction for nitrite (i.e. a pink colour) shall be eliminated, since C.perfringens consistently gives an intense and immediate reaction.