MOTILITY NITRATE MEDIUM, BUFFERED (ISO 7937:2004)

Clostridium perfringens food poisoning is one of the most common type of human foodborne illness. The foods usually involved are cooked meat or poultry products containing large number of viable cells. Clostridium perfringens is a gram-positive, rod shaped anaerobic, spore-forming bacteria that produces enterotoxin. This toxin if ingested, can cause food poisoning. Motility Nitrate Medium, Buffered formulated in accordance with FDA and APHA, is recommended for the detection of C.perfringens on the basis of motility and nitrate test. Motility Nitrate Medium, Buffered with a slightly lower pH is recommended by the ISO Committee for isolation and detection of C.perfringens on the basis of motility and nitrate test.
Growth of motile organisms extends out from the line of inoculation. The medium contains 0.5% each of glycerol and galactose to improve the consistency of the nitrate reduction reaction with different strains of the organisms. Potassium nitrate serves as a base for nitrate reduction. A red or orange colour formation on addition of nitrate reagents indicates reduction of nitrate to nitrite. Motility is indicated by turbidity extending out from the line of stab inoculation. Non-motile organisms grow only in the inoculated area. After 3-8 hours of incubation, a small puffball of motility may be seen around the line of inoculation. If this is not observed, tubes should be re-incubated for 24-48 hours and compared for turbidity to an un-inoculated tube. Negative motility reactions should be confirmed by a hanging drop preparation.
In the nitrate reduction test, a pink to red color develops after addition of the reagents if nitrite is present. Colour development indicates that nitrate reduction has occurred in the tube. Some organisms further reduce nitrite to ammonia that can be detected by the addition of a small amount of zinc dust to the tubes exhibiting no colour. A pink colour in this part of the test indicates no nitrate reduction. A colourless reaction indicates that nitrates have been completely reduced.
Inoculate 2 grams of food sample in 15 to 20 ml of Chopped Liver Broth or Tryptone Glucose Yeast Extract Broth. After an incubation at 35-37?C for 20-24 hours, isolate on Perfringens Agar Base (TSC/SFP Agar Base). Presumptive C.perfringens colonies are confirmed biochemically by inoculating into Motility Nitrate Medium, Buffered to detect motility and nitrate reduction.

for isolation and detection of Clostridium perfringens on the basis of motility and nitrate test