a) Presumptive Test

i.

Macconkey broth may be used. Inoculate a series of fermentation tubes with

appropriate measured quantities of nutritive ingredients in the mixture should
be sufficient and according to the requirements. 10 ml and above aliquote should
be inoculated in double strength and 1 ml and its should be inoculated into
single strength medium.

ii.

Incubate all the tubes at 370 C for 24 hours to 48 hours. Examine each tube at the
end of 24(+,-) 2 hours. Examine for gas production and if no gas has been formed
again re-incubate for another 24 hours and at the end of 48 hours 3 hours
examine again. Record the presence of or absence of gas, at each examination of
the tubes regardless of the amount.

iii.

Formation of gas within 48(+-)3 hours in any amount in the inner fermentation
tubes, constitutes a possible presumptive test. The absence of gas formation at
the end of 48(+-)3 hours of incubation constitute negative test.

b.

Confirmed Test: - The medium used for confirmed test is brilliant green bile
lactose broth (BGLB)

i.

Submit all primary fermentation tubes showing any amount of gas at the end of
24 hours and 48 hours incubation to the confirmed test. Use a sterile metal loop, 3
to 4 mm in diameter to transfer one or two loopful of medium from the
presumptive positive tube to a tube of BGLB broth. When making such transfers,
gently shake the tube first or mix by rotating. Incubate the inoculated tubes at
370C for 48(+-) 3 hours.

ii.

The formation of gas in any amount in the Durhams tubes of BGLB tube at any
time within 48(+-) 3 hours constitute a positive confirmed test.