Materials and methods

Five microorganisms Escherichia coli, Salmonella typhimurium, Klebsiella

pneumonia, Proteus mirabilis and Pseudomonas aeruginosa which including
were chosen to undergo the identification that listed below:
Selective media
Lines were drawn on the bottom of the plate of the two Mac Conkey Agar
plates and 2 Deoxycholate Citrate Agar to produce equal sizes of sectors.
Each sector with microorganism was labelled with their name. Some culture
of Escherichia coli from a nutrient agar slant were streaked onto the sector of
the plate by using an inoculation loop. The steps were repeated with
Salmonella typhimurium, Klebsiella pneumonia, Proteus mirabilis and
Pseudomonas aeruginosa. The cultures were incubated at 37 C for 24 hours
and the plates were kept at 4 C after the incubation period.

Oxidation-fermentation test
The oxidation-fermentation medium was inoculated with an inoculating
needle by stabbing once down the center of the gel to approximately one-
fourth inch from the bottom of the tube. Duplicate tubes were inoculated and
one tube overlaid with 1 ml sterile paraffin. The open tube with the cap
loosened and the closed tube with paraffin overlay were incubated at 25-37
C for 5 days. The daily colour changes was examined.

Fermentation test
Each microorganism was inoculated into tubes which containing Andrade
peptone water with different carbohydrates. The inoculated microorganisms
were incubated at 37C for 5 days.

Oxidase test
A good-sized amount of inoculum was picked from a plate culture or slant
culture and placed on a piece of filter paper. One drop of the reagent was
added.

Methyl Red test

Methyl red- VP broth was inoculated with your culture and incubated at 37 C
for one to two days, five drops of methyl red indicator were added to the
broth culture. The colour of the dye was observed and recorded immediately.
Urease test
The whole surface of Christensens agar slant was inoculated with a heavy
inoculum and incubated at 35 C for two days.

VP test
The MRVP broth was inoculated with the culture and incubated at 37 C for
one to two days. 0.6 ml of -napthol solution, 0.2 ml of 40 % KOH was added
to each test tube and mixed gently. The test tubes were then let undisturbed
for 10-15minutes. The development of the colour red for a positive reactive
was observed and recorded.

Hydrogen sulfide (H2S Production)

Triple sugar ion (TSI) was inoculated in each tube by making a single stab
through the agar. The cultures were incubated at 35 C for two days and the
colours of the agar after two days were observed.

Indole production
Tryptone water broth was inoculated with the cultures and were incubated at
37 C for 24 hours. Three drops of Kovacs reagents were added into each
tube. The layer of the surface was observed after adding the reagent.

Citrate utilization
Each microorganism was inoculated onto the Simmons citrate agar and was
incubated at 37 C for five days. The colour of the agars were observed after
five days.