CLIBAC LAB

BIOCHEMICAL TESTS Interpretation of result

▪ K/A = glucose was fermented
▪ K/A + G = glucose was fermented + gas
▪ K/K = no sugar was fermented
▪ A/A + G = 2-3 sugars were fermented, + gas
A/A + G + H2S = glucose & lactose/sucrose
Gas and H2S production

Visible result on TSI

Biochem Tests:
1.TSI
2.SIM
3.LIA
4.Urease
5.SCA
6.MRVP

l- TSI
▪ -triple sugar iron
▪ -butt slant medium
▪ 3 sugars :
a. lactose
b.glucose
c. Sucrose
pH indicator: phenol red
Acid : yellow (A)
Alkaline: red (K)
H2S indicator : sodium thiosulfate and
Ferrous sulfate

Determines:
1.Fermentation of sugar
2.H2S production
3.Gas production
1. Displacement of medium
2. Cracks
3. Bubbles

Gas /H2S production

 CLIBAC LAB
III-LIA ▪ (+) blue slant
▪ Lysine Iron Agar
▪ Decarboxylase test
▪ Butt slant
▪ Glucose
▪ Decarboxylase basal medium
▪ 3 Amino acids
a.Lysine
b.ornithine
c.Arginine
Indicator : Bromcresol purple
Acid : yellow
Alkaline: purple
MEDIA:
LIA (Lysine Iron Agar), Moeller’s medium or Falkow’s
decarboxylase basal medium with Indicator + sugar V- Urease Test
(glucose) + an amino acid ( lysine, Ornithine or ▪ Urea broth/ Christensen’s Urea agar
arginine) ▪ Indicator : phenol red
Acid – yellow
Principle Alkaline – red
▪ Some organisms can decarboxylate amino acid thru Principle :
the enzyme decarboxylase and convert it to lysine to Some organisms produce the enzyme urease which
CADAVERINE. hydrolyzes urea to NH4OH (alkaline)
▪ Decarboxylation produce enough alkalinity over the Urease Test
acid produced from the fermentation of glucose. PROCEDURE:
Lysine > cadaverine 1.Label the urea broth
Arginine > ornithine 2. Using aseptic technique, inoculate the tube with
Ornithine > putrescine bacterium by means of loop inoculation.
3.Incubate the tube for 24-48 hours at 35 C
4.Examine the urea broth for positive result
RESULTS:
Positive= RED (pH>8.4) Negative= YELLOW (pH<6.8)
Urease test result
▪ (+) alkaline – red/pink
▪ (-) acid – yellow

IV-SCA
▪ -Simmon’s Citrate Agar
▪ slant
▪ Indicator : Bromthymol blue
Acid – yellow
Alkaline- blue
Neutral –green
SCA principle
▪ Some organisms can utilize citrate as the sole
Source of carbon releasing Ammonia (NH3) in the

Process. Organisms which hydrolyzes urea

 CLIBAC LAB
In 24 hrs: When dextrose is fermented some organisms
▪ 1. Proteus Produce not only acid but also a compound known As
▪ 2.Morganella acetylmethyl carbinol or acetoin, in which the
▪ 3.Providencia rettgeri presence of KOH will be oxidized to dimethyl
Slow Carbinol (end product), which in turn will react with
▪ 1.Klebsiella the guanidine compound present in the broth to
▪ 2. Enterobacter produce the deep red color
▪ 3. Citrobacter PROCEDURE:
1.Use the ½ aliquot from the methyl red test. Add
V1- MRVP 0.6ml Of Barritt’s solution A and 0.2ml of solution B
▪ Methyl Red Voges Proskauer Medium or Clark to the culture, shake vigorously to aerate.
And Lubs Dextrose Broth Medium or Buffered (alternatively, about 15 drops of reagent A followed
Peptone Glucose Broth by 5 drops of reagent B works fairly well and avoids
▪ broth pipetting.) positive reaction occur at once or within
▪ Indicator : Methyl red 20 minutes.
Acid – red Positive result:
Alkaline – yellow Red layer on top in 10 minutes (remove plus or cup:
MRVP Principle oxygen needed for reaction.
▪ Organism fermenting glucose produce a large Negative= No color, or color other than red
amount of acid and overcome the neutralizing effect ▪ Most enterobacteriaceae give opposite MR and VP
of the buffer. reactions, as increasing alkalinity (negative MR test)
▪ This is based on the final hydrogen Ion is due to the production of acetoin (acetylmethy
concentration reached by the carbinol), giving a positive VP test.
Culture.
▪ (+) pH 4.5 and below- bright red
▪ (-) pH above 4.5 -yellow
MRVP
Methyl Red
Procedure:
1. Label the MR-VP broth medium tube.
2. Using aseptic technique, inoculate the tube with
the
appropriate bacterium by means of a loop
inoculation
3. Incubate all tubes at 35’C for 24-48 hours. For slow
fermenters it may take 4 to 5 days.
4. Transfer 1/3 of the culture into an empty test tube
and
set this aside for the Voges-Proskauer test.
5. To the 2/3 of the culture remaining in the tube, add
0.2ml (about 4-5drops) of methyl red indicator.
Positive result: formation of deep red color

Voges Proskauer
Principle: