OXIDATIVE-FERMENTATIVE TEST (HUGH AND LEIFSON):

PRINCIPLE:
- Saccharolytic microorganisms degrade glucose either fermentatively or
oxidatively. The end products of fermentation are relatively strong mixed
acids that can be detected by the colour change in the medium.
- Motility can also be observed in this medium.
PROCEDURE:
Two tube with OF medium stabbed with the inocumlum. One tube covered with
liquid paraffin over the medium.
Incubate
OPEN TUBE COVERED TUBE METABOLISM
Acid (yellow) Alkaline (green) Oxidative
Acid (yellow) Acid (yellow) Fermentative
Alkaline (green or blue) Alkaline (green or blue) Nonsaccharolytic
RESULT:
Fermenter- E. coli DIAG:-
Oxidiser Pseudomonas aeruginosa
Nonsaccharolytic Moraxella spp.

TRIPLE SUGAR IRON TEST:

PRINCIPLE:
TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone
This test is used to determine whether a GNB utilizes glucose and lactose or
sucrose fermentatively and forms H2S.
PROCEDURE:
Inoculate TSI with a straight wire Incubate Observe
RESULT:
DIAG:-
ACID SLANT/ACID BUTT (A/A): E. coli
ALALINE SLANT/ ACID BUTT (K/A) WITH H2S: Salmonella typhi
ALKALINE SLANT/ NO CHANGE: Pseudomonas aeruginosa
PHENYLALANINE DEAMINASE TEST:
PRINCIPLE:
This test is used to determine the ability of an organism to oxidatively deaminate
phenylalanine to phenyl-pyruvic acid.
PROCEDURE:
Inoculate phenylalanine slant with I drop of 24hr BHI broth
Incubate
Add 4 to 5 drops of 10% aqueous ferric chloride
Green colour POSITIVE
INTERPRETATION: DIAG:-
POSITIVE: Proteus vulgaris
NEGATIVE: E. coli

BILE ESCULINE AGAR TEST:

PRINCIPLE:
- Gram-positive bacteria other than some streptococci and enterococci are
inhibited by the bile in the medium.
- Some Enterococci and streptococci can grow in the presence of 40% bile and
hydrolyse esculine.
- They subsequently turn the indicator ferric ammonium citrate dark brown.
- This dark brown colour results from the combination of esculetin with ferric
ions to form a phenolic iron complex.
PROCEDURE:
- The BEA slant is inoculated Incubate Colour change blackening of
the slant
- RESULT:
- Positive: Enterococcu faecalis
- Negative: Streptococcus mitis

AUTOMATED METHODS
BacT Alert VITEK-2
MALDI-TOF

GENOTYPIC METHODS
COLONY BACTERIAL GENE AMPLIFICATION IDENTIFICATION
PCR

REAL-TIME PCR