This document provides instructions for several biochemical tests used to identify Gram-negative bacteria, including the indole test, methyl red test, Voges-Proskauer test, citrate utilization test, urease test, oxidase test, H2S production test, motility test, Kligler iron agar test, sugar fermentation test, ONPG test, oxidation fermentation test, and phenyl pyruvic acid test. For each test, the document explains the principle, media or reagents used, and method for performing and interpreting the test results.

1. 4/1/2013
Sharq Elneil College
School of Medical Laboratory Sciences
Department of Microbiology
Medical Bacteriology course
Biochemical reaction of Gram
negative bacteria
U.Mahadi Hassan Mahmoud
Bsc, Msc, MIBMS Microbiology
INDOLE TEST
Principle:
 Certain bacteria break down Amino acid called
Tryptophan to Give Indole. Indole react with
Covac’s reagent to give Red ring.
Method:
 Sub-culture on media contain Tryptophan (e.g.:
Peptone water, Incubate at 37ºC for 24 hrs.
 Add few drops of Covac’s Reagent (4-
paradimethyle amino benzaldehyde in amyl
alcohol and HCL).
 Examine for development of Red Ring.
 +ve E. coli, -ve Klebsiella
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Methyl Red (MR)
Principle:
 Certain bacteria ferment Glucose to give high
amount of Lactic acid which react with Methyl
Red to give Red colour.
Method:
 Sub-culture on Glucose Phosphate peptone
water, incubate at 37ºC for 24 hrs.
 Add few drops from MR and see development
of Red colour.
 +ve E. coli, -ve Klebsiella.
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Vogous Proskaur (V.P)
Principle:
 Some organism Breakdown Glucose to give
Acetyl Methyl carbinol which oxidized by air to
give Diacetyl methyl carbinol react with -
naphthol in Alkaline pH (KOH) to give red
colour.
Method:
 Subculture in Glucose Phosphate peptone
water incubate at 37ºC for 24 hrs.
 Add 1 ml of 40% KOH and 3 ml - naphthol
and examined for red colour with 2-5 min.
 Listeria Monocytogenes V.P +ve and MR +ve.
Citrate Utilization test
Media content:
 Koser’s citrate: (Broth media contain
ammonium citrate and bromothymol blue)
 Simon Citrate (Koser’s citrate + agar)
Principle:
 Some organism Utilize citrate as a source of
carbon allow the ammonium ion free (Alkaline
pH) this give blue colour. Method:
 Subculture on media contain ammonium
citrate and incubate then read after 24-48 hrs.
may take 96 hrs.
 Proteus Citrate +ve, E. coli Citrate –ve.
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Urease test
Principle:
 Some organism secrete Urease which
breakdown urea into ammonia (Alkaline) which
change the colour of phenol red into red colour.
Method:
 Subculture on christensen’s urea broth or urea
agar slop, incubate for 4 hrs and see the
development of red colour.
 Proteus is rapidly urease +ve, E. coli is Urease
-ve
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Oxidase test
Principle:
 Certain organism produce oxidase enzyme that
oxidize oxidase reagents to give purple colour.
Methods:
Filter paper method:
 Test requirements:
Freshly prepared 1% Oxidase reagent (tetramethyl-p-
phenylene diamine).
Filter paper.
Wood stick or glass rods.
 Take a colonies and put it in filter paper, add drop of
oxidase reagents and examined for purple colour.
 Oxidase +ve like Neisseria and Pseudomonas.
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H2S production
Principle:
 H2S react with Ferric ammonium citrate to
give black colour.
Method:
 Subculture the organism in any media (solid
or broth) and put on the cover filter paper
impregnated on Ferric ammonium citrate,
Examined for black colour after 24 hrs
incubation.
 Proteus and Salmonella H2S +ve.
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7. 4/1/2013
Motility test
Kligler Iron agar
Principle:
 This test used to detect the ability of the organism to
ferment Glucose, or Glucose and Lactose with the
production of Acid only or with Acid Which react with
ferric chloride to give blacking.
 Glucose fermentation give lactic acid which oxidized
by air and return back to glucose on the top of the
medium.
Media content:
 Nutrition, Ferric chloride, Phenol red, Glucose and
Lactose.
Method:
 Subculture using straight loop till reach the bottom
(Butt) and get out making Zigzag on the surface,
incubate and read.
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Sugar fermentation
Principle:
 The ability of the organism to ferment sugar depends
on production of two enzymes:
 Permiase----Allow the penetration of sugar.
 β Galactosidase----Ferment the sugar.
Media content:
 Nutrient broth, Sugar (Glucose, Maltose,
sucrose,….), Durham tube, Andrad’s indicator (Acid
Fuchsin neutralized by NaOH).
Method:
 Subculture, incubate, read.
 Acid---- Yellow colour.
 Alkaline---- Red colour.
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ONPG
Principle:
 Some organism produce β Galactosidase
only (Late lactose fermenter). This enzymes
detected by ONPG (O-NitroPhenyl-β-D-
Galactoside) test in which β Galactosidase
break down β Galactoside allow the O-
NitroPhenyl free which have yellow colour.
Method:
 Subculture the organism on broth media
contain O-Nitrophenyl-β-D-Galactoside,
incubate and read for formation of Yellow
colour.
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Oxidation Fermentation
Principle:
 The test depends on fermentation of carbohydrate on
anaerobic condition of oxidation of it in aerobic
condition.
Media content:
 2 media each contain nutrition, sugar and bromothymol
blue. One of them closed from air by paraffin oil.
Results:
 Oxidative ferment sugar on open tube (Yellow colour).
 Fermentative but anaerobically give yellow on closed
tube.
 Facultative anaerobic ferment CHO on both tube
(Yellow).
 Non oxidative- Non fermentative– give Blue colour on
both tube.
Phenyl Pyruvic Acid (PPA)
Principle:
 Some organism deaminate Phenyl alanine to
Phenyl Pyruvic acid which react with Ferric
chloride to give green colour.
Method:
 Subculture the organism on media contain
Phenyl alanine and incubate at 37C for 24
hrs, add 10% ferric chloride on the surface
and examined for green colour.
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