This document provides information on various biochemical tests performed for the identification of gram-negative bacteria, including catalase, cytochrome oxidase, indole, methyl red, Voges-Proskauer, citrate utilization, urea hydrolysis, triple sugar iron, sugar fermentation, oxidation-fermentation, nitrate reduction, and decarboxylase tests. It describes the principles, procedures, reagents, and expected results for each test as well as example organisms that would produce positive or negative reactions. Quality control organisms are also listed to ensure the validity of the test results.

1. BIOCHEMICAL REACTIONS
FOR
GRAM NEGATIVE ORGANISMS
DR.SABA NAAZ
FIRST YEAR POST GRADUATE
M D MICROBIOLOGY
KAKATIYA MEDICAL COLLEGE

2. IDENTIFICATION Of BACTERIA:
Identification of bacteria can be done by several
methods such as:
1. Conventional methods for culture and identification
2. Automated culture techniques
3. Serology
4. Molecular methods

3. FOR GRAM-NEGATIVE BACILLI:
The following are the common biochemical tests done routinely:
• Indole test
• Methyl red test (MR)
• Voges Proskauer (VP)
• Citrate utilization test
• Urea hydrolysis test
• Triple sugar iron test (TSI).
• Sugar fermentation test
• Oxidation fermentation test (OF)
• Nitrate reduction test
• Decarboxylase test
• Phenyl pyruvic acid test (PPA)

4. Catalase
Principle
Catalase is an enzyme that decomposes hydrogen peroxide (H2O2) into water
and oxygen.
2H2O2 → 2H2O + O2 (gas bubbles)
Reagents
A. Hydrogen peroxide 3%
B. Glass rod
Procedure:-
Add 1ml of 3% H2O2 directly to an 18-24h inoculated pure agar slant culture and
observe for immediate effervensens and record the results.

5. Results
The rapid and sustained appearance of bubbles or
effervescence constitutes a positive test.
Quality Control
Positive control: Staphylococcus aureus ATCC 25923
Negative control: Streptococcus species ATCC 19615
.
CATALASE POSITIVE CATALASE NEGATIVE
Staphylococci Streptococci
Listeria monocytogenes Enterococci
Corynebacteria Lactobacillus
Micrococcus Gardnerella vaginalis
Gonococci Ekienella corrodens
Enterobacteriaceae Kingella kingae
Actinomycetes

6. Cytochrome OxidaseTest
Principle
It detects the presence of cytochrome oxidase enzyme in bacteria, which catalyzes the
oxidation of reduced cytochrome by atmospheric oxygen.
REAGENTS
Tetramethyl-p-phenylenediamine dihydrochloride, 1%
Dimethyl-p-phenylenediamine dihydrochloride, 1%
PROCEDURE
two methods:
(1) the direct plate technique
(2) Modified oxidase test

7. RESULT
Bacterial colonies having cytochrome oxidase activity develop a deep blue color
at the inoculation site within 10 seconds
Quality Control
Positive control: Pseudomonas aeruginosa ATCC 27853
Negative control: Escherichia coli ATCC 25922
OXIDASE POSITIVE OXIDASE NEGATIVE
Neisseria Enterobacteriaceae
Vibrio Staphylococcus
Campylobacter Acinetobacter spp
Pseudomonas Bordetella
Aeromonas

8. INDOLE TEST:
Principle
It detects the ability of certain bacteria to produce an enzyme
tryptophanase that breaks down amino acid tryptophan present in
the medium into indole.
Tryptophan Indole + Pyruvic acid + Ammonia
Indole + p- di methyl amino benzaldehyde RED coloured product
REAGENT:-Kovac’s reagent
Ehrlich’s reagent
Procedure
Inoculate tryptophan broth with the test organism and incubate at 35°C for 18–24 hours. At
the end of this time, add 5 drops of reagent down the inner wall of the tube

9. .
Results
The development of a bright fuchsia red color at the interface of the
reagent and the broth within seconds after adding the reagent is indicative
of the presence Of indole and is a positive test.
Quality Control
Positive control: Escherichia coli ATCC 25922
Negative control: Klebsiella pneumoniae ATCC 70063

10. INDOLE POSITIVE INDOLE NEGATIVE
E.Coli K. Pnemoniae
K.Oxytoca Shigella species
Citrobacter koseri Salmonella species
Edwardsiella tarda Citrobacter freundii
Proteus vulgaris Enterobacter aerogenes
Morganella morganii Enterobacter cloacae
Providencia Hafnia
Proteus mirabilis
Pseudomonas aeruginosa

11. METHYL RED (MR) TEST:
 Principle
This test detects the production of sufficient acid during fermentation of glucose by bacteria and
sustained maintenance of a pH below 4.5.
 Organisms that can maintain the low pH after prolonged incubation (48- 72 hrs )
overcoming the pH buffering system of the medium can be called methyl red positive.
Methyl red : pH pH
6.0 yellow 4.4 red
COMPONENTS
 METHYL RED TEST BROTH- GLUCOSE PHOSPHATE PEPTONE WATER
 Peptone : 5 g
 K2HPO4 : 5 G (dipotassium phosphate)
 Water : 5 L
 Glucose (10%) : 50 mL

12. Methyl red indicator solution :
Methyl red : 0.1 g
Ethanol : 300 ml
Distilled water : 200 ml .
Procedure
The test organism is inoculated in glucose phosphate broth and incubated at
37°C for 48-72hrs
Then add five drops of 0.04% solution of methyl red, mix well and
read the results immediately
Results
The development of a stable red color in the surface of the medium
indicates sufficient acid production to lower the pH to 4.4 and constitutes a
positive test

13. Quality Control
Positive control: Escherichia coli ATCC 25922
Negative control: Klebsiella pneumoniae ATCC70063
MR POSITIVE MR NEGATIVE
E. Coli K. Pnemoniae
K. Ozaenae Enterobacter spp
K. Rinoscleromatis
K. Ornitholytica
Salmonella species
Proteus mirabilis

14. VOGES-PROSKAUER (VP) TEST ORACETOIN PRODUCTION TEST:
 Principle
 This test depends on the production of acetyl methyl carbinol (acetoin)
from pyruvic acid in the media. In the presence of alkali and atmospheric
oxygen, acetoin is oxidised to diacetyl which reacts with a-naphthol to give
red colour
 MEDIA :- methyl red–Voges-Proskauer (MR/VP) broth
 Reagent 1 :
α-Naphthol 5 g
Absolute ethyl alcohol 100 mL

15. . Reagent 2
Potassium hydroxide 40 g
Distilled water 100 mL
Procedure
• Inoculate a tube of MR/VP broth with a pure culture of the test organism.
Incubate for 24 hours at 35°C.
• At the end of this time, aliquot 1 mL of broth to a clean test tube.
• Add 0.6 mL of 5% α naphthol, followed by 0.2 mL of 40% KOH.
• It is essential that the reagents be added in this order.
• Shake the tube gently to expose the medium to atmospheric oxygen and
allow the tube to remain undisturbed for 10–15 minutes

16. VP POSITIVE VP NEGATIVE
K. Pnemoniae E. Coli
Serratia Salmonella
Vibrio cholera Proteus mirabilis
Aeromonas sobria Yersinia
Results
A positive test is represented by the development of a red
color 15 minutes or more after addition of the reagents.
Quality Control
Positive control: Klebsiella pneumoniae ATCC70063
Negative control: Escherichia coli ATCC25922

17. Citrate Utilization Test
Principle
To identify organisms capable of using sodium citrate as the sole
carbon source & inorganic ammonium salts as the sole nitrogen
source
 Citrate sodium bicarbonate + ammonia
Bromothymol blue (yellow) bromothymol blue (Blue)
Ph-6.9 ph- 7.6
MEDIA :- Simmon’s citrate medium
 Koser’s citrate medium (liquid medium)

18.  Procedure
 A well-isolated colony is picked from the surface of a primarily isolation medium and inoculated as a
single streak on the slant surface of the citrate agar tube. The tube is incubated at 35°C for 24–48
hours
Results
A positive test is represented by the development of a deep blue color within 24–48 hours
A positive test may also be read without a blue color if there is visible colony growth along the
inoculation streak line
In koser’s citrate medium presence of turbidity is noted due to growth of bacteria.

19. Quality Control
Positive control— Klebsiella pneumoniae ATCC70063
Negative control—Escherichia coli ATCC 25922
CITRATE POSITIVE CITRATE NEGATIVE
Salmonella paratyphi E. Coli
Citrobacter Shigella
Klebsiella Hafnia
Enterobacter Yersinia
Serratia

20. UREA HYDROLYSIS TEST
 Principle
To determine the ability of organism to split urea into two molecule
of ammonia by action of enzyme urease, ammonia reactes in solution to
form ammonium carbonate with resulting alkalinity And increase in ph of
medium.
Indicator:- Phenol red Phenol red
PH-6.8 PH-8.4
MEDIA:-Stuart’s urea broth
Christensen’s urea agar

21. Procedure
 The broth medium is inoculated with a loopful of a pure culture of the test
organism
The surface of the agar slant is streaked with the test organism
and both incubated at 35°C for 18–24hours.
 Results
Organisms that hydrolyze urea rapidly may produce positive reactions by change in color
original yellow color changes to pink in alkaline medium.
.

22. Quality Control
Positive control— Klebsiella pneumoniae ATCC70063
Negative control—Escherichia coli ATCC 25922

23. TRIPLE SUGAR IRON (TSI) AGAR TEST:
 Principle
 TSI detects three properties of bacteria, which includes fermentation of sugars to produce acid
and/or gas and production of H2S
 Media:-
 TSI medium contains three sugars—glucose, sucrose and lactose in the ratio of 1:10:10 parts.
 Phenol red
 Ferrous sulphate
Procedure:-
with the help of inoculating wire which contain isolate of pure colony, stab 3-5
mm and then streak on TSI agar , and incubate at 37c for 18-24hrs.

24.  Result:- Reaction in TSI Example
Acidic slant/acidic butt  > 2 sugars fermented
2.lactose/sucrose
A/A, gas production, no H2S Escherichia coli
Klebsiella pneumoniae
Alkaline slant/acidic butt Only glucose fermenter group
K/A no gas, no H2S shigella
K/A, no gas,H2S produced(small
amount)
Salmonella Typhi
Proteus mirabilis
K/A, gas produced, H2S
produce(abundant)
Salmonella paratyphi B
K/A, gas produced, no H2S Salmonella paratyphi A
Alkaline slant/alkaline butt Non-fermenters group
K/K, no gas, no H2S Pseudomonas, Acinetobacter

25. QUALITY CONTROL :
A/A + gas production : E.coli ATCC 25922
K/A +/- gas production, H2S + : Salmonella enterica sub sp.enterica serovar
typhimurium ATCC 14028
K/K : P. aeruginosa ATCC 27853
K/A, H2S + : Proteus mirabilis ATCC 12453
K/A : Shigella flexneri ATCC 12022

26. CARBOHYDRATE (SUGAR) FERMENTATION TEST:
Principle:-
It detects the ability of an organism to ferment a specific carbohydrate (sugar)
incorporated in a medium producing acid with/without gas. Glucose, lactose,
sucrose, mannitol, maltose and xylose are widely used for sugar fermentation.
Media:- broth containing specific sugar 1% with Andrade’s indicator
Procedure:-
Test organism is inoculated in sugar medium and incubated at 37C for 18-24hr.
Interpretation:-
Positive:- pinkish red
Negative:- yellow

27. Quality controle:-
Glucose
Positive control
with gas- Escherichia coli ATCC25922
without gas- shigella flexneriATCC12022
Negative control: Alcaligenes faecalis ATCC8750
Lactose
Positive control -Escherichia coliATCC25922
Negative control – proteus vulgaris ATCC6380
Glucose and lactose fermenters:-E.coli
Klebsiella spp
• Glucose and mannitol fermenters:-Salmonella sp

29. OXIDATION-FERMENTATION TEST (OF TEST):
 PRINCIPAL
 Hugh and Leifson OF test differentiates between fermenters and non-fermenters (that utilize
sugars oxidatively).
 MEDIA AND REAGENTS
 OF medium differs from ordinary sugar fermentation medium by containing:
• Agar (0.3%)—making the medium semisolid that permits the diffusion of acids from the surface
to throughout the medium changing the color of the bromothymol blue indicator to yellow
• Increased carbohydrate concentration from 0.5% to 1%
• Decreased peptone concentration from 1% to 0.2%.

30. OF Media of Hugh and Leffson media:-
Peptone – 2g
Sodium chloride -5g
D-glucose -10g
Bromothymol blue – 0.03g
Agar -3g
Dipotassium phosphate - 0.30
Distilled water – 1L
PH- 7.1
Medium autoclaved in a flask at 121C for 15min
Carbohydrate to be added is sterilized separately and added to give a final concentration of 1%
Procedure:-
Two tubes are required for the OF test, each inoculated with the unknown organism, using a
straight needle, stabbing the medium three to four times halfway to the bottom of the tube.

31.  One tube of each pair is covered with a 1-cm layer of sterile mineral oil or melted paraffin,
leaving the other tube open to the air. Incubate both tubes at 35°C and examine daily for
several days.
RESULT
 Open Tube Covered Tubes Metabolism
Acid (yellow) Alkaline (green) Oxidative
Acid (yellow) Acid (yellow) Fermentative
Alkaline (green or blue) Alkaline (green or blue) Nonsaccharolytic

32. CONTROL E.COLI Psudomonas Alcaligenes
QUALITY CONTROL
Glucose fermenter: Escherichia coli
Glucose oxidizer: Pseudomonas aeruginosa
Nonsaccharolytic: Alkaligenes fecalis

33. NITRATE REDUCTION TEST:
 principle
 This test detects the presence of an enzyme nitrate
reductase in the organism, which reduces nitrate present in
the medium (nitrate broth) to nitrite or free nitrogen gas.
 REAGENTS
 Nitrate Broth or Nitrate Agar (Slant)
 Beef extract 3 g
 Peptone 5 g
 Potassium nitrate (KNO3 ) 1 g
 Agar (nitrite-free) 12 g
 Distilled water 1 L

34. Reagent A
α-Naphthylamine 5 g
Acetic acid (5 N), 30% 1 L
Reagent B
Sulfanilic acid 8 g
Acetic acid (5 N), 30% 1 L
Procedure:
Inoculate the nitrate medium with a loopful of the test organism isolated in pure
culture on agar medium, and incubate at 35°C for 18–24 hours
At the end of incubation, add 1 mL each of reagents A and B to the test medium, in
that order

35.  RESULT
 The development of a red color within 30 seconds after adding the test reagents
indicates the presence of nitrites and represents a positive reaction for nitrate reduction
 adding zinc dust indicates the presence of residual nitrates and
confirms a true negative reaction.
 Free nitrogen gas is detected by using a Durham’s tube
QUALITY CONTROL
A.Positive control: Escherichia coli ATCC25922
B.Negative control: Acinetobacter baumaniiATCC17978

36. DECARBOXYLASE TEST;
 PRINCIPLE
It detects the presence of substrate specific decarboxylase enzyme in the bacteria that break
down amino acids, such as lysine, arginine and ornithine to produce alkaline by-products
which change the color of the indicator to purple.
Each decarboxylase enzyme is specific for an amino acid
 Lysine, ornithine, and arginine are the three amino acids routinely tested in the
identification of the Enterobacteriaceae.
 The specific amine products are as follows:
Lysine → Cadaverine
Ornithine →Putrescin
Arginine →Citrulline

37. REAGENTS
Moller Decarboxylase Broth Base
Peptone 5 g
Beef extract 5 g
Bromcresol purple 0.01 g
Cresol red 0.005 g
Glucose 0.5 g
Pyridoxal 0.005 g
Distilled water 1 L
Final pH 6.0
Amino Acid
Add 10 g of the L (levo) -form of the amino acid (lysine,
ornithine, or arginine).
Double this amount if the D (dextro) -form is to be used,
because only the L-form is active

38. PROCEDURE
inoculate two tubes of Møller decarboxylase medium, one
containing the amino acid to be tested and the other to be
used as a control tube devoid of amino acid.
 RESULT
 Conversion of the control tube to a yellow color indicates that the organism is viable and that
the pH of the medium has been lowered sufficiently to activate the decarboxylase enzymes.
Reversion of the tube containing the amino acid to a blue-purple color indicates a positive
test owing to the formation of amines from the decarboxylation reaction
 QUALITY CONTROL
 Amino Acid Positive Control Negative Control
 Lysine Enterobacter aerogenes Enterobacter cloacae
 Ornithine Enterobacter cloacae Klebsiella pneumoniae
 Arginine Enterobacter cloacae Enterobacter aerogenes

39. PHENYLALANINE DEAMINASE TEST:
PRINCIPLE
To determine the ability of an organism to deaminate phenylalanine to phenyl
pyruvic acid (PPA) and ammonia. This test is also commonly called as PPA test.
MEDIA AND REAGENTS
Phenylalanine agar is poured as a slant into a tube
DL-Phenylalanine- 2g
Yeast extract- 3g (serves as the carbon and nitrogen)
Sodium chloride – 5g
Disodium phosphate – 1g
Agar – 12g
Distilled water- 1L
PH- 7.3

40.  Reagents- ferric chloride -12g
concentrated HCL-2.5ml
distilled water-100ml
PROCEDURE
 The agar slant of the medium is inoculated with a single colony of the
test organism isolated in pure culture of primary plating agar. After
incubation at 35°C for 18–24 hours, 4 or 5 drops of the ferric chloride
reagent are added directly to the surface of the agar
 RESULT
 The immediate appearance of an intense green color indicates the presence of
phenylpyruvic acid and is positive test

41. QUALITY CONTROL
Positive control: Proteus mirabilis ATCC12453
Negative control: Escherichia coli ATCC25922
PPA positive:- Proteus spp,
Morganella spp
Providencia spp
PPA negative :- All members of enterobacteriaceae.