This document provides an overview of biochemical methods used for bacterial identification. It discusses why bacterial identification is important, the typical identification scheme involving isolation, staining, culturing, and biochemical/molecular tests. Several common biochemical tests are described in detail, including their principles, media used, and how to interpret results. These tests analyze bacterial metabolism of carbohydrates, proteins, lipids and other compounds. Automated identification systems that can rapidly identify bacteria based on biochemical profiles are also mentioned.

1. SAKEENA ASMI T
MAHATMA GANDHI UNIVERSITY
BIOCHEMICAL METHODS
FOR IDENTIFICATION OF
BACTERIA

2. • To distinguish harmless microbes from
pathogenic microbes.
• Characterize an outbreak of disease and
determine the source.
• Verify the authenticity of pathogenic strain
for quality control purposes.
• Determine appropriate antimicrobial therapy.
• Basically for the prevention, control and
treatment of a disease.
WHY IS IDENTIFICATION IMPORTANT?

3. IDENTIFICATION SCHEME
SAMPLE
(Soil, water,
food, etc…)
ISOLATION
(Spread plate, pour
plate, streak plate)
STAINING
MOTILITY
(Hanging drop method)
CULTURING
(Special media)
BIOCHEMICAL
REACTIONS
MOLECULAR
TECHNIQUES

4.  Biochemical tests are the tests used for
identification of bacteria species based on the
differences in the biochemical activities of
different bacteria.
 Differences in carbohydrate metabolism,
protein metabolism, fat metabolism, production
of certain enzymes, ability to utilize a particular
compound etc.
Each species of bacteria have a well defined set
of metabolic activities different from all other
species
These biochemical fingerprints are properties
controlled by the bacterial enzymes.
WHAT ARE BIOCHEMICAL TESTS?

5. 1. Indole test
2. Methyl red test
3. Voges-Proskauer test
4. Citrate utilisation test
5. Carbohydrate fermentation test
6. Triple sugar iron(TSI)test
7. Nitrate reduction test
8. Urease test
9. Oxidase test
10. Catalase test
11. Oxidation-fermentation(O/F)test
12. O-Nitrophenyl galactosidase(ONPG)test
IMViC test
Intracellular
Enzyme
Activity

6. 1. Coagulase test
2. Gelatinase test
3. Starch hydrolysis test
4. Lipid hydrolysis test
5. Deoxyribonuclease test(DNase
test)
Extracellular
Enzyme
Activity

7. INDOLE TEST
To determine the ability of microbe to degrade the amino acid tryptophan.
MEDIA : Tryptophan 1% or peptone broth
REAGENT : Kovacs’ reagent (Dimethylamine benzaldehyde)
PRINCIPLE:
Tryptophanase
Tryptophan Indole + pyruvic acid + ammonia
Indole + Kovacs reagent Rosindole + H2O
(Cherry red colour
compound)
Hcl
Butanol
POSITIVE : Cherry red coloured ring at the
interface of reagent and broth.
Eg: E.coli
NEGATIVE : No colour change is observed.
Eg: Klebsiella

8. SPOT INDOLE
To perform this test, a test colony is smeared in a
piece of filter paper saturated with Kovac’s
reagent. The appearance of red colour indicates a
positive test.

9. METHYL RED TEST (MR TEST)
To determine the ability of microbes to oxidize glucose with production
and stabilization of high content of acid end products.
MEDIA : MR broth – peptone, glucose, dipotassium phosphate &
distilled water.
REAGENT : MR reagent – MR 0.1g in 300ml of 95% ethanol & DW 200ml.
PRINCIPLE:
Glucose Mixed acids (pH less than 4.4) + Methyl red
Red colour
POSITIVE : Red colour is observed. Eg: E.coli
NEGATIVE : Yellow colour is observed.
Eg: Klebsiella

10. VOGES-PROSKAUER TEST (VP TEST)
To determine the ability of microbes to produce non acidic or neutral end
products.
MEDIA : VP broth ( same as MR broth)
REAGENT : Barrits A – Alpha naphthol 5%
Barrits B – 40% Potassium hydroxide
PRINCIPLE :
Glucose Pyruvate Acetoin 2, 3-butanediol
Acetoin + α-napthol (0.6ml) Diacetyl
(Pink coloured complex)
40% KOH (0.2ml)
POSITIVE : Pink colour is observed.
Eg: Klebsiella
NEGATIVE : No colour change is observed.
Eg: E.coli

11. CITRATE UTILISATION TEST
To determine the ability of the microbes to ferment citrate as sole carbon
source.
MEDIA : Simmons citrate medium
INDICATOR : Bromothymol blue
PRINCIPLE:
Sodium citrate Pyruvic acid + Oxaloacetic acid+ CO2
Excess sodium from + CO2 + H2O Na2CO3 (pH ) (green blue)
sodium citrate
Citrate permease
POSITIVE : Change of colour from green to blue
Eg: Klebsiella
NEGATIVE : No colour change is observed.
Eg: E.coli

12. CARBOHYDRATE FERMENTATION TEST
To determine the ability of microbes to ferment specific carbohydrates with the
production of acid and/or gas.
MEDIA : Nutrient broth with sugars (Glucose, lactose, sucrose, maltose)
INDICATOR : Phenol red or Bromocresol purple to detect acid & Durham's tube to
detect gas.
PRINCIPLE :
Carbohydrate Organic acids + CO2 + H2
 Acid lowers the pH which can be detected by pH indicators.
 Gas production (CO2) can be detected in Durham’s tube.
POSITIVE : Acid only – colour change to yellow (A)
Eg: S.aureus
Acid & Gas – colour change to yellow
& bubble in durham's tube(A/G)
Eg: E.coli, Klebsiella
NEGATIVE : No colour change is observed.
Eg: Pseudomonas
fermentation

13. TRIPLE SUGAR IRON TEST (TSI TEST)
To differentiate among and between the members of Enterobacteraceae and
screen for enteric pathogens based on carbohydrate fermentation and H2S
production.
MEDIA : TSI agar - glucose 0.1%, lactose & sucrose 1% concentration, protein
source(peptone), NaCl, Sodium thiosulfate, Ferric ammonium citrate.
INDICATOR : Phenol red (pH indicator)
PRINCIPLE :
Carbohydrates Acid + CO2
Peptones NH3 (makes medium alkaline)
Phenol red
 Acid lowers the pH and ammonia increases the pH which can be detected by
pH indicators.
 Gas production (CO2) can be detected by cracks in the medium.
Yellow
Red
acid
alkali

14. Bacteria + Sodium thiosulfate H2S gas
H2S gas + Fe3+ FeS (black precipitate)
 H2S can be detected by black precipitate formation in the medium.
acid environment

15. Result interpretation:
• Red slant, red butt, no gas, no H2S - K/K no H2S
• Red slant, yellow butt, no gas, no H2S - K/A no H2S
• Yellow slant, yellow butt, gas, no H2s - A/ A no H2S
• Yellow slant, yellow butt, gas, H2s - A/ A H2S
• Red slant, yellow butt, gas, H2S - K/ A H2S
 Alkaline slant /acidic butt only glucose is fermented
 Acidic slant / acidic butt glucose, sucrose, lactose all 3 sugars
are fermented
 Bubbles or cracks present gas production
 Black precipitate present H2S production

16. NITRATE REDUCTION TEST
To determine the ability of some microbes to reduce nitrate(NO3
- ) to nitrites(NO2
-)
or beyond the nitrite stage.
MEDIA : Nitrate broth – Potassium nitrate
REAGENTS : Alpha-naphthylamine (Reagent A) & Sulfanilic acid (Reagent B)
PRINCIPLE :
NO3
- NO2
- N2
 If nitrite(NO2
-) is formed, then
NO2
-
Nitrate reductase Other enzymes
+ reagent A + reagent B Sulfobenzene azo-alpha
(colorless) naphthylamine
Eg: E.coli, S. aureus (red coloured)
 If nitrogen gas(N2) is formed Colourless reaction
 If the organism is non reducer Colourless reaction
To differentiate these two cases, add zinc powder.
Zinc powder can reduce nitrate to nitrites.

17. • If organism reduced nitrate to nitrogen gas, then there are no nitrates present
and the addition of zinc dust will have no effect. Hence the test is positive and
gives colourless reaction the organism is a REDUCER
• If nitrates were not reduced then zinc will reduce them to nitrites. Nitrites will
react with 2 reagents and give a red colour. Test is negative and organism is NON
REDUCER. Eg: N.gonorrhoeae
Nitrate broth
Zinc powder
Nitrogen gas
/non reducer
Nitrogen gas
Reducer
Non reducer

18. UREASE TEST
To determine the ability of microbes to degrade urea by urease.
MEDIA : Christensen’s Urea agar
REAGENTS : Phenol red
PRINCIPLE :
Urea + 2 H2O CO2 + H2O + 2 NH3
Ammonia increases the pH which can be detected by pH indicator
Urease
POSITIVE : Pink colour is observed
Eg: Proteus
NEGATIVE : No colour change
Eg: E.coli

19. OXIDASE TEST
To determine the ability of microbes to produce oxidase enzyme.
REAGENT : 1% Kovacs reagent (tetra methyl para phenylenediamine dihydrochloride)
PRINCIPLE :
• Cytochrome C oxidase is an enzyme that facilitates transfer of electrons to
oxygen in aerobic bacterial respiratory transport system.
• Here the oxidase reagent substitutes as the electron acceptor.
Kovacs reagent Indophenol
(colourless oxidase reagent) (purple colour)
Cytochrome oxidase
POSITIVE : Purple colour is observed
Eg: Pseudomonas
NEGATIVE : No colour change
Eg: E.coli, S. aureus

20. CATALASE TEST
To determine the ability of microbes to produce catalase enzyme
REAGENT : Hydrogen peroxide
PRINCIPLE:
• During respiration many microbes produce byproducts that are toxic to cells
like Hydrogen peroxide(H2O2)
• To neutralize this toxin microbes produce an enzyme CATALASE
2 H2O2 2 H2O + 02 (gas bubbles)
POSITIVE : Presence of effervescence
Eg: E.coli, S.aureus etc
NEGATIVE : No effervescence
Eg: Streptococcus
Catalase

21. OXIDATIVE – FERMENTATIVE TEST (OF TEST)
To differentiate between oxidative and fermentative bacteria
MEDIA : Hugh Leifson’s OF media (high glucose & low peptone)
INDICATOR : Bromothymol blue
PRINCIPLE :
• Under anaerobic condition(overlay with a layer of oil), fermentative organisms
covert glucose mixed acids.
• Under aerobic condition, non-fermenting(oxidative) organisms produce small
amount of weak acids.
• The decrease amount of peptones and increase amount of glucose facilitates
detection of weak acids.
• Acid decreases the pH which can be detected by pH indicator which changes
the colour from green to yellow.
Examples: Oxidative – Pseudomonas aeruginosa
Fermentative - E.coli
Non saccharolytic - Alcaligens faecalis

22. Open
(Aerobic tube)
Covered
(Anaerobic tube)
Metabolism
Acid
(Yellow)
Alkaline
(Green)
Oxidative
Acid
(Yellow)
Acid
(Yellow)
Fermentative
Alkaline
(Green)
Alkaline
(Green)
Non saccharolytic
A
B
C

23. ONPG TEST FOR β-GALACTOSIDASE
To determine the presence or absence of the enzyme β-galactosidase
MEDIA : ONPG broth
PRINCIPLE :
• β – galactosidase is an enzyme that converts
Lactose Galactose + Glucose
• O – Nitro – β – D – galactopyranoside is structurally similar to lactose except that
orthonitro phenol has been substituted for glucose.
Onpg(colourless) Galactose + orthonitro phenol(yellow)
galactosidase
galactosidase
POSITIVE : Yellow colour observed
Eg: E.coli
NEGATIVE : No colour change
Eg: Proteus vulgaris

24. COAGULASE TEST
This test is used to identify Staphylococcus aureus (positive) from Coagulase
negative S.aureus and other microbes.
• Coagulase is an enzyme that convert fibrinogen in plasma fibrin
S.aureus
SLIDE TEST
• Heavy suspension of organism made on glass slide and mixed with a drop of
plasma.
COAGULASE POSITIVE : Macroscopic clumping in 10 seconds or less in coagulated
plasma drop and no clumping in saline.
Bound coagulase (SLIDE TEST)
Free coagulase (TUBE TEST)

25. COAGULASE NEGATIVE : No clumping in either drop.
• Negative results should be confirmed with tube coagulase test.
TUBE TEST
• A suspension of organism is suspended and incubated with plasma at 370C
COAGULASE POSITIVE : Clot of any size is observed.
Eg: S.aureus
COAGULASE NEGATIVE : No clot (plasma remains wholly liquid or shows only a
flocculant or ropy precipitate)
Eg: S.epidermidis, E.coli

26. GELATINASE TEST
To determine the ability of an enzyme to produce the enzyme gelatinase that
hydrolyze gelatin
MEDIA : Gelatin medium – gelatin, peptone, beef extract
PRINCIPLE :
Gelatin Polypeptides Amino acids
• If the organism produce gelatinase, then it will liquefy the medium.
gelatinase gelatinase
POSITIVE : Partial or complete
liquefaction
Eg: Aeromonas hydrophila
NEGATIVE : No liquefaction
Eg: E.coli

27. STARCH HYDROLYSIS TEST
To determine the ability of an organism to produce the enzyme amylase and
hydrolyze starch.
MEDIA : Starch agar – nutrient agar + starch
INDICATOR : Iodine solution (Grams iodine)
PRINCIPLE :
 The starch molecule consists of two constituents:
Amylose, an unbranched glucose polymer
Amylopectin, a large branched polymer
 Both amylose and amylopectin are rapidly hydrolyzed by certain bacteria using
enzymes called α-amylases.
Starch Dextrin + Maltose + Glucose
(Amylose + Amylopectin)
α - amylase

28. POSITIVE : If the organism has produced amylase, will hydrolyze starch and a
clear area appears after adding grams iodine.
Eg: Bacillus subtilis, B.megaterium
NEGATIVE: Absence of clearing after adding grams iodine, no starch hydrolysis.
Eg: Staphylococcus epidermidis, S.agalactiae

29. API STRIPS – RAPID TESTS
(Analytical Profile Index)
• Commercial miniaturized biochemical test panels – cover a significant number
of clinically important groups of bacteria as well as food and water associated
microorganisms.
• Different test panels are prepared in dehydrated forms which are reconstituted
upon use by action of bacterial suspensions.
• After incubation, positive test results are scored as a 7 digit number (profile).
• Identity of the bacterium is then easily derived from the database with the
relevant cumulative profile code book or software.

30. MOST USED API SYSTEM

31. AUTOMATED IDENTIFICATION SYSTEM
• Offer greater accuracy and increased speed identification over manual
methods.
• MicroScan Walkaway & Vitek 2 – two very popular automated identification
system.
• Both have used convectional panels with instruments that can read and
interpret panel results, perform antibiotic susceptibility tests and print results
without human intervention.
VITEK - 2

32. MICROSCAN WALKAWAY

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THANK