The document discusses various biochemical tests used to identify bacteria based on their reactions. It describes tests like catalase, oxidase, indole, citrate utilization, urea hydrolysis, and triple sugar iron that are used to differentiate bacteria based on their ability to break down substrates or produce certain enzymes. These tests help identify both gram-positive and gram-negative bacteria at the genus or species level based on whether their reactions are positive or negative in the various biochemical tests.

1. Biochemical Reaction
Dr Avinanda Biswas
Tutor of Microbiology

2. Biochemical Identification
Based on the type of colony morphology and Gram staining appearance observed in culture smear , the appropriate
biochemical tests are employed.
 Initially, catalase and oxidase tests are done on all types of colonies grown on media .
 For gram-negative bacilli: The following are the common biochemical tests done routinely , abbreviated as ‘ICUT’ :
 Indole test
 Citrate utilization test
 Urea hydrolysis test
 Triple sugar iron test
 For gram-positive cocci :The useful biochemical tests are as follows :
 Coagulase test ( for Staphylococcal aureus )
 CAMP test ( Christine – Atkins – Munch – Peterson) for group B Streptococcus .
 Bile esculin hydrolysis test (for Enterococcus)
 Inulin fermentation test and bile solubility test ( for pneumococcus)
 Antimicrobial susceptibility tests are done for bacterial identification are as follows :
 Optochin susceptibility test – done to differentiate pneumococcus (sensitive) from viridans streptococci.
 Bacitracin susceptibility test – done to differentiate group A ( sensitive) from group B (resistant) Streptococcus .

3. CatalaseTest
When a colony of any catalase producing bacteria is mixed with a drop of
hydrogen peroxide (3 % H2O2) placed on a slide , effervescence or bubble
appear due to breakdown of H2O2 by catalase to produce oxygen .
Catalase test is primarily used to differentiate between staphylococcus
(catalase positive) from Staphylococcus ( catalase negative) .
It is also positive for members of the families Enterobacteriaceae ,
Vibrionaceae , Pseudomonas etc .

5. OxidaseTest
It detects the presence of cytochrome oxidase enzyme in bacteria , which catalyzes
the oxidation of reduced cytochrome by atmospheric oxygen .
When a filter paper strip or disk , soaked in oxidase reagent is smeared with a
bacterial colony producing cytochrome oxidase enzyme , the smeared are turns
deep purple within 10 seconds due to oxidation of the dye to form a purple –
colored compound indophenol blue .
Examples :
 Oxidase positive (deep purple) : Pseudomonas ,Vibrio , Neisseria , Bacillus ,
Haemophillus etc .
 Oxidase negative ( no color change ) : members of family Enterobacteriaceae ,
Acinetobacter etc .

6. Oxidase test

7. IndoleTest
 Inoculate the test organism into tryptophane broth
 Incubate at 37°C for 24 hours
 After incubation interval, add 1 ml Kovacs reagent which contain 4 (p) –
dimethylamino
• benzaldehyde, shake the tube gently and read immediately
Method

8. Contd.
Result:
 A bright pink color in the top layer indicates the presence of indole
 The absence of color means that indole was not produced i.e. indole is negative .
Significance:
Indole Positive : A red-colored ring is formed near the surface of the broth . Example :
Escherichia coli , Proteus vulgaris , Vibrio cholerae etc .
Indole Negative : Yellow – colored ring is formed near the surface of the broth , e.g.,
Pseudomonas , Salmonella etc .

9. Positive for
Escherichia coli
Negative for
Klebsiella pneumoniae
IndoleTest

10. Citrate UtilizationTest
It detects the ability of a few bacteria to utilize citrate as the sole source of
carbon for their growth , with production of alkaline metabolic products .Test
is performed on Simmon’s citrate medium. Citrate utilizing bacteria produce
growth and a color change , i.e., original green color changes to blue .
Citrate test is positive for Klebsiella , Pneumonia , Citrobacter , Enterobacter
etc .
The test is negative for Escherichia coli , Shigella etc .

11. Principle:
Citrate Na2CO3
Alkaline,↑pH
Bromothymolblue
CO2 + Na + H2O
Pyruvate
Simmone’s Citrate media
ContainsCitrate as a sole of C source
Positivetest
CITRATETEST

12. Positive Negative
Citrate UtilizationTest

13. Urea HydrolysisTest
Urease producing bacteria can split urea present in the medium to produce
ammonia that makes the medium alkaline .
Test is done on Christensen’s urea medium , which contains phenol red indicator
that changes to pink color alkaline medium
Urease test is positive for : Klebsiella pneumoniae, Proteus species , helicobacter
pylori etc.
Urea test is negative for : Escherichae coli , Shigella , Salmonella etc .

14. Urea HydrolysisTest
Negative Positive

15. Triple Sugar Iron (TSI)Test
• TSI contains
– Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched
Peptone provide the nitrogen, carbon, and vitamins required for organism growth.
– Three different types of sugars
• Glucose (1 part)
• Lactose (10 part)
• Sucrose (10 part)
– Phenol red (acidic: Yellow)
• TSI dispensed in tubes with equal butt & slant

16. Contd.
• Principle
• – To determine the ability of an organism to attack a specific carbohydrate
incorporated into a basal growth medium, with or without the production of
gas, along with the determination of possible hydrogen sulphide production.
• When the carbohydrates are fermented, acid production is detected by the Phenol
Red pH indicator.
• SodiumThiosulfate is reduced to hydrogen sulfide, and hydrogen sulfide reacts with
an iron salt yielding the typical black iron sulfide.
• Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator. Sodium Chloride
maintains the osmotic balance of the medium. Agar is the solidifying agent

17. Contd.
• Method:
– Inoculate TSI medium with an organism by inoculating
needle by stabbing the butt and streaking the slant
– Incubate at 37°C for 24 hours

18. Reaction on TSI
Result
Example
Butt
colo
r
Slan
t
colo
r
H2S
Red Red Negative
Alk/Alk/-
(Noaction on sugars)
Nonfermenter
e.g.
Pseudomonas
Yellow Red
Negative A/Alk/-
(Glucose
fermented
withoutH2S)
LNF
e.g. Shigella
Yellow Red
Positiv
e black
in butt
A/Alk/+
(Glucose
fermented
withH2S)
LNF
e.g. Salmonella &
Proteus
Yellow Yellow
Negative
A/A/-
(threesugars
are
fermented)
LF
e.g. E. coli,
Klebsiella,
Enterobacter
Result

19. Triple Sugar IronTest