BIOCHEMICHAL

REACTIONS
Coagulase test
Principle:
 This test is primarily done for the identification of S.aureus
(coagulase positive) to separate it from the coagulase
negative non-pathogenic form (S .epidermidis ).The
coagulase positive character of S.aureus is recognized by its
ability to clot citrated or oxalated human or rabbit plasma.
Two forms of coagulase could be shown by S.aureus:
● Production of a surface-associated protein known as
clumping factor or bound coagulase that reacts with
fibrinogen.
● Production of an extracellular enzyme, coagulase, which
converts plasma fibrinogen into fibrin, aided by an activator
present in plasma.
Procedure:
A)-Slide method ( for bound coagulase) :
  A small drop of normal saline was placed on slide,
then a loopful of bacterial growth was emulsified in
the saline until it appears milky. A loopful of citrated
or oxalated human plasma was then added and
mixed with the emulsion thoroughly.
Positive result: positive test is recognized within 5-10
seconds by the formation of small flocks or clumps
visible with naked eye.(e.g. Staphylococcus aureus )
Negative result : no clumps was formed (e.g.
Staphylococcus epidermidis)
Positive result
B)-Tube method ( for free coagulase):  
    This method is more sensitive than the slide method. It however,
takes longer time to perform.
1.Emulisify a single colony from a 24-hour blood agar culture of the
organism in 0.5 ml of citrated plasma (fresh plasma recommended ).

2. Incubate at 35ºC in a water bath.

3. Examine the tube by gentle inversion (do not shake). A positive result
is seen within a few hours (1 then 4 hours) as a distinct clot, if no clot
was formed, examine within 24 hour.  
Positive result: positive test is shown by a clot or coagulum formation.
                              
 Negative result : absence of clotting is a negative test.    
Catalase activity
Principle:
Bacterial cells produce hydrogen peroxide during aerobic respiration. If hydrogen
peroxide accumulates in the cell, it becomes toxic. For this reason, most aerobic and
facultatively anaerobic bacteria posses an enzyme called catalase, which is capable of
splitting hydrogen peroxide to release free oxygen. The release of oxygen gas can be
seen readily by the appearance of white froth when a few drops of H2O2  was added to
the cultured colonies.
2H2O2      catalase      2H2O   +   O2

Procedure:
   A part of colony was put on a slide then 1-2 drops of 3% H2O2 was added and
observed, immediate production of gas signifies positive test.
 
1- Do not use blood agar which contains catalase.
2- Do not use  iron loop for inoculation.
Positive result: added H2O2      catalase       H2O + O2  production of air bubbles (e.g.
 Staphylococci )
Negative result: added H2O2     no catalase    H2O2  no air bubbles was formed (e.g.
Streptococci)
Oxidase test
Principle:
     The oxidase test demonstrates the presence of oxidase which catalyzes transport of electrons between
electron donors and a redox dye (tetramethyl phenyl diamine ).
Electron donors      Oxidase       Redox dye (violet to deep purple colour)
Procedure:
  Fresh filter paper was soaked in dye then 1 to 2 colonies was placed and smeared over it. Positive result
was indicated as colonies become purple.
Positive result:
* cytochrome c(oxidized)+oxidase reagent (reduced) colorless                      cytochrome c (reduced) +
oxidase reagent (oxidized) dark purple
Example : Pseudomonas  aeruginosa

Negative result:
* cytochrome c (reduced) + oxidase reagent (reduced) colorless                  oxidase reagent (reduced)
colorless
Example: Escherichia  coli
Urease test
Principle:
        Some organisms have the ability to utilize urea as their only source of nitrogen
because they produce the urease enzyme which splits urea and liberates ammonia
and carbon dioxide. As a result of ammonia production, the urea- containing
medium becomes alkaline (pH raises) and causes the phenol red indicator to
become pinkish red.
Urea  +   H2O             Urease          2NH3   +   CO2
Procedure:
   Christensen urea slope medium with phenol red was inoculated with specified
organism and incubated at 37 ºC for 24 h.
Positive result:
  Urea (yellow-orange)       urease       NH3 (pH  ) pinkish-red + CO2
A positive reaction is seen by the change of the colour of the medium to pinkish-red
(e.g. Proteus vulgaris )
Negative result:
Urea (yellow-orange)      no urease      Urea (yellow-orange)   
A negative result show no change in slope colour (remain yellow) e.g.
E. coli
negative positive
 BIOCHEMICAL TESTS (IMViC)
Indole
Methyl Red/Voges Proskauer
Citrate
 
Triple Sugar Iron Agar (TSI) test
H2S production in SIM
Lactose fermentation
Sucrose fermentation
Glucose fermentation & gas production
Indole Test
Principle:
Indole test is performed to determine the ability of the organism to
split tryptophan molecule into Indole. Indole is one of the metabolic
degradation product of the amino acid tryptophan
Bacteria that possess the enzyme tryptophanase are capable of
hydrolyzing and deaminating tryptophan with the production of
Indole, Pyruvic acid and ammonia.
Property it tests for:
This test is performed to help differentiate species of the family
Enterobacteriaceae.

Media and Reagents Used:

Tryptone broth contains tryptophan.
Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.
•

Indole test
Procedure:
‐Inoculate Tryptone broth with the test organism and incubate for 18 to 24 hrs at 37c
‐Add 15 drops of Kovac’s reagent down the inner wall of the tube

Interpretation:
‐Development of bright 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

Indole Positive:
E.coli
Proteus vulgaris

Indole Negative:
Salmonella spp.
Klebsiella spp.
Enterobacter aerogens
Methyl Red/Voges‐Proskauer (MR/VP)
Properties these test for: Both tests are used to differentiate
species of the family Enterobacteriaceae.
Media and Reagents Used:
•Glucose Broth
•Methyl Red indicator for MR test
•Voges Proskauer reagents‐A: 5% Alpha‐Naphthol & ethanol,
B: Potassium Hydroxide; (3:1 ratio) & Deionized Water.
•Principle of MR test:
•To test the ability of the organism to produce and maintain
stable acid end products from glucose fermentation and to
overcome the buffering capacity of the system
•This is a qualitative test for acid production.
MR test
Procedure:
‐Inoculate the MR/VP broth with a pure culture of the test
organism and incubate at 35°for 48 to 72 hrs.
-Add 5 drops of MR reagent to the broth
Result interpretation:
-Positive result is red (indicating pH below 6)
-Negative result is yellow (indicating no acid production)

Left: negative/Right: positive

MR Positive: E. coli
MR Negative:
Enterobacteraerogenes
Enterobactercloacae
Klebsiella spp.
Voges Proskauer Test (acetoin production)
Principle:
To determine the ability of the organisms to produce neutral end product acetyl methyl
carbinol(acetoin) from glucose fermentation
Procedure:
Innoculatepure culture of the test organism into MR/VP broth and incubate for 24 hrs at
37°c
Aliquot 1 ml of the broth to a sterile test tube and add 0.6ml of VP(A) followed by
0.2ml of VP(B)
Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube
to remain undisturbed for 10 to 15 mins
Intrepretation:
Positive : Pinkish red color at the surface of the medium
Negative : Yellow color at the surface of the medium
VP: left + and right –
Positive: Klebseilla pneumoniae, Enterobacter,            
Negative: E.coli
Citrate Utilization test:
This test is one of several technique used to assist in the identification of enterobacteria. The test is
based on the ability of an organism to use citrate as its only sole source of carbon and ammonia as
its only source of nitrogen.
Principle:
The test organism is cultured in a medium which contains sodium citrate, an ammonium salt and the
indicator bromothymol blue. Growth in the medium is shown by turbidity and a change in colour of
the indicator from light green to blue, due to alkaline reaction following citrate utilization.
citrate (green)     citrase  oxaloacetic acid + acetic   Oxaloacetic acid  pyruvic
acid + CO2
CO2 + Na+                         Na2CO3 (pH increases   (blue and growth )
Example: Enterbacter  aerogenes

Procedure:
Inoculum is streaked over the slant of Simmon’s citrate
agar in a tube and incubated for 24 ‐48 hrs.
Result interpretation:
Growth on the slant and change in colour
to blue of the medium indicates positive result.
Left‐Positive: Klebsiella pneumoniae
Right‐Negative: E. coli
Triple Sugar Iron Agar (TSI) test
Principle:
       TSI slants are specially useful as a first step in the identification
of Enterobacteriaceae. Growth of the organism on TSI slant can indicate
the type of sugar fermented (glucose, sucrose, or lactose) and in addition
identifies the hydrogen sulphide (H 2S) producer, with acid production (by
fermenters). Sodium thiosulfate is the source of sulfure for H2S
production in this media. TSI contain ferrous sulfate, which reacts with
H2S to form a black precipitate called ferrous sulfide. The pH indicator in
both media is phenol red. Phenol red is red at the initial pH of 7.4 but
turns yellow at an acidic pH and dark red at an alkaline pH.  
Method:
1.With a straight inoculating wire, touch the top of a well isolated colony.
2.Inoculate TSI by first stabbing through center of the medium to the
bottom of the tube and then streaking the surface of the agar slant.
3.Leave the cap on loosely and incubate the tube for 18 ‐24 hours at 35oC in
an incubator.
Triple Sugar Iron Agar (TSI) test
Result interpretation:
1)-No carbohydrate fermentation or hydrogen sulfide
production (Alk/ Alk or Alk/ NC): No acids are produced
in the slant or butt to cause a drop in the pH of the medium.
The slant and butt remain red or turn dark red due to the
production of alkaline products from peptone.
2)-Glucose fermentation only(Alk/A):Glucose fermentation
results in the formation of acids that lower the pH of the
butt. The butt turns yellow while the slant remain red or
turns a dark red.
 
3)-Glucose fermentation only with hydrogen sulfide
production(Alk/ A, H2S): If glucose fermentation occurs
with hydrogen sulfide production, the butt turns black and
yellow while the slant remains red.
4)-Lactose and/ or sucrose and glucose fermentation
(A/A): Lactose and/ or sucrose and glucose fermentation
results in the formation of acids that lower the pH of the
slant and butt. The slant and butt turn yellow.
5)- Lactose and/ or sucrose and glucose fermentation
with hydrogen sulfide production (A/A, H2S): If
lactose and/ or sucrose and glucose fermentation occurs
with hydrogen sulfide production, the butt turns black and
yellow while the slant turns yellow.
Sugar fermentation test
1-Lactose Fermentation
Property it tests for: This tests for the bacteria’s ability to
ferment lactose.
Media and Reagents Used: Lactose broth contains beef
extract, gelatin peptone, and lactose. A phenol red indicator
is added to indicate acid production from fermentation.
How to Perform Test:
Inoculate lactose broth with inoculating loop.
Results:
A positive result is yellow after indicator is added
(indicating lactose fermentation)
A negative result will have no color change or will be
reddish.
2-Sucrose Fermentation
Property it tests for: This test is done to help differentiate
species of the family Enterobacteriaceae. This tests the
bacteria’s ability to ferment sucrose and production of acid
end‐product
Media and Reagents Used: Sucrose broth contains beef
extract, gelatin peptone, and sucrose. Phenol red indicator is
added to indicate an acid end‐product.
How to Perform Test: Inoculate sucrose broth with
inoculating loop.
Results:
A positive result is yellow after indicator is added
(indicating sucrose fermentation)
A negative result has no color change or is reddish.
3-Glucose Fermentation & Gas Production
Property it tests for: This test is done to help differentiate species of the
family Enterobacteriaceae. This tests for the bacteria’s ability to ferment
glucose and produce gas and/or an acid end ‐product..

Media and Reagents Used: Glucose broth contains beef extract, gelatin
peptone, and glucose. A phenol red indicator is added to indicate an acid
end‐product. A Durham tube is added to indicate gas production.

How to Perform Test: Inoculate broth with inoculating loop.

Results:
•A positive result for acid is yellow after indicator is added (indicating
glucose fermentation)
•A positive result for gas is a bubble in the Durham tube.
•A completely negative result has no color change or reddish color and no
bubble.
Sugar Fermentation Tests
Tube 1: Negative acid /Negative gas
Tube 2A: Must incubate longer (ambiguous result)
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas