Biochemical
Tests
Uses: use in identification of
microorganisms.
Classification:
1. Test for metabolism of carbohydrates:
sugars fermentation test and O/F test.
2. Test for specific break down of
product: MR & VP.
3. Test to show ability to utilize a specific
substrate: citrate utilization test.
4. Test for metabolism of proteins and
amino acids: H2S production test
indole production test
5. Test for enzymes: catalase, oxidase &
1. Oxidase test
Aim: This test assist in the identification of
gram negative rods also to differentiate
family of enterobacteriaceae oxidase –ve
from pseudomonasceae and vibrionceae
oxidase +ve also to detect ability of
bacteria to produce oxidas enzyme
Principle: The enzyme oxidase (cytochrom
oxidas oxidize phenylenediamine found
in oxidas reagent to give deep purple
colour.
Requirement: * oxidase reagent
(Tetramethyl-p-phenylenediamine
dihydrochloride). Filter paper .disc or
strip, petri dish , wooden stick, over night
Method: filter paper or plate and
strip method :-
Place apiece of filter paper in a
clean petri-dish and add 2 – 3
drops of freshly prepared oxidase
reagent.
Using a piece of wooden stick or
glass (not an oxidized wire loop)
remove a colony of the test
organism and smear it on the
filter paper.
No change within 1o second
……….. -ve test.
Positive result: vibrio, Pseudomonas
& Neisseria
Negative result………Salmonella
& Shigella ( all enterobacteriaceae
oxidase negative )
* Acidity inhibit oxidase result …….?
2. Kligler Iron Agar (KIA):
Aim: This test assist in the identification of gram negative
rods. To differentiate between LF and non LF
To differentiate between H2S production and non H2S
production :- Principle: based on fermentation of glucose
and lactose, Gas production, and hydrogen sulfide is
production.
Fermentation of sugar(s) change a pH , so indicator
(phenol red) will change the colour of the media from red
to yellow. The presence of a black color indicates that H2S
was produced. H2S reacts with the ferrous sulfate in the
media to make ferrous sulfide, which is black color .
Requirement: * KIA.(kligler iron adar )
Or triple sugar iron (TSI)
Component of KIA:- source of CHO glucose
1gm and lactose 10 gm
Source of aminoacid (peptone, yeast and
beef extract ,cystine
Source of sulpher ( sodium thio sulphate
Indicator (phenol red ) H2S indicator ferric
citrate and frrrus sulphate
Method:
Inoculate, using a straight wire loop
to stab agar butt, close to the
opening and then streak the top
slope (zigzag).
Incubate the medium at 37oC for 24
hrs.
Look for color change on the
Results:
Gas H2S Lactose Glucose
production production fermentatio fermentatio
n n
Cracks and Black tube Yellow Yellow butt
bubbles end slope
slope Butt or stab
3. Indole production test:
Aim: This test assist in the identification of
gram negative rods).
and differentiate between
enterobactriaceaea
to differentiate between two genus ,tow
spp and two serotype
also to detect ability of bacteria to break
down of tryptophan by tryptophanase to
indole
Principle: The organism break down the
treptophan and produce pyrovic acid,
Requirments: * Peptone water(broth
C M) or semi sold culture media )
e.g _MIU ,MIO,MIL,SIM
* kovac,s reagent
(paradimethyl aminobenzaldehyde
_ 0r paradimethyl
aminocinnmaldehyde in case of
indol spot method
Method:
Using sterile loop inoculate the
tested organism into 2 ml
peptone water or tryptone
water.
Incubate the tube at 37oC for 24 hrs.
Add 0.5 ml of kovac,s reagent , shake gently and
examine for red colour ring within 10 mins.
Results:
Red surface layer ………….+ve test.
No red surface layer ……..…-ve test.
Positive result : …………..… E. coli. Proteus vulgais
.shigella dysentry-2 .vibrio
Negative result………………Klebsiela .P.
mirabilis .pseudomomas and shigella dysentry -1
i
MIU method and indol spot
Using sterile loop inoculate the
tested organism into media dy
stabbing
Then insert strip which
impregnated with kovac reagent
Incubate at 37 c observe the
resultes
Indol spot method
In filter paper impregnated with
paradimethyl aminocinnmalehyde
by sterile loop or wooden stick
4. Citrate utilization test
Aim: This test assist in the identification of gram
negative rods
to determine the ability of bacteria to utilize Na citrate
as only source of carbon an ammonum phosphate as
only source of nitrogen
Principle: It is depend on the ability of the organism to
use citrate as its only source of carbon and ammonia
as its only source of nitrogen. If it can not use citrate
and ammonia then it will not grow. If it can use
citrate and ammonia, then the bacteria will grow and
the media will turn a bright blue as a result of an
increase in the pH of the media.
Requirements: * Koser’s Citrate broth
* or Simmo’n citrate agar
Bromothymole blue as inicator
hrs.
Look for color change and/or turbidity
on the medium.
Results:
Blue color and/or turbidity in the medium…
+ve test.
No change in the
medium……………………... -ve test.
Positive result :Klebsiela .pseudomonas.
proteus .citrobacter,enterobacter.serria
spp
Negative result..………E.coli. S.
typhi .and S.P.A
5. Urease production test
Aim: This test assist in the identification of gram
negative rods to determine the ability of
bacteria to break down of urea by broduction
of ureas enzyme
Principle: The enzyme urease hydrolyzes urea,
producing ammonia (alkaline pH), which
changes the colour of phenol red indicator to
red-pink colour.
Requirement: * Christensen's urea medium. Or
urea agar
Method:
Using sterile straight wire inoculates the test
organism into slope surface of Christensen's
urea medium.
Incubate the medium at 37oC for 24 hrs.
Results:
Pink color on the medium………+ve test.
No pink color on the medium…… -ve test.
Positive result : ………………… Klebsiela.
Yersinia enteroclotica . proteus spp . (2-4 hrs)
H. pylori (30-60 second ) and brucella 1l2 hrs
Negative result……………………E.coli.
S.typhi. Shigella spp
6.7 MR VP(Methyl Red-Vogues
Proskauer)test:
Aim: This test assist in the identification of gram – ve
rods).
To identify bacteria which produce stable acid by
mechanism of mixed acid fermentation pathway
(MR)
To identify bacteria which produce neutral end product
(acetoin ) by mechanism of acetion pathway (VP)
Principle: The MR test is used to determine if glucose
can be converted to acidic products (lactatic acid ,
Requirments: *2 tubes glucose 6 phosphate
peptone water or buffered glucose peptone
water or M.R-V.P CM
* Methyl red indicator.
* 40% KOH. (for VP)
* 5% alpha naphthol(VP TEST)
• Method:
• Using sterile loop inoculate the tested
organism into 2 ml glucose 6 phosphate
peptone water.
• Incubate at 37oC for 24 hrs.
A. MR test:
• Add methyl red.
• Look for colour change.
B. VP test:
• add alpha-naphthol
• add 40% KOH
• look for colour development within 15
minutes.
Results:
A. MR test:
Red colour………………………………
+ve test.
Yellow colour ……………………..….. -
ve test.
B. VP test
Red colour within 15 minutes ……
+ve test.
1 MR (-ve)
2 MR
(+ve)
Notes
Generally enterobacteriaceae are M
R +ve and VP –ve except
Proteus mirabilis ,hafnia and proteus
myxofaciens are MR +ve and VP +ve
In case of pseudomonas aeroginosa
MR –ve and VP –ve
Motility test -8
Type of motility
1- active ,progressive or true motility
The movement of bacteria in many
direction
2- pasive or brownian motility
The move ment of bacteria in one
direction
Method to detect the
motility
A- wet preparation
Wet preparation method
In clean dry slide place drop of
normal salin
By sterile loop take small abortion
from organism under testing and
mix well
Place the cover glass and see
under microscope
Record your report
Hanging
Technique drop technique
Place drop of liquid bacterial in center of
cover class
Place of water at each conner of cover glas
Invert modified slide over the cover glass
The cover glass will stick to the slide and th
slied inverted again the drop of bacteria
suspended in the well after that see under
microscope
Result
Positive result zigzag or tumbling movemen
Negative result no movement or vibration o
Motility test semi sold CM (Non-Biochemical
Tests):
Aim: This test assist in the identification of gram negative
rods.
Principle: This test use to check the ability of bacteria to
migrate away from a line of inoculation via flagella.
Requirement: * semi solid media (motility media ) (MIU,
MIO, MIL, SIM), loop and over night culture
Method:
1. Inoculate into motility media using straight wire loop,
stab the media in as straight a line as
And withdraw the loop very carefully to
avoid destroying the straight line.
2. Incubate at 37oC for 24 hrs.
Results:
Migration away from the original line of
inoculation motile organism.
Lack of migration away from the line of
inoculation non motile organism.
mot Non
ile mot
ile
Aim: This test assist in the identification of
gram negative rods.
Principle: Bacteria produce acidic products
when they ferment certain carbohydrates,
this lower the pH, and change indicator
colour. If there is no fermentation the
media remains without change. If gas is
produce as a by product of fermentation,
then the Durham tube will have a bubble in
Requirement: * (Set of sugar )
Method:
• Using sterile loop inoculate the tested
organism in the media.
• Incubate at 37oC for 24 hrs.
Results:
Look for colour change*
(fermentation)
Look for bubble in the Durham tube
10- Oxidation Fermentation test (OF test)
Aim: This test assist in the identification of gram negative
rods. To differentiate between those bacteria that
oxidize carbohydrates from bacteria that ferment
carbohydrates
Principle: Oxidation: (utilization of carbohydrate in the
presence of oxygen). Fermentation (utilization of
carbohydrate in the presence or absence of oxygen.
Carbohydrates utilizaton produce acid, this lower the
pH, and change indicator colour.
Requirement: * 2 tubes and oxidation fermentation
media which include :- carbohydrate ,pepton ,sodium
Method:
Inoculate, using a straight wire loop to
stab agar butt, close one tube by sterile
wax.
Incubate the medium at 37oC for 24 hrs.
Look for color change on the medium.
Results:
Example Opened Closed Reaction
Tube Tube
Pseudomonas Yellow Green Oxidation
sp.
E. coli Yellow Yellow Fermentati
on
Alkaligenes Blue Green No sugar
feacalis utilization
Green Yellow Technical
error
11. Analytical Profile Index (API )
IMViC Reactions
I: Indole; M: Methyl red; Vi: Voges-Proskauer; C:
Citrate
I M Vi C
Escherichia coli + + – –
Edwardsiella tarda + + – –
Proteus vulgaris + + – –
Klebsiella pneumoniae – – + +
Klebsiella oxytoca + – + +
Enterobacter spp. – – + +
Serratia marcescens – – + +
Citrobacter freundii – + – +
Citrobacter koseri + + – +
IViC Reactions
I: Indole; P: ; Vi: Voges-Proskauer; C: Citrate
I P Vi C
Eschericia + – – –
Shigella +/– – – –
Yersinia +/– – – –
Edwardsiella + – – –
Salmonella – – – +
Citrobacter – – – +
Klebsiella +/– – + +
Enterobacter – – + +
Serratia – – + +
Proteus +/– + +/– +
Morganella + + – +
Providencia – + – +
