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IDENTIFICATION OF BACTERIA
BIOCHEMICAL REACTION
• The most widely used tests in microbiology
laboratories.
BIOCHEMICAL TESTS
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Sugar fermentation
Iodole production
Methyl red test
Voges-Proskauer test
Citrate utilization
Urease test
Catalase production
Oxidase reaction
Triple sugar iron medium
OTHER TESTS
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Litmus milk
Nitrate reduction
Production of ammonia
Hydrogen sulphide production
Methylene blue reduction
Egg yolk reaction
Growth in presence of KCN
SUGAR FERMENTATION
• Tested in sugar media
• Acid production is shown by change in color of
the medium to pink/red
• Gas produced get collected in Durham’s tube
SUGAR FERMENTATION
UREASE
Urea-is a diamide of carbonic acid
All amides are easily hydrolysed with the release of
ammonia and CO2 – by the organisms that can
hydrolyse urea.
The amonia reacts in solution to form ammonium
carbonate,resulting in alkalinization of the medium
Step 1:Urea+water→(in the presence of urease)
ammonia + carbondioxide
Step 2: phenopthaline(colourless PH<8.1) →in
alkaline media due to ammonia become pink
red(PH >8.1)
Media and reagents
• Stuart’s urea broth:
– Yeast extract
– Monopotassium phosphphate
– Disodium phosphate
– Urea
– Phenol red
– Distilled water
– PH-6.8
Media and reagents
• Christensen’s urea agar:
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Peptone
Glucose
Sodium chloride
Monopotassium phosphate
Urea
Phenol red
Agar
Distilled water
PH – 6.8
• POSITIVE CONTROL: proteus species, klebsiella
• Negative control: escherichia coli
• 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
inoculated at 35deg C for 18 to 24 hrs
• RESULT : organism that hydrolyse urea rapidly may
produce pisitive reaction within 1-2 hrs
• Less active species may take 3 or more days.
• In stuarts broth:red colour through out the media
indicates alkalinazitation and urea hydrolysis
• In christensen’s media:
– 1. rapid urea spliters- (proteus species) produce red
colour throughout the media.
– 2. slow urea splitters(klebsiella species) red colour
initially in slant only ,gradually converting the entire
tube.
– 3. no urea hydrolysis – media remain original yello
colour.
UREASE TEST
EXAMPLES
• POSITIVE:Proteus
Klebsiella
H.pylori
• NEGATIVE: E.Coli
Salmonella
COMPOSITE MEDIA(TSI)
• COMPOSITE MEDIA-different properties of
the bacterium can be studied and interpreted
using a single medium.
• TSI(Triple sugar iron agar):
• a)tests whether the bacterium utilizes
glucose only , or lactose and sucrose also
either by oxidative or fermentative method.
• b)gas formation present or not
• C)H2S production present or not.
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Triple sugar iron agar
Medium
Beef extract – 3g
yeast extract – 3g
peptone 20g
glucose 1g
lactose 10g
sucrose 10g
ferric chloride ,Nacl – 5g
sodium thiosulphate 0.3g
agar- 12g
phenol red, 0.2 %solution 12ml
distilled water -1 liter
heat to dissolve the solids add the indicator solution mix and tube
sterile at 121 degree Celsius for 15 min and cool to form slop with
deep 3 cm butts.
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TSI – triple sugar iron agar:
Kligler iron agar:
Standard test for testing H2S production
It is a multi-test medium
Disadvantage chemical interaction, acid
production from fermentable sucrose may
inhibit blackening of the iron indicator.
• Method :
• Streak a heavy inoculum over the surface of
the slope and stab into the butt, incubate
aerobically at 37 degree Celsius for 24 hrs.
• TSI MEDIA-Consist of a butt and slant
• INTERPRETATION:
• After inoculation,if slant remains red and butt
becomes yellow it indicates that all the three
sugars are fermented.(eg)E.coli
• Bubbles in the butt indicates gas
production.(eg)Klebsiella spp
• Blackening of the medium indicates H2S
production.(eg)Proteus spp
TSI MEDIA
Oxidative fermentative (OF)
test: Principle, procedure and results
• The oxidative-fermentative (OF) test was
developed by Hugh and Leifson in 1953. They
developed OF media to differentiate between
oxidative bacteria (that produces acid from
carbohydrates under aerobic condition only)
and fermentative bacteria (that produces acid
both under aerobic and anaerobic conditions).
• Saccharolytic microorganisms degrade glucose
either fermentatively or oxidatively.
• The end products of fermentation are relatively
strong mixed acids that can be detected in a
conventional fermentation test medium.
However, the acids formed in oxidative
degradation of glucose are extremely weak and
less, and the more sensitive oxidation
fermentation medium of Hugh and Leifson’s OF
medium is required for the detection. The
medium was made by increasing the amount of
glucose above that found in medium used to
detect fermentation and by decreasing the
amount of peptone.
• The OF medium of Hugh and Leifson differs
carbohydrate fermentation media as follows:
• The concentration of agar is decreased to 2% from 3%,
making it semisolid in consistency (This assists in
the determination the motility of the organism).
• The concentration of peptone is decreased from 11%
to 2%. (decreasing the amount of alkaline product
produced by the metabolism of peptone; thus reducing
the neutralizing effect of these products).
• Carbohydrate concentration is increased by 0.5%
to 1.0% (The increased concentration of glucose in the
medium enhances the production of these weak acids
to a level that can be detected by bromthymol blue
indicator.)
• Principle:
• The oxidative-fermentative test determines if certain gramnegative rods metabolize glucose by fermentation or aerobic
respiration (oxidatively). During the anaerobic process of
fermentation, pyruvate is converted to a variety of mixed
acids depending on the type of fermentation. The high
concentration of acid produced during fermentation
will turn the bromthymol blue indicator in OF media from
green to yellow in the presence or absence of oxygen .
• Certain nonfermenting gram-negative bacteria metabolize
glucose using aerobic respiration and therefore only produce
a small amount of weak acids during glycolysis and Krebs
cycle. The decrease amount of peptone and increase
amount of glucose facilitates the detection of weak acids
thus produced.
• Dipotassium phosphate buffer is added to further promote
acid detection.
• Uses:
OF Test is used to determine if gram-negative bacteria metabolize
carbohydrates oxidatively, by fermentation, or are nonsacchrolytic
(have no ability to use the carbohydrate in the media).
• Media:
Hugh and Leifson’s OF basal medium; the constituents are as follows:
• Sodium chloride : 5.0 g
• Di-potassium phosphate : 0.3 g
• Peptone : 2.0 g
• Bromthymol blue:0.03 g
• Agar: 3.0 g
• Glucose : 10 g
• Water : 1000 ml
• The pH should be adjusted to 7.1 prior to autoclaving. After the
medium is autoclaved at 121°C for 15 minutes, a filter sterilized
solution of 10% solution of carbohydrate is aseptically added to the
medium to a final concentration of 1%.
• Procedure
• Inoculate two tubes of OF test medium with the test
organism using a straight wire by stabbing “half way to the
bottom” of the tube.
• Cover one tube of each pair with 1 cm layer of sterile
mineral oil or liquid paraffin (it creates anaerobic condition
in the tube by preventing diffusion of oxygen), leaving the
other tube open to the air.
• Incubate both tubes at 35oC for 48 hours (Slow growing
bacteria may take 3 to 4 days before results can be
observed)
• Interpretation
• Acid production is detected in the medium by the
appearance of a yellow color. In the case of oxidative
organisms; color production may be first noted near the
surface of the medium.
Oxidative Fermentative Test
Following are the reaction patterns:
Open (Aerobic) Tube
Covered (Anaerobic)
Tube
Metabolism
Acid (Yellow)
Alkaline (Green)
Oxidative
Acid (Yellow)
Acid (Yellow)
Fermentative
Alkaline (Green)
Non saccharolytic (glucose
not metabolised)
Alkaline (Green)
• Fermentative result: Acid production on both (open and covered)
tubes. The acid produced changes the pH indicator, bromthymol
blue, from green to yellow. e.g. Escherichia coli
• Oxidative result: Acid production in the open tube (aerobic) and not
the oil-covered tube (anaerobic) indicates an oxidative
result. Nonfermenting bacteria that metabolize glucose via oxidative
metabolism give an oxidative result. e.g. Pseudomonas aeruginosa
• Non saccharolytic (Negative OF result): Nonsacchrolytic bacteria
give a negative OF result. The negative result is indicated by no color
change in the oil-covered tube and in some cases an increase in pH
(pH 7.6) changing the bromthymol blue from green to blue in the
top of the open tube. The increase in pH is due to amine production
by bacteria that break down the peptone (protein) in the medium.
e.g. Alcaligenes faecalis.
• Quality Control
• Glucose Fermenter: Escherichia coli
• Glucose oxidizer: Pseudomonas aeruginosa
• Nonsaccharolytic: Moraxella species