CATALASE TEST

AIM: To demonstrate the availability of catalase in test organisms.

PRINCIPLE:
The enzyme catalase is present in most cytochrome containing aerobic and
facultative anaerobic bacteria. It catalyzes decomposition of hydrogen
peroxidase enzymes instead of catalase.
Catalase enzymes is reported to serve a protective function in the cell by
breaking down hydrogen peroxide, which is an oxidative end product of the
aerobic breakdown of sugar. Hydrogen peroxide, if accumulates can be toxic to
the cell (organism).
The test is used mainly to differentiate streptococcus spp (-ve) from
micrococcus andf staphyloccus spp (+ve). It differentiate Bacillus (+ve) from
clostridium spp (-ve), and also listeria monocytogenes (+ve) and/or
croynebacterium (+ve) (except C. pyogenes and C. haemolyticum) from
Erysipelothrix (-ve)

PROCEDURE
1. PLATE METHOD
Pipette 0.5 ml hydrogen peroxide directly onto the bacterial colony
See, if any bubbles evolve.

2. INOCULATION LOOP METHOD

Aseptically pick up some bacterial growth with an inoculation
loop/straight wire.
Pipette out a drop of hydrogen peroxide onto the bacterial growth
adhering to the loop.
See if any bubbles evolve (sometimes to see the small bubbles, a
binocular sterenomicroscope may be required).

3. SLIDE METHODS
With a straight wire, pick up bacterial growth from the center of 18-24
hr pure colony and place it on a clean glass slide .
Add a drop of H2O2 over the organisms on the slide (do not reverse
the order of procedure, otherwise false positive results may occur).

OBSERVATION AND INFERENCE

RESULT

POST VIVA QUESTIONS :

1 Describe a procedure commonly performed in your lab to do the
catalyst
2 Name four bacteria which are typically catalase-positive
3 Hydrogen peroxide is split by the enzyme catalase into water and
oxygen. (T/F)
4 Most anaerobic bacteria have the enzyme catalase.(T/F)
5 All mycobacterium spp are positive for catalse test, expert some
isoniazid (INH) resistance Mycobacterium tuberclosis strains which
may have decreased or no catalase activity. (T/F)
6 Catalase test can be done on colonies, which are 48hr old, may give
false negative results.
7 Hydrogen peroxide can be stored in transparent glass bottles.

Oxidase Test
AIM: To determine the oxygen requirement of the bacteria.
i. To demonstrate the presence of oxidase enzyme.
ii. To demonstrate metabolic nature of bacteria.

PRINCIPLE
Oxidase enzymes play an important role in the operation of the electron
transport system during aerobic respiration. Cytochrome oxidase (aa3 type)
uses O2 as an electron acceptor during the oxidation of reduced cytochrome c to
form water and oxidized cytochrome c.
The ability of bacteria to produce cytochrome oxidase can be determined by the
addition of the oxidase test reagent or test strip (tetramethyl-pphenylenediamine dihydrochloride or an Oxidase Disk, p-aminodimethylaniline) to colonies that have grown on a plate medium. Or, using a
wooden applicator stick, a bacterial sample can either be rubbed on a Dry Slide
Oxidase reaction area, on a KEY test strip, or filter paper moistened with the
oxidase reagent. The light pink oxidase test reagent (Disk, strip, or Slide) serves
as an artificial substrate, donating electrons to cytochrome oxidase and in the
process becoming oxidized to a purple and then dark purple compound in the
presence of free O2 and the oxidase. The presence of this dark purple coloration
represents a positive test. No color change or a light pink col- oration on the
colonies indicates the absence of oxidase and is a negative test.

REQUIREMENTS
Culture plate with growth (to be identified)
Cultures of E.coli (as a negative control strain) and Pseudomonas
aeruginosa (as a +ve control strain)
Filter paper.
Inoculation Loop/Straight wire (preferably platinum and not
nichrome.

 One percent tetramethyl-p-phenylenediamine dihydrocloride ( freshly

prepared solutions).

PROCEDURE
FILTER PAPER METHOD
Moisten a piece of filter paper with a few drops of freshly prepared
1% solution of oxidase reagent.
Aseptically with the help of a wooden stick transfer the suspected
growth from the agar medium and smear it on the moistened paper.
Immediately observe for change of colour. The development of purple
colour within 10 sec indicates a positive test. Reactions occurring
between 10 and 60 sec indicates a delayed reaction
Record your findings.

Direct Plate Method

Add a few drops of the freshly prepared 1% solution of reagent on a
few suspected colonies on the plate medium. Do not invert or flood
the plate.
Look for colour changes within but less sensitive.

OBSERVATION AND INFERENCE

RESULT

POST VIVA QUESTIONS

1 Describe the procedure commonly done in your lab to be the oxidative
test
2 Name two bacteria for which the oxidase test is the most often done.

TRUE OR FALSE
1 The oxidase reagent most often used is tetramethyl-p-phenylenediamine
tetrachloride
2 In the oxidase test the dye gets reduced .
3 In oxidase test the positive colour change occurring after 90 sec in
significant.
4 A platinum inoculation loop or a wooden stick is preferably used to do the
oxidase test.
5 V.cholerae is oxidase positive.

Carbohydrate Fermentation Test

Aim:
Principle:
A metabolic process performed by almost all types of bacteria is known as
fermentation. This will result in the production of ATP, the ultimate energy source
of the organism. This will happen either in the presence or absence of atmospheric
oxygen. Bacteria utilize the nutrients in their environment to produce ATP for their
biological processes such as growth and reproduction.

Phenol red carbohydrate broth

Phenol red broth is a general purpose fermentation media comprising of trypticase,
sodium chloride, phenol red and a carbohydrate. When the bacterium is inoculated
into the tube, the bacterium which ferments the sugar will result in the production
of acid that will change the color of phenol red. Fermentation reactions often begin
with glycolysis. Glucose acts as an electron donor in the fermentation reaction,
pyruvate, and metabolic product of glucose act as an electron acceptor. The other
disaccharides and polysaccharides are hydrolyzed into glucose or converted into
glucose and then the fermentation reaction will occur. Finally the reaction will
result in the end products such as acid, ethanol, Hydrogen and Carbon dioxide and
other compounds. This depends on the species of bacteria. Phenol red broth is a
test is differential for gram negative bacteria. When the organism ferments
carbohydrates, acidic organic by products (Lactic acid, formic acid or acetic acid)
is accumulated which turns the medium into yellow color with reduction in the pH
(acidic). The inverted Durham tubes will detect the presence of gas. The
degradation of peptones in the broth may result in the production of which will
change the broth color to pink often at the top of the tube.alkaline end products.

Materials Required:
1. Phenol Red Carbohydrate Fermentation Broth.
2. Bacterial culture.
3. Inoculation loop.
4. Incubator(370 C).

Procedure:
I. Preparation of Carbohydrate Fermentation Broth
1. Weigh and dissolve trypticase, Sodium chloride, and Phenol red in
100 ml distilled water and transfer into conical flasks.
2. Add 0.5% to 1% of desired carbohydrate into all flasks.
3. Insert inverted Durham tubes into all tubes, the Durham tubes should
be fully filled with broth.
Sterilize at 1150 C for 15 minutes.
Important: Do not overheat the Phenol red Carbohydrate
fermentation broth. The overheating will result in breaking down of
the molecules and form compounds with a characteristic color and
flavour. The process is known as caramelization of sugar (the
browning of sugar).
Transfer the sugar into screw capped tubes or fermentation tubes and
label properly.
Ingredients of the Fermentation Broth:
1. Trypticase: 1g
2. Carbohydrate: 0.5g
3. Sodium Chloride: 0.5g
4. Phenol red : 0.0189mg

II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth
1. Aseptically inoculate each labeled carbohydrate broth with bacterial
culture.(keep uninoculated tubes as control tubes).
2. Incubate the tubes at 18-24 hours at 37oC.
3. Observe the reaction.
Precautions:
1. After inoculation into a particular sugar, sterilize the loop in order to
avoid cross contamination of the tube with other sugars.
2. Keep uninoculated sugar tubes as control tubes.
3. Do not use the tubes with Durham tubes that are partially filled or
with bubbles.
4. Over incubation will help the bacteria to degrade proteins and will
result give false positive results.
Results of carbohydrate fermentation test:
Acid production: Changes the medium into yellow color- organism
ferments the given carbohydrate and produce organic acids there by
reducing the ph of the medium into acidic.
Acid and Gas production: Changes the medium into yellow color-organism
ferments the given Carbohydrate and produce organic acids and gas. Gas
production can be etected by the presence of small bubbles in the inverted
durham tubes.
1. Absence of fermentation: The broth retains the red color. The organism
cannot utilize the carbohydrate but the organism continues to grow in the
medium using other energy sources in the medium.

POST VIVA QUESTIONS

1) The indicator used in the Phenol red carbohydrate fermentation test is
a) Brilliant green

b)Phenol red

c)Methylene blue

d)Phenolphthalein

2) The importance of indicator in the Phenol red carbohydrate fermentation broth

a)Maintains the pH of the medium as 7.3

b)Red in acidic condition

c)Yellow in alkaline condition

d)Both b and c

3) The ability of microorganism to ferment carbohydrate mainly depends on

a)The concentration of the carbohydrate
b)The time of incubation of the inoculated carbohydrate broth
c)The enzyme systems of the bacteria
d)None of the above
4) Fermentation will result in ---------------a)ATP production
b)Alkaline end products
c)Acidic end products with or without gas production
d)All the above
5) After the incubation of the culture, the phenol red broth remains unchanged.
This is because
a)The carbohydrate is fermented to organic acids
b)The bacteria cannot utilize the given carbohydrate
c)The pH of the medium is acidic
d)Lack of incubation

6) In the acidic pH the phenol red changes its color from

a)Pink to red

b)Yellow to red

c)Red to yellow

d)Pink to yellow

7) Acid and gas production during fermentation indicates------------a)The organism uses the given carbohydrate and produced organic acids and
gas
b)The organism is anaerobic and did not use the carbohydrate
c)Increases the pH of the media
d)The peptones in the medium are degraded
8) Phenol red carbohydrate fermentation broth is
a)Enriched media

b)Selective media

c) General purpose media

d)None of the above

9) The production of gas during fermentation can be determined by

a)Yellow color of the broth
b)Checking the pH of the broth
c)Checking the bubbles in the inverted durham tubes
d)Both b and c
10) The concentration of sugar added in the Phenol red Carbohydrate fermentation
test is
a)10 % to 15%

b)0.5% to 1%

c)0.1% to 0.5%

d)0.5%to10%

11) Carbohydrates can be sterilized at 121 degree Celcius

a)True

b) False

12) The application of high temperature for sugar sterilization will result in --------a)Browning effect of sugar
a)Hydrolyze sugar

b)Caramelization
d)Both a and b