Catalase is an enzyme, which is produced by microorganisms that live in oxygenated

environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme
neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally
lack the catalase enzyme.

Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water. To find out
if a particular bacterial isolate is able to produce catalase enzyme, small inoculum of bacterial
isolate is mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration
of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble
production.

Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have
the ability to respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only
ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci).

Tube Catalase Test-Procedure and Results

1. Add 4 to 5 drops of 3% H2O2 (Hydrogen peroxide) to in a test tube

2. Using a wooden applicator stick, collect a small amount of organism from a well-
isolated 18- to 24-hour colony and place into the test tube (Note: Be careful not to
pick up any agar (esp if using Blood Agar).- Explanation in precaution below)
3. Place the tube against a dark background and observe for immediate bubble
formation (O2 + water = bubbles) at the end of the wooden applicator stick

Results:

 Catalase Positive reactions: Evident by immediate effervescence (bubble

formation)
 Catalase Negative reaction: No bubbleformation (no catalase enzyme to hydrolyze
the hydrogen peroxide)
 Methyl Red (MR) test determines whether the microbe performs mixed acids
fermentation when supplied glucose. Types and proportion of fermentation
products produced by anaerobic fermentation of glucose is one of the key
taxonomic characteristics which help to differentiate various genera of enteric
bacteria.
 Fig:
Methyl Red (MR) Test Reaction

 Mixed acid fermentation is one of the two broad patterns, 2-3-butanediol fermentation
being another. In mixed acid fermentation, three acids (acetic, lactic and succinic) are
formed in significant amounts. The mixed acid pathway gives 4 mol of acidic products
(mainly lactic and acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of
CO2, and 1 mol of H2 per mol of glucose fermented.
 These large amounts of acid results significant decrease in the pH of the medium below
4.4. This is visualized by using pH indicator, methyl red (p-dimethylaminoaeobenzene-
O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4.
 The pH at which methyl red detects acid is considerably lower than the pH for other
indicators used in bacteriologic culture media. Thus, to produce a color change, the test
organism must produce large quantities of acid from carbohydrate substrate being used.
 MR Positive: When the culture medium turns red after addition of methyl red, because of
a pH at or below 4.4 from the fermentation of glucose.
 MR Negative: When the culture medium remains yellow, which occurs when less acid is
produced (pH is higher) from the fermentation of glucose.

Procedure for Methyl Red (MR) Test

MR-VP broth is used for both MR Test and VP test. Only the addition of reagent
differs, and both tests are carried out consecutively.

1. Inoculate two tubes containing MR-VP Broth with a pure culture of the
microorganisms under investigation.
2. Incubate at 35 °C for up to 4 days.
3. Add about 5 drops of the methyl red indicator solution to the first tube (for Voges-
Proskauer test, Barrit’s reagent is added to another tube).
4. A positive reaction is indicated, if the colour of the medium changes to red within a
few minutes.
Methy Red Test
Left: Negative
Right: Positive

Expected results:
The development of a stable red color in the surface of the medium indicates sufficient acid
production to lower the pH to 4.4 and constitutes a positive test. Because other organisms may
produce smaller quantities of acid from the test substrate, an intermediate orange color between
yellow and red may develop. This does not indicate a positive test.

1. Escherichia coli: MR test positive- appearance of red color after the addition of
methyl red reagent.
2. Enterobacter aerogenes: MR test Negative- the lack of color change after the
addition of methyl red.

Voges-Proskauer is a double eponym, named after two microbiologists working at the

beginning of the 20th century. They first observed the red color reaction produced by appropriate
culture media after treatment with potassium hydroxide. It was later discovered that the active
product in the medium formed by bacterial metabolism is acetyl methyl carbinol, a product of the
butylenes glycol pathway.

Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further
metabolized through various metabolic pathways, depending on the enzyme systems possessed
by different bacteria. One such pathways result in the production of acetion (acetyl methyl
carbinol), a neutral-reacting end product.
Results of Voges-Proskauer (VP) Test

Organisms such as members of the Klebsiella-Enterobacter-Hafnia-Serratia group produce

acetoin as the chief end product of glucose metabolism and form smaller quantities of mixed
acids. In the presence of atmospheric oxygen and 40% potassium hydroxide, acetoin is converted
to diacetyl, and alpha-naphthol serves as a catalyst to bring out a red complex.

Media and Reagents

1. Media: The medium is MR/VP broth

2. Reagents:
1. Alpha-naphthol, 5% color intensifier
1. Alpha Naphthol-5g
2. Absolute ethyl alcohol- 100 mL
2. Potassium Hydrooxide, 40%, oxidizing agent
1. Potassium hydroxide 40g
2. Distilled water to: 100 mL

Quality Control
Positive and negative controls should be run after preparation of each lot of medium and after
making each batch of reagent. Suggested controls include the following:

 Positive Control: Enterobacter aerogenes

 Negative Control: Escherichia coli

Procedure of Voges Proskauer Test

1. Inoculate a tube of MR/VP broth with a pure culture of the test organism.
2. Incubate for 24 hours at 35oC
3. At the end of this time, aliquot 1 mL of broth to clean test tube.
4. Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH. (Note: It is
essential that the reagents be added in this order.)
5. Shake the tube gently to expose the medium to atmospheric oxygen and allow the
tube to remain undisturbed for 10 to 15 minutes.

Results and Interpretation

A positive test is represented by the development of a red color 15 minutes or more after the
addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin . The
test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures
may produce a copper like color, potentially resulting in a false positive interpretation.

The oxidase test is designed for specifically detecting the

presence of the terminal enzyme system in aerobic respiration
called cytochrome C oxidase or cytochrome a3. Cytochrome C
oxidase is the terminal or last H2 electron acceptor in aerobic
respiratory mechanism which is composed of a number of
enzymes which alternatively oxidize and reduce each other by
donating or accepting electrons derived from H2.
The ability of an organism to produce the cytochrome C oxidase
can be determined by using the reagent tetramethyl-p-
phenylenediamine dihydrochloride impregnated in filter disk.
The reagent serves as an artificial substrate donating electrons
and thereby becoming oxidized to a deep purple compound in the
presence of the enzyme oxidase and free O2. Development of
pink, then maroon and finally dark purple colouration after
rubbing the organism in the oxidase disc containing the reagent
indicates positive reaction. The positive reaction involve the
conversion of colourless, reduced tetramethyl-p-
phenylenediamine to oxidized form into deep purple colour in
presence of Cytochrome C oxidase. No colour change is
indicative of the negative test result.
Procedure of Oxidase Test
1. Take a filter paper soaked with the substrate tetramethyl-p-
phenylenediamine dihydrochloride.
2. Moisten the paper with a sterile distilled water.
3. Pick the colony to be tested with wooden or platinum loop
and smear in the filter paper.
4. Observe inoculated area of paper for a color change to deep
blue or purple within 10-30 second.
Or
1. Take a commercially available oxidase disc containing the
reagent.
2. Pick the isolated colony to be to be tested and rub in the
disc.
3. Observe for colour change within 10 seconds.
Result Interpretation of Oxidase Test

Positive test: Development of deep purple color within 10

seconds
Negative test: Absence of color

Quality Control of Oxidase Test

Positive: Pseudomonas aeruginosaATCC27853
Negative: Escherichia coli ATCC 25922
Gelatin is a protein derived from the animal protein collagen– component of
vertebrate connective tissue. It has been used as a solidifying agent in food for a long
time. Gelatin hydrolysis test is a great way to highlight proteolysis by bacteria

Principle of Gelatin hydrolysis test

Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase
(proteolytic enzyme) that liquefy gelatin. Hydrolysis of gelatin indicates the presence of
gelatinases.

This process takes place in two sequential reactions. In the first reaction, gelatinases degrade
gelatin to polypeptides. Then, the polypeptides are further converted into amino acids. The
bacterial cells can then take up these amino acids and use them in their metabolic processes.

Uses of Gelatin Hydrolysis test

Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus,
Clostridium, Proteus, Pseudomonas, and Serratia.

It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from the gelatinase-

negative, non-pathogenic S. epidermidis . Gram-positive, spore-forming, rodshaped, aerobic or
anaerobic bacteria such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridium
perfringens and Clostridium tetani, are also positive for gelatin hydrolysis.

The test can also be used to differentiate genera of gelatinase-producing bacteria

such Serratia and Proteus from other members of the family Enterobacteriaceae.

Procedure /Method of Gelatin hydrolysis test

There are several methods for determining gelatinase production, all of which make use of
gelatin as the substrate. The standard and most commonly employed method is the nutrient
gelatin stab method.

1. Inoculate a heavy inoculum of test bacteria (18- to 24-hour-old) by stabbing 4-5

times (half inch) on the tube containing nutrient gelatin medium.
2. Incubate the inoculated tube along with an uninoculated medium at 35°C, or at
the test bacterium’s optimal growth temperature, for up to 2 weeks.
3. Remove the tubes daily from the incubator and place in ice bath or refrigerator
(4°C) for 15-30 minutes (until control is gelled) every day to check for gelatin
liquefaction.(Gelatin normally liquefies at 28°C and above, so to confirm that
liquefaction was due to gelatinase activity, the tubes are immersed in an ice bath
or kept in refrigerator at 4°C).
4. Tilt the tubes to observe if gelatin has been hydrolyzed.
Expected results
Positive: Partial or total liquefaction of the inoculated tube (uninoculated control medium must
be completely solidified) even after exposure to cold temperature of ice bath or refrigerator (4°C)

Gelatin Hydrolysis Test: Above tube: Positive

Below Tube: Negative

Negative: Complete solidification of the inoculated tube even after exposure to cold temperature
of ice bath or refrigerator (4°C)

Common bacteria and their reactions to the gelatin hydrolysis test performed on
nutrient gelatin.

Species Growth Liquefaction

Bacillus subtilis + +

Clostridium perfringens + +

Escherichia coli + –

Proteus vulgaris + +

Serratia liquefaciens + +

Staphylococcus aureus + +

Control organisms

 Positive Control: Proteus vulgaris

 Negative Control: Enterobacter aerogenes