Coagulase Test

Coagulase is a protein enzyme produced by several microorganisms that converts (soluble) fibrinogen in plasma to (insoluble)
fibrin and causes clotting of plasma. The coagulase enzyme acts as a virulence factor in some organisms as it interacts with the
fibrinogen present on the host’s cell surface.

Uses Of Coagulase Test  

 Coagulase test is an important test that differentiates the species of the genus Staphylococci into two groups; Coagulase
positive Staphylococci and Coagulase Negative Staphylococci (CONS).
 The coagulase test is used to determine the production of coagulase by different microorganisms.

Objectives of Coagulase Test

 To detect free coagulase and bound coagulase produced by different organisms.
 To differentiate coagulase-positive Staphylococci from coagulase-negative Staphylococci.
 To identify and differentiate S. aureus from other Staphylococcal species.

Types

Staphylococcus aureus produces two forms of coagulase, bound and free.

Bound Coagulase

It is also known as clumping factor. It is attached to bacterial cell wall and reacts directly with fibrinogen. This is shown by
formation of visible mass. It doesn’t require coagulase reacting factor (CRF). It can be detected rapidly in the slide test, but this
test requires several colonies and lacks sensitivity.

Free coagulase
 It is an extracellular enzyme (released from the cell).
 Free coagulase is different from bound coagulase in that the clotting mechanism of free coagulase requires the activation of
a plasma coagulase-reacting factor (CRF), which is a modified or derived thrombin molecule, to form a coagulase-CRF
complex.
 The plasma added to the tube is preferably rabbit plasma which acts as a binding factor.
 The complex then reacts with fibrinogen to form the fibrin clot in a test tube.

Principle

Reagent
Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors and is safe. It is treated with anticoagulant EDTA to
interrupt the normal clotting mechanism
Human plasma is commonly not used for the test, as it is less sensitive and potentially infectious with human pathogenic viruses.

Procedure
Slide Coagulase Test Procedure (to detect bound coagulase)
1. Place a drop of physiological saline on each end of a clean and grease-free glass slide, or on two separate slides.
2. Using a sterile inoculating loop, emulsify a portion of the isolated colony in each drop to make two thick smooth milk-
colored suspensions.
3. Add a drop of human or rabbit plasma to one of the suspensions, and mix gently.
 4. Look for immediate coarse clumping of the organisms within 10 seconds.
5. No plasma is added to the second suspension to differentiate any granular appearance of the organism from true coagulase
clumping.

Tube Coagulase Test Procedure (to detect free coagulase)

1. Dilute the plasma 1 in 10 in physiological saline (mix 0.2 ml of plasma with 1.8 ml of saline).
2. Take three small test tubes and label:
T _ Test organism (18–24 h broth culture)* Pos _ Positive control (18–24
h S. aureus broth culture)*
Neg _ Negative control (sterile broth)*
3. Pipette 0.2 ml of plasma into each tube.
4. Add 0.8 ml of the test broth culture to tube T.
Add 0.8 ml of the S. aureus culture to the tube labelled ‘Pos’.
Add 0.8 ml of sterile broth to the tube labeled ‘Neg’.
5. After mixing gently, incubate the three tubes at 35–37 _C. Examine for
clotting after 1 hour.
6. Examine at 1, 2 and 4 hours for clot formation by tilting the tube through
90°.If no clotting has occurred, examine after 3 hours. If the test is still negative,
leave the tube at room temperature overnight and re-examine again
(This step is essential, for some strains of S. aureus, including many MRSA,
produce a delayed clot which is rapidly lysed at 37°C by the organism’s
staphylokinase.)

Result Interpretation
Clumping with in 10 sec - Stap. areus
No clumping - negative
Examples

Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria like  S. pseudintermedius, S.
intermedius, S. schleiferi,  S. delphini, S. hyicus, S. lutrae, S. hyicus

Coagulase Negative organisms: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc.

LIMITATION

 Methicillin-resistant S. aureus can be deficient in bound coagulase, which results in a negative slide test.

 S. intermedius and S. hyicus may be positive in the tube test; these species are generally found only in dogs and pigs,
respectively, but are as infectious as S. aureus when they infect humans.
 S. lugdunensis and S. schleiferi produce slide coagulase, but the reaction is more efficient if the human plasma is used
rather than rabbit plasma.
 Citrated blood should not be used as false-positive results can occur.
 Colonies from a Mannitol Salt Agar (MSA) culture are not suitable for coagulase testing. The organism must first be
cultured on Nutrient Agar or Blood Agar.
  All coagulase negative slides must be confirmed using a tube coagulase test as the definitive test for S. aureus. 

CATALASE TEST

Introduction
 The catalase test is a biochemical test for aerobic organisms that detects the production of catalase an enzyme that catalyses
the release of oxygen from hydrogen peroxide (H2O2) in the organism. It is an essential enzyme in pathogenic organisms as it
protects the organism from oxidative damage from the reactive oxygen species.
  The enzyme neutralizes the bactericidal effects of hydrogen peroxide, and its concentration in bacteria has been correlated
with the pathogenicity of the organism.
 Normally 3% H2O2 is used for the routine culture while 15% H2O2 is used for detection of catalase in anaerobes.

Uses Of Test
 The morphologically similar Enterococcus or Streptococcus (catalase negative) and Staphylococcus (catalase positive) can
be differentiated using the catalase test.
 Also valuable in differentiating aerobic and obligate anaerobic bacteria.
 Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.
 It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are
positive.
 Catalase test can be used as an aid to the identification of Enterobacteriaceae.
 Catalase test is essential for differentiating catalase-positive Micrococcaceae and Staphylococcaceae from catalase-negative
Streptococcaceae.
 The test also allows differentiating aerobic and obligate anaerobic organisms.
 The value of the test has been found in the presumptive differentiation among certain Enterobacteriaceae.
 Thisis an important test for the differentiation of aerotolerant strains of Clostridium, which are catalase-negative,
from Bacillus, which are catalase positive.
 This test has been used to differentiate between genera and is also valuable in the speciation of certain gram-positive
organisms such as Aerococcus and

Objective Of Test
 To detect the ability of organisms to produce the catalase enzyme.
 To differentiate catalase-positive organisms like micrococci and staphylococci from catalase-negative organisms like
streptococci.

Principle
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a
bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen
bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24
hours old.

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of
aerobic carbohydrate metabolism.

Reagents
Hydrogen peroxide reagent
 30% H2O2 for Neisseria
 15% H2O2 for anaerobes
 3% H2O2 for other bacteria (purchase or dilute 30% 1:10 in deionized water prior to use)

Procedure
Tube Method

1. Pour 1-2 ml of hydrogen peroxide solution into a test tube.

2. Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism and immerse in the
hydrogen peroxide solution.
3. Observe for immediate bubbling.
Slide Method

1. Use a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a clean, dry glass slide.
2. Place a drop of 3% H2O2 in the glass slide.
 3. Observe for the evolution of oxygen bubbles.

Result Interpretation
Positive: Copious bubbles produced, active bubbling
Negative: No or very few bubbles produced.

Example
Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family
Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella,
Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi.

Limitation
 Culture should be 18 to 24 hours old. Because the enzyme is present in viable cells only, colonies that are older than 24
hours should not be used. Older cultures may give false-negative results.
 Hydrogen peroxide must be fresh as it is very unstable.
 Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the reaction to be weakly
positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction.
 RBCs contain catalase, and thus, in order to avoid false-positive results, the test organisms should not be taken from blood
agar culture.
 The test should not be tested from Mueller-Hinton agar.
 The reagent and the colony should not be mixed.
 Some strains of S. aureus may appear catalase-negative by drop method so the test should be repeated with the tube
method.
 30% H2O2 is extremely caustic to the skin. If contact occurs, wash immediately with 70% ethyl alcohol, not water.
 Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal. However, platinum loops do
not yield false-positive results.
Deoxyribonuclease (DNase) Test:
Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is way too large to enter the cell membrane. In order to
utilize external DNA, bacteria cells secrete exoenzymes (DNases) outside of the cell that hydrolyze DNA into nucleotides. 
The nucleotides can then move across the cell membrane via transport proteins to be utilized. The cell can use nucleotides
to make nucleic acids and to use as a source of nitrogen, phosphate and carbon. Extracellular DNases have been reported
in a relatively small subset of prokaryotes, including a number of human pathogens. DNA hydrolysis is tested by growing
an organism on a DNase Test Agar plate (providing nutrients and DNA) and then checking the plate for hydrolysis. DNA
Hydrolysis test or Deoxyribonuclease (DNase) test is thus used to determine the ability of an organism to hydrolyze DNA
and utilize it as a source of carbon and energy for growth. It helps in the detection of deoxyribonuclease activity of bacteria
and fungi, and especially for identification of pathogenic Staphylococci.
Deoxyribonuclease (DNase) Test is a biochemical test performed to differentiate organisms on the basis of
their ability to produce the DNase enzyme.

DNA hydrolysis test or Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and
utilize it as a source of carbon and energy for growth.

An agar medium; DNase agar, a differential medium is used to test the ability of an organism to produce deoxyribonuclease or
DNase.

This medium is pale green in color because of the DNA-methyl green (indicator) complex (Note: Methyl green is a cation that binds
to the negatively-charged DNA). It also contains nutrients for the bacteria.
If the organism that grows in the medium produces Deoxyribonuclease, it breaks down DNA into smaller fragments.  When the
DNA is broken down, it no longer binds to the methyl green, and green color fades and the colony is surrounded by a
colorless zone.
 Deoxyribonucleic acid (DNA) is a large molecular-sized polymer composed of multiple nucleotides monomer that is
large in size and thus, cannot enter the bacterial cell membrane.
 Microorganisms produce the deoxyribonuclease enzyme to breakdown the DNA into smaller monomers which can
then be taken into the cell easily.
 The nucleotides are used to make nucleic acid for the bacteria as well as a source of nitrogen, phosphorus, and
carbon.
 Some microorganisms can even produce extracellular DNase that breaks down larger DNA into smaller monomeric
units so that they can be taken into the cell via the transport proteins present on the cell membrane.
 The degradation of DNA is also considered a virulence factor as it causes the degradation of the host’s DNA.
 The ability to produce DNase can thus be used to differentiate between different pathogenic microorganisms.
 DNase test is an important test for the presumptive identification of Staphylococcus aureus and differentiates it
from other Staphylococcal species.

Objectives of Deoxyribonuclease (DNase)

 To determine the ability of an organism to produce the DNase enzyme.
 To differentiate and identify S. aureus from other Staphylococcal species.

Principle
 DNases are enzymes that hydrolyze DNA and release free nucleotides and phosphate.
 The deoxyribonuclease enzyme produced by bacteria is extracellular endonucleases that break down DNA, yielding
a high concentration of oligonucleotides.
 The media used to detect these enzymes can be made by using various indicators (toluidine blue or methyl green)
or no indicators to detect the hydrolysis of DNA.
 The first method is performed with no indicator. The hydrolysis of DNA is indicated by the clearing of the agar after
the addition of HCl (the oligonucleotides dissolve in acid causing a clear zone, but DNA salts are insoluble).
 When methyl green indicator is added, DNA combines with the methyl green to produce a green color.
 The complex is released when the DNA is hydrolyzed, and the freed methyl green is colorless at pH 7.5.
  When toluidine blue O (TBO) is added, a complex is formed with the DNA, which changes its structure when DNA
is hydrolyzed, resulting in a bright pink color.
 The media with dyes can inhibit some microbial growth. Using a heavy inoculum prevents this problem and makes
the test more rapid, as it detects preformed enzymes.
 Staphylococcus aureus possesses a heat-stable enzyme, a thermonuclease. To detect this enzyme, first, the
organisms are destroyed by heat, and then the free DNase reacts with the medium.

 Uses of DNase Test: 

1. It is used to differentiate S.aureus (DNase +ve) from other Staphylococci that do not produce such enzyme. The DNase
test is particularly useful when plasma is not available to perform a coagulase test or when the results of a coagulase test
are difficult to interpret.
2. DNase test distinguishes M. catarrhalis from all other gram-negative diplococci (e.g. Neisseria gonorrhoeae & Neisseria
meningitidis) of human origin.

Requirements:
1. Media: DNase Agar or DNase agar with Methyl green indicator.
2. Reagent: Hydrochloric acid (1mol/L) only when DNase agar without an indicator is used
3. Others: Inoculating loop, Bunsen burner
Media Used
 DNase Test Agar Base is used for testing the production of the DNase enzyme. The medium can also be added
with indicators like methyl green or toluidine blue O.
 The composition of the DNase Test Agar Base is given below:
S.N Ingredients Gram/liter

1. Tryptone 15.0
 2. Soya peptone 5.0

Deoxyribonucleic
3. 2.0
acid (DNA)

4. Sodium chloride 5.0

Bacteriological
5. 15.0
agar

Final pH at 25°C: 7.3 ±0.2

Procedure of DNase (DNA hydrolysis test)

1. Dry the surface of agar plates before use. Each plate may be divided into sections by drawing lines on the bottom of the
plate.
2. Inoculate the test agar medium: There are two types of inoculation that can be done.

Spot Inoculation

 Touch a colony of the organism under test with a loop and inoculate it onto a small area of the DNase test agar plate, in
the middle of one of the marked sections to form a thick plaque of growth 5-10 mm in diameter after incubation.
 Incubate the plate at 37°C for 18-24hr.

Band or line streak inoculation

  Use a heavy inoculum and draw a line 3-4 cm long from the rim to the center of the DNase test agar plate
 Incubate the plate at 37°C for 18-24hr.
Expected results:
1. Positive: When DNA is hydrolyzed, methyl green is released turning the medium colorless around the test organism.
2. Negative: If there is no degradation of DNA, the medium remains green.
Test results
1. DNase Test positive organisms: 
1. Serratia marcescens
2. Staphylococcus aureus
3. Campylobacter jejuni (some strains)
4. M. Catarrhalis 
2.  DNase test negative organisms: 
1. Staphylococcus epidermidis
2. Neisseria gonorrhoeae 

Limitation of DNase Test

1. Some MRSA strains do not give positive DNase test result and some strains of the coagulase-negative staphylococci
such as Staphylococcus capitis may give weak reactions.
2. Serratia and Moraxella species also produce deoxyribonuclease.
3. 1N HCl is bactericidal for Staphylococci. Once the HCl has been applied, the test must be read within 5 minutes and
cannot be continued by re-incubation.