THE CATALASE TEST

The catalase test facilitates the detection of the enzyme catalase in bacteria. It is essential for
differentiating catalase-positive Micrococcaceae from catalase-negative Streptococcaceae.

The catalase test is also valuable in differentiating aerobic and obligate anaerobic bacteria,
as anaerobes are generally known to lack the enzyme

In this context, the catalase test is valuable in differentiating aerotolerant strains

of Clostridium, which are catalase negative, from Bacillus, which are catalase positive

1
 THE CATALASE TEST
THEORY

The catalase enzyme serves to neutralize the bactericidal (killing ) effects of hydrogen
peroxide

Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen
(2H2O2 + Catalase → 2H2O + O2). This reaction is evident by the rapid formation of
bubbles

2
 THE COAGULASE TEST

PURPOSE
The coagulase test differentiates strains of Staphylococcus aureus from S. epidermidis and
other coagulase-negative species.

S. aureus strains are usually capable of coagulating EDTA-treated plasma in the tube test
and will produce clumps of cells in the slide test.

3
 THEORY
The coagulase test can be performed using two different procedures.

1. The slide test is simple, giving results within 10 seconds, but it can give false negatives.

2. The tube test is the definitive test, however, it can take up to 24 hours to complete.

For both tests, clumping or clots of any size indicate a positive response Likewise, the
speed of clotting is not related to the virulence of the bacteria

4
 THE SLIDE COAGULAZE TEST
The slide test is performed by preparing a suspension of bacterial cells mixed into a drop of
EDTA-treated rabbit plasma on a microscope slide.

If bound coagulase is present on the bacterial cells, then the presence of plasma will cause the
bacterial cells to clump.

The clumping will occur because the clumping factor causes the cells to bind to fibrinogen in the
plasma.

This will result in visible clumping of bacterial cells on the microscope slide.

Figure 1 illustrates the visible clumping of cells on the microscope slide

5
 THE SLIDE COAGULAZE TEST CONT.
Demonstrating bound coagulase, otherwise known as clumping factor

Materials
Microscope slide
1. Overnight plate culture of the organism to be tested, grown on noninhibitory agar
2. Control organisms, usually include S. epidermidis (negative control) and S.
aureus (positive control)
3. Water or physiological saline
4. Nonsterile dropper
5. Reconstituted coagulase plasma
6. Sterile dropper
7. Marking pen or wax pencil

Method of transferring bacteria from plate (such as a wire loop and flame, sterile wooden
applicator stick, or sterile disposable loop)

Discard container for contaminated materials

6
 The slide coagulaze test
Procedure
1. Draw two circles on the slide using the marking pen. Draw the circles as far apart as
possible so there will be room to keep the suspensions separate and to mix each of the
suspensions. Identify where the water (or saline) and plasma will be placed. If control strains
will be utilized, additional slides will be required to test them using the procedure outlined
below.

2. With the nonsterile dropper, place a small drop of water or saline into the circle on the
appropriate end of the slide. The saline or water is a control to verify that the strain does not
autoagglutinate and clump with itself instead of being easily emulsified.

3. Using the sterile dropper, place a small drop of reconstituted plasma into the circle on the
opposite end of the slide.

7
 The slide coagulaze test
Procedure cont.
4. With the loop or applicator stick, collect cells from one colony and emulsify the cells in
the water (or saline) and then in the drop of plasma. The order is important to prevent
carryover of small amounts of plasma into the control drop.

If using a disposable loop or applicator stick, carefully place it into the discard
container. If using a reusable loop, sterilize the loop before proceeding.

5. Watch for clumping within 10 seconds of adding the bacterial cells to the plasma.

The clumping will become more visible if the slide is rocked gently in a figure 8
motion. The control drop, saline or water, should show no clumping of bacterial cells.

8
 Fig 1
Slide coagulaze test

Figure 2 is a comparison of how the cells in Figure 1 appeared when fixed, stained
with methylene blue, and observed at 1,000X magnification in the light microscope.
9
 THE TUBE COAGULASE TEST
The tube coagulase test is performed by mixing bacterial cells into a larger volume of
plasma in a small test tube.

As the bacteria multiply in the plasma, they secrete staphylocoagulase.

Staphylocoagulase initiates blood coagulation by activating prothrombin.

Staphylocoagulase adheres to fibrinogen, forming a complex that cleaves fibrinogen into

fibrin, bypassing the blood clotting cascade and directly causing a clot of fibrin to form.

Formation of a clot will be noted within 24 hours for a positive response. Figure 3 shows a
negative reaction and a positive reaction.

10
 The tube coagulase test
Three results are possible.

❖ If the strain shows clumping in the saline control and is difficult to emulsify, it exhibits
autoagglutination, and no results can be obtained from the slide test. This strain must be
tested in the tube test.

❖ If the strain shows clumping only when emulsified in the plasma and there is no
autoagglutination, this is a positive slide test. This strain is not further tested in the tube test.

❖ If the strain shows no clumping in either saline or plasma, it has tested negative on
the slide coagulase test and should be further tested using the tube coagulase test.

The positive control species should show clumping only when emulsified in the plasma and
the negative control species should not show clumping in either water (saline) or plasma.

11
Tube Coagulase Test
Demonstrating staphylocoagulase production

Materials

Small sterile test tubes (for example, 12 X 75-mm snap-capped tubes)

Sterile wooden applicator stick or transfer loop
Discard container for contaminated materials
Test tube rack
Culture of organism to be tested, must be fresh (less than 24 hours) on noninhibitory
media
Control organisms, usually include S. epidermidis (negative control) and S. aureus (positive
control)
Watch or timer
Incubator set at approximately 37oC
Pipettor (to transfer 0.5 ml volume)
Sterile tips
Marking pen or wax pencil
Reconstituted coagulase plasma

12
 Tube coagulaze test
Procedure

1. Label the test tube with the organism to be tested.

2. Using the pipettor, aseptically transfer 0.5 ml of the reconstituted plasma into the test tube.

3. Select two or three isolated colonies of the bacteria to be tested and collect them using the
sterile loop or applicator stick.

4. Emulsify the bacteria in the 0.5 ml of plasma and place in the incubator. Discard
the disposable loop or applicator stick into the discard container or sterilize the reusable loop
before proceeding.

13
 TUBE COAGULAZE TEST
PROCEDURE CONT.
5. Note the time when the test began. At intervals over the next 4 hours, observe the
culture, looking for evidence of a clot. Any clot formation is a positive result.

6. If no clot is observed by the end of 4 hours, then the test may be continued with an
overnight incubation at room temperature and a final observation at 24 hours.

7. The positive control organism should exhibit clotting by the end 24 hours, while the
negative control organism should not.

8. When the test is completed, dispose of the contaminated materials appropriately.

14
 MANNITOL SALT AGAR (MSA)
Purpose

Mannitol salt agar (MSA) is both a selective and differential medium used in the isolation of
staphylococci. It contains 7.5% sodium chloride and thus selects for those bacteria which can
tolerate high salt concentrations.

MSA also distinguishes bacteria based on the ability to ferment the sugar mannitol, the only
carbohydrate in the medium.

16
 MSA THEORY
Staphylococci can withstand the osmotic pressure created by 7.5% NaCl, while this
concentration will inhibit the growth of most other gram-positive and gram-negative bacteria .

Additionally, MSA contains mannitol and uses phenol red as a pH indicator (pK = 7.8) in the
medium.

At pH levels below 6.9, the medium is a yellow color. In the neutral pH ranges (6.9 to 8.4) the
color is red; while above pH 8.4, the color of phenol red is pink

When mannitol is fermented by a bacterium, acid is produced, which lowers the pH and results
in the formation of a yellow area surrounding an isolated colony on MSA.

A nonfermenting bacterium that withstands the high salt concentration would display a red to
pink area due to peptone breakdown (15).

In clinical samples, mannitol positive isolates are suggestive of Staphylococcus aureus and should
be tested further.

17
 MSA PROTOCOL

Streak a plate of mannitol salt agar with appropriate culture using the quadrant streak plate
method to obtain isolated colonies.

Well-isolated colonies will provide the best results in the biochemical differentiation bacteria
using MSA.

18
 MSA

(A) Staphylococcus aureus, (B) Staphylococcus epidermidis, and (C) Escherichia coli streaked
on a mannitol salt agar plate. The mannitol fermenting colony (yellow) is S. aureus, while
themannitol nonfermenting colony (pink) is S. epidermidis. The growth of E. coli was
inhibited by the high salt concentration.

19
 White blood cells called neutrophils have a two-pronged defense against bacteria:
They can swallow and destroy them or they can release neutrophil extracellular
traps (NETs). The fibrous NETs are made up of DNA and toxic compounds that can
catch and kill pathogenic microbes.

But some bacteria nimbly evade NETs,. One possible explanation has been that
those bacteria produce an enzyme that degrades the traps.

Disease-producing bacteria seem to make more of an enzyme called DNase than

benign microbes do, "There's been speculation for a long time that DNases could be
virulent,

20
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

21
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 performed a coagulase test or when the results of a coagulase test are difficult to
interpret.
DNase test can be used for other Gram negavive bacteria
.

22
Dry the Surface of agar plates before use. Each plate may be divided into sections by
drawing lines on the bottom of the plate.
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 5-10mm 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.
When using DNase agar without indicator,
Flood the plate with 1N Hydrochloric Acid.
Leave the plate to stand for a few minutes to allow the reagent to absorb into the plate.
Decant excess hydrochloric acid and then examine the plate within 5 minutes against a dark
background.
23
24
 DNase medium with 1N HCL as indicator

1. In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing

of the agar after addition of HCL (oligonucleotides dissolves in acid but DNA salts
are insoluble). The acid precipitates unhydrolyzed DNA making the medium opaque.
Therefore, DNase producing colonies hydrolyze DNA and produce a clear zone
around the growth.

25
 DNase medium with methyl green as indicator
2. In case of DNase agar with methyl green, DNA combines with methyl green (act as
cation) to produce mint green color. When the DNA is hydrolyzed, the complex is released
and the free methyl green is colorless at pH 7.5. So the clear halo is appeared around
the areas where DNase producing organism grow.

26
DNase medium with Toluidine blue as indicator

When toluidine blue O (TBO) is added to the DNase agar, a complex is formed
with the DNA, which changes structure when DNA is hydrolyzed, resulting in a
bright pink color.

27