An overview of biochemical test used in Microbiology and the source of presentation is greatly acknolwledge

1. Biochemical Tests for Gram
Positive Bacteria

2. Catalase Test
• This test is used to differentiate between
catalase producing organisms,
(Staphylococci) and non catalase producing
organisms, (Streptococci).
Principle
– Catalase acts as a catalyst in the breakdown of
hydrogen peroxide to oxygen and water.
– An organism is tested for catalase production by
bringing it into contact with hydrogen peroxide.
– Bubbles of oxygen are released if the organism
is a catalase producer.

3. Catalase Test
Requirements
– Hydrogen peroxide, 3%, sterile wooden stick or glass rod
Method
– Pour 2-3 mL of the hydrogen peroxide solution into a test tube
– Using a sterile wooden stick or a glass rod, remove several colonies of
the test organism and immerse in the hydrogen peroxide solution
– Look for immediate bubbling
Results
– Active bubbling ………………………….. Positive
– No bubbles ………………………………... Negative
Controls
– Positive ……………………………………. Staphylococcus spp
– Negative …………………………………... Streptococcus spp

4. Catalase Test
Negative Positive

5. Catalase Test
• Important
– The wire loop must not be used, as false
positives may occur
– Performing test on slide is not recommended,
because of the potential risk of aerosol
production
– If a slide test has to be performed, then place
slide in a petri dish, and add hydrogen peroxide
to the test organism suspension, and
immediately cover the petri dish

6. Coagulase Test
This test is used to identify the Staphylococcus
aureus, which produces the enzyme coagulase
• Principle
– Coagulase causes plasma to clot by converting fibrinogen
to fibrin. Two types of coagulase are produced by most
strains of Staphylococcus aureus
• Free coagulase which converts fibrinogen to fibrin by
activating a coagulase reacting factor present in plasma. Free
coagulase is detected by clotting in the tube test
• Bound coagulase (clumping factor) which converts fibrinogen
directly to fibrin without requiring a coagulase reacting factor.
It can be detected by the clumping of bacterial cells in the
rapid slide test

7. Coagulase Test
• Requirements
– EDTA anti-coagulated human plasma or rabbit plasma, slide, wire loop
• Slide test method
– Place a drop of distilled water on each end of a slide
– Emulsify a colony of the test organism in each of the drops
– Add a loopful of plasma to one of the suspensions, and mix gently
– Look for clumping of the organisms within 10 seconds
• Results
– Clumping within 10 seconds ……………….. Staphylococcus aureus
– No clumping within 10 seconds ………………. No bound coagulase
• Controls

8. Coagulase Test
• Tube Test Method
– Take 3 test tubes and label them as T, P, and N
– Pipette 0.2 mL of plasma into each tube
– Add 0.8 mL of the test broth culture to tube T
– Add 0.8 mL of the Staphylococcus aureus culture to the tube P
– Add 0.8 mL of the sterile broth to the tube N
– After mixing gently, incubate the three tubes at 37° C
– Examine for clotting after one hour
– If no clotting has occurred, examine after 3 hours. If still no clotting, then leave
tubes overnight
• Results
– Clotting of tube contents or clot in tube ………………….. Staphylococcus aureus
– No clotting …………………….………………………………..……. Negative test

9. Coagulase Test
Positive Negative

10. Coagulase Test
• Important
– A tube test must always be performed when the result of a slide test is not
clear, or when the slide test is negative and Staphylococcus has been
isolated from a serious infection
– The plasma used should be preferably pooled
– Oxalate or heparin plasma can also be used
– Do not use citrated plasma because citrate utilizing bacteria e.g.
Enterococci, Pseudomonas and Serratia may cause clotting of the plasma
in tube test
– Occasionally human plasma may contain inhibitory substances which can
interfere with test results. It is therefore essential to test the plasma using
a known coagulase positive Staphylococcus aureus
– Virulent strains of Yersinia pestis are also coagulase positive

11. DNase Test
• This test is used to identify Staphylococcus aureus which produces DNase
enzyme
• It 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
• Principle
– DNase hydrolyzes DNA.
– The test organism is cultured on a medium which contains DNA.
– After overnight incubation, the colonies are tested for DNase production by flooding
the plate with a weak HCL solution.
– The acid precipitates unhydrolyzed DNA.
– DNase producing colonies are therefore surrounded by clear areas due to DNA
hydrolysis.

12. DNase Test
• Requirement
– DNase agar plate, HCL 1N
• Method
– Divide a DNase plate into the required number of areas and label them
– Using a sterile loop or swab, spot inoculate the test and control organisms
– Incubate the plate at 37° C overnight
– Cover the surface of the plate with 1N HCl solution. Tip off the excess acid
– Look for clearing around the colonies within 5 minutes of adding the acid
• Results
– Clearing around the colonies ……. DNase positive strain
– No clearing around the colonies ….. DNase negative strain
• Controls

13. DNase Test

14. Bile Solubility Test
• This test helps to differentiate Streptococcus pneumoniae, which is soluble in
bile and bile salts, from other alpha hemolytic Streptococci (Viridans) which
are insoluble
• Principle
– A heavy inoculum of the test organism is emulsified in physiological saline and the
bile salt sodium deoxycholate is added
– This dissolves Streptococcus pneumoniae as shown by a clearing of the turbidity
within 10-15 minutes
– Viridans and other Streptococci are not dissolved and therefore there is no clearing
of the turbidity
• Requirement
– Sodium deoxycholate, 100 g/L
– Physiological saline

15. Bile Solubility Test
• Tube Method
– Emulsify several colonies of the test organism in a tube containing 2 mL sterile
saline, to give a turbid suspension
– Divide the organism suspension between two tubes
– To one tube, add 2 drops of the sodium deoxycholate reagent and mix
– To the other tube (negative control), add 2 drops of sterile distilled water and mix
– Leave both tubes for 10-15 minutes at 35-37° C
– Look for a clearing of turbidity in the tube containing the sodium deoxycholate
• Results
– Clearing of turbidity …………………………… Probably S. pneumoniae
– No clearing of turbidity ………………………. Probably Not S. pneumoniae
• Controls
– Bile solubility positive control ………………….. S. pneumoniae
– Bile solubility negative control …………………. E. faecalis

16. Bile Solubility Test

17. Bile Solubility Test
• Important
– Bile solubility test can be performed by testing
colonies directly on a culture plate or on a slide
– The tube method is recommended because the
results are easy to read
– Some strains of S pneumoniae are not dissolved
by bile salts
– Occasionally some strains of viridans
streptococci give a positive test

18. Litmus Milk De-colorization Test
• This test is a rapid inexpensive technique to assist in the
identification of Enterococci
• It is based on the ability of most strains of Enterococcus spp
to reduce litmus milk by enzyme action which is shown by
decolorization of litmus
• Principle
– A heavy inoculum of the test organism is incubated for up to 4
hours in a tube containing litmus milk
– Reduction of the litmus milk is indicated by a change in color of
the medium from mauve to white or pale yellow
• Requirements
– Litmus milk medium

19. Litmus Milk De-colorization Test
• Method
– Using a sterile loop, inoculate 0.5 mL of sterile litmus milk
medium with the test organism
– Incubate at 35-37° C for up to 4 hours, examining at half hour
intervals for a reduction reaction
– The reduction is shown by a color change from mauve to white or
pale yellow
• Results
– White or pale yellow-pink color …… Suggestive of Enterococcus
– No change or a pink color …….. Probably not Enterococcus
• Controls
– Positive control …….. Enterococcus spp