Biochemical Tests for Gram Positive Bacteria

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.

Catalase TestRequirements

– 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

Catalase Test

Negative

Positive

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

Coagulase TestThis 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

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

– Positive coagulase control ………………………… Staphylococcus aureus– Negative coagulase control ………………………. Staphylococcus epidermidis

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

Coagulase Test

Positive

Negative

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

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.

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

– Positive DNase control …….. Staphylococcus aureus– Negative Dnase control …… Staphylococcus epidermidis

DNase Test

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

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

Bile Solubility Test

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

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

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– Negative control …… Viridans streptococci