Biochemical Tests

With the advent of microbial identification with the MALDI-TOF Mass spectroscope many of these simple biochemical tests have become redundant. However, we still include them in this manual because not all laboratories have access to a MALDI-TOF MS. For many of the tests, commercial kits are available and techniques will vary somewhat between brands. Hence the actual detail of the procedure may differ from that in the videos. Always check the pack inserts for correct procedure. 

On this page:

CATALASE TEST

OXIDASE TEST

COAGULASE TEST

BILE SOLUBILITY TEST

NITRATE REDUCTASE TEST

PYR TEST

X AND V FACTOR TESTING FOR HAEMOPHILUS SPP.

SERUM SUGARS for Neisseria species

Other tests:

• Oxidation/fermentation
• Urease
• ONPG
• Methyl red/Voges Proskauer test

Details of these tests can be found in the media section.

CATALASE TEST

The catalase test is extremely useful in the differentiation of bacteria, particularly gram positive bacteria. It is based on the possession of the enzyme, catalase, which catalyses the reaction:

2H₂O₂ ® 2H₂O + O₂

The oxygen is given off as a gas which creates bubbles in the reagent. Hence a positive catalase test results in bubbling of the reagent (H₂O₂), while a negative test does not show bubbling. The enzyme is involved in the detoxification of hydrogen peroxide, which is formed from oxygen radicals.

For further information go to http://www.microbelibrary.org/index.php/library/laboratory-test/3226-catalase-test-protocol

REAGENT

3% H₂O₂, prepared as follows:
H₂O₂ stock solution, 30% w/v 10ml
MQ water 90ml

Store at 4^oC in a light proof bottle.

METHOD

1. Pick up a portion of a colony with a loop and smear it on a clean dry slide. Add a drop of catalase reagent and observe for bubbles. Red blood cells also possess catalase, so care must be taken when testing colonies from blood agar plates, so that false positives do not occur.
2. Alternatively, a drop of reagent can be added directly to a colony on a nutrient agar plate.

RESULTS

Gas Bubbles Observed         Positive
No Gas Bubbles                    Negative

CONTROL ORGANISMS

Positive Staphylococcus aureus
Negative Streptococcus pyogenes (group A Streptococci)

Catalase test video

OXIDASE TEST

This test detects the presence of the enzyme, cytochrome oxidase, which is the terminal enzyme in the electron transport chain and indicates that the bacteria which possess this enzyme are capable of aerobic respiration for the production of energy. Organisms which are oxidase positive may be able to grow anaerobically if they possess alternative anaerobic fermentative pathways for energy production, but when growing in air, these organisms will preferentially use oxidative pathways to produce energy. Organisms which are oxidase negative, cannot utilise oxidative phosphorylation to produce energy, but can use anaerobic fermentative pathways and may be able to grow either aerobically or anaerobically depending on whether oxygen is toxic to them or not. This usually depends on the presence of catalase and superoxide dismutase enzymes.
The test is useful in the differentiation of gram negative bacilli and relies on the development of a colour change in the reagent by oxidase positive organisms.

REAGENT

N,N,N’,N’, tetramethyl-p-phenylenediamine, made up as a 1% w/v aqueous solution, is used to soak filter paper strips (approximately 1x3 cm) which are then dried and stored in a dark container. The reagent is subject to auto-oxidation and colour change if exposed to light.

METHOD

Remove a filter paper strip from the container using forceps. Fold the end at right angles to the rest of the strip. Touch the paper onto a colony on the plate, lift off and observe for a deep purple colour to develop within 5-10 seconds for a positive result. If the paper does not turn purple within this time, the test is negative, even though the paper may turn purple after a few seconds more. Ultimately the reagent will auto-oxidise and turn purple with time, and it will also auto-oxidise if the colonies are inoculated to the paper with a nichrome wire loop.
Dispose of the strip appropriately as it is contaminated.

For further information go to http://www.microbelibrary.org/index.php/library/laboratory-test/3229-oxidase-test-protocol

RESULTS

Colour change to purple within 5-10 seconds     Positive
No or slow colour change                                   Negative

CONTROL ORGANISMS

Positive Pseudomonas aeruginosa
Negative Escherichia coli

Oxidase test video

NOTES

• Do not perform an oxidase test on colonies growing on media containing glucose. Glucose fermentation will inhibit oxidase enzyme activity, resulting in false negatives. Suitable media include nutrient and blood agar plates, but not MacConkey or XLD plates.
• False negatives may occur if a culture contains both Pseudomonas and Neisseria spp. Oxidase-positive pseudomonads produce an inhibitor of Neisseria oxidase. Be aware of this in respiratory specimens which may contain both genera.
• An alternative method for the test is to pick up colonies from the plate and smear them on the test strip. This avoids possible contamination of the plate. However, nichrome in the standard loops will auto-oxidise the reagent so wooden applicators or platinum loops MUST be used.

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COAGULASE TEST

Production of coagulase is an important method for distinguishing Staphylococcus aureus from other Staphylococci. S. aureus produces 2 forms of coagulase: bound and free. The tube coagulase test detects both bound and free coagulase, while the slide test detects bound coagulase or clumping factor. Not all S. aureus strains produce the bound form of the enzyme and hence the tube test is considered definitive. The basis of the test is that the enzyme initiates the coagulation cascade in plasma to produce visible clumps (in the slide test) or a gel of the plasma (in the tube test). Most laboratories now utilise a latex co-agglutination test to detect coagulase positive Staphylococci. See the section on commercial identification tests.

REAGENT

Rabbit plasma (citrated), diluted 1/5 in sterile saline for the tube test, and undiluted in the slide test.

METHOD

1. TUBE TEST

Add 0.5 ml of diluted citrated rabbit plasma to a small glass test tube. Emulsify the colony of interest in the plasma. Incubate at 37^oC in a water bath or heating block. Examine at 30 minute intervals until gelling occurs. Test by tipping the tube at 45^o to determine if the plasma has solidified or not. Leave for up to 6 hours before discarding as negative.

2. SLIDE TEST

Emulsify a colony of interest in a drop of sterile saline on a clean microscope slide. Mix in a loopful of undiluted rabbit plasma. Observe for visible clumping.
Some strains of Staphylococci may not emulsify completely and appear clumped before the addition of plasma. These strains need to be tested by the tube method.

RESULTS

Gelling or clumping in plasma     Positive
No gelling or clumping                Negative

CONTROL ORGANISMS

Positive Staphylococcus aureus
Negative Staphylococcus epidermidis

For further information and photographs of tube and slide tests go to: http://www.microbelibrary.org/index.php/library/laboratory-test/3220-coagulase-test-protocol

BILE SOLUBILITY TEST

This test is used to differentiate Streptococcus pneumoniae from other alpha haemolytic Streptococci such as “viridans” species. Colonies of S. pneumoniae are lysed by solutions of bile salts such as sodium desoxycholate whereas other species of Streptococci are not affected.

REAGENT

10% w/v sodium desoxycholate

METHOD

Add a drop of the reagent directly to suspect colonies on an agar plate. Incubate at 37^oC for 15 minutes.

RESULTS

Colonies dissolve (disappear)     Positive
No change to colonies                Negative

CONTROL ORGANISMS

Positive Streptococcus pneumoniae
Negative “viridans” Streptococci

Bile solubility test video

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NITRATE REDUCTASE TEST

Many micro-organisms can reduce nitrate to nitrite and many can further reduce the nitrite to nitrogen gas or other nitrogenous compounds.

NO^3- → NO^2- → N₂

Nitrate reduction is a characteristic feature of the members of the Enterobacteriaceae and is a useful differentiating test for other non fastidious gram negative bacilli.
The test initially relies on the detection of nitrite by the addition of the 2 colour generating reagents (nitrate A and B). If however, this is negative, it may be either because nitrate has not been reduced OR nitrite has been completely consumed in producing nitrogen gas. To distinguish between these 2 possibilities, zinc powder is added, which in the acidic conditions, will reduce any residual nitrate remaining to generate nitrite, which in turn will produce a colour change.

REAGENTS

0.2% w/v aqueous sulphanilamide - acidified (nitrate A)
0.1% w/v aqueous naphthylethylene diamine dihydrochloric acid (nitrate B)
zinc powder
nutrient broth containing 0.1% w/v KNO₃

METHOD

Grow the organism of interest at 37^oC overnight in the nitrate broth.
Add 2 drops of nitrate A reagent (which contains acid) followed by 2 drops of B reagent. Observe for the development of a deep pink colour. If no colour has developed within 5 minutes, add a small amount of zinc powder. Allow the powder to settle and observe for the deep pink colour again.

RESULTS

Colour change after addition of nitrate A and B only = nitrate reductase positive
No colour change after reagents or zinc = nitrate reductase positive and nitrite reductase positive
No colour change after addition of nitrate A and B but colour change develops after addition of zinc = nitrate reductase negative

CONTROL ORGANISMS

Positive Escherichia coli
Negative Acinetobacter baumannii

PYR TEST

This test detects the presence of the enzyme, pyrrolidonyl-peptidase and is useful in the differentiation of Enterococci (positive) from Lancefield group D Streptococci (negative).
In the test, the substrate for the enzyme, L-pyrrolidonyl-beta-naphthylamide is cleaved and the resultant naphthylamine forms a red coloured compound on addition of the developer containing p-dimethylamino-cinnamaldehyde.

REAGENTS

PYR reagent 0.7%w/v solution of L-pyrrolidonyl-beta-naphthylamide.
Developer 0.01%w/v p-dimethylamino-cinnamaldehyde

PROCEDURE

1. Place a clean filter paper disc presoaked in PYR reagent in the lid of a petri dish. Moisten with sterile saline.
2. Pick up a colony to be tested and inoculate to the filter paper.
3. Leave at room temperature for 5-10 minutes.
4. Place a drop of colour developer on the disc.
5. Leave for a further 10 minutes.

RESULTS

POSITIVE Pink colour develops
NEGATIVE no colour change or yellowish

CONTROL ORGANISMS

POSITIVE Enterococcus faecalis
NEGATIVE Streptococcus agalactiae (Lancefield group B)

PYR test for Streptococci video

X AND V FACTOR TESTING FOR HAEMOPHILUS SPP.

Species of bacteria in the genus Haemophilus have a growth requirement for either X (haem) or V (NAD/NADP) factor or both. Dependence on these factors is used to distinguish between the various species in the genus. Both factors are supplied in chocolate agar and hence this medium is not suitable for this test. Some X factor may be present in blood agar plates (there are always some lysed red cells present) and hence this medium is not suitable either. The test must be carried out on Columbia agar plates without added blood.

1. Make a suspension of the bacteria to be tested in 1 ml sterile saline using several colonies. The suspension should be visibly turbid.
2. Dip a swab into the suspension and use it to make a lawn of bacteria on the plate. Dispose of the swab appropriately. Alternatively, add 0.1ml of the suspension to the plate and make a lawn with a flame sterilized spreader.
3. Add a disc of X factor, a disc of V factor and a disc of X+V factor to the plate in a triangle with the discs about 3cm apart.
4. Incubate the plate overnight at 37^oC in the CO₂ incubator.

RESULTS

 
You should not get growth around all 3 discs with the same organism. If you do, it may indicate a contaminant.

X and V factor testing video

SERUM SUGARS

Used to differentiate between species of Neisseria. No longer available as a commercial product. Previously consisted of a set of four tubes containing glucose, lactose, maltose and sucrose respectively in a base medium of serum enriched agar. Contains a pH indicator to indicate fermentation of the sugar via a colour change. Organisms are inoculated to the slope of the agar and stabbed into the base of the tube, then incubated at 37^oC overnight. Colour change from pink to yellow indicates a positive.

Serum Sugars video