OXIDASE

Oxidase

The oxidase test identifies organisms that produce the enzyme cytochrome oxidase by Gram- negative bacteria. Cytochrome oxidase involves in the electron transport chain by transferring electrons from a donor molecule to oxygen. It is a hallmark test for the Neiserria. It is also used to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other Enterobacteriaciae.
The test can be carried out simply by swabbing some of the test culture into one of the boxes on an oxidase dry slide. If there is a colour change of purple or blue at 30seconds – 1 minute; then the result is positive.



microbiology.scu.edu.tw/micromicrobe-exp/exp_website/Exp_30.htm

http://web.fccj.edu/~lnorman/unknowns.htm?index=2

CATALASE

Catalase

Catalase test is done to detect the presence of catalase enzymes. This enzyme is responsible for protecting bacteria from the accumulation of hydrogen peroxide (H2O2), which can occur during aerobic breakdown of sugars. If allowed to accumulate, it is highly toxic to the organism and can result in death. Catalase will break down H2O2 into water and O2.
Chemical equation for the breakdown of hydrogen peroxide:
2H2O2 → 2H2O + O2
Catalase test can be done by smearing a small amount of the test organism onto the lid of a petri plate/ culture dish. Then add a drop of hydrogen peroxide to the smear. If bubbles can be seen, which is O2, then the test is positive and it can be concluded that the organism makes catalase. A lack of bubbles shows that there is an absence of catalase.

www.usi.edu/Science/biology/ceseeley/cases/catc.html
The positive result is above, top. The negative result is above, below the positive.


http://web.fccj.edu/~lnorman/unknowns.htm?index=2

Bubbling upon the addition of hydrogen peroxide is indicative of the presence of catalase for this organism.

GELATINE UTILIZATION

Gelatine Utilization

Gelatine Utilization is used to test if bacteria can digest the Protein Gelatine. An enzyme called the Gelatinase must be produced by the bacteria to digest Gelatin. A transfer needle should be used to stab the gelatine, to inoculate the media. The inoculated media should be incubated for at least 48 hours. Then it should be transferred to the refrigerator. The media should be completely chilled before it is taken out for observation. If the media is solid after refrigeration it means that the result is negative. This means that the bacteria did not digest the gelatine. However, if the media is still in a liquid state after refrigeration, the test result is positive. This means that the bacteria has digested the gelatine.

http://web.fccj.edu/~lnorman/images/gelatin.jpg

The 'Serratia marcescens' on the left is positive for gelatinase production, as evidenced by the liquidation of the media. The 'Salmonella typhimurium' on the right is negative, as evidenced by the solidity of the media.

STARCH HYDROLYSIS

Starch Hydrolysis

Starch hydrolysis is a test used to detect the enzyme Amylase, which breaks down starch. After incubation, the plate is treated with Gram’s iodine. If the starch has been hydrolysed, then a reddish colour of clear zone will be formed around the bacterial growth. However, if the starch has not been hydrolysed, a blue or black area will be formed indicating the presence of starch. This test can be easily conducted by using an inoculating loop to spread bacteria on the plate surface. After it is incubated at an appropriate temperature, iodine will be added to the surface. Then, the colour change should be noted to know the results.

http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Starch%20Hydrolysis%20Medium.html
A. Absence of starch hydrolysis is indicated by the blue colour that completely surrounds the colony.

http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Starch%20Hydrolysis%20Medium.html
B. Starch hydrolysis is indicated by the reddish colour of clear zone.

CARBOHYDRATE UTILIZATION

Carbohydrate Utilization

When carbohydrates are fermented by bacteria, they produce acidic products. A change in pH can be detected when fermentation of a given carbohydrate has occurred. Acids lower the pH of the medium which will cause the pH indicator of Phenol Red to turn Yellow. When bacteria does not ferment the carbohydrate, the media remains red. Sometimes during fermentation, gas is produced. The Durham tube will then have a gas bubble trapped within it. There are three carbohydrate tests that can be performed. They are the Glucose (Dextrose) test, Lactose Test and the Sucrose Test. In all of these tests, the bacteria will be inoculated to the medium using a transfer loop. The results obtained will be similar to that illustrated in the picture below. The left tube shows less acid formation than the far right tube, but gas is still produced in this case. The Centre tube shows no carbohydrate utilization as there is neither any acid or gas produced. The medium remains the same. The Right tube shows acid is produced as evidenced by the yellow colouration; and gas is produced (an air bubble is trapped in the Durham tube).

http://web.fccj.edu/~lnorman/images/carbohydrate.jpg

www.blinn.edu

UREA

Urea

This is a test to detect the enzyme Urease which breaks down urea into ammonia. The liquid media is inoculated with a transfer loop. Since Ammonia is base, it will change the pH of the media. This change can be detected by using a pH indicator. In this case, the indicator being used is phenol red. Positive Results: The medium’s colour changes from yellow to bright pinkish-red; which indicates the presence of the enzyme Urease. Negative Results: The medium remains yellow.

http://www.bch.msu.edu/

www.bch.msu.edu

METHYL RED TEST

Methyl Red Test

This is a test to determine if glucose can be converted into acidic products. Bacteria is inoculated into a tube with a sterile transfer loop. The tube is incubated at 35°C between 2-5 days. After incubation, 2.5ml of the medium is transferred to another tube and five drops of the pH indicator methyl red is added to this tube. Thereafter, it is gently stirred, and any colour changes is noted down. Positive Results: Red colouration (Glucose is converted to acidic products). Negative Results: Yellow-orangish colour (Glucose is converted to neutral products)

http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/imvic.html

INDOLE PRODUCTION

Indole Production

This is a test to determine if bacteria can breakdown amino acid tryptophan into Indole.
SIM media or TSB (Tryptic soy broth) is inoculated using a transfer loop. Kovac's reagent is added to media after 48 hours of incubation, to determine if Indole is produced by the bacteria. Positive Results: A Red layer forms on top of media (It signifies that the bacteria is able to break down the tryptophan to form Indole). Negative Results: No red layer will be formed.

http://www.microbiology.scu.edu.tw/

www.microbiology.scu.edu.tw

CITRATE UTILIZATION

Citrate Utilization:

This tests for the ability of bacteria to convert Citrate, which is an intermediate of the Kreb’s Cycle into Oxaloacetate, which is another intermediate of the Kreb’s Cycle. In the media being used, Citrate is the only Carbon source available for usage for the survivial of the bacteria. If Citrate is not utilized, the bacteria will not grow. However, if the Citrate is utilized, the bactaria will grow on the media slant, and cause it to turn bright blue; as a result of the increase in pH. A transfer loop has to be utilized to transfer the bacteria onto the medium.

http://web.fccj.edu/~lnorman/images/citrate.jpg

http://web.fccj.edu/~lnorman/images/citrate.jpg