Biochemical tests for Staphylococcus aureus

Biochemical tests for identification of bacteria

Biochemical tests are actually those tests that are used for the identification of species of bacteria. These tests identify bacterial species on the basis of their biochemical activities. Here, you are going to learn different Biochemical tests for Staphylococcus aureus

Biochemical tests for Staphylococcus aureus:

There is numerous biochemical test that we use to confirm staphylococcus auras. Some of them are;

1- Coagulase test:

Principle:

We use this test to distinguish positive S. auras from negative coagulase staphylococcus. Coagulase is basically an enzyme that is produced by staphylococcus aureus that transforms fibrinogen which is present in plasma into fibrin. This is one of the most important Biochemical tests for Staphylococcus aureus

Procedure:

  • Firstly, we have to emulsify a colony of Staphylococcus in a water drop on a grease-free and clean slide along with minimum spreading.
  • Then make likely suspension of negative and positive strains to confirm suitable plasma reactivity.
  • Incline a flamed and cooled wire into pure plasma then withdraw and mix adhering traces of plasma into staphylococcal suspension on the slide.
  • If clumping appears, then the test is positive otherwise negative.

Biochemical tests for Staphylococcus aureus

Observation:

A clumping of cocci visible to naked eyes starts to appear.

Result:

It indicates the presence of staphylococcus aureus.

2- Catalase test:

Principle:

We usually perform a catalase test to regulate whether gram + cocci is staphylococci or streptococci. Catalase is an enzyme that transforms HO into H2O and O. This is also one of the most important Biochemical tests for Staphylococcus aureus

Procedure:

  • At first, bacteria are gently mixed with hydrogen peroxide
  • Because of the production of oxygen, if a bubble seems to appear, the test is positive.
  • On the other hand, if no bubble appears, the test is negative or it is said to be catalase-negative.

Biochemical tests for Staphylococcus aureus

Observation:

After mixing, the bubble was seeming to appear.

Result:

It means that gram-positive cocci are staphylococcus.

3- Indole production test:

Principle:

Indole production test determines the capability of specific bacteria to decay amino acid to indole which gathers in the medium. Indole is produced by reductive deamination from tryptophan through indole pyruvic acid.

Procedure:

  • First, you have to take sterilized test tubes having 4 milliliters of tryptophan broth.
  • Then aseptically inoculate the tube by taking growth from eighteen to twenty-four hours culture.
  • Set at incubation at 37 degrees centigrade for 24-28 hours.
  • Then add 1/2ml of Kovac’s reagent to broth culture and see the presence or absence of the ring.

Biochemical tests for Staphylococcus aureus

Observation:

The ring starts to appear.

Result:

It indicates the presence of staphylococcus aureus.

 4- Methyl red test:

Principle:

We use this test to determine if the bacteria has the ability to employ glucose along with the creation of stable acid then the color of the methyl orange turns from yellow to red when added to broth culture.

Procedure:

  • First, let the medium equilibrate to room temp. before inoculation
  • Then lightly inoculate the medium by using organisms that were taken from 118-24 hrs. pure culture and aerobically incubate at 37 degrees centigrade.
  • After incubation, place 1ml of broth to clean the test tube and incubate again the remaining broth for 24 hrs.
  • Then add 1-2 drops of methyl red indicator to the test tube containing broth.
  • Then observe the changes in color

Biochemical tests for Staphylococcus aureus

Observation:

The orange color of the methyl indicator starts changing from yellow to red.

Result:

The red color signifies a positive test and indicates the presence of staphylococcus aureus.

5- Citrate test:

Principle:

This test is performed to determine the capability of organisms to employ citrate as the only carbon source.

Procedure:

  • First, by touching the tip of a needle for 24 hrs. old colony, inoculate Simmons citrate agar lightly on a slant, and then incubate it at 37 degrees centigrade.
  • Then after incubation, observe the changes in color.

Biochemical tests for Staphylococcus aureus

Observation:

The intense Prussian blue color starts to develop.

Result:

This indicates the presence of s. aureus.

6- Gram staining test:

Principle:

We use this test to determine the capability of the bacterial cell wall to retain crystal violet dye all through solvent treatment. The cell wall of gram-negative bacteria has lower peptidoglycan and higher lipid content than gram-positive bacteria. The cell wall of bacteria is stained by crystal violet.

Procedure:

  • Firstly, you have to make a slide of the cell sample to be stained and then heat fix it through the Bunsen burner 3 times.
  • The second step is to add a primary stain to the slide and incubate it for 1 min.
  • After rinsing the slide with a gentle stream of water, add mordant as gram’s iodine for 1 min.
  • Then again rinse the slide with alcohol for 3s and wash with a gentle stream of water. If the sample is gram-positive then alcohol decolorize the sample
  • After adding secondary stain (safranin), incubate the sample for 1 min and wash again with water for 5s.
  • If a bacterium is gram-negative, it will lose the primary stain and change into a secondary stain causing it to appear red when observing under a microscope.
Biochemical tests for Staphylococcus aureus
Gram Staining Procedure

Observation:

It is observed that bacteria will not lose their primary stain.

Result:

It means it’s a gram-positive staphylococcus bacterium.

7- Oxidase test:

Principle:

The oxidase test works on the principle that few bacteria form indophenol blue from the oxidation of dimethyl-p-phenylenediamine and alpha naphthol. Cytochrome oxidase, the N-dimethyl-p-phenylenediamine oxalate, and alpha naphthol react to indophenol blue.

Procedure:

  • First, take a filter paper that is saturated with substrate tetramethyl-p-phenylenediamine dihydrochloride and moisten it with sterile water.
  • Then with a wooden loop, pick the colony which is to be tested and smear it in filter paper and observe the color change of the inoculated area of the paper

Biochemical tests for Staphylococcus aureus

Observation:

Deep blue color starts to appear within 10-20s.

Result:

It signifies a positive test and indicates the presence of S. aureus.

8- Nitrate reduction test:

Principle:

We use this test to detect nitrite and determine its ability to produce a red compound after reaction with sulfanilic acid to make a complex that further reacts with alpha naphthylamine to produce a red precipitate.

Procedure:

  • In the first step, there is the inoculation of nitrate broth with the suspension of bacteria.
  • Then tubes are incubated at 37 degrees centigrade for 24 hrs.
  • Before adding reagent, we will look for nitrogen gas to produce.
  • Then we will add 6-8 drops of nitrite reagent A&B and observe the color change.
  • We will add zinc powder if no color develops.

Nitrate reduction test

Observation:

We will observe that the cherry red color starts to develop.

Result:

It indicates the presence of staphylococcus aureus.

9- Urease test:

Principle:

We use this test for the identification of those bacteria that has the ability to hydrolyze urea to generate carbon dioxide and ammonia.

Procedure:

  • At first, we will streak urea agar slant surface with part of the well-isolated colony.
  • Then incubate it at 35-37 degrees centigrade for 48 hrs. to seven days.
  • Then we will examine the color change for as long as seven days.

Urease test

Observation:

We will notice that within seven days, the pink color starts to develop.

Result:

It indicates the presence of S. aureus.

10- Voges Proskauer test:

Principle:

We use this test to determine the ability of organisms to produce acetyl methyl carbinol from the fermentation of glucose.

Procedure:

  • Before inoculation, let the medium equilibrate at room temperature.
  • Then lightly inoculate the medium by using an organism taken from pure culture and aerobically incubate at 37 degrees centigrade.
  • After incubation, place 2ml broth to clean the test tube and reintubate the remaining broth.
  • Then add six drops of 5% alpha-naphthalol and gently mix to aerate.
  • After adding two drops of 40% KOH and again mix to aerate.
  • Then shake vigorously and observe the development of pink color.

Voges Proskauer test

Observation:

The pink color starts to develop.

Result:

The pink-red color at the surface indicates the presence of s. aureus.

See also How does PCR works?

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