Nematodes

Microbiological tests for Bacterial classification or identification

1. Gas production

In this test, hydrogen sulfide will be produced if the test is positive

Procedure

• Add lactose/sugar solution in a test tube.
• Sterilize sugar with tinala/water bath.
• Inoculate bacteria in it.
• Place an inverted Derham tube in the test tube for trapping gas bubbles.
• Incubate for 18-24 hours at 37oC.

Result:

If gas is produced test is positive.

2.  Eijkman test

• Inoculate bacteria in a medium at 42-44oC.
• This test is specific for E.coli because E.coli has potential to grow at this range of temperature.

3. Casein test

• Take casein milk agar.
• Inoculate bacteria in it (inoculate only 1-2 colonies)
• Incubate at 37oC.

Result

If the milky appearance of agar disappears then bacteria will be casein positive, otherwise, it is negative.

 4. Catalase test

Staphylococcus aureus is positive for this test but Streptococcus aureus is negative for this test.
Catalase convert hydrogen peroxide into water and oxygen molecule.

Slide test

• Take 1% hydrogen peroxide (1-2 drops).
• Take bacterial colonies and inoculate it.

Result

After 10-15 seconds, if bubble formation starts, bacteria is positive for this test, otherwise, it is negative. (Bubbles are of oxygen molecule).

Test tube test

•  Inoculate bacteria in the Tween-80 agar solution.
• Add 0.5 ml of 1% hydrogen peroxide. 

Result

After 10-15 seconds, if bubble formation starts, bacteria is positive for this test, otherwise, it is negative. (Bubbles are of oxygen molecule).

 5. Coagulase test

 This test is specific for Staphylococcus aureus.

Slide test

• Take serum and inoculate bacteria in it.

Result

If agglutination occurs, test will be positive,, otherwise it is negative.

6. Gelatin test

 This test is used to check the motility of bacteria.
In initial stages of this test practice, gelatin was used which is solidifying material. Now we use bacterial agar instead of gelatin. Staphylococcus aureus decomposes gelatin and makes it liquid. All bacteria will mix in it and we can not identify the motility of bacteria. This is why nowadays we use bacteria agar.

Procedure

• Pour gelatin in a test tube (solid or semi-solid).
• Inoculate bacteria in it.
• Incubate bacteria in it at room temperature for 18-24 hours and check the bacterial growth.

Result

If gelatin turns into liquid, bacteria is positive for gelatin.
If remains in solid or semi-solid form, bacteria is negative for gelatin.

7. Indole test

• Make a solution of peptone water.
• Inoculate bacteria in it.
• Incubate it for 24 hours at room temperature.
• Then add acid Kovac reagent in it.

Result

If pink color appears after the addition of reagent, then bacteria is indole positive, otherwise it is negative.

 8. Lipolytic activity

• Take tributyrin agar.
• Inoculate bacteria in it
• Incubate for 24 hours at room temperature.

Result

  If the media becomes black, then the test is positive. Otherwise, it is negative.

 9. Methyl red test

• Prepare media of peptone water and glucose.
• Inoculate bacteria in it.
• Incubate for 24 hours at room temperature.
• Add methyl red in it as an indicator.

 Result

 If the color becomes red after adding an indicator, then the test is positive otherwise the test is negative.
Also, check the end product either acidic or basic.
 MR positive test is inversely proportional to the VP negative test.
Red color => acidic product and pH is less than 7 = MR positive
 VP+  = High pH => basic product and pH is greater than 7

 10. VP/ Voges-Proskauer

•  Prepare glucose and peptone water media.
• Incubate bacteria in it.
• Incubate for 24 hours at room temperature.
• Add indicator: Berrot reagent in it.

Result

If yellow or pink color is produced, test is positive, otherwise it is negative.

  11. Citrate test

• Take Simon citrate agar.
• Inoculate bacteria on it.
• Incubate for 24 hours at room temperature.

Result

If metallic color around the colony is produced, then test is positive, otherwise it is negative.

 12. Starch hydrolysis test

• Take starch agar.
• Inoculate bacteria in it.
• Incubate for 18-24 hours at room temperature (37oC).
• Add indicator; Iodine

Result

If there is a clear zone around the streaking then the test is positive, otherwise, it is negative.

Indicator

Basically, it is used for a liquid medium. Put iodine on a clear zone after inoculation. If blue color appears on the plate except for surrounding streaks then test is positive, otherwise, it is negative.