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Gram Stain
Bacteria have their unique morphological features which helps them to distinguish from one another. This is better accomplished by techniques that play an important role. These are nothing but staining techniques. Various methods of staining exist.
The first to be dealt is Gram Staining. This technique was invented by a Danish bacteriologist, Christian Gram and is most popular. This method includes the utility of sequence of dyes that enable certain bacteria to become purple while others pink. The bacteria which become purple color on staining are known as Gram –positive while that become pink colored are known as Gram-negative (Tami port, 2010).
The bacterial cell wall is responsible for the stain color to develop(Tami port, 2010). The materials required are Slides, Crystal Violet (the primary stain), water, Acetone alcohol, safranain, staining rack, oil immersion, compound microscope.
Methods
The steps are as follows: Add slowly drops of crystal violet on the surface of the slide completely and for 1 minute allow it to stand. Fill the slide surface with Gram’s iodine and wait for 1 minute.
Following that the slide is rinsed with water. Next, the slide is flooded with iodine and allowed to stand for1 minute. It is then rinsed with water. The slide is flooded with Acetone Alcohol and after standing for 15 seconds, it is rinsed with water. Finally, the slide is flooded with safranin and allowed to stand for 1 minute and then rinsed with water. After this slide should be allowed to dry and observed under compound microscope using an oil immersion (1000x TM).
Result
The Gram positive cells develop purple color, indicating that they have taken up the primary stain. The Gram negative cells develop pink indicating that they have taken up counter stain.
In this test Pseudomonas ssp was identified and it is Gram negative. Therefore, the gram staining procedure helps in identifying the bacteria that may be gram positive and gram negative.
Catalase Test
This test is performed to identify the catalase enzymes by their catalytic action on Hydrogen peroxide to yield oxygen and water.Hydrogen peroxide is generally produced by the bacteria as an oxidative final product of sugars that are degraded aerobically. When hydrogen peroxide gets stored in bacterial cells, the consequence is cell demise.catalse generally, degrades hydrogen peroxide or enables oxidation of secondary substrates. Catalase does not exert oxidizing action on different peroxides.
Materials
Microscope slide, sterile loop (made of nichrome), Bacterial colonies, 3-6% Hydrogen peroxide solution, clean capped test tubes. Procedure: Take a slide and with the help of a sterile loop pick few colonies. These are smeared onto the glass slide. Next, a drop of Hydrogen peroxide solution is added.
The slide is later observed for vigorous air bubbles within 10 -15 seconds. Result: Bubbles generated reflect the presence of catalase in bacterial cells whereas no bubbles reflect a negative test. Therefore, the presence or absence of air bubbles from the bacterial cells placed on the glass slide in a smear form helps us to distinguish the bacteria that are catalase positive or catalase negative.
Oxidase Test
Microorganisms identified by this test yield cytochrome oxidase). This enzyme is involved in the process of electron transfer process by the transport of electrons to oxygen from a molecule that acts like donor. The oxidase reagent possesses a chromogenic reducing agent. This substance when undergoes oxidation changes its color.
When th organism generates cytochrome oxidase, in 15 seconds the oxidase reagent takes up blue or purple or blue color.
Materials
Petri dish, filter paper, N, N, N,N-tetramethylparaphenylene-diamine hydrochloride, sterile loop or wooden stick.
Procedure
Obtain a petridish and keep a small filter paper exactly at the bottom. Add few drops of N,N,N,N-tetramethylparaphenylene-diamine hydrochloride such that the paper turns wet.
Take the sterile loop and pick few colonies. Place them on the filter paper by rubbing. Observe for blue color within 1 – 2 minutes.
Result
Positive culture is detected by color difference.
API system
The API stands for Analytical Profile Index. This test identifies various microorganisms. There are several API products available commercially. Some of them are API 20E, API 20 NE, API 20 Rapid 20 E, API NH, API 20 A etc. The API includes strips which contain small miniaturized tubes.
Materials
Agar plates containing bacterial species, – sterile mineral oil, saline tube, sterile Pasteur pipettes + bulbs, 0.85% NaCl solutions (5ml), API 20E test strip (for oxidase – gram negative rods). API test strip incubation chamber. Kovac’s reagent, 10% FeCl3, Barrett’s reagents A and B, Result sheets.
Methods
Take a saline tube and prepare a suspension of the bacteria. Now, take a large colony with the help of a sterile loop and inoculate bacterium (pure culture) into the 0.85% NaCl solution. Maintain that the suspension is homogenous and devoid any floating bacterial clumps.
In order to quantify the suspension, employ McFarland barium sulfate standard No. 3. for API strip inoculation: Take the strip and handle above the table at a small angle, with the pipette introduce the bacterial suspension into well. At the sides of the cupule touch the pipette end. This enables capillary pressure into well such that fluid enters inside.This is achieved by gentle squeezing of the bulb ,which eliminates bubble formation in wells. Here, care should be taken such that each well filled till the neck.
Incubation of the strip in its chamber
In this step, take incubation chamber and pour water to fill. In the bottom, incubation chamber possesses contains small holes.
The filling of chamber is such that the indentations are just filled. Next, keep the strip in the bottom of the chamber. Take care to avoid much water as the API strip may totally get wet. Now at the bottom keep incubation chamber top and name it. Keep the strip in incubation at 37° C for 18-24 hours (www.biotech.univ.gda).
Reading the strips/interpretation
With the help of supplied result sheets, the results are read. This would create an API number profile on the results sheet. This information can be recorded in a computer or entered in a laboratory manual.
References
- Tami Port. Bacteria Gram Stain Reaction. Test for Gram-positive and Gram-negative Bacterial Identification. Web.
- Common Laboratory Techniques. Web.
- Sputum culture. Web.
- https://biology-forums.com/index.php?acti%20on=dlattach;topic=573.0;attach=253
- Oxidase Test. Web.
- Analytical Profile Index. Web.
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