Question

Suppose that you have an unknown culture that is growing within your culture dish. You wish to stain it in order to identify it. Describe, in full detail, the process you would go through to make a slide for that culture, and which staining technique you would use. (Hint: which stain would give you the most information about morphology and specific properties)

Answer 1

Staining techniques are done to visualise the morphological characters of microorganisms under a microscope.

For identification of unknown bacteria, Gram Staining is widely used. Gram staining is essential procedure used in identification of bacteria and is frequently the only method required for studying morphology. The Gram stain differentiate bacteria into two broad groups. Gram positive bacteria are those that resist decolourisation and retain primary Stain, appearing violet. Gram negative bacteria are decolourised by organic solvent and therefore taking counter stain appear red.

Gram staining Procedure :

​​​​Materials required

1. Clean glass slides

2. Inoculation loop

3. Bunsen burner

4. Bibulous paper

5. Microscope

6. Lens paper and lens cleaner

7. Immersion oil

8. Distilled water

9. Culture of organism.

Reagent for Gram Staining

1. Primary Stain - Crystal violet

2. Mordant - Grams iodine

3. Decolourised - Ethyl alcohol

4. Secondary Stain - Safranin

Procedure:

A. Preparation of the glass microscopic slide:

Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides with alcohol. After cleaning, dry the slides and place them on laboratory towel until ready for use.

B. Labelling of the slides

Drawing a circle on the underside of the slide using a glassmarker pen may be helpful to clearly designate the area in which you will prepare the smear. Care should be taken that the label should not be in contact with the staining reagents.

C. Preparation of Smear

With a sterile cooled loop, place a drop of sterile water or saline solution on the slide. Sterilize and cool loop again and pick up a very small sample of a bacterial colony and gently stir into the drop of water/ saline on the slide . Spread the smear with the loop to make it thin.

It is very important to prevent preparing thick, dense smears which contain an excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass through, thus making it difficult to visualise the morphology of single cells. An effective smear appears as a thin whitish layer or film after heat fixing.

D. Heat fixing

Heat fixing kills the bacteria in the smear, firmly adhere to slide, and allows the sample to more readily take up stains. Allow the smear to stir dry, after hold the slide at one end and pass the entire slide through the flame of a Bunsen burner two or three times with the smear side up. Now the smear is ready to be stained.

Take care to prevent over heating the slide because protein in the specimen can coagulate causing cellular morphology to appear distorted.

E. Gram stain procedure

1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with Crystal violet and let stand for 1 minute

3. Tilt slide slightly and gently rinse with tap water.

4. Gently flood the smear with Gram's iodine and let stand for 1 minute.

5. Tilt the slide slightly and gently rinse with tap water . The smear will appear as a purple colour on the slide.

6. Decolorize using 95% Ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over decolorize.

7. Immediately rinse with distilled water.

8. Gently flood with Safranin to counter and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water.

10. Blot dry the slide with bibulous paper.

11. View the smear using a light microscope under oil immersion.