How do you do Gram staining and why is it useful Why is surf

How do you do Gram staining and why is it useful? Why is surface area to volume ratio important? How does this affect the size of a cell?

Solution

Grams straining procedure:

The method was developed by the Danish scientist Hans Christian Gram.

1. Preparation of heat fixed slide :
Bacterial suspensions : With a sterile cooled loop, place a loopful of the broth culture on the slide. Spread by means of circular motion of the inoculating loop A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.
Plate cultures: With a sterile cooled loop, place a drop of sterile water or saline solution on the slide. Sterilize and cool the 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 to create an emulsion.
Swab Samples: Roll the swab over the cleaned surface of a glass slide.

Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the sample to more readily take up stains.
  
2. Gently flood smear with crystal violet and let stand for 1 minute.

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

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 or distilled water using a wash bottle. The smear will appear as a purple circle 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 water.

8. Gently flood with safranin to counter-stain and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

10. Blot dry the slide with blotting paper.

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

Uses: It is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.

Post the other question as a seperate one.