Question

Step by step procedure of gram staining.

Answer 1

Materials Required:

1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Lens paper and lens cleaner
7. Immersion oil
8. Distilled water
9. 18 to 24 hour cultures of organisms

Reagents:

1. Primary Stain - Crystal Violet
2. Mordant - Grams Iodine
3. Decolourizer - Ethyl Alcohol
4. Secondary Stain - Safranin

Procedure: Part 1: 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 spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until ready for use.

Part 2: Labeling of the slides

Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to clearly designate the area in which you will prepare the smear. You may also label the slide with the initials of the name of the organism on the edge of the slide. Care should be taken that the label should not be in contact with the staining reagents.

Part 3: Preparation of the smear

• Bacterial suspensions in broth: 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 to about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.
• Bacterial 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.

Please note: 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 visualize the morphology of single cells. Smears typically require only a small amount of bacterial culture. An effective smear appears as a thin whitish layer or film after heat-fixing.

Part 4: Heat Fixing

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.

• Allow the smear to air dry.
• After the smear has air-dried, hold the slide at one end and pass the entire slide through the flame of a Bunsen burner two to three times with the smear-side up.

Now the smear is ready to be stained.

Please Note: Take care to prevent overheating the slide because proteins in the specimen can coagulate causing cellular morphology to appear distorted.

Part 5: 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 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 bibulous paper.
    
11. View the smear using a light-microscope under oil-immersion.