Practical 06: Gram Staining and microscopic examination of microbes.

Objective:

This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and (2) Gram-negative microorganisms that take the color of the counterstain (usually Safranin).

Principle of Gram Stain

The two major groups of bacteria can be divided into gram-positive and gram-negative. The Gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups.

Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a complex within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue.

The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal violet and iodine, the CV-I complexes are not trapped within the peptidoglycan. Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink.

Materials Required

Clean glass slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope, Lens paper and lens cleaner, Immersion oil, Distilled water, 18 to 24 hour cultures of organisms

Reagents:

1. Primary Stain - Crystal Violet

2. Mordant - Grams Iodine

3. Decolorizer - 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 the 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

• Place slide with heat-fixed smear on staining tray.

• Gently flood smear with crystal violet and let stand for 1 minute.

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

• Gently flood the smear with Gram’s iodine and let stand for 1 minute.

• 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.

• 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.

• Immediately rinse with water.

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

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

• Blot dry the slide with bibulous paper.

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

Limitations

1. Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results.

2. Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult. Gram-negative organisms may not decolorize properly.

3. Cultures older than 16 to 18 hours will contain living and dead cells. Cells that are dead will be deteriorating and will not retain the stain properly.

4. The stain may form a precipitate with aging. Filtering through gauze will remove excess crystals.

5. Gram stains from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity due to the successful treatment.

6. Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will not grow in culture. Some strains are obligate anaerobes.

7. Faintly staining Gram-negative organisms, such as Campylobacter and Brucella, may be visualized using an alternative counterstain (e.g., basic fuchsin).