Gram Staining Method
Introduction
This staining technique helps to determine gross morphology of the bacteria and differentiation of bacteria into two groups (a) Gram positive and (b) Gram negative. This differentiation is helpful in determining the subsequent biochemical tests and media for their culture in the laboratory.
The staining technique involves following steps – (a) Preparation of smear (b) Fixing of the smear by heating (c) Staining with crystal violet (d) Treatment with mordant (e) Treatment with alcohol or acetone (f) Staining with counter stain (safranin or basic fuchsin).
Principle
- The air dried fixed smear of bacteria, picks up stain and looks purple, when treated with the crystal violet stain (Gram stain) and iodine. Iodine enhances the staining reaction between the dye (crystal violet) and the internal cellular contents of bacteria.
- The alcohol (or acetone) treatment decolorizes Gram negative bacteria while Gram positive bacteria retain the color (purple).
- Counterstaining with safranin (or basic fuchsin) stains the Gram negative bacteria red.
- Under oil immersion microscopic examination, Gram negative organisms appear red & Gram positive organisms appear purple in color.
Requirements
- Different types of specimens and laboratory cultures which contain Gram negative and Gram positive bacteria can be useful for Gram staining method.
- Glass slides and cedar wood oil.
- Nichrome loop and Bunsen burner.
- Microscope.
Reagents
- Crystal violet stain
- Solution A
- Crystal violet : 2 g
- Ethyl alcohol : 20 ml
- Solution B
- Ammonium oxalate : 0.8 g
- Distilled water : 80 ml
- Solution A
Mix solutions A and B. Keep for 24 hours and filter. Store in an amber colored dropping bottle.
2. Gram’s iodine solution
- Iodine : 1.0 g
- Potassium iodide : 2.0 g
- Distilled water to : 100 ml.
Store in amber-colored dropping bottle.
3. Decolorizer
Mix 95% alcohol and acetone in equal proportion. (if alcohol is not available rectified spirit can be used.) Store in a white dropping bottle.
4. Safranin solution
- Safranin ‘O’ : 0.34 g
- Absolute alcohol : 10 ml.
- Distilled water : 90 ml
Filter and store in an amber colored dropping bottle.
Stability of the Reagents
All the reagents are stable at room temperature (25°C + 5°C).
Procedure of Gram Staining Method
- Smear preparation
- Take a grease-free dry slide and make an oval shaped mark at the center by using a glass marker.
- Sterilize the inoculating (nichrome) loop on the flame of a bunsen burner.
- Transfer a loopful of culture (or specimen) by the sterile nichrome loop and make a smear in the premarked area on the slide (smear should not be very thin as well as very thick).
- Allow the smear to dry in the air.
- Fix the dry smear by passing the slide 3 to 4 times through the flame quickly with the smear side facing up.
- Gram Staining Procedure
- Place the slide on the staining glass rods.
- Cover the smear with crystal violet stain and leave for 1 minute.
- Wash carefully under running tap water.
- Flood the smear with Gram’s iodine solution and wait for one minute.
- Drain off the iodine.
- Decolorize the smear with alcohol-acetone (or rectified spirit) for 20 to 30 seconds. Continue till purple stain just stops coming on the slide.
- Gently wash the slide under running tap water and drain completely.
- Counterstain the smear with safranin for 10 seconds or with dilute (1:20) basic fuchsin for about 1 minute.
- Drain the staining solution and allow the stained smear to dry in air (or dry it carefully by using a blotting paper).
- First observe for uniform stained area under low power objective, afterwards under high power objective, and finally under oil immersion objective.
Precautions
It is necessary to standardize the time for steps 5 and 6. Variations in these timings may lead to false results.
Results
The staining results of Gram stain are as follows:
| 1 | Gram Negative Bacteria | Pale to Dark Red |
| 2 | Gram Positive Bacteria | Dark Purple |
| 3 | Yeast cells | Dark Purple |
| 4 | Nuclei of pus cells | Red |
| 5 | Epithelial cells | Pale red |