Gram staining procedure: Staining steps, Result, Gram Positive & Negative bacteria

How to prepare and fix smears

1. Make a smear of 15- 20mm diameter by inserting Specimen in the middle of a slide.
2. In the case of sputum, with the help of a clean stick, take a little sputum from the pot in the middle of the slide and make a thin smear. Smear boats should be made with suspected sputum such as the blood-stained part of the sputum.
3. In case of purulent specimen, thin smear should be made with sterile loop.
4. If specimen is watery (e.g. CSF, synovial fluid, ascetic fluid) Smear should be made by centrifuge with sediment.
5. Smear should be air-dried after making.
6. Smear well dried, fixed by passing smear over the flame For 3-4 times). 
7. Before staining, you have to dip in slide cool.

Gram stain test procedure:

(WHO current guidelines)

• Materials and reagents
• Microscope
• Crystal Violet
• Microscopic Slide
• Spirit Lamp
• Slide Rack
• Lugols lodine
• Et Acetone-Alcohol decolourizer
• Neutral red 1g / l or carbol Fuchsin (1 in 10 distilled water)

Principle of Gram staining:

Crystal violet dyes all bacteria purple and iodine solution fixes that color better. Acetone-Alcohol Decolorizer, all the bacteria that are not well stained in iodine

decolorizes bacteria. Neutral red or carbol fuchsine counter decolorized bacteria in bright pink color.

Gram Staining method:

1. Smear should be made on a microscopic slide with specimen (Sputum, Throat swab. Pus, Conjunctival swab, Prostatic smear, CSF).
2. After drying the smear, you have to fix the smear with a little heat of spirit lamp.
3. Cover the smear with crystal violet and keep it for 60 seconds.
4. Slide wash with clean water.
5. Cover the smear with Lugol's iodine and keep for 60 seconds
6. wash Slide with clean water.
7. Call the smear with Acetone-Alcohol decolorizer on the smear 2-3
8. Seconds must be kept.
9. Slide smear on neutral red (0.1%) or carbol fuchsine (1 in10 distilled water) and keep for 2 minutes.
10. Slide wash with clean water and slide air dry in slide rack.
11. Smear with an immersion oil and look at the microscope at 100x objective.

Gram Staining Results:

Gram positive organism: Dark-purple can be seen in Gram positive organism. (eg Streptococci, Color Staphylococci, Micrococci, Pneumococci, Enterococci, Diphtheria bacilli, Anthrax Bacilli)

Gram negative organism: Gram negative organisms can be seen red color after gram staining. (eg. Gonococci, Meningococci, Coliform Bacilli, Shigella, Salmonellae, Cholera, Vibrious)

Crystal violet preparation:

Crystal violet ---------------- 0.5g
Distilled water -------------- 100 ml

Lugol's iodine preparation:

iodine -------------------- 1g
Potassium iodine ------ 2g
Distilled water ----- 100ml

Neutral red, 1g / 1 (0.1%) solution Preparation:

Neutral red ----------------- lg
Distilled water ---------- 1000 ml

Carbol fuchsin preparation:

Basic fuchsin ------------------- 1g
Absolute alcohol --------------- 10ml
5% Phenol solution ------------ 1000ml

Acetone-alcohol decolorizer (For gram stain):

Acetone ------------------------------ 200 ml
Absolute ethanol of methanol --- 475ml
Distilled water ---- --- 200ml ----- 25 ml