1.

Gram Staining Procedure

Gram's Stain is one of the most common bacteria identification processes and is used in
hospitals on a regular basis. It is a primary test that separates bacteria rapidly and cost-
effectively into one of two types: Gram-Positive or Gram-Negative.

The main dye, the crystal violet stain, is made from violet blue smear. The gram's iodine is a
mordant, contributing to the penetration and adhesion of the crystal violet into the gram-
positive cell. The combination of acetone and alcohol is a coloring agent that is able to erase
the stain from all of the gram-positive species. Safranine is the counter-stain that stains
anything in gram-negative species that have been decolorized..

1. Apply a smear of bacteria on to a slide. Air dry and then heat fix by passing it through a
flame a few times. Make sure you air dry the bacteria before heat fixing.
2. Add about 5 drops of Hucker’s Crystal Violet to the culture. Let stand for one minute.
Bacteria will stain purple. Wash briefly with water and shake off excess.
3. Add about 5 drops of iodine solution to the culture. Let stand for 30 seconds, wash
briefly with water and shake off excess.
4. Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color stops
running. Be careful not to over-decolorize. Wash immediately (within 5 seconds) with
water and shake off excess. 
5. Add about 5 drops of Safranine O. Let stand for one minute, wash briefly with water and
shake off excess.
6. Examine under microscope at both 400x and 1,000x oil immersion.

2. Acid Fast Staining Procedure

    A. Zeehl Nielsen

1. Prepare bacterial smear on clean and grease free slide, using sterile technique.
2. Allow smear to air dry and then heat fix.
Alcohol-fixation: This is recommended when the smear has not been prepared from
sodium hypochlorite (bleach) treated sputum and will not be stained
immediately. M. tuberculosis is killed by bleach and during the staining process. Heat-
fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation
is bactericidal.
3. Cover the smear with carbol fuchsin stain.
4. Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow
the heated stain to remain on the slide for 5 minutes.
Heating the stain: Great care must be taken when heating the carbol fuchsin especially
if staining is carried out over a tray or other container in which highly
fiammable chemicals have collected from previous staining. Only a small fiame should
be applied under the slides using an ignited swab previously dampened with a few drops
 of acid alcohol or 70% v/v ethanol or methanol. Do not use a large ethanol soaked swab
because this is a fire risk.
5. Wash off the stain with clean water.
Note: When the tap water is not clean, wash the smear with filtered water or clean
boiled rainwater.
6. Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently
decolorized, i.e. pale pink.
Caution: Acid alcohol is fiammable, therefore use it with care well away from an open
fiame.
7. Wash well with clean water.
8. Cover the smear with malachite green stain for 1–2 minutes, using the longer time when
the smear is thin.
9. Wash off the stain with clean water.
10. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry
(do not blot dry).
11. Examine the smear microscopically, using the 100 X oil immersion objective.

    B. Kinyoun Method

1. Make smear on a slide with organisms

o Clean slide, wax label slide, spread organism, air dry for 10 minutes, heat fix
2. Dip slide into Carbol Fuchsin for 20 minutes.
3. Rinse slide
4. Dip slide into Acid-alcohol for 3-5 seconds.
5. Rinse Slide
6. Dip slide into Methylene blue for 30 seconds.
7. Rinse Slide
8. Blot slide dry with bibulous paper.
9. Observe under Microscope.

3. Spore Staining 

1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting
paper or toweling cut to fit the slide.
3. Saturate the blotting paper with malachite green stain solution and steam for 5
minutes, keeping the paper moist and adding more dye as required. Alternatively, the
slide may be steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.
6. Examine the slide under microscope for the presence of endospores. Endospores are
bright green and vegetative cells are brownish red to pink.
4. Capsular Staining Procedure

1. Prepare thin smears of bacterial culture on a microscope slide.

2. Allow the smear to only air-dry. Do not heat-fix as this will cause the capsule to shrink or
be destroyed.
3. Apply 1% crystal violet and allow it to remain on the slide for 2 minutes.
4. With the slide over the proper waste container provided, gently wash off the crystal
violet with 20% copper sulfate. Caution: Do not wash the copper sulfate and stain
directly into the sink.
5. Blot the slide dry with bibulous paper.
6. Observe with the oil immersion lens.

Reference:

Aryal, S., & Kalaivani. (2021, February 04). Capsule stain- Principle, procedure and RESULT
INTERPRETATION. Retrieved February 16, 2021, from
https://microbenotes.com/capsule-stain-principle-procedure-and-result-interpretation/

Aryal, S., Michelle, Gmario, J., Saad, A., Abdullahi, M., Nyakanga, E., . . . Adekeye, J. (2018,
June 12). Acid-Fast stain- principle, PROCEDURE, interpretation and examples. Retrieved
February 16, 2021, from https://microbiologyinfo.com/acid-fast-stain-principle-procedure-
interpretation-and-examples/

Aryal, S., Celestina, Katherine, & Tanaka, M. (2018, June 12). Endospore STAINING-
PRINCIPLE, Reagents, procedure and result. Retrieved February 16, 2021, from
https://microbiologyinfo.com/endospore-staining-principle-reagents-procedure-and-result/

How to do a gram's stain. (n.d.). Retrieved February 16, 2021, from

https://www.microscope.com/education-center/how-to-guides/grams-stain/

Kinyoun stain. (2021, January 17). Retrieved February 16, 2021, from
https://en.wikipedia.org/wiki/Kinyoun_stain