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Gram Staining

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17 views4 pages

Gram Staining

Uploaded by

alirehan2030
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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PRACTICAL #2

STAINING:
Staining simply means coloring of the microorganisms with the dye that emphasizes and elucidate
different important structures of microorganisms including bacteria, virus, protozoa and etc.

IMPORTANCE OF STAINING:

1. In microbiology the concept of staining is very important because it highlights the structures of
microorganisms allowing them to be seen under a microscope (simple and electron microscope).

2. It is also used to differentiate different microorganisms.

3. Used for the identification of microorganisms like bacteria which may b either gram positive or gram
negative.

TYPES OF STAINING TECHNIQUES:

There are three types of staining techniques as mentioned below;

1. Simple stains

2. Differential stains

3. Special stains
SIMPLE STAIN PROCEDURE:
Procedure:

1. Clean and dry microscope slides thoroughly.


2. Flame the surface in which the smear is to be spread.
3. Flame the inoculating loop.
4. Transfer a loop full of tap water to the flamed slide surface.
5. Reflame the loop making sure the entire length of the wire that will enter the tube has been heated to
redness.
6. Remove the tube cap with the fingers of the hand holding the loop.
7. Flame the tube mouth.
8. Touch the inoculating loop to the inside of the tube to make sure it is not so hot that it will distort the
bacterial cells; then pick up a pinhead size sample of the bacterial growth without digging into the agar.
9. Reflame the tube mouth, replace the can, and put the tube back in the holder.
10. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area
the size of a dime. It should be a thin, even smear.
11. Reflame the inoculating loop to redness including the entire length that entered the tube.
12. Allow the smear to dry thoroughly.
13. Heat fix the smear cautiously by passing the underside of the slide through the burner flame two or
three times. Test the temperature of the slide after each pass against the back of the hand. It has been
heat sufficiently when it feels hot but can still be held against the skin for several seconds. Overheating
will distort the cells.
14. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered
with the stain for the time designated below.
Methylene blue - 1 minute
Crystal violet - 30 seconds
Carbol fuchsin - 20 seconds
During the staining the slide may be placed on the rack or held in the fingers.
15. At the end of the designated time rinse off the
excess stain with gently running tap water. Rinse
thoroughly.
16. Wipe the back of the slide and blot the stained
surface with bibulous paper or with a paper towel
17. Place the stained smear on the microscope stage
smear side up and focus the smear using the 10%
obiective.
18. Choose an area of the smear in which the cells are
well spread in a monolayer. Center the area to be
studied, apply oil directly to the smear, and focus the
smear under oil with the 100X objective.
19. Draw the cells observed.
DIFFERENTIAL STAINING:
Gram staining:
1. Fixation of clinical materials to the surface of the microscope slide either by heating or by using
methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is
especially useful for examining bloody specimen material).
2. Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple
3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine
(CV-I) complex; all cells continue to appear blue.
4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.
5. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-
walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, remain blue.
gram-positive bacteria. The gram-negative bacteria appear colorless and gram-positive bacteria.
6. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative cells
red/pink; the gram-positive bacteria remain blue.

ACID FAST STAINING:


1. Prepare bacterial smear on clean and grease free slide, using sterile technique.
2. Allow smear to air dry and then heat fix.
3. 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.
4. Cover the smear with carbol fuchsin stain.
5. 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.
6. 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 flammable chemicals have collected from
previous staining. Only a small flame 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.
7. Wash off the stain with clean water.
8. Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.
9. Cover the smear with 3% /v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e.
pale pink.
10. Caution: Acid alcohol is frammable, therefore use it with care well away from an open fiame.
11. Wash well with clean water.
12. Cover the smear with malachite green stain for 1-2 minutes, using the longer time when the smear is
thin.
13. Wash off the stain with clean water.
14. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot
dry).
15. Examine the smear microscopically, using the 100 X oil immersion objective.

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