staining

Lab five :- Medical M icrobiology
1
Prepared by D.Nebras AL- Aldabagh
Staining
It’s a process of adding a dye to a microbial culture .
Types of staining methods:
Simple staining : is composed of one type of stain e.g, crystal
violate, safranine. methylene blue.
Differential staining : It gives different color to different bacteria e.g.
Gram stain, Albert s stain, and Ziehl neelsen stain.
Special staining : like Capsule stain , flagella , spores )
Preparing heat fixed bacterial smear:-
A heat fixed smear is a thin layer of the bacterial specimen dried and fixed onto the slide.
Procedure:
1- A circle should be marked on the under side of a slide with a glass marker. Several circles can be located on the same slide.
2- To prepare a smear from a suspended culture, aseptically transfer 2- 3 loopfuls of the culture (after shaking), place directly on the slide and spread.
To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the circled area. After aseptically removing material from a culture it is them mixed with the drop of water.
3- Air dry or dry over the flame.
4- heat fixation: by passing the slide three quick passes through the flame.
Purpose from Heat fixation:-
Heat fixation accomplishes three things: (1) it kills the organisms; (2) it causes the organisms to adhere to the slide; and (3) it alters the organisms so that they more readily accept stains (dyes). If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed.
If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps.
If you heat-fix too much, , and you will see distorted cells and cellular remains
Lab five :- Medical M icrobiology
2
Figure 1(Smear preparation)
Negative stain
The method consists of mixing the microorganisms in a small amount of
nigrosine or india ink , these two pigments are not really bacterial stains , they
do not penetrate the micoorganisms ; instead they obliterate the background ,
leaving the organisms transparent and visible in a darkened field . The method
is useful for studying spirochetes that don’t stain readily with ordinary dyes .
Lab five :- Medical M icrobiology
3
Figure 2 : negative staining procedure
capsule stain : this can easily achieved by combining negative and simple staining
techniques . The problem with trying to stain capsules is that if you prepare a heat –
fixed smear of the organism by ordinary methods , you will destroy the capsule ; and ,
if you do not heat – fix the slide , the organism will slide off the slide during washing
.
Gram stain:
First devised by Hans Christian Gram during the late nineteenth century, the gram
stain can be used to divide most bacterial species effectively into tow large groups:
those that take up the basic dye, Crystal violet (gram – positive), and those that allow
the crystal violate dye to wash out easily with the decolorizer alcohol or acetone
(gram – negative).
Staining Procedure.
Slides with heat – fixed smears.
gram staining kit and washing bottle.
1- Fix smear by heat.
2- Cover with crystal violet.
3- Wash with water. Do not blot.
4- Cover the smear with iodine solution for one minute.
5- decolorize for 10-30 seconds with gentle agitation with 95% ethyl alcohol.
6- Wash with water.
7- Cover the smear with safranin for 10-30 seconds.
8- Wash gently with water for a few seconds, air dry, or blot with absorbent
paper.
9- Examine the slide under oil immersion (Figure 3).
Results:
Lab five :- Medical M icrobiology
4
Organisms that retain the violet-iodine complexes after washing in ethanol stain
purple and are termed Gram- positive, those that lose this complex stain red from
safranin counter stain are termed Gram negative.
Figure 3 : Gram stain method
Ziehl- Neelsen stain:-
Most bacteria in the genus Mycobacterium contain very high percentage lipid in
their cell wall.
This stain is used in the identification of the tuberculosis bacillus , Mycobacterium
tuberculosis , and the leprosy organism , Mycobacterium leprae . after decolorization ,
methylene blue (counter stain) is add to the organisms any material that is not acid –
fast Thus stained slide of mixture of acid – fast organisms , tissue cells and non-acidfast
bacteria will reveal blue. acid – fast bacteria will reveal red rods .
Lab five :- Medical M icrobiology
5
Figure 4 (Ziehl- Neelsen stain)
Procedure:-
Ziehl- Neelsen acid- fast stain
1- Fix smear ( sputum) by heat. Heating helps in penetration of dye(carbol
fuchsin) through waxy coat surrounding the cell wall of Mycobacteria.
2- Cover with carbol fuchsin, steam gently for 5 minutes over direct flame (or for
20 minutes over a water bath).
3- Wash with water.
4- Decolorize in acid- alcohol until only a faint pink color remains.
5- Wash with water.
6- Counter stain for 10-30 seconds with methylene blue.
7- Wash with water and let dry.
8- Examine the slide under oil immersion ( Figure 4)
Albert’s stain :This stain was used to diagnosis Corynebacterium ,
Procedure:-
1- prepare a smear and fix it by gently heat over a flame .
2- Cover the smear with Albert stain staining solution . Allow it for 5 minutes .
3- Pour off the stain after 5 minutes . Do not wash .
4- Cover the smear with Albert iodine for two minutes staining solution .
5- Pour off the stain after 2 minutes . do not wash .
6- Blot to dry the smear .
Lab five :- Medical M icrobiology
6
Examine the slide under oil immersion, it was examined under microscope
showed bacilli with numerous metachromatic granules.
Note: You might have noticed that we do not wash the stain unlike other staining
methods because malachite green is highly soluble in water and quality of stain fades if
we wash the stain.
Results:-
Metachromatic granules Bluish- black
Bacillary body or protoplasm Green
Uses:-
1- Albert staining is useful in the demonstration of metachromatic granules.
2- Presence of metachromatic granules helps to distinguish C.diphtheriae
from diphtheriods which lack them.