BACTERIAL STAINING

PREPARATION OF SMEARS
• Smear is a distribution of bacterial cells on a slide for
the purpose of viewing them under the microscope.
The goal of preparing a good smear are:
• Making smear is a precondition to facilitate staining
and further observation of microorganisms under
microscope.
• Without making appropriate smear, which is thin
enough to make a single layer of bacteria, it is
difficult to observe and read different staining reactions
of bacteria.
• When ever possible, smears should be spread on
slides which have one end frosted for labeling
How to make smears
• Smears should be spread evenly covering an area
of about 15-20 mm diameter on a slide
Smearing techniques used for different
specimen:
• Purulent specimen – (which contain pus/ dead WBC)
using a sterile wire loop, make a thin preparation. Do not
centrifuge a purulent fluid.
E.g. C.S.F containing pus cells
• Non purulent fluid specimens - centrifuge the fluid and
make a smear from a drop of the well mixed sediment.
• Culture – Emulsify a colony in sterile distilled water and
make a thin preparation on a slide. When a broth culture,
transfer a loop full to a slide and make a thin preparation.
• Sputum- Use a piece of clean stick to transfer and
spread purulent and caseous material on a slide. Soak
the stick in a phenol or hypochlorite disinfectant before
discarding it.
• Swabs- Roll the swab on a slide. This is particularly
important when looking for intracellular bacteria such as
N. gonorrhoea (urethral, cervical or eye swab). Rolling
the swab avoids damaging the pus cells.
• Feaces (stool) – Use a piece of clean stick to transfer
pus and mucus to a slide and spread to make a thin
preparation.
The thickness of the smear should allow to read a text
when placed under the smear.
Materials for bacteriological Smear preparation

• Microscope slide
• Swabs (are stick with a cotton roll at one tip)-, for
making smear taking sample from body parts or
culture media
• Applicator stick- for taking sample from & make
smear on slide
• Heat, alcohol or other chemical – for fixing the
smear
• Lead pencil – (not grease pencil or ink) – for putting
marks on slides for identification
 Every slide must be labeled clearly with the
patients name, serial number, and date.
Method of collecting and making skin smear of M.
Leprea

• A smear for the examination of M. leprae must be

collected using aseptic and safe technique.

• The site sampled should be the edge of a leprosy

lesion.

1. Explain the procedure to the patient

2. Fit a new sterilized scalpel blade (surgical blade) in

its scalpel holder.
3. Wearing gloves, cleanse the area from where the smear is
to be taken, using a cotton wool swab, moistened with
70% v/v ethanol. Allow the area to dry.

4. Pinch the skin tightly between the thumb and index finger
until it becomes pale due to loss of blood.

NB. The area should be kept bloodless.

5. Using the sterile blade, make a small cut through the skin
surface, about 5mm long and deep enough in to the
dermis (2-3mm) where the bacteria will be found, continue
to hold the skin tightly.
6. Turn the scalpel blade until it is at a right angle to
the cut, using the blunt edge of the blade, scrape
firmly two or three times along the edges and bottom
of the cut to collect a sample of tissues juice and cell
(not blood)

7. Transfer the sample to a slide. Make a small circular

smear, covering evenly an area measuring 5 – 7
mm in diameter.

8. Cover the cut with small dressing.

Drying and fixing smears

• After making a smear, leave the slide

in a safe place for the smear to air-dry protected

from direct sun light.

• When a smear requires urgent staining, it can be

dried quickly using gentle heat.

• The purpose of fixation is to preserve (kill)

microorganisms and to prevent smear being
washed from slides during staining.
Heat Fixation

• This is widely used but can damage organisms and

alter their staining reactions especially when
excessive heat is used.

• Heat fixation also damages leukocytes and is

therefore unsuitable for fixing smears which may
contain intracellular organisms such as N.
gonorrhoeae and N. meningitides.

• When used, heat fixation must be carried out with

care.
The following techniques are
recommended:
1. Allow the smear to air-dry completely

2. Rapidly pass the slide, smear upper

most, three times through the flame of
a sprit lamp or pilot flame of a Bunsen
burner.

NB: do not heat smears excessively

3. Allow the smear to cool before staining.

Alcohol Fixation
• This form of fixation is far less damaging to
microorganisms than heat. Cells, especially pus
cells, are also well preserved.
- Alcohol fixation is more bactericidal than heat. (E.g.
M. tuberculosis is rapidly killed in sputum smears
after applying 70% v/v alcohol.
• Alcohol fixation is recommended for fixing smears
for Gram negative intracellular diplococcic (N.
gonorrhea or N. meningitides), fix with one or two
drops of absolute methanol or ethanol.
• Leave the alcohol on the smear for a minimum of 2
minutes or until the alcohol evaporates.
Other Chemical Fixation

• For dangerous organisms, to ensure all the

organisms are killed.

- 40g/l potassium permanganate is recommended

for fixing smears containing Anthrax bacilli.

- Formaldehyde vapor is sometimes recommended

for fixing smears Mycobacterium species.
Formaldehyde fixed smears, however, tend to stain
poorly and the chemical it self is toxic with an
injurious vapor.
Precautions to take when staining smears
• Use a staining rack. Do not immerse slides in
containers of stain because this can lead to
contamination of stain and transfer of organisms
from one smear to another.
• Do not attempt to stain a smear that is too thick.
This is one of the commonest causes of poor
staining and incorrect reporting of smears.
• When washing smears of CSF; which can be easily
washed, don’t pour water directly on a slide,
instead direct the water on the back of the slide.
• After staining, place the slides at an angle in a
draining rack for the smears to air dry. Do not blot
smears to dry with filter or blotting paper.
 PRINCIPLE OF STAINING IN BACTERIOLOGY

• Even with the microscope, bacteria are difficult to

see unless they are treated in a way that increases
contrast between the organisms and their
background.

• The most common method to increase contrast is to

stain part or all of the microbe.
Definition
• Stains (Dyes) are coloured chemical compounds
that are used to selectively give (impart) colour to
the colourless structures of bacteria or other cells.

• Staining is the process of imparting colour to the

colourless structures (cell wall, spore, etc) of the
bacteria in order to make it visible under the
microscope.
Uses of staining
1. To observe the morphology, size and arrangement
of bacteria

2. To differentiate one group of bacteria from the other

group.

 Staining reactions are made possible because of the

physical phenomena of capillary osmosis, solubility,

adsorption, and absorption of stains or dyes by cells

of microorganisms.
 Basic principle of staining: The cellular
components of mammalian as well as microbial cell
are different.

 For example the nuclei of cell is negatively charged

because of the presence of acidic component (DNA)
hence it combines with positively charged
compounds (basic dyes), and the cytoplasm parts
of a cell is generally positively charged therefore
combines with negatively charged compounds
(acidic dyes).
Why are stains not taken up by every micro-organism?
Factors controlling selectivity of microbial cells are:
1. number and affinity of binding sites
2. rate of reagent uptake
3. rate of reaction
4. rate of reagent loss (differentiation or regressive
staining)
• Differentiation is the process of removing excess
dye from cells in order to highlight a structure which
retains the dye.
• Regressive staining means that the cells is
deliberately overstained and then destained
(differentiated) until the proper endpoint is reached.
Type of stain in Microbiology

There are three kinds of stains:

1. Basic stains

2. Acidic stains

3. Neutral stains

NB: This classification is not based on the pH of stains

A. Basic Stains

 Are stains in which the colouring substance is

contained in the base part of the stain and the acidic
part is colourless.

 The bacterial cells can easily stain with basic dyes

e.g. Methylene blue stain, Safranin, Genetian violet,

Carbolfuchsin etc
B. Acidic Stains

• Are stains in which the colouring substance is

contained in the acidic part of the stain and the base
part is colourless.

• Acidic dyes are mainly used in histology laboratory.

• It is not commonly used in microbiology laboratory;

e.g. eosin.
C. Neutral Stains

 Are stains in which the acidic and basic components

of stains are coloured.

 Neutral dyes stain both nucleic acid and cytoplasm.

these stains are commonly used in hematology
laboratory. e.g. Giemsa’s stain, Wright’s stain.
Type of staining methods:

1. Simple staining method

2. Differential staining method

3. Special staining method

1. Simple staining method

• only a single dye is used.

• For the purpose of viewing bacterial shapes and
arrangements

• There are two kinds of simple staining methods

A. Positive staining

B. Negative staining

A. Positive staining: The bacteria or its parts are

stained by the dye. e.g. Methylene blue stain, Crystal
violent stain
Procedure

1. Make a smear and label it

2. Allow the smear to dry in air

3. Fix the smear over a flame

4. Apply a few drops of positive simple stain

5. Wash off the stain with water

6. Air -dry and examine under microscope

Result-all the bacterial surface is stained.

B. Negative staining-

• The dye stains the background and the bacteria

component remain unstained. e.g. Indian ink stain.

2. Differential staining method

• A method in which multiple stains (dye) are used to

distinguish different group of bacteria. e.g. Gram’s
stain, Ziehl-Neelson stain.
 GRAM’S STAIN
• This method was developed by the Danish
bacteriologist Christian Gram in 1984.

Basic concepts:
• Most bacteria are differentiated by their gram
reaction due to differences in their cell wall
structure.
• The surface of bacterial cell has got a negative
charge due to the presence of polysaccharides and
lipids (PG) this has made the surface of the bacteria
to have affinity to cationic or basic dyes
Principle

• The principle of Gram’s stain is that cells are first

fixed to slide by heat or alcohol and stained with a
basic dye (e.g. crystal violate), which is taken up in
similar amounts by all bacteria.

• The slides are then treated with an Gram’s iodine

(iodine KI mixture) to fix (mordant) the crystal violet
stain on Gram positive bacteria, decolorized with
acetone or alcohol, and finally counter stained with
Safranin.
• Gram positive bacteria: - stain dark purple with
crystal violet (CV) and are not decolorized by
acetone or ethanol. The following are some
important examples of gram positive bacteria.
• Staphylococcus,
• Streptococcus,
• Clostridium
• Bacillus
• Corynebacterium … etc
– Note!. The reason for the retention of the primary
stain (CV) by the gram positive bacteria after
decolorization is due to the presence of more acidic
protoplasm (PG layer) of these organisms which
bind to the basic dye.
• Gram negative bacteria: - stain red because after
being stained with crystal violet they are decolorized
by acetone or ethanol and take up red counter
stain. (Neutral red, Safranin or dilute carbol fuchsin).

• The following are some important gram negative

bacteria:-

• Nesseria spp.

• Haemophilus spp.

• Salmonella, shigella, vibrio, Klebsilla…etc.

Required reagents

• Crystal violet

• Gram’s Iodine

• Acetone-Alcohol or 95% Alcohol

• Safranin or Neutral red

Gram’s stain reagents preparation

1.Crystal violet stain

• Crystal violet …………………………. 20g

• Ammonium oxalate …………………… 9g

• Ethanol or methanol absolute ……….. 95ml

• Distilled water ……………………….. to 1 litre

3. Acetone-alcohol
To make 1 litre
• Acetone …………………………… 500ml
• Ethanol or methanol absolute ….. 475 ml
• Distilled water ……………….……. 25ml

N.B: some workers prefer to use acetone by itself or

ethanol 95% v/v as decolorizing solution.
• A mixture of acetone and alcohol is recommended
because it decolorizes more rapidly than ethanol
95% v/v and is less likely to over decolorize smears
than acetone with out alcohol added.
2. Gram’s iodine

• Potassium iodide ………………….. 20 g

• Iodine …………………….………… 10 g

• Distilled water ……………………… to 1 litre

Should be stored in a brown bottle

4. Safranin
1. Prepare a stock solution
 Safranine O ----------------------------------------------- 2.5g
 Ethanol (95%) --------------------------------------------100ml
 Mix until all the safranine is dissolved. Transfer the
solution to a glass – stoppered bottle.
 Label the bottle ( Safranine stock solution) and write
the date.
2. Prepare a working solution in a glass stoppered bottle
 Stock solution ----------------------------------------------10ml
 Distilled water ----------------------------------------------90ml
 Label the bottle ( Safranine working solution) and
write the date
5. Neutral red; 1g/l (w/v)
To make 1 litre
• Neutral red ……………. 1g
• Distilled water ………… 1 litre

N.B: Neutral red is selected as the counter stain

because it stains well gonococci and meningococci.
Safranin can also be used.
• The dilute carbolfuchsin (1 in 10) is recommended
for staining Vincent’s organisms. Yersinia,
Haemaophilus, campylobacter, and vibrio species.
Procedure of Gram’s Stain
1. Fix the dried smear with heat by gently passing it
over
sprit lamp or Bunsen burner.
– Note: When the smear is for the detection of
gonococci or meningococci, it should be fixed
with methanol for 2 minutes
2. Cover the fixed smear with crystal violet stain for 30
– 60 seconds
3. Rapidly wash off the stain with clean water
4. Tip of all the water, and cover the smear with
Lugol’s iodine for 30 – 60 seconds
5. Wash off the iodine with clean water
6. Decolorize rapidly with acetone-alcohol for 30
seconds. wash immediately with clean water.
7. Cover the smear with Neutral red or Safranin for 2
minutes
8. Wash off the stain with clean water.
9. Wipe the back of the slide clean, and place it in a
draining rack for the smear to air dry
10. Examine the smear microscopically, first with the
40x objective to check the staining and to see the
distribution of material, and then with oil immersion
objective to report the bacteria and cells.
Results
• Gram positive bacteria …..…………….. Purple(blue)
• Yeast cells ………………………………. Dark purple
• Gram negative bacteria …….………….. Pale to red
• Nuclei of pus cell …….………….……… Red
• Epithelia cells …………………………... Pale red
Reporting of Gram’s stained smear
The report should include the following:
1. Number of bacteria present whether many, moderate,
few or scanty
2. Gram reaction of the bacteria whether Gram positive or
Gram negative
3. Morphology and arrangement of the bacteria whether
cocci, diplococci, streptococci, rods, or coccobacili;
also whether the organisms are intracellular.
4. Presence and number of pus cells.
5. Presence of yeast cells and epithelia cells.
Example
• A Gram’s stain of urethral smear report might read as:
– “Moderate number of gram negative intercellular
diplococci and many pus cells “
 Factors which contribute to false Gram’s staining
result

I. False Gram positive reaction

• Preparation of too tick smear – the stain will not be

fully washed.

• Presence of sediment in crystal violet - this can be

avoided by filtering the stain before use.

• Decolourizing time - when insufficient decolourization

time is used or (when not fully decolorized).
II. False Gram Negative reactions
• Making smear from old culture.
• Cell wall damage due to antibiotic.
• Excessive heat fixation of smear.
• Over decolourization of smear (decolourization for longer
time).
• Use of an iodine solution which is too old (i.e. yellow in
stead of brown in colour) (its mordant effect will be
decreased).

Solution for the above problem

• Always check new batches of stain and reagent for
correct staining reaction using a smear containing known
gram positive and gram negative organism as a control.
 Ziehl-Neelson (Acid Fast Bacilli-AFB) staining
method

• Developed by Paul Ehrlich in1882, and modified by

Ziehl and Neelson

• Ziehl-Neelson stain is used for staining

mycobacteria which are hardly stained by Gram‘s
staining method.

• Once the Mycobacteria is stained with primary stain

it can not be decolorized with acid, so named as
acid-fast bacteria.
• Mycobacteria typically are slightly bent or curved
slender rods. About 2-4 m long and 0.2-0.5 m
wide.
• The most striking chemical feature of mycobacteria is
their extra ordinary high lipid content in the cell wall (up
to 60% of its dry weight). This high lipid content
probably accounts for some of the other unusual
properties of mycobacteria.
 E.g. Relative impermeability to stains, acid
fastness, unusual resistance to killing by acid and
alkali.
• The cell wall of Mycobacteria also contains a peptido-
glycan layer, glycolipids, protein and Mycolic acid (This
is unique to mycobacteria, nocardiae and
corynebacteria).
Principle of Ziehl-Neelson (Acid fast) staining method
 Sputum smear is heat –fixed, flooded with a solution of
carbolfuchsin (a mixture of basic fuchsin and phenol) and
heated until steam rises. The heating which facilitate
penetration (entrance) of the primary stain into the
bacterium.
 After washing with water, the slide is covered with 3%
HCl (decolourizer).
 Then, washed with water and flooded with methylene blue
(Mycobacterium tuberculosis) and malachite green
(Mycobacterium leprae).
Materials for AFB staining
• Sputum container (for M. tuberculosis) – for sample
collection
• Wire loop or applicator stick – to spread sputum on the
microscope slide
• Microscope slide – for making smears
• Marking pen – to put identification number on the
microscope slide
• Forceps- to hold smeared slide
• Bunsen burner or sprit lamp – to fix the smeared slide
and to flame the smear during staining.
• Staining racks (staining rods) – for staining.
• Slide rack – to place stained slide to dry in the air
• Ziehel-Neelson (AFB) stain (Carbolfuchsin, 3% Acid
alcohol, and Methylene blue (malachite green)
Ziehl Neelson stain reagents preparation

A. Carbolfuchsin
- Solution A (saturated solution of basic fuchsin)
Basic fuchsin ………….. 3gm
Ethanol …………………..100ml

- Solution B (phenol aqueous solution, 50g/L ( 5%)

Phenol ……………………..... 10gm
Distilled water ……………… 200ml

- Mix 10 ml of solution A with 90ml of solution B.

- Transfer resulting mixture to a glass stoppered amber
bottle and label.
B. 3% Acid –alcohol (decolourizer)

Hydrochloric acid (conc) …………… 3ml

Ethanol ………………………………..97ml

Label the bottle.

N.B: It is recommended to use 25% H2SO4 as
decolorizing solution

• Content per liter:

• sulfuric acid (minimum 95%)........250 ml

• water ...............................................750 ml
Preparation:

1. Measure 750ml of water into a 2-liter flask

2. Measure 250mL of concentrated sulphuric acid in
a cylinder
3. Pour it slowly into the flask containing the water,
directing the flow of acid gently along the inner
side of the flask
4. Stop and swirl flask regularly as a lot of heat is
generated until all acid is added
5. Mix well and allow to cool before use
C. Methylene Blue Stain

• Methylene blue chloride .............................. 0.5g

• Distilled water......................................... 100ml

D. Malachite green
• Malachite green …………………. 0.5gm
• Distilled water ……………………. 100ml

- Using a pestle and mortar, grind the malachite green

crystal to a powder. Dissolve the grind powder in
100ml distilled water and store in a dark brown bottle.
Hot and cold Ziehl-Neelson technique
• In the hot Zn technique the phenolic– carbol fuchsin stain
is heated to enable the dye to penetrate the waxy
mycobacterial cell wall.

• Techniques that do no heat the stain are referred to as

‘cold’ techniques. In these, a penetration of the stain is
usually achieved by increasing the concentration of basic
fuchsin and phenol.

• Comparison between the ‘hot’ and ‘cold’ method has

shown that both M. leprae and M. tuberculosis stain less
well by the cold method.
Difference between Acid fastness of
Mycobacterium species

• M. tuberculosis and M. ulcerance are strongly acid

fast, and a 3% v/v acid solution or 25% H2SO4 is
used to decolorize the smear.

• M. lepreae is only weakly acid fast. A 1% v/v acid or

5% H2SO4 decolorizing solution is therefore used
for M. lepreae smears and also different staining
and decolorizing times.
Procedure
1. Use blood – specked, purulent and caseous sputum
for smear preparation (do not use saliva)
2. Heat fix the dried smear,
Alcohol fixation: This is recommended when the
smear has not been prepared from sodium
Hypochlorite (bleach)
- Heat fixation of untreated sputum will not kill M.
tuberculosis where as alcohol – fixation is
bactericidal.
 Since M. tuberculosis can infect almost any organ in
the body, the laboratory could receive a variety of
extra – pulmonary specimen. E.g. body fluid, tissue,
pus, CSF, urine etc.
3. Cover the smear with carbol-fuchsin stain
4. Heat the stain until vapor just begins to rise. Do not
over heat – Allow the heated stain to remain on the
slide for 5 minutes.
5. Wash off the stain with clean water
6. Cover the smear with 3% acid alcohol for 5 minutes
or until the smear is sufficiently decolorized. i.e. pale
pink.
7. Wash well with clean water
8. Cover the smear with methylene blue for 1 – 2
minutes.
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
100x oil immersion object.
12. Examine a minimum of 300 fields systematically
before the smear is reported as negative.
Results
• AFB............. Red, straight or slightly curved rods,
occurring single or in a small groups
• Cells......................................... Blue
• Background Material ……….... Blue
REPORTING OF SPUTUM SMEAR
• When any definite red bacilli are seen report the smear as
AFB positive and give an indication of the number of
bacteria present as follows:
When
1-9 AFB/100 fields……………..report the exact number
10–100 AFB/100 fields………...report +
1 – 10 AFB/field…………………report ++
More than 10 AFB/field…………report +++
N.B: When only one or two AFB are seen, request further
specimen for examination.
• When no AFB are seen after examining 300 fields report
as ‘ No AFB seen’ do not report as “Negative” because
organisms may be present but not seen in those field
examined.
Sensitivity of direct microscopy
• Studies have shown that between 5000 to 10,000 tubercle
bacilli per millilitre of sputum are required for direct
microscopy to be positive. So sufficiently large number of
acid fast bacilli should be present to be detected by direct
microscopy
• The sensitivity can further be improved by examination of
more than one smear from a patient. So up to three
specimens (spot– morning- spot) are required and this
can significantly increase the sensitivity.
• Other way of increasing sensitivity is sodium hypochlorite
(bleach) centrifugation technique to concentrate AFB.
NB: Studies have shown that centrifugation of bleach treated sputum
increased the sensitivity of direct microscopy from 43.4% to 76.3%,
increasing the number of smear-positive patients detected.
Sensitivity is significantly increased in HIV/AIDS patients.
Sodium hypochlorite centrifugation technique to
concentrate AFB

1. Transfer 1-2 ml of sputum to a screw - cap container of

15-20 ml capacity

2. Add an equal volume of concentrated sodium

hypochlorite (bleach) solution and mix well.

3. Leave at room temperature for 10-15 minutes, shake at

intervals to break down the mucus in the sputum.
4. Add about 8 ml of distilled water, and mix well.
5. Centrifuge at 3000g for 15 minutes.
N.B: When centrifugation is not possible, leave the NaOCl treated
sputum to sediment overnight.

6. Using a glass Pasteur pipette, remove and discard the

supernatant fluid. Mix the sediment.
7. Transfer a drop of the well mixed sediment to a clean
scratch free glass slide. Spread the sediment to make a
thin preparation and allow to air dry.
8. Heat fix the smear and stain it using Ziehl Neelson
technique and examine microscopically.
Factors which bring false positive results

• Utilization of slides that have been positive. These

should be discarded.

• Using scratched slides on which deposits of stain

may look like bacilli

• Using unfiltered fuchsin which may contain crystals .

• Carelessness in heating the fuchsine, allowing it to

dry and crystallize on the smear

• Inadequate decolorizing of the smear.

 Fluorescence staining for Mycobacteria

• Fluorescence microscopy uses illumination from

either a quartz halogen lamp or a high pressure
mercury vapor lamp.

• The advantage of fluorescence microscopy is that a

low magnification is used to scan smears, allowing a
much larger of the smear to be seen and resulting in
more rapid examination.
 Reagents
Auramine O
• Auramine-----------------------------------------------0.1gm
• 95% Ethanol-------------------------------------------10 ml
• Dissolve Auramin with Ethanol --------------Solution1
Phenol
• Phenol crystal------------------------------------------3.0
gm
• Distilled water-----------------------------------------87ml
• Dissolve phenol with distilled water------Solution 2

 Mix solution 1 and 2 and store in amber bottle away

from light and heat.
Decolorizing solution
• 3% acid alcohol
Counter stain
• Either Potassium permanganate or acridine orange
can be used
Potassium permanganate
Potassium permanganate (KMnO4) ---------------0.5g
Dis. Water----------------------------------------------100ml
Dissolve and store in amber bottle
Acridine orange
• Acridine orange----------------------------------------0.01g
• Anhydrous dibasic sodium phosphate (NA2HPO4) -
0.01g
• Distilled water------------------------------------------100ml

 Dissolve sodium phosphate in distilled water. Add

Acridine orange. Store in amber bottle.
Procedure

1. Place the dried labeled smear on staining rack

2. Flood entire smear with Auramine O and allow to

stain for 15min ensuring that staining solution
remains on smear

3. Rinse with distilled water and drain. Tap water

contains chlorine which may interfere with
fluorescence.

4. Decolorize with 3% acid alcohol for two minutes

5. Rinse with distilled water and drain

6. Flood the smear with Potassium permanganate or
acridine orange and allow to counter stain for two
minutes. Time is critical with potassium
permanganate because counter staining for longer
time may quench the fluorescence of acid fast bacilli.

7. Rinse with distilled water and drain

8. Allow the smear to air dry. Do not blot. Read as soon

as possible. Examine fluorescence stained smear
within 24 hours as the fluorescence may fade with
time.
Figure: Fluorescent stained mycobacteria under fluorescence
microscope
3. Special Staining method

• These are stains, which are used to stain capsules

and spores.

There are two types of special staining methods

A. Capsule Staining method

B. Spore staining method

A. Capsule staining method
• This technique is used for showing the presence of
capsules around bacteria

Required reagents
• Crystal Violet ………………………..10g/l (1% w/v)
stain
• Copper sulphate……………………200g/l (20%w/v)
Procedure
1 Fix the direct smear using alcohol
Note: when preparing the smear, mix the organism suspension with a
drop of normal serum this will help to show the capsules
2 Cover the smear with crystal violet stain and heat gently
until the steam just begins to raise, leave to stain for 1
minute.
3 Wash off the stain with the copper sulfate solution
N.B don’t use water
4 Wipe the back of the slide clean and place in a draining
rack for smear to air dry
5 Examine the smear microscopically first with the 40x, then
with the 100 x oil immersion objective to look for
capsulated bacteria.
Result

• Bacterial cell ----------------------------------- dark purple

• Capsule outline -------------------------------- Pale blue

B. Spore staining method

 It is based on the binding of the malachite green and

the permeability of the spore vs. cell wall. The
steaming helps the malachite green to permeate the
spore wall.

• The primary dye malachite green is a relatively weakly

binding dye to the cell wall and spore wall. In fact, if
washed well with water, the dye comes right out of the
cell wall. That is why there does not need a
decolourizer in this stain.
Materials needed:
– Dye kit (malachite green, Safranin)
– Stain rack
– Hot plate
– Paper towel (cut the size of the slide)
Procedure
1. Make a smear, air-dry and heat-fix.
2. Put a beaker of water on the hot plate and boil until steam
is coming up from the water. Then turn the hot plate down
so that the water is barely boiling.
3. Place the wire stain rack over the beaker which now has
steam coming up from the boiled water.
4. Cut a small piece of paper towel and place it on top of the
smear on the slide. The towel will keep the dye from
evaporating too quickly, thereby giving more contact time
between the dye and the bacterial walls.
5. Flood the smear with the primary dye, malachite green,
and leave for 5 minutes. Keep the paper towel moist
with the malachite green. DO NOT let the dye dry on
the towel.

6. Remove and discard the small paper towel piece.

7. Wash really WELL with water and move the slide and
wire rack from the boiling water to the regular stain tray
to finish up the last step in the procedure.

8. Place the smear in the stain jar or flood the smear with
the counter stain dye, Safranin, and leave for 1 minute.
The cold method spore stain
• Without heat you have to really rough up the spore
wall to get in the dye.
• Heat fix the smear by running the slide through the
flame about 20 times, and leave malachite green on
for 20 minutes during the stain process.
Interpretation
• Spores will be a light green:
• Vegetative cell walls will pick up the counter stain
safranin and become red (Red)
• Notice: the position of the spore could be --terminal,
central, or subterminal and this may be helpful in the
identification of the unknown species of bacteria.
 REFERENCE
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