CHAPTER IV

BACTERIAL
STAINING

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Learning objectives
At the end of this chapter, the student will be able to:
1.Describe smear preparation for bacterial examination
2.Define stains and bacterial staining.
3.Describe the uses of staining solutions.
4.List the types of staining methods that are used to stain bacteria.
5.List the different types of bacterial staining techniques.
6.Discuss the principles of Gram’s stain and Ziehl - Nelseen stain.
7.Describe the factors that contribute for false Gram’s stain and Zihel -
Nelseen stain results.
8.Prepare the Gram’s stain and Zihel -Nelseen stain reagents.

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PREPARATION OF SMEARS FOR BACTERIOLOGICAL
DIAGNOSIS

ØWhy we need to make smears?

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. .

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Cont..
Materials for bacteriological Smear preparation
Microscope slide
Swab, for making smear taking sample from body parts or culture
media
Applicator stick- for taking sample from & make smear on slide
Lead pencil – (not grease pencil or ink) – for putting marks on
slides for identification
Heat, alcohol or other chemical – for fixing the smear on the slide
to prevent from being washed during staining procedure and for
killing the bacteria on the slide.

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Preparing smear
If smears are to provide reliable information they must be
prepared, labeled and fixed correctly prior to being stained.

Labeling Slides
Every slide must be labeled clearly with the date and the patients
name and number. When ever possible, smears should be spread
on slides which have one end frosted for labeling
◦ A lead pencil should be used for writing on the
frosted area. Because pencil marks unlike grease
pencil marks, will not be washed off during the
staining processes.

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Cont…
How to make smears
Smears should be spread evenly covering an area of about
15 – 20 mm diameter on a slide
◦ NB. Careless handling of pathological material and cultures may
result in the contamination of the out side of vessels, the laboratory
bench, or floor. Microorganisms can then be transferred from these
surfaces to the finger and hands, enter blood stream through cuts
and scratches, or be transferred to the mouth and eyes. so when ever
smears are made every precaution and aseptic technique should be
followed strictly!

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ØSome of the techniques used to make smears from different specimens are as follows.
Purulent specimen – (Purulent – samples 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- (are stick with a cotton roll at one tip)- 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
(decontaminate the stick before discarding it) spread to make a thin preparation.

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The thickness of the smear should allow to read a text when
placed under the smear.

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Cont…
Method of collecting and making skin smear of M.Leprea
A smear for the examination of M. leprae must be collected
using on asceptic and safe technique. The site sampled
should be the edge of a leprosy lesion.
1. Explain the procedure to the patient
2. Fit anew scalpel blade (surgical blade) in its scalpel holder.
Sterilize the blade by wiping it carefully with a piece of
absorbent cotton wool soaked in 70% v/v ethanol and
flaming it for 2-3 seconds in the flame of a sprit lamp. Allow
the blade to cool.

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Cont…
3. Wearing protective rubber 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.

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Cont…
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.

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Cont…
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 microorganisms and
to prevent smear being washed form slides during staining.
Smears are fixed by heat, alcohol, or occasionally by other
chemicals.

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Cont..
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.

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Cont.…
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.

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 Cont.….
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 therefore recommended for fixing smears
when looking for Gram negative intracellular diplococcic.
- Alcohol fixation is more bactericidal than heat. (E.g. M.
tuberculosis is rapidly killed in sputum smears after applying 70%
v/v alcohol.

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Cont.…
The following technique is recommended.
Allow the smear to air dry completely
Depending on the type of smear, alcohol fix as follows.
- For the detection of intracellular Gram negative
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.

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Cont.…
Other Chemical Fixation
Other chemicals are some times necessary to fix smears,
which contain particularly dangerous organisms, to ensure
all the organisms are killed.
- 40g/l potassium permanganate is recommended for fixing
smears, which may contain Anthrax bacilli.
- Formaldehyde vapor is sometimes recommended for
fixing smears which may contain Mycobacterium species.
Formaldehyde fixed smears, however, tend to stain poorly
and the chemical it self is toxic with an injurious vapor.

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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 C.S.F sediment and specimens which can
be easily washed from a slide, direct the water from a wash bottle
on the back of the slide, not directly on the smear.
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.

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 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.

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Definition
ØStains (Dyes) are coloured chemical compounds that
are used to selectively give (impart) colour to the
colourless structures of bacteria or other cells.

ØBacterial 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.

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Uses of staining
1. To observe the morphology, size and arrangement of bacteria
2. To differentiate one group of bacteria from the other group.
3.To help in the primary level of diagnosis

ØStaining reactions are made possible because of the

Physical phenomena of capillary osmosis, solubility,
adsorption, and absorption of stains or dyes by cells of
micro-organisms.

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Cont…
§Basic principle: 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).
§Individual variation in the cell wall constituents among different groups
of bacteria will consequently produce variations in colours during
microscopic examination.

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Cont…

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)

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Cont…
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

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Cont.…
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

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Cont.…
B. Acidic Stains
ØAre stains in which the colouring substance is contained
in the acidic part of the stain and the base part is colourless.
ØAcidic dyes are mainly used in histology laboratory. It
is not commonly used in microbiology laboratory ;
e.g. eosin.

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Cont.…
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.

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Cont…
Type of staining methods
1. Simple staining method
2. Differential staining method
3. Special staining method

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1. Simple staining method
It is a type of staining method in which only a single dye is used.

There are two kinds of simple staining methods

A. Positive staining
B. Negative staining

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Cont…
A. Positive staining: The bacteria or its parts are
stained by the dye. e.g. Methylene blue stain,
Crystal violent stain
ØBasic dyes are Positively charged
ØThese dyes get attached to negatively
charged cytoplasm of microbial organism

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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.

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B. Negative staining-
Ø The dye stains the background and the bacteria
component remain unstained. e.g. Indian ink stain.
ØThey give a contrast back ground

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B. Differential Staining

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.

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 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
(when the colouring part is contained in the basic part.)

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Cont…
Principle
ØThe 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.
ØThe 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.

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Cont…
ØGram positive bacteria: - stain dark purple with crystal
violet and are not decolorized by acetone or ethanol. The
following are some important examples of gram positive
bacteria.
◦ Staphylococcus,
◦ Streptococcus,
◦ Clostridium
◦ Bacillus
◦ Corynebacterium … etc
◦ N.B. 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.

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Cont…
ØGram 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, Coliforms …etc.

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Cont…
Required reagents
Crystal violet
Gram’s Iodine
Acetone-Alcohol or 95% Alcohol
Safranin or Neutral red

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Cont…
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

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Cont..
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.

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Cont…
2. Gram’s iodine
Potassium iodide ………………….. 20 g
Iodine …………………….………… 10 g
Distilled water ……………………… to 1 litre
Should be stored in a brown bottle

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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

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Cont…
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.

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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.

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 Cont…
7. Cover the smear with Neutral red or Safranin for 1 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.

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Cont…
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

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Figure: Gram positive and Gram negative bacteria

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3/11/2023 48
Cont…

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.

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Cont…
4. Presence and number of pus cells.
5. Presence of yeast cells and epithelia cells.

Example of a gram stain teport

A Gram’s stain of urethral smear report might read as:
◦ “Moderate number of gram negative intercellular diplococci and
many pus cells “

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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.
Application of crystal violet - 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).

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Cont.…
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.

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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.

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 Cont…
- Mycobacteria typically are slightly bent or curved slender rods.
About 2um- 4 um long and 0.2 um – 0.5 um 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).

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 Cont…
Photogenic Mycobacteria
1. Tubercle bacilli
- M. tuberculosis (human tubercle type)
- M. bovis (bovine type)
2. Leprosy bacilli
- M. leprae
- M. lepraemurium
3. Environmental mycobacteria (atypical, anonymous)
mycobacteria
- M. avium- intercellulare
- M. xenopi etc

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Cont…
M. tuberculosis
Most infections with M. tuberculosis are caused by inhaling
cough droplets or dust particles containing tubercle bacilli
which become lodged in the lung forming a small
inflammatory lesion and cause Pulmonary tuberculosis.
Infected droplets remain air born for considerable time, and
may be inhaled by susceptible persons
Tuberculosis is a disease of global importance. One third of
the world’s population is estimated to have been infected
with M. tuberculosis and eight million new cases of
tuberculosis arise each year.

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Cont…
M. bovis
Is found mainly as a pathogen in a cattle and occasionally in
other animals.
Humans become infected by close contact with infected
cattle or by ingesting the organisms in raw un treated milk.
Person to person transmission of bovine strains may also
occur.

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Cont…
Other forms of tuberculosis
Tuberculous meningitis – when tubercle bacilli reach the
meninges through blood and affect the meninge.
Miliary tuberculosis: - Wide spread miliary infection (liver,
spleen & lymph glands)
Renal and urogental tuberculosis
Bone and joint tuberculosis

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Cont…
M. Leprae
causes leprosy, a chronic infectious disease that affects the skin,
peripheral nerves, mucosa of the upper respiratory tact and the eye.
It is mainly transmitted via the respiratory tract or skin and has a long
period of incubation(2 – 5 years) or latency

Environmental Mycobacteria –
are the most frequent causes of pulmonary infections resembling tuberculosis, and
disseminated disease and also cause lymphadenitis (mainly in children).
Such species are acid fast but differ from the M. tuberculosis complex by being
opportunistic pathogens, with limited distribution and acquired from the environment.
E.g Soil or water.

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Cont…
Principle of Ziehl-Neelson (Acid fast) staining method
§Sputum smear is heat –fixed, flooded with a solution of
carbilfusin (a mixture of basic fuschin 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).

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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
- Methylene blue (malachite green)

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Ziehl Neelson stain reagents preparation

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

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

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

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

- Transfer resulting mixture to a glass stopperd amber bottle and
label.
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B. 3% Acid –alcohol (decolorizer)
Hydrocloric acid ( conc) …………… 3ml
Ehanol ………………………………..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

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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

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C. Methylene Blue Stain

Methlene 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.

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Cont…
Hot and cold Ziehel Nelson 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 basicfuchsin
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.

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Cont…
Difference between Acid fastness of Mycobacterium
species
M. tuberculosis and M. ulcerance are strongly acid fast,
when staining specimens for these species, 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.

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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 form 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.
NB: The benefit of microscopy on the specimens is limited and it is
recommended that extra pulmonary specimens be referred for culture.
However if there is no option the fluid (non purulent) should be centrifuged
and smears prepared form the sediment.

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3. Cover the smear with carbolfuchsin 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.

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Cont..
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.

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Results
AFB............. Red, straight or slightly curved rods,
occurring single or in a small groups
Cells......................................... Blue
Background Material ……….. Blue

Fig. AFB under the microscope

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3/11/2023 73
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:
1 to 9 AFB per 100 fields report the exact number
1+ 10 to 99 AFB per 100 fields
2+ 1 to 10 AFB per field (count at least 50 fields)
3+-more than 10 AFB per field (count at least 20 fields)
N.B: AFB means number of acid fast bacilli seen.
when very few AFB are seen E.g When only one or two AFB are seen, request further
specimen for examination.
When no AFB are seen after examining 300 fields report the smear as ‘ No AFB seen’ do not
report as “Negative” because organisms may be present but not seen in those field examined.

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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 two specimens
(spot–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.

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Sodium hypochlorite centrifugation technique to concentrate
AFB
1. Transfer 1-2 ml of sputum to a screw - cap container of 15m-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.

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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.

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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.

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Fluorescence staining for Mycobacteria

Fluorescence microscopy uses illumination from either a

quarth 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.

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 Cont…
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 Distiied water---------------Solution 2

Mix solution 1 and 2 and store in amber bottle away from light and heat.

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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.

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 CONT…
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

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Cont..
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.

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Figure: Fluorescent stained mycobacteria under fluorescence microscope

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Cont…
3. Special Staining method
Special stains are used to color and isolate specific parts of
microorganisms, such as endospores and flagella, and to reveal
the presence of capsules.
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

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Capsule staining is more difficult than other types of staining procedures because
➢ capsular materials are soluble in water and
➢ may be dislodged or removed during rigorous washing.
Mix the bacteria in a solution containing a fine colloidal suspension of colored
particles (usually India ink or nigrosin) to provide a contrasting background and
then stain the bacteria with a simple stain, such as safranin

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Cont..
Because of their chemical composition, capsules do not accept most
biological dyes, such as safranin, and thus appear as halos surrounding
each stained bacterial cell.

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 II. Endospore (Spore) Staining

An endospore
➢ A special resistant, dormant structure
➢ Endospores cannot be stained by ordinary
methods, such as simple staining and Gram
staining
because the dyes do not penetrate the wall of the
endospore.
Highly refractive, they can be detected under the light
microscope when unstained

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Cont…
The most commonly used endospore stain is the
Schaeffer- Fulton endospore stain.
Malachite green, the primary stain, is applied to a
heat fixed smear and heated to steaming for about
5 minutes.
The heat helps the stain penetrate the
endospore wall.

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Cont..
Then the preparation is washed for about 30
seconds with water to remove the malachite
green from all of the cells’ parts except the endospores.
Safranin (1 min), a counterstain, is applied to the smear to
stain portions of the cell (Vegetative) other than endospores.
the endospores appear green within red or pink cells.

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