This document provides instructions for preparing and fixing bacterial smears for microscopy. Smears should be made by transferring samples from various specimens, such as purulent fluids, cultures, sputum, and swabs, onto labeled microscope slides using sterile loops or sticks. The smears are then air dried before being fixed through heat, alcohol, or chemical fixation. Alcohol fixation is recommended for intracellular bacteria as it preserves cell structure better than heat fixation. Smears can then be stained for microscopic examination.

1. Microbiology
practical (Lab 3)
Preparation and fixation of
bacterial smears

2. How to prepare and fix smears
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Labeling slides Every slide must be labeled
clearly with the date and the patient’s name
and number.
spread on slides which have one end frosted for
labeling. With the increased use of such slides in
recent years, their price is now little more than
slides without a frosted end.

3. •
A lead pencil should be used for writing on the
frosted area because pencil marks, unlike and grease
pencil marks, will not be washed of smears should be
spread evenly covering an
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area of about 15–20 mm diameter on a slide.
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. The techniques used to make smears from different
specimens are as follows:

4. •
Purulent specimen: Using a sterile wire loop,
make a thin preparation. Do not centrifuge a
purulent fluid, e.g. c.s.f. containing pus cells.
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● Non-purulent fluid specimen: Centrifuge the
fluid and make a smear from a drop of the well-
mixed sediment.
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● Culture: Emulsify a colony in sterile distilled
water and make a thin preparation on a slide.
When a broth culture, transfer a loopful to a
slide and make a thin preparation.

5. •
● Sputum: Use a piece of clean stick to transfer
and spread purulent and caseous material on a
slide
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● Swabs: Roll the swab on a slide. This is
particularly important when looking for
intracellular bacteria such as N. gonorrhoeae
(urethral, cervical, or eye swab). Rolling the swab
avoids damaging the pus cells.
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● Faeces: Use a piece of clean stick to transfer
pus and mucus to a slide.
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.

6. Drying smears
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After making a smear, leave the slide in a safe
place for the smear to air-dry, protected from
dust, flies and direct sunlight.

7. fixing smears
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Smears are fixed by heat, alcohol, or occasionally by
other chemicals.

8. Heat fixation
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This is widely used but can damage organisms and
alter their staining reactions especially when
excessive heat is used.
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Heat fixation also damages leucocytes and is
therefore unsuitable for fixing smears which may
contain intracellular organisms such as N.
gonorrhoeae and N. meningitidis. When used, heat
fixation must be carried out with care. The following
technique is recommended:

9. •
1-- Allow the smear to air-dry completely.
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2-- Rapidly pass the slide, smear uppermost,
three times through the flame of a spirit lamp or
pilot flame of a Bunsen burner.

10. Alcohol fixation
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This form of fixation is far less damaging to
microorganisms than heat.
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Cells, especially pus cells, are also well
preserved. Alcohol fixation is therefore
recommended for fixing smears when looking for
Gram negative intracellular diplococci.
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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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A method of alcohol fixing smears is as follows:

11. •
1-- Allow the smear to air-dry completely.
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2-- Depending on the type of smear, alcohol-fix as
follows: –
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For the detection of intracellular Gram negative
diplococci (N. gonorrhoeae or N. meningitidis), fix
with one or two drops of absolute methanol or
ethanol. – For the detection of other organisms
including M. tuberculosis, fix with one or two drops
of 70% v/v methanol or ethanol (absolute methanol
can also be used but a 70% v/v solution is adequate).
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3-- Leave the alcohol on the smear for a minimum
of 2 minutes or until the alcohol evaporates.

12. Other chemical fixatives
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Other chemicals are smears which contain
particularly dangerous organisms to ensure all
the organisms are killed, e.g. 40 g/l potassium
permanganate is recommended for fixing smears
which may contain anthrax bacilli.
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Formaldehyde vapour is sometimes
recommended for fixing smears which may
contain Mycobacterium species.

13. •
Thanks