1
Preparing a Bacterial Smear
-Since bacterial cells are so small, special problems arise in the preparation
of a good smear
-There are 3 principal precautions that you must take to avoid problems:
1. Do not use scratched microscope slides since scratches in the glass
slide can be confused with microorganisms.
2. Be sure that you are working with a very clean slide.
3. Avoid making smears that are too thick or too thin.
A dirty slide may be greasy or may be laden with dirt and dust; such a
slide will result in an unsatisfactory smear for 3 reasons:
1. The smear containing the desired microbes will wash off the slide
during the staining process.
2. When you deposit the bacterial suspension on the microscope
slide, it will coalesce, i.e., it will not remain spread out.
3. Dirt, dust and other debris can be mistaken for microbes.
-the slide must be so clean that a drop of water spreads out on it
*water coalescing/beading on the slide indicates the presence of an oily film that
clumps bacteria
*even a small amount of oil from a fingerprint leaves a film
*Another difficulty in making a good smear is to get the right amount of
bacteria on the slide.
a good smear should neither be too thick nor too thin
1. What are the consequences if a smear is too thick? (too many cells on
the slide)
-does not allow the proper penetration of light through the smear
-bacterial cells are often packed too closely together
*You are most likely to make thick smears when your cells are obtained with a
loop from solid culture media (e.g. slant and agar plate)
2. What are the consequences if a smear is too thin? (only a few cells are
deposited on the slide)
-searching for bacterial cells is time-consuming
*thin smears usually result when the smears are made from broth cultures
-LABELING is as important in making smears for stained slides as it is for
cultures
labeling should be done at one end of the slide
-AIR DRYING of the slides should be done in a flat position
-HEAT FIXATION kills the microorganisms, coagulates the protoplasm of
the cells and causes them to adhere to the slide
adequate heat fixation shrinks cells slightly, but it helps bacteria
adhere to the slide through several rinses
excessive heat warps cells; applying heat to the smear before it is
completely dry also distorts cells
-Remember to PRACTICE ASEPTIC TECHNIQUE
-Smears from Broth Cultures:
disperse the bacteria that have settled to the bottom of the broth
culture tube
-Smears from Growth on Solid Media:
*bacteria from the solid agar must be put into a liquid before they can be spread
out on the slide
add a small amount of NSS/water (2-3 loopfuls or 1 small drop) to
the center of the slide
-a large drop is undesirable because it takes much longer for the
slide to dry
aseptically remove only a small amount of bacteria (colony) from
the culture
mix and spread out
Staining Bacterial Smears
-The Chemistry of Staining:
based on the principle that unlike charges attract; similar charges repel
-a bacterial cells carries electrical charges:
in an aqueous environment, with the pH at approx. 7, the net
electrical charge produced by most bacteria is negative
the (-) charged cells attract molecules carrying (+) charges; repel
those that are (-) charged
-each dye in a salt containing 2 ionsone with a (+) charge (cation), and the
other with a (-) charge (anion)
either one of the two ions can be the chromophore (part of the
molecule that is brightly colored)
-the most frequently employed microbiological dyes are called basic dyes
their chromophores are (+) charged
the chromophores are attracted to, and subsequently color bacteria
examples of basic dyes: methylene blue, crystal violet, safranin
acid dyes (e.g. nigrosine) are generally repelled by bacteria because
of the (-) charges on their chromophores (acidic dyes color the
background and leave the cells colorless)
Gram Staining
Table 1.1 The Gram Stain Procedure
Step
Primary Stain
Reagent
Crystal Violet
Time
1 min
Color
Gram (+)
Gram (-)
Purple
Purple
Grams Iodine
1 min
Purple
Purple
95% Ethanol
Brief
Purple
Colorless
Safranin
30 sec- Purple
1 min
Pink/Red
Brief H2O rinse
Mordant
Brief H2O rinse
Decolorizer
Brief H2O rinse
Counter Stain
Brief H2O rinse
Table 1.2 How to Limit Variables that Affect Gram Stain Results
1. Use actively growing cells. Old cells lose their ability to hold the stain; they
appear gram-negative.
2. Prepare thin smears and adjust decolorization time. Where cells are
3.
4.
5.
6.
crowded, they resist decolorization. Bacteria in thin smears decolorize much faster
than bacteria in thick smears.
Examine well-dispersed cells; avoid stain crystals. The Gram reactions of
clumped bacteria or cells adjacent to crystals of dye are unreliable.
Avoid overheating the cells during heat fixation. Excessive heat disrupts
cell walls making gram-positive bacteria appear gram-negative.
Use fresh staining reagents. Old reagents give variable results and form
crystals.
Adjust timing to your reagents. For example, many laboratories decolorize
with fast-acting acetone-alcohol instead of 95% ethanol.
7. Do not rinse too long. Water is a decolorizer.
8. Remember, gram-positivity is a characteristic that can be lost.
Acid Fast Staining
-in 1882 Paul Ehrlich developed the Acid-Fast Stain while working with
Mycobacterium tuberculosis
-the Acid-fast procedure is a differential stain
one that distinguishes one group of bacteria from another
-Mycobacterium and many Nocardia species are called acid-fast because
during the staining procedure, they retain the primary dye despite
decolorization with the powerful solvent acid-alcohol
nearly all other genera are non acid-fast
-Ehrlichs method was improved by later microbiologists including Ziehl
and Neelsen
both the Ehrlich and the Ziehl-Neelsen techniques require heat to
drive a primary dye past the cell wall
Kinyoun acid-fast Procedure, a wetting agent (detergent) makes
heating unnecessary
-Cell wall structure and Acid-Fast Staining:
in the cell walls of Mycobacterium and many Nocardia species are
lipoidal mycolic acids
*normally, these lipids prevent dye chromophores from coloring the cell
*some mycobacteria, including Mycobacterium tuberculosis contain so
much mycolic acid that they are nearly impossible to stain with Gram
Staining
*others e.g. M. smegmatis (originally isolated from smegma) and M. phlei
(isolated from hay and grass) are protected by less mycolic acid
one theory concerning the ability of the acid-fast stain to differentiate
bacteria holds that mycolic acids are somewhat permeable to dye that is
dissolved in alcohol and phenol and applied with heat (or detergent)
-after the bacteria are cooled (or the detergent is rinsed off), mycolic acids
in cell walls coalesce, forming a barrierthe lipids prevent acid-alcohol
from decolorizing the protoplasm
-Medical Significance of Acid-fast Bacteria and Acid-fast Staining
Table 1.3 The Ziehl-Neelsen Acid-Fast Stain Procedure
Procedure
Reagent
Time
Cell Color
Acid-Fast
Primary Dye
Carbolfuchsin
3-5
Brief H2O rinse (contains basic fuchsin,
phenol, ethanol and H2O)
mins.
Decolorizer
Brief
Acid-alcohol
Non Acid-Fast
Red
Red
Red
Colorless
Brief H2O rinse (3% conc. HCl in 95%
ethanol)
Counterstain
Brief H2O rinse
Methylene Blue
30-45 Red
sec.
Blue
