SIMPLE STAINING OF
BACTERIA
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AIM
• To observe size, shape and arrangements of given bacterial
sample using simple staining
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PRINCIPLE
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INTRODUCTION
• Visualization of microorganisms in the living state is quite difficult, not
only because they are minute, but also because they are transparent
and practically colorless when suspended in an aqueous medium.
• To study their properties and to divide microorganisms into specific
groups for diagnostic purposes, biological stains and staining procedures
in conjunction with light microscopy are used.
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STAINS
• Chemically, a stain (dye) may be defined as an organic compound containing
a benzene ring plus a chromophore and an auxochrome group.
• The ability of a stain to bind to macromolecular cellular components
such as proteins or nucleic acids depends on the electrical charge
found on the chromogen portion, as well as on the cellular component
to be stained.
Chromatophore groups have
conjugated double bonds such as
C=O, C=N, N=N which gives dyes
their colour. 5
ACIDIC AND BASIC STAINS
• Acidic stains are anionic, which means that, on ionization of the stain,
the chromogen portion exhibits a negative charge and therefore has a
strong affinity for the positive constituents of the cell.
• Proteins, positively charged cellular components, will readily bind to and
accept the color of the negatively charged, anionic chromogen of an
acidic stain.
• Examples - Picric acid, Rose bengal, Eosin, Acid fuchsin produce an
anionic chromogen.
• Basic stains are cationic, because on ionization the chromogen
portion exhibits a positive charge and therefore has a strong affinity
for the negative constituents of the cell.
• Nucleic acids, negatively charged cellular components, will readily bind to
and accept the color of the positively charged, cationic chromogen of a
basic stain.
• Examples - Methylene blue, Crystal violet, Safranin, Malachite green,
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Basic fuchsin produce a cationic chromogen.
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• Basic stains are more commonly used for bacterial staining.
• The presence of a negative charge on the bacterial surface
acts to repel most acidic stains and thus prevent their
penetration into the cell.
• Numerous staining techniques are available for visualization,
differentiation, and separation of bacteria in terms of
morphological characteristics and cellular structures.
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PREPARATION OF BACTERIAL SMEARS
• Bacterial smears must be prepared before execution of any of the
staining techniques.
• Bacterial smear is a dry preparation of bacterial cells on a glass slide.
• Although not difficult, the preparation requires adequate care.
• In a good bacterial smear properly processed :
• The bacterial cells are evenly spread out on the slide such that they are adequately
separated from one another.
• The bacteria are not washed off from the slide during staining
• Bacterial form is not distorted.
• Following are the steps in smear preparation.
• 1. Preparation of the glass microscope slide
• 2. Labeling of slides
• 3. Preparation of smear
• 4. Heat fixation
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1. PREPARATION OF THE GLASS MICROSCOPIC SLIDE
• Clean slides are essential for the preparation of microbial smears.
• Grease or oil from the fingers on slides must be removed by washing the
slides with soap and water, followed by a rinse of 95% alcohol.
• After cleaning, dry the slides and place them on blotting sheets until
ready for use.
• Remember to hold the clean slides by their edges.
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2. LABELING OF SLIDES
• Proper labeling of the slide is essential.
• The initials of the organism can be written on either end of the slide with
a glassware marking pencil on the surface on which the smear is to be
made.
• Care should be taken that the label does not come into contact with
staining reagents.
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3. PREPARATION OF SMEAR
• It is crucial to avoid thick, dense smears.
• A thick or dense smear occurs when too much of the culture is used in
its preparation, which concentrates a large number of cells on the slide.
• This type of preparation diminishes the amount of light that can pass
through and makes it difficult to visualize the morphology of single
cells.
• Smears require only a small amount of the bacterial culture.
• A good smear is one that, when dried, appears as a thin whitish
layer or film.
• The print of your textbook should be legible through the smear.
• Different techniques are used depending on whether the smear is made
from a broth or solid medium culture.
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SMEAR PREPARATION FROM BROTH CULTURES
• Resuspend the culture by tapping the tube
with your finger.
• Depending on the size of the loop, one or two
loopfuls should be applied to the center of the
slide with a sterile inoculating loop and
spread evenly over an area about the size of
a coin.
• Set the smears on the laboratory table and
allow to air-dry.
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SMEAR PREPARATION FROM SOLID MEDIUM CULTURES
• Organisms cultured in a solid medium produce thick, dense
surface growth and are not amenable to direct transfer to
the glass slide.
• These cultures must be diluted by placing one or two
loopfuls of water on the center of the slide in which the cells
will be emulsified.
• Transfer of the cells requires the use of a sterile inoculating
loop.
• Only the tip of the loop or needle should touch the culture to
prevent the transfer of too many cells.
• Suspension is accomplished by spreading the cells in a
circular motion in the drop of water with the loop or needle.
• This helps to avoid cell clumping.
• The finished smear should occupy an area about the size of
a coin and should appear as a translucent, or
semitransparent, confluent whitish film.
• At this point the smear should be allowed to dry completely.
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• Do not blow on slide or wave it in the air.
FIXATION
• The stained cells seen under the microscope should resemble the
living cells as closely as possible.
• Fixation is a process by which internal and external structures
of cells and microorganisms are preserved and fixed.
• It inactivates the enzymes that might disrupt the cell
morphology and tuffens the cell structure so that they do not
change during staining and observation.
• A microorganism is usually killed and attached firmly to the
slide during fixation.
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TYPES OF FIXATION
• Heat fixation – it is routinely used for prokaryotes. Typically a film
of cells or smear is gently heated by passing the slide to the
flame. Heat fixation preserves overall morphology but not
structures within the cells.
• Chemical fixation – it is used to protect fine cellular sub-structure
and the morphology of larger, more delicate microorganisms. The
chemical fixative penetrates cells and reacts with cellular
components usually proteins and lipids to render them inactive
and immobile.
• Common fixative mixture contain components such as ethanol,
acetic acid, mercuric chloride, formaldehyde and glutaraldehyde.
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4. HEAT FIXATION
• Unless fixed on the glass slide, the bacterial smear will wash
away during the staining procedure.
• This is avoided by heat fixation, during which the bacterial
proteins are coagulated and fixed to the glass surface.
• Heat fixation is performed by the rapid passage of the air-dried
smear two or three times over the flame of the Bunsen burner.
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PROPER SLIDE PREPARATION
• Before any staining or visualization of a bacterial sample can take place, a
proper smear must be prepared.
• A smear that is too thick may give a false result due to retention of dye that
should have been rinsed away or because the thickness may prevent dye
penetration.
• A smear that is too thin may have too few cells, increasing the time and
energy to find the bacteria under magnification.
• Inconclusive results due to improperly prepared slides may have an impact
on patient treatment and outcomes.
• Good smears are those which allow newsprint to be read through the smear.
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SIMPLE STAINING
• In simple staining, the bacterial smear is
stained with a single reagent, which produces
a distinctive contrast between the organism and
its background.
• Basic stains with a positively charged
chromogen are preferred because bacterial
nucleic acids and certain cell wall
components carry a negative charge that
strongly attracts and binds to the cationic
chromogen.
• The purpose of simple staining is to elucidate
the morphology and arrangement of bacterial
cells.
• The most commonly used basic stains are
methylene blue, crystal violet, safranin and
carbol fuchsin.
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QUICK AND SIMPLE STAIN
• Simple stains are relatively quick and useful methods of testing for
the presence of, determining the shape of, or determining the
numbers of bacteria present in a sample.
• Generally involving a single staining step, simple staining
methods are not considered differential or diagnostic and
have limited uses.
• However, this is a quick procedure for determining whether a
clinical sample has the presence of a foreign bacterial pathogen.
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MATERIALS
REQUIRED
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MATERIAL REQUIRED
• Cultures - 24-hour nutrient agar plate cultures of Escherichia coli,
and Staphylococcus.
• Equipment - Glass microscope slides, Bunsen burner, inoculating
loop, glassware marking pencil. staining tray, microscope, lens
paper, bibulous (highly absorbent) paper.
• Reagents - Crystal violet.
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PROCEDURE
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PROCEDURE – SMEAR PREPARATION
1. Label two clean slides with the initials of the organism.
2. Using a loop, place one to two loops of water on each slide.
3. With a sterile loop, touch the entire loop to the culture ([Link]) and
emulsify the cells in water on Slide 1
4. Then, with a sterile loop, just touch the tip of the loop to the culture
(Staphyloccocus) and emulsify it in the water on Slide 2.
5. Allow both slides to air-dry completely.
6. Heat fix the preparation. Pass the air dried slide through the outer portion
of the Bunsen flame to prevent overheating, which can distort the
morphology through plasmolysis of the cell wall.
7. Examine each slide for the confluent, whitish film or haze.
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PROCEDURE FOR SIMPLE STAINING
1. Place the slides on the staining tray and flood the smear with crystal
violet for 45 seconds.
2. Gently wash the smears with tap water to remove excess stain.
3. During this step, hold the slide parallel to the stream of water; in this way
you can reduce the loss of organisms from the preparation.
4. Using blotting paper, blot dry but do not wipe the slide.
5. Examine all stained slides under the microscope using the 100 X oil
immersion lens.
6. Record your observations.
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RESULTS AND
DISCUSSION
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EXPECTED RESULTS OF SIMPLE STAINING
E. coli Staphylococcus Bacillus
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SIMPLE STAINING
Observations and Results
Color of the background
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Magnification
PRECAUTIONS FOR SIMPLE STAINING
1. The smear should not be too thick or thin.
2. When heat-fixing a smear, always make sure that the smear is on the
top of the slide as you pass it through the flame.
3. Bacteria growing on solid media tend to cling to each other and must be
dispersed sufficiently by diluting with water. If this is not done, the smear
will be too thick and uneven. Be careful not to use too much paste in
making the smear. It is easy to ruin your results by using too many
bacteria.
4. Always wait until the slide is dry before heat-fixing.
5. The inoculating loop must be relatively cool before inserting it into any
broth. Always flame the inoculating loop after using it and before setting
it down.
6. Avoid overstaining and understaining.
7. When rinsing with water, direct the stream of water so that it runs gently
over the smear. 30