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Lab 3 Objectives
Bacterial Morphology:
Bacteria are unicellular procaryotes belonging to the Kingdom Procaryotae
(Monera). These cells are usually smaller than eucaryotic cells, with most
only 0.5 to 2.0 μm (micrometers) in diameter and 0.5 to 10.0
μm in length. Bacteria exhibit a variety of
shapes and
cell arrangements if they
remain together after cell division (binary fission). This week in lab you
will be preparing your own simple stain and negative stain slides for observing
bacterial morphology. Because it is critical for accurately identifying
bacteria in clinical applications, be able to recognize and properly name the
various shapes and arrangements that bacteria exhibit.
Simple Staining
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Background and Purpose:
With this technique only a single staining reagent is used on a bacterial smear,
making the cells more visible when viewed microscopically. This is useful
when one wants to determine the basic morphology of cells and the presence or
absence of various types of granules within a cell's cytoplasm. The dyes
used for this procedure contain color-bearing ions known as chromophores.
If the chromophores are positively-charged (cationic), the dye is classified as
a basic stain. Because chemicals of opposite charge are attracted
to each other, basic stains are preferred in simple staining because they are
strongly attracted to nucleic acids and parts of the cell wall that bear
numerous negative charges. The most commonly used basic dyes are methylene
blue, malachite green, basic fuchsin, crystal violet, and safranin. Since these basic dyes
color the cells and not the background, simple staining can be regarded as a
positive staining technique. In contrast to basic stains, acidic
stains contain chromophores that are negatively-charged (anionic).
Since these dyes tend to be repelled by cells, they are more useful in
negative staining procedures that stain the background (see below).
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General Procedure:
A basic stain is applied to a smear for a specified time and then
washed off with water.
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Materials:
basic stain (e.g., methylene blue, crystal violet, safranin),
staining rack, slide holder, disposable latex gloves, wash bottle,
bibulous paper
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Negative Staining
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Background and Purpose:
This method colors the background, while the bacteria remain unstained or
colorless against this darkened field. The agent used, nigrosine, does not
color the bacteria because of ionic repulsion between the stain and the cells,
both of which are negatively charged. Since this technique does not
require heat-fixing the slide, cells do not shrink or become distorted.
This is particularly useful when accurate determination of cell size and shape
is needed. It also allows for observation of spirochaetes or spirilli that
do not stain readily by other staining methods.
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General Procedure:
Bacterial cells are mixed in a small drop of nigrosine on a
microscope slide. A second slide is used to spread the suspension into a
thin film across the slide. After air-drying, the slide can be observed
under oil immersion.
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Materials:
Microscope slides, nigrosine, sterile toothpicks or an inoculating
wire
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