Experiment 2
Microscopy: Simple staining, Gram stain and cell fractionation
2.1 Introduction
Most living microorganisms are generally colorless and almost invisible because of
their lack of contrast with the water in which they may reside, staining is necessary in
order to make them readily visible for observation of intracellular structures as well as
overall morphology. This lab exercise has been designed to give the student expertise
in staining and slide preparation, an appreciation for the morphology of some
microorganisms. Delicate transparent living organisms can be more easily observed
with darkfield microscopy (Figure 2.1) than with conventional brightfield
microscopy.
Figure 2.1 Transparent living microorganisms, such as the
syphilis spirochaete, can be seen much more easily when
observed in a dark field.
2.2 Objectives
1.
2.
3.
4.
5.
6.
7.
To learn how to properly prepare slides for microbiological examination
To learn how to clean and dispose of used slides
To learn how to prepare smears from solid and liquid cultures
To learn how to perform wet-mounts and/or hanging drop preparations
To learn how to perform simple staining and Gram stain
To learn how to record microscopic observations
To learn the idea of cell fractionation
2.3 Materials required
Light microscope (compound microscope, binocular microscope)
Stereomicroscope (dissecting microscope)
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Glass slides and cover slips
Ocular and stage micrometers
Prepared slides
Lens paper
2.4 Negative staining
Negative, indirect, or background staining is achieved by mixing bacteria with an
acidic stain such as nigrosin, India ink, or eosin, and then spreading out the mixture
on a slide to form a film. The above stains will not penetrate and stain the bacterial
cells due to repulsion between the negative charge of the stains and the negatively
charged bacterial wall. Instead, these stains either produce a deposit around the
bacteria or produce a dark background so that the bacteria appear as unstained cells
with a clear area around them (Figure 2.2).
Figure 2.2 India Ink Stain of Bacillus megaterium (×1,000). Notice the
dark background around the clear bacterial cells.
1. Use an inoculating loop to apply a small amount of bacteria to one end of a clean
microscope slide.
2. Add 1 to 2 loops of nigrosin, India ink, or eosin solution to the bacteria and mix
thoroughly.
3. Spread the mixture over the slide using a second slide. The second slide should be
held at a 45° angle so that the bacteria-nigrosin solution spreads across its edge.
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2.5 Bacterial smear preparation
A bacterial smear is a dried preparation of bacterial cells on a glass slide. In a
bacterial smear that has been properly processed, (1) the bacteria are evenly spread
out on the slide in such a concentration that they are adequately separated from one
another, (2) the bacteria are not washed off the slide during staining, and (3) bacterial
form is not distorted.
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For the broth culture, shake the culture tube and, with an inoculating loop, aseptically
transfer 1 to 2 loopfuls of bacteria to the center of the slide. Spread this out to about a
3 cm2 area. When preparing a smear from a slant or plate, place a loopful of water in
the center of the slide. With the inoculating needle, aseptically pick up a very small
amount of culture and mix into the drop of water. Spread this out as above. Allow the
slide to air dry. Pass the slide through a Bunsen burner flame three times to heat-fix
and kill the bacteria.
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2.5 Simple staining
1. Place the three fixed smears on a staining loop or rack over a sink.
2. Stain one slide with alkaline methylene blue for 1 minute; one slide with
carbolfuchsin for 5 to 10 seconds; and one slide with crystal violet for 20 to 30
seconds.
3. Wash stain off slide with water for a few seconds
4. Blot slide dry with bibulous paper. Be careful not to rub the smear when drying the
slide because this will remove the stained bacteria.
5. Examine under the microscope to see if your slides are overstaining or
understaining.
2.6 Gram stain
In 1884 the Danish bacteriologist Christian Gram developed a staining technique that
separates bacteria into two groups: those that are gram-positive and those that are
gram-negative. The procedure is based on the ability of microorganisms to retain the
purple color of crystal violet during decolorization with alcohol. Gram-negative
bacteria are decolorized by the alcohol, losing the purple color of crystal violet.
Gram-positive bacteria are not decolorized and remain purple. After decolorization,
safranin, a red counterstain, is used to impart a pink color to the decolorized
gram-negative organisms.
Note that crystal violet, the primary stain, causes both gram-positive and
gram-negative organisms to become purple after 20 seconds of staining. When
Gram’s iodine, the mordant, is applied to the cells for one minute, the color of
gram-positive and gram-negative bacteria remains the same: purple. The function of
the mordant here is to combine with crystal violet to form a relatively insoluble
compound in the gram-positive bacteria. When the decolorizing agent, 95% ethanol,
is added to the cells for 10–20 seconds, the gram-negative bacteria are leached
colorless, but the gram-positive bacteria remain purple. In the final step a counterstain,
safranin, adds a pink color to the decolorized gram-negative bacteria without affecting
the color of the purple gram-positive bacteria.
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Figure 2.6.1 Color changes that occur at each step in the Gram-staining process
Figure 2.6.2
Gram-staining procedures
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2.7 Cell fractionation
The basic principle for all microscopes is that the cell is composed of smaller physical
units, the organelles. Definition of the organelles is possible with microscopy, but the
function of individual organelles is often beyond the ability of observations through a
microscope. We are able to increase our chemical knowledge of organelle function by
isolating organelles into reasonably pure fractions.
A host of fractionation procedures are employed by cell biologists. Each organelle has
characteristics (size, shape and density) which make it different from other organelles
within the same cell. If the cell is broken open in a gentle manner, each of its
organelles can be subsequently isolated. The process of breaking open cells is
homogenization and the subsequent isolation of organelles is fractionation. Isolating
the organelles requires the use of physical chemistry techniques, and those techniques
can range from the use of simple sieves, gravity sedimentation or differential
precipitation, to ultracentrifugation of fluorescent labeled organelles in computer
generated density gradients.
Figure 2.7.1 Animal Cell organelles
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Figure 2.7.2
Table 2.7.1
Plant Cell organelles
Size and density of some typical organelles*
Organelle
Diameter (µm)
Density (g/ml)
Nuclei
5 – 10
1.4
Mitochondria
1-2
1.1
Ribosomes
0.02
1.6
Lysosomes
1-2
1.1
2.7.1 Cell Fractionation Procedure
Instructor will homogenize fresh pea pods in a blender and filtered the homogenate
through filter paper. Before centrifuging, examine the homogenate with the
microscope. Observe many liberated starch grains, intact cells containing starch grains,
and intact cells from the pod walls that contain bright green chloroplasts.
If you centrifuge the filtrate at low speed, you will pellet cell wall fragments, starch
grains, nuclei, and some chloroplasts. If you centrifuge the same tube at high speed,
you will pellet chloroplasts on top of the starch layer. Sample the supernatant, the
green pellet layer, and the white pellet layer, see what they contain, and test each with
Lugol’s solution.
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