MBG 274

Microbiology Laboratory
Lecture 4

Dr. İrem Yalım Camcı

MICROSCOPIC SLIDE TECHNIQUES-II

Visualization of microorganisms in the living state is quite difficult, not only because they are very small, 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 have become major tools in microbiology.
Numerous staining techniques are available for visualization, differentiation, and separation of bacteria in
terms of morphological characteristics and cellular structures. A summary of commonly used procedures is
outlined in Figure 1.

Staining
Techniques

Simple Differential
Staining
Staining

Negative
Direct Simple Differential Structural
(Indirect Simple)
Staining Staining Staining
Staining

Figure 1: Staining techniques

Differential Staining is a staining method with more than one dye. It is a procedure that takes advantage of
differences in the physical and chemical properties of different groups of bacteria. It allows us to differentiate
between different kinds of bacterial cells or different parts of a bacterial cell (Gram staining).

Structural staining is possible to visualize endospores, capsules, cell walls, flagella, metachromatic granules
and other intracellular constituents of microbial cells. The cellular component to be studied in this exercise is
the endospore.
Before staining bacteria, you must first understand how to “fix” the organisms to the glass slide. If the
preparation is not fixed, the organisms will be washed off the slide during staining. A simple method is that
of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms
through the flame of a gas burner. The heat coagulates the organisms’ proteins, causing the bacteria to stick to
the slide.
Experiment 1: Gram Stain
The most important differential stain used in bacteriology is the Gram stain, named after Dr. Hans Christian
Gram. It divides bacterial cells into two major groups, gram-positive and gram-negative, which makes it an
essential tool for classification and differentiation of microorganisms. The Gram stain reaction is based on the
difference in the chemical composition of bacterial cell walls (Figure 2).

Figure 2: Gram positive and Gram negative cell wall

Gram-positive cells have a thick peptidoglycan layer, whereas the peptidoglycan layer in gram-negative cells
is much thinner and surrounded by outer lipid containing layers. Peptidoglycan is mainly a polysaccharide
composed of two chemical subunits found only in the bacterial cell wall. These subunits are N-
acetylglucosamine and N-acetylmuramic acid. With some organisms, as the adjacent layers of peptidoglycan
are formed, they are cross-linked by short chains of peptides by means of a transpeptidase enzyme, resulting
in the shape and rigidity of the cell wall.
The Gram stain uses four different reagents. Descriptions of these reagents and their mechanisms of action are
as follow. Figure 3 shows the microscopic appearance of cells at each step of the Gram staining procedure.

Figure 3: Microscopic observation of cells at each steps of the Gram staining procedure
Primary Stain
Crystal Violet This violet stain is used first and stains all cells purple.
Mordant
Gram’s Iodine This reagent serves not only as a killing agent but also as a mordant, a substance that increases
the cells’ affinity for a stain. It does this by binding to the primary stain, thus forming an insoluble complex.
The resultant crystal-violet–iodine (CV-I) complex serves to intensify the color of the stain. At this point, all
cells will appear purple-black.
Decolorizing Agent
Ethyl Alcohol, 95% This reagent serves a dual function as a protein-dehydrating agent and as a lipid solvent.
Its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer
in bacterial cell walls. In gram-negative cells, the alcohol increases the porosity of the cell wall by dissolving
the lipids in the outer layers. Thus, the CV-I complex can be more easily removed from the thinner and less
highly cross-linked peptidoglycan layer. Therefore, the washing-out effect of the alcohol facilitates the release
of the unbound CV-I complex, leaving the cells colorless or unstained. The much thicker peptidoglycan layer
in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are
made smaller due to the dehydrating effect of the alcohol. Thus, the tightly bound primary stain complex is
difficult to remove, and the cells remain purple.
Note: Be careful not to over-decolorize the smear with alcohol.
Counterstain
Safranin This is the final reagent, used to stain pink those cells that have been previously decolorized. Since
only gram-negative cells undergo decolorization, they may now absorb the counterstain. Gram-positive cells
retain the purple color of the primary stain. The preparation of adequately stained smears requires that you
bear in mind the following precautions:
1. The most critical phase of the procedure is the decolorization step, which is based on the ease with which
the CV-I complex is released from the cell. Remember that over-decolorization will result in loss of the
primary stain, causing gram-positive organisms to appear gram negative. Under-decolorization, however, will
not completely remove the CV-I complex, causing gram-negative organisms to appear gram positive.
Strict adherence to all instructions will help remedy part of the difficulty, but individual experience and
practice are the keys to correct decolorization.
2. It is imperative that, between applications of the reagents, slides be thoroughly washed under running water
or water applied with an eyedropper. This removes excess reagent and prepares the slide for application of the
subsequent reagent.
3. The best Gram-stained preparations are made with fresh cultures, that is, not older than 24 hours. As cultures
age, especially in the case of gram-positive cells, the organisms tend to lose their ability to retain the primary
stain and may appear to be gram-variable; that is, some cells will appear purple, while others will appear
pink.

Procedure
✓ Prepare the bacterial smears from 24-hour pure E. coli, Staphylococcus aureus, B. cereus and Candida
albicans cultures.
✓ Gently flood smears with crystal violet and let stand for 1 minute.
✓ Gently wash with tap water.
✓ Gently flood smears with the Gram’s iodine mordant and let stand for 1 minute.
✓ Gently wash with tap water.
✓ Decolorize with 95% ethyl alcohol. Note: Do not over-decolorize. Add reagent drop by drop until the
alcohol runs almost clear, showing only a blue tinge.
✓ Gently wash with tap water.
✓ Counterstain with safranin for 30 seconds.
✓ Gently wash with tap water. Blot dry with tissue paper and examine under oil immersion.
✓ Find the painted area at 4X magnification of the microscope. Then, determine the examined area at 10X
and 40X magnification. Finally, investigate stained cells by dropping immersion oil at 100X
magnification.
(Please draw the image with colored pencils in your report)
Experiment 2: Spore Stain
Members of the anaerobic genera Clostridium and Desulfoto maculum and the aerobic genus Bacillus are
examples of organisms that have the capacity to exist either as metabolically active vegetative cells or as
highly resistant, metabolically inactive cell types called spores. When environmental conditions become
unfavorable for continuing vegetative cellular activities, particularly with the exhaustion of a nutritional
carbon source, these cells have the capacity to undergo sporogenesis and give rise to a new intracellular
structure called the endospore, which is surrounded by impervious layers called spore coats. As conditions
continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an
independent cell called a free spore. Because of the chemical composition of spore layers, the spore is resistant
to the damaging effects of excessive heat, freezing, radiation, desiccation, and chemical agents, as well as to
the commonly employed microbiological stains. With the return of favorable environmental conditions, the
free spore may revert to a metabolically active and less resistant vegetative cell through germination. It should
be emphasized that sporogenesis and germination are not means of reproduction but merely mechanisms
ensure cell survival under all environmental conditions.
Primary Stain
Malachite Green Unlike most vegetative cell types that were stained by common procedures, spores, because
of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat
is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will
appear green.
Decolorizing Agent
Water Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only
the excess primary stain. The spore remains green. On the other hand, the stain does not demonstrate a strong
affinity for vegetative cell components; the water removes it, and these cells will be colorless.
Counterstain
Safranin This contrasting red stain is used as the second reagent to color the decolorized vegetative cells,
which will absorb the counterstain and appear red. The spores retain the green color of the primary stain.

Procedure
✓ Prepare the bacterial smear from 2-3 days old pure B. cereus culture.
✓ Place the slides on the staining rack and cover the smears with malachite green. Allow to stand
for 30 and 60 seconds before heating.
✓ Heat the preparations gently by passing the Bunsen burner under the slides. Continue heating
until you observe a steam when the flame is removed.
✓ Maintain steaming for minutes. Add more dye as needed to prevent the smear from drying out.
✓ Allow the slide to cool and rinse (front and back) in slowly running water.
✓ Cover the upper surface of the bacterial smear preparation with the safranin by a Pasteur pipette
and wait for 60 seconds.
✓ After, 60 second, wash the over stain with dH2O.
✓ Allow the stained bacterial smear to dry.
✓ After drying, examine the staining under a microscope.
✓ Find the painted area at 4X magnification of the microscope. Then, determine the examined area at 10X
and 40X magnification. Finally, investigate B. cereus cells and endospore at 100X magnification after
dropping immersion oil.
(By drawing what you see with colored pencils at report, please evaluate)

Experiment 3: Capsule Staining

A capsule is a gelatinous outer layer that is secreted by the cell and that surrounds and adheres to the cell wall.
It is not common to all organisms. Cells that have a heavy capsule are generally virulent and capable of
producing disease, since the structure protects bacteria against the normal phagocytic activities of host cells.
Chemically, the capsular material is composed mainly of complex polysaccharides such as levans, dextrans,
and celluloses.
The clinically important bacteria such as Streptococcus pneumoniae, Klebsiella pneumoniae and Neisseria
meningitis have capsules. The capsule is considered to be a virulence factor because it protects the bacteria
against phagocytosis.

Capsule staining is more difficult than other types of differential staining procedures because the capsular
materials are water-soluble and may be dislodged and removed with vigorous washing. Smears should not be
heated because the resultant cell shrinkage may create a clear zone around the organism that is an artifact that
can be mistaken for the capsule. Capsules are resistant to staining. Therefore, they are visible under the
microscope only when stained with a special method.
Procedure
✓ In this staining method, there is no need for heat fixation.
✓ Place a small drop of nigrosin close to one end of a clean slide.
✓ Using aseptic technique, place a loopful of inoculum from the Klebsielle pneumonia culture (24 Hours) in
the drop of nigrosin and mix.
✓ With a clean glass slide, spread the mixture over the entire surface of the slide to create a very thin smear.
✓ Allow smears to air-dry.
Note: Do not heat fix
✓ Cover the upper surface of the bacterial smear preparation with the safranin by a Pasteur pipette
and wait for 30 seconds.
✓ After, 30 second, wash the over stain with dH2O.
✓ Remove excess stain and dH2O with filter paper and allow to dry.
✓ After drying, examine the staining under a microscope.
✓ Find the painted area at 4X magnification of the microscope. Then, determine the examined area at 10X
and 40X magnification. Finally, investigate stained the Klebsielle pneumonia cells at 100X magnification
by dropping immersion oil.
(Draw the images with colored pencils at report)

Note: Apply all producedures to the unknown bacteria which is given to your group in order to
identify it.