EXERCISE 4

MICROSCOPIC OBSERVATION OF MICROORGANISMS

Most types of cells do not have much natural pigment and are therefore difficult to see
under the light microscope unless they are stained. Several types of stains are used to make
bacterial cells more visible. In addition, specific staining techniques can be used to determine the
cells’ biochemical or structural properties, such as cell wall type and presence or absence of
endospores. This type of information can help scientists identify and classify microorganisms, and
can be used by health care providers to diagnose the cause of a bacterial infection.

One type of staining procedure that can be used is the simple stain, in which only one stain
is used, and all types of bacteria appear as the color of that stain when viewed under the
microscope. Some stains commonly used for simple staining include crystal violet, safranin, and
methylene blue. Simple stains can be used to determine a bacterial species’ morphology and
arrangement, but they do not give any additional information. Scientists will often choose to
perform a differential stain, as this allows them to gather additional information about the bacteria
they are working with. Differential stains use more than one stain, and cells will have a differe nt
appearance based on their chemical or structural properties. Some examples of differential stains
are the Gram stain, and endospore stain.

Gram staining procedure has been widely used by microbiologists everywhere to obtain
important information about the bacterial species they are working with. Knowing the Gram
reaction of a clinical isolate can help the health care professional make a diagnosis and choose the
appropriate antibiotic for treatment. Gram stain results reflect differences in cell wall compositio n.
Gram positive cells have thick layers of a peptidoglycan (a carbohydrate) in their cell walls; Gram
negative bacteria have very little. Gram positive bacteria also have teichoic acids, whereas Gram
negatives do not. Gram negative cells have an outer membrane that resembles the phospholip id
bilayer of the cell membrane. The outer membrane contains lipopolysaccharides (LPS), which are
released as endotoxins when Gram negative cells die. This can be of concern to a person with an
infection caused by a gram negative organism.

There are a variety of staining procedures used to identify specific external or interna l
structures that are not found in all bacterial species. Endospore staining, capsule staining, and
flagellar staining are special staining techniques that allow for the differentiation of specific
bacterial structures found either inside or outside of cells. Bacterial endospores are dormant
structures produced by a variety of bacterial species and which are highly resistant to heat,
desiccation, and toxic chemicals. Endospores resist staining, but once stained are highly resistant
to being decolorized and counterstained (much like acid-fast cells). A single bacterium can contain
only one endospore, the shape and location of which is usually characteristic of the species. The
degree to which an endospore changes the shape of the sporangium (spore-containing cell) is also
a feature useful in identification. Not all cells present within a population will contain endospores,
and older cells will often degenerate leaving their spores (now called exospores) behind in the
environment.
 Some bacteria secrete a polysaccharide-rich structure external to the cell wall called a
glycocalyx. If the glycocalyx is thin and loosely attached, it is called a slime layer; if it is thick
and tightly bound to the cell, it is called a capsule. The glycocalyx can protect the cell from
desiccation and can allow the cell to stick to surfaces like tissues in the body. They may also
provide cells with protection against detection and phagocytosis by immune cells and contribute
to the formation of a biofilm: in this way a glycocalyx can act as a virulence factor; (contributes
to the ability of an organism to cause disease). Capsules can be detected using a negative staining
procedure in which the background (the slide) and the bacteria are stained, but the capsule is not
stained. The capsule appears as a clear unstained zone around the bacterial cell. Since capsules are
destroyed by heat, the capsule staining procedure is done without heat-fixing the bacteria.

Flagella (long whip-like structures used for bacterial motility) and some bacteria (e.g.
spirochetes) are too thin to be observed with regular staining procedures. In these cases, a silver
stain is used. Silver nitrate is applied to the bacteria along with a special mordant; the silver nitrate
precipitates around the flagella or the thin bacteria, thus thickening them so they can be observed
under the light microscope.

On the other hand, hanging drop and wet mount techniques allow for observation of living
organisms. The wet mount tends to dry out quickly under the heat of the microscope light; it is
simpler to perform than the hanging drop, but it is useful for short-term observation only. This
method is the simplest and quickest way to determine motility. It is also useful for determining
cellular shape and arrangement which is sometimes destroyed during the staining process.

The hanging drop preparation is a more complex technique, but it allows for longer- term
observation and more reliable observation of motility. These techniques are usually performed
without the addition of any stains; therefore, the organisms can be difficult to see. Reduce the
illumination on your microscope as much as you can while still allowing yourself enough light to
observe the organism.

Learning Objectives

At the end of the exercise, the student should be able to:

a. learn the differences between simple staining and differential staining technique;
b. state the differences between gram positive and gram negative bacteria;
c. learn how to perform the gram stain procedure;
d. use microscopy to examine gram stained cells;
e. prepare specimen preparation such as wet mount and hanging drop method; and
f. learn about some special staining procedures and specimen preparation, and view examples
of these under oil immersion.
 WET MOUNT PREPARATION

A. Materials

● Glass slides and coverslips

● Live Saccharomyces culture
● Pond water sample (if available)
● Microscope

B. Procedure

These slides will be prepared with live microbes on them. Since the liquid is left on the
slide, cover slips are needed to prevent this liquid from touching the lenses.

1. Live protozoa: use a dropper to place 1-2 drops of a pond water sample onto a clean
microscope slide. Take a cover slip and place it down over the water as shown in the
diagram below (try to avoid air bubbles).

2. Preparation of a live yeast culture: place one drop of the yeast (Saccharomyces) suspension
and one drop of methylene blue onto a clean microscope slide, and prepare a wet mount as
shown.

3. Observe these slides under the microscope (use the magnifications suggested by your
instructor).

Figure 1. Wet mount preparation set-up

See these links for the actual demonstration of the wet mount preparation:

HANGING DROP METHOD

A. Materials

● Clean glass depression slides/normal glass slides

● Petroleum jelly
● Parafilm (for normal glass slides)
● Cover slips
● Bacterial culture (preferably 24 to 48-hr broth culture)
● Toothpick
● Inoculating loop
● Microscope

B. Procedure

The examination of microorganisms in a hanging drop method involves the following

protocol:

1. Take grease-free or clean coverslip and take one glass depression slide and clean the well
with a piece of dry tissue.

2. Place a thin parafilm around the corner of the slide. Note: This is done when you are using
a normal glass slide. But if you are using a depression slide already, there is no need to
place a parafilm.

3. Gently shake the bacterial broth culture (optimally from a young broth culture) until it is
evenly suspended. If your culture is from a solid or a semi-solid media, you should add a
drop of water first onto the cover slip before mixing it with the culture. Using good aseptic
technique, sterilize the wire loop or toothpick, remove the cap of the tube, and take up a
loopful of culture.

4. Place a loopful of culture in the center of the cover slip.

5. Dab all four corners of the coverslip with petroleum jelly.
6. Turn the depression slide upside down over the cover slip and then press it down gently so
that the petroleum jelly adheres to the slide.
7. Now turn the slide over. You should have a sealed wet mount, with the drop of culture
hanging in the well.
8. Place the slide on the microscope stage, cover glass up. Start your examination with the
low-power objective to find the focus.
9. Continue your examination with the high-power and oil-immersion objectives (Please be
very careful not to break the cover slip).

Figure 2. Hanging drop preparation

See this link for the actual demonstration of the hanging drop preparation:
https://www.youtube.com/watch?v=OA6oZfvDoYg&t=33s
 SIMPLE STAINING

A. Materials

● Clean glass slide

● Stains: methylene blue, safranin, crystal violet
● Alcohol lamp
● Inoculating loop
● Bacterial culture from broth or solid medium (preferably 24 to 48-hr broth culture)

B. Procedure

Bacterial smear preparation from a culture grown in broth medium

1. Obtain a glass slide and label it appropriately at one end.

2. Ensure that the bacterial cells in the mixed broth culture are fully mixed.
3. Using a sterilized inoculating loop, transfer a loopful of broth from the bacterial
culture to be stained to the glass slide. Be sure to sterilize the loop between each
transfer.
4. Using the microbiological loop, make a bacterial smear by spreading the broth
such that the liquid is about the size of a quarter.
5. Sterilize the bacteriological loop and place it aside.
6. The wet smear must now be dried.
7. The dry slide now needs to be heat fixed.

Bacterial smear preparation from a culture grown in solid medium

1. Obtain a glass slide and label it appropriately at one end.

2. Place one drop of water from your loop on the center of the glass slide.
3. Transfer a small amount of solid inoculum into the drop of water and spread
thoroughly.
4. Spread both into a thin area and allow smear to air-dry.
5. When the smear is completely dry, heat-fix by quickly passing the smear over the
flame two to three times.

Simple staining of a bacterial smear

Perform the following staining procedure using the heat-fixed, bacterial smears.

1. Obtain the vial of the stain (could be crystal violet, methylene blue, and safranin)
and flood the area of the smear. Allow the stain to remain for one minute.
2. Lifting the slide at an angle, use the water bottle on the bench to rinse both sides
of the slide until the dye has been removed.
3. Gently blot (do not rub or scrape) the slide dry with bibulous paper or paper towel.
 4. Examine the slide under the microscope using both the high power and immers io n
oil objective lenses.

See this link for the actual demonstration on how to do simple staining:
https://www.youtube.com/watch?v=kaOJDG_PnDQ

GRAM STAINING

A. Materials

● Clean glass slides and coverslips

● Staining trays and newspaper
● Gram stain reagents: crystal violet, Gram’s iodine, safranin, 95% ethanol
● Water bottle (for rinsing)
● Bacterial cultures

B. Procedure

Perform this gram staining procedure using this link:

https://learn.chm.msu.edu/vibl/content/gramstain.html?fbclid=IwAR3s-
_HRifdGIVvqnd559G-KGFxFFAoskhHVwgKGYbqCiW9LJjmdu_l7Pm8

1. After opening the link, click the “module” hyperlink.

2. Another tab will open then click the “Start” hyperlink to begin the virtual module.
3. From a liquid culture, take a loopful of bacteria and emulsify it in a small drop of water or
saline on the slide. This should be a thin, not milky, suspension or it will not stain properly.
Air dry the slide. This is done automatically in the virtual module.
4. Then start performing the gram staining process through the following steps:
a. Heat fix the slide: click on the Bunsen burner, pass the slide gently two or three
times (1-2 seconds) through the flame. Do not overheat - this will cause distortion
of the cells.
b. Flood the slide with crystal violet for 1 minute.
c. Rinse with water.
d. Flood the slide with iodine for 1 minute.
e. Rinse with water.
f. Decolorize with alcohol for 5-10 seconds.
g. Rinse with water.
h. Flood the slide with safranin for 1 minute.
i. Rinse with water.
j. View slide under the microscope.
5. The “slide” contains E. coli and S. aureus.
6. When you are finished with the exercise, click on “Examine Examples” to see actual
micrographs of several bacteria that have been gram stained.
 SPECIAL STAINING

Endospore staining

A. Materials

● Malachite green
● Safranin
● Tap water
● Glass slides and coverslips
● Blotting paper
● Bacterial culture

B. Procedure

1. Take a clean grease free slide and make a smear.

2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper
or toweling cut to fit the slide.
3. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes,
keeping the paper moist and adding more dye as required. Alternatively, the slide may be
steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water then blot dry.
6. Examine the slide under a microscope for the presence of endospores. Endospores appear
bright green and vegetative cells are brownish red to pink.

See this link for the actual demonstration of the endospore staining:
https://www.youtube.com/watch?v=4tylE4f-kM0

Nigrosin capsule staining

A.Materials
● Crystal violet
● Nigrosin
● Glass slides and coverslips
● Bacterial culture (capsule-forming bacteria such as Klebsiella, Streptococcus, Azotobacter,
etc.)
● Microscope

B. Procedure
1. Place a small drop of a Nigrosin stain or India ink on the slide.
2. Using sterile technique, add a loopful of bacterial culture to the slide, smearing it in
the dye.
 3. Use the other slide to drag the ink-cell mixture into a thin film along the first slide
and let it stand for 5-7 minutes.
4. Allow to air dry (do not heat fix).
5. Flood the smear with crystal violet stain (this will stain the cells but not the
capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a 45
degree angle and let stain run off until it air dries .
6. Examine the smear microscopically for the presence of encapsulated cells as
indicated by clear zones surrounding the cells.

See this link for the actual demonstration of the capsule staining:
https://www.youtube.com/watch?v=0_cEdk1gx2A

Flagellar staining

A. Materials
● Bacterial culture on blood agar media (preferably grown for 16-24 hours)
● Glass slides and coverslips
● RYU flagella stain (Remel, Lenexa, Kansas)
● Microscope

B. Procedure

1. Grow the organisms to be stained at room temperature on blood agar for 16 to 24

hours.
2. Add a small drop of water to a clean glass slide.
3. Dip a sterile inoculating loop into sterile water.
4. Touch the loopful of water to the colony margin briefly (this allows motile cells to
swim into the droplet of water).
5. Touch the loopful of motile cells to the drop of water on the slide.
6. Cover the faintly turbid drop of water on the slide with a cover slip. Note: A
proper wet mount has barely enough liquid to fill the space under a cover slip.
Small air spaces around the edge are preferable.
7. Examine the slide immediately under 40x for motile cells.
8. If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes.
9. Apply 2 drops of RYU flagella stain gently on the edge of the cover slip. The
stain will flow by capillary action and mix with the cell suspension.
10. After 5 to 10 minutes at room temperature, examine the cells for the presence,
absence, location, and number of flagella per bacterial cell.

See this link for the actual demonstration of flagella staining:

https://www.youtube.com/watch?v=BKrjktrVtHo&t=48s
 REFERENCES

Brown, A. E., & Smith, H. (2016). LooseLeaf Benson’s Microbiological Applications Laboratory
Manual--Concise Version.

Bykowski, T., & Stevenson, B. (2008). Aseptic technique. Current protocols in

microbiology, 11(1), A-4D.

Fernandez, W. L., Dalmacio, I. F., Raymundo, A. K., Zamora, A. F., Mendoza, B. C. (2008).
Laboratory Manual in General Microbiology. Seven Lakes Printing Press.
James, C., & Natalie, S. (2014). Microbiology. A laboratory manual. Pearson education.
Madigan, M. T., Bender, K. S., Buckley, B., Sattley, W. M., & D. Stahl, A. (2019). Brock biology
of microorganism. 15th ed. Boston, MA: Pearson Publisher.

Kumar, A., Murthy, L.N., Jeyakumari A. Sterilization technique used in microbiolo gy.
http://drs.cift.res.in/bitstream/handle/123456789/4540/Sterlization%20technique%20used%
20in%20microbiology.pdf?sequence=1&isAllowed=y Accessed on October 13, 2021.

QUESTIONS TO ANSWER

Briefly answer the following questions and input your answers to this Google Form link:
https://docs.google.com/forms/d/1dZciVaw-
1yC3B253tPf2g0WChfCIT0rC1l8QOJFgB8Y/edit?usp=sharing

1. Explain the major differences between the gram positive and gram negative cell wall.
2. What are the functions of the individual stains and reagents used in Gram staining?
3. Explain how bacterial cells would look in the gram staining procedure if the following
mistakes were made:
a. Decolorizer left on too long
b. Decolorizer not left on long enough
c. Slide not heat-fixed before staining
4. What are the two things that are stained in a capsule stain?
5. Why is it important to leave the malachite green on the slide for at least 5 minutes in the
endospore-staining procedure?
6. Why do you think an infection caused by an endospore former might be harder to treat than
one caused by a non-spore former?