Microbiology and Parasitology Laboratory Manual

Novee Jane A. Ceriño BS Nursing Level 1 B- SERVICE

Laboratory Exercise 1: Introduction to Microscopy

Objectives:

1. acquainted with the basic principles of compound light microscopy.

2. able to properly use the low power, high power, and oil immersion objectives.
3. able to exercise the steps necessary for proper care of a microscope.

A decent microscope is a must-have piece of equipment for every microbiology lab. Although
there are many different types of microscopes, the compound microscope is the most effective in
diagnostic work. It magnifies and illuminates microscopic items such as bacteria and other
microbes that would otherwise be undetectable to the naked eye using a set of lenses and a
powerful light source. Throughout your laboratory course, this sort of microscope will be
utilized. You'll understand how exactly it is and how useful it is for researching microorganisms
in clinical specimens and cultures as you have more experience with it.

A good microscope should have three properties:

1. Good resolution: Resolution power refers to the ability to produce separate images of
closely placed objects so that they can be distinguished as two separate entities.

The resolution power of:

• Unaided human eye is about 0.2 mm (200 µm)

• Light microscope is about 0.2 µm
• Electron microscope is about 0.5 nm Resolution depends on refractive index. Oil has a
higher refractive index than air.

2. Good contrast: This can be further improved by staining the specimen.

3. Good magnification: This is achieved using concave lenses.

There are two sets of lenses that make up the magnification system in a compound light
microscope. The objective lenses provide the initial magnification of the specimen. This "real
image" is then projected up through the body tube to the ocular lens, which magnifies the real
image 10X. This is the image that is seen by your eyes. Microscopes for bacteriological use are
usually equipped with at least three objectives:

(1) low power (10X magnification)

(2) high power (40-45X)

(3) oil immersion (100X)

The desired objective is rotated into place by a revolving nosepiece.

To calculate the total magnification, the power of the ocular lens (10X) is multiplied by the
power of the objective being used (10X, 40X, or 100X).

STUDY QUESTIONS

1. What conclusion can you make about the relationship between the size of the microscopic
field (average number of organisms per field) and the magnification used?

- Because the total magnification and the diameter of the field of view have an inverse
relationship – that is, as magnification increases, the diameter of the field decreases in
proportion – the diameter of the field of view at various magnifications can be calculated
mathematically using the formula.

2. How do you determine the actual total magnification of the specimen you are looking at?
(Show your calculations for each of the three objectives.)

- Total magnification is the total number of times the specimen you are viewing is magnified.
To determine the total magnification, you multiply the ocular lens magnification times the
objective lens magnification. On your microscopes, the ocular lens always magnifies 10X.
Example: 10X (ocular) x 4X (objective) = 40X (total magnification)

- To calculate the magnification, simply multiply the ocular lens (10x) by the objective lens.
With this microscope you can obtain four different magnifications: 40x, 100x, 400x and
1000x.

(1) Low power 10X(10X) =100X magnification

(2) High power 40X (10X) = 400X magnification

(3) Oil immersion 100X (10X) = 1000X magnification

3. Why do you have to use the oil-immersion objective to view bacteria?

- At high magnifications, it's better to use an oil-immersed objective since the oil
compensates for the short focal lengths that come with bigger magnifications. The oil has a
refractive index that is identical to that of the glass slides and slipcovers.

4. Give the different types of Microscopes with their descriptions for example.

The Compound Light Microscope

Commonly binocular (two eyepieces), the compound light microscope, combines the power
of lenses and light to enlarge the subject being viewed.
Typically, the eyepiece itself allows for 10X or 15X magnification and when combined with
the three or four objective lenses, which can be rotated into the field of view, produce higher
magnification to a maximum of around 1000X generally.
The compound light microscope is popular among botanists for studying plant cells, in
biology to view bacteria and parasites as well as a variety of human/animal cells.
light microscope objective lenses
It is a useful microscope in forensic labs for identifying drug structures.
Compound light microscopes are one of the most familiar of the different types of
microscopes as they are most often found in science and biology classrooms.
For this reason, simple models are readily available and are inexpensive.
As well, several microscopy imaging techniques benefit scientists and researchers using the
compound microscope and are worth exploring.

The Digital Microscope

Step into the 21st century with a digital microscope and enter a world of amazing detail.
The digital microscope, invented in Japan in 1986, uses the power of the computer to view
objects not visible to the naked eye.
Among the different types of microscopes, this kind can be found with or without eyepieces
to peer into.
It connects to a computer monitor via a USB cable, much like connecting a printer or mouse.
The computer software allows the monitor to display the magnified specimen. Moving
images can be recorded or single images captured in the computer’s memory.
An advantage of digital microscopes is the ability to email images, as well as comfortably
watch moving images for long periods.
The popularity of the digital microscope has increased at schools and among hobbyists.

5. Draw and label the parts of the microscope.

Laboratory Exercise 2: The Gram Stain

Objectives:

1. Explain the technique and theory of the Gram Stain.

2. Describe bacterial cell morphology.
3. Explain the importance of the Gram stain as an important step in the identification of a
bacterial species.
4. Properly perform a Gram stain

Individual bacterial cells exhibit morphology typical of their species: size, shape, and
arrangement of cells. These can be demonstrated by making a smear on a glass slide, then
staining the smear with a suitable dye. The use of a stained smear permits microscopic
examination of the smear with the oil immersion lens, which gives the greatest magnification,
revealing the size, shape, and arrangement. The study of individual bacterial cells is thus
frequently one of the first steps in the identification of bacteria.

In this exercise, you will use the Gram stain. This is called a differential stain because it not only
shows bacterial morphology but allows differentiation of different bacterial types since different
species react differently to the stain. The differential Gram stain gives information about the
bacterial cell wall, which may be gram-positive or gram-negative. Gram-positive bacteria will
appear purple, the color of the primary stain, crystal violet. Gram-negative bacteria will appear
pink-red, the color of the counterstain, safranin. The Gram stain is especially useful as one of the
first steps in the identification of a bacterial species since it reveals both the morphology and the
Gram reaction of the bacteria.

The bacteria may show the following shapes: coccus/cocci(spherical), bacillus/bacilli(rod-

shaped), or spirillum/spirilla(curved or spiral). The cells may assume a characteristic
arrangement: some occur singly, others appear in pairs (Diplo-), chains (strepto-), or clusters
(staphyloma-).
 STUDY QUESTIONS

1. Fill in the following table:

The appearance of the bacterial cells after each cell.
Steps Gram-positive cell Gram-negative cell
Crystal violet Purple Purple
Gram’s iodine Purple Purple
95% alcohol Purple Colorless
safranin Purple Red

2. Explain the major differences between the gram-positive and gram-negative cell walls.
- Gram-negative bacteria have a thin peptidoglycan cell wall that is surrounded by an outer
lipopolysaccharide-containing membrane. Gram-positive bacteria do not have an outer
membrane, but are surrounded by layers of peptidoglycan that are several times thicker than those
seen in Gram-negative bacteria..

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

Answer:

a) would take away too much color and make it hard to see

b) wouldn't get rid of enough excess dye so would be hard to differentiate

c) would cause bacteria to move and be hard to find and see

4. Why must fresh bacterial cultures be used in a Gram stain?

Even though the species is gram-positive, older bacterial cells may have cell wall damage
that causes them to seem gram-negative. As a result, for Gram staining, it is ideal to utilize
fresh bacterial cultures. Second, mistakes like keeping the decolorizer on for too long might
have an impact on the outcome.

5. Draw and label examples of Escherichia coli and Staphylococcus epidermidis.

Laboratory Exercise 3: Special Staining Techniques: Acid-fast Bacilli Stain, Capsule Stain,
Endospore Stain, and Flagella Stain

Objectives:

1. Identify special bacterial structures: capsules, flagella, and endospores.

2. Explain the significance of these bacterial structures in the diagnosis and identification of
disease.
3. Perform or describe the techniques that identify these special structures.
4. Identify acid-fast bacilli on a stained preparation.
5. Explain the significance of acid-fast bacilli in a specimen.

Some bacteria possess cell walls and other structures that are best demonstrated by methods
other than the Gram Stain. This exercise deals with a differential stain for the special type of
waxy cell walls possessed by Mycobacterium and with methods used to demonstrate endospores,
capsules, and flagella. In addition to their value in the identification of certain bacteria,
demonstration of these structures is important for your understanding of the basic structure and
function of bacterial cells in disease processes.

Bacterial Capsules

The capsule is a gelatinous, slimy material surrounding the bacterial cell. In many cases, the
capsule helps protect the cell against phagocytosis. Thus, potential pathogens are protected from
the body's natural defenses and are more likely to cause disease than non-capsulated strains. The
capsule also allows bacteria to adhere to surfaces, such as mucous membranes and teeth. Other
functions of a capsule include protection from dehydration and loss of nutrients. In this exercise,
capsules are demonstrated by the negative stain, in which the capsule shows up as a clear area or
halo surrounding the cell against the dark background of the nigrosin stain.

Bacterial Flagella

Flagella are structures that enable bacteria to be motile. They may occur singly at one end, in
tufts at one or both ends, or arranged all around the cell.

monotrichous = a single flagellum

amphitrichous = a single flagellum at both ends of the cell

lophotrichous = two or more flagella at one or both ends of the cell

peritrichous = flagella distributed over the entire cell

The number and arrangement of flagella can be used to help identify bacteria. Flagella are
demonstrated by special stains using mordants that increase the width of the flagella and are then
stained with carbol-fuchsin so that they may be seen with the microscope. NOTE: The pink color
of the microbes is due to the color of the primary carbon-fuchsin stain, and is NOT an indication
of a gram reaction, as in the Gram stain procedure.

Bacterial Endospores

Endospores are very resistant structures that are formed by certain bacteria under adverse
conditions.

Two genera of gram-positive bacilli (rods) are endospore-formers: Bacillus and Clostridium.
Endospores enable the organism to survive drying and lack of nutrients, so they can exist in dust
and soil for many years. Endospores are the most resistant form of life known. Their presence in
dust accounts for much of the laboratory contaminants. The very thick spore wall does not stain
easily, so the endospores will appear in Gram stains as unstained areas inside the cell. To stain
the spores themselves, carbon-fuchsin stain is heated so that it will be absorbed by the wall of the
endospore so that they appear red. The vegetative part of the cell will decolorize upon rinsing
with 95% ethanol and can then be counterstained with methylene blue or brilliant green for
contrast.

Study question
1. What is the importance of performing these special stains? What information do they
give you?
2. Is a bacterium that possesses a capsule always considered a pathogen? What are the
functions of a capsule?
- Yes it is because Capsule (also known as K antigen) is a major virulence factor
of bacteria. Bacterial capsules, largely composed of polysaccharides, act as a virulence
factor. A key function of the capsule is to aid in protection from phagocytosis ,
enabling the bacterium to establish infection successfully.

3. Why are endospores important to a bacterial cell? Under what conditions are they
formed? Give an example of the genus and species of four (4) pathogenic bacteria that
produce bacterial endospores.
- endospore are important for it enables the bacteria to develop a dormant and extremely
resistant cell in order to protect the cell's genetic material under tremendous stress.
Endospores are resistant to environmental stresses that would typically kill the bacteria.
They are formed when essential nutrients are depleted or when water is unavailable.
Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Clostridium botulinum, and
Clostridium tetani are examples of bacteria that produce bacterial endospores.

4. What genera is the acid-fast stain used to identify? Name two diseases that can be
diagnosed with the aid of the acid-fast stain.
- This stain is used to detect Mycobacterium tuberculosis, the TB causative agent. At
normal temperature, acid-fast organisms have a waxy lipoid capsule with a high
molecular weight. As a result, aqueous-based staining solutions cannot penetrate the
organism. Leprosy, a once feared, but now a rare and easily treatable disease that affects the
nerves, eyes, and skin.

5. Write a brief explanation of why each of the following bacterial structures requires a
"special" staining technique to be observed. (Explain why they cannot be demonstrated
using a Gram Stain.) a. capsule- If a pathogen loses its ability to form
capsules, it can become avirulent. Bacterial capsules are non-ionic, so neither acidic nor
basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to
stain the background using an acidic stain and to stain the cell itself using a basic stain
b. Endospore- Endospores are highly resistant to environmental conditions
such as heat, chemicals (also stains and dyes) and therefore require special
techniques for staining
c. acid-fast bacilli- Acid-fast organisms are characterized by wax-like, nearly
impermeable cell walls; they contain mycolic acid and large amounts of fatty acids,
waxes, and complex lipids. This type of cell wall is resistant to most compounds,
therefore acid-fast organisms require a special staining technique