Exercise 3 Preparation of Smears and Simple Staining

1. Which bacterium is a rod?

Answer:

Bacillus megaterium

2. Of what value is a simple stain?

Answer:

The value of simple stain is to allow us the determination of cell morphology, size and arrangement of
organisms with the application of only one reagent such as Methylene blue to stain all bacteria similarly.

3. What is the purpose of fixing the smear?

Answer:

The purpose of fixing the smear is to cause the bacterial enzymes to denature, killing the organism or
cause autolysis and so that the coagulated proteins from the cells will cause them to adhere to the slide.

4. What are the two methods of fixing a smear?

Answer:

The two methods of fixing a smear are heat fixing and chemical fixing. Heat fixing is performed by
passing the dry smear through the Bunsen burner several times. Chemical fixing is done by covering the
smear with 95% methyl alcohol.

5. In heat fixing, what would happen if too much heat were applied?

Answer:

In heat fixing, if too much heat were applied, the cell membrane will undergo autolysis preventing its
accurate and proper observation in the microscope.

Exercise 4 Negative Staining

1. Which cell is a rod? How does its appearance differ from the rod you stained with methylene
blue (simple stain)?

Answer:

The cell that is a rod is Bacillus subtilis. Its appearance is clearer thatn the one stained with simple stain
because negative staining does not stain the bacteria. It stains only the background providing greater
contrast.

2. Why is the size more accurate in a negative stain than in a simple stain?
Answer:

The size is more accurate in a negative stain than in simple stain because there is no heat fixing or strong
chemicals used to distort the microorganisms. In addition, only air-dry is used and the microorganisms
do not pick up the stain.

3. Could any dye be used in place of nigrosin for negative staining? What type of dyes are used for
negative staining? Briefly explain?

Answer:

Yes, acidic dyes with negative charge can be substituted such such as eosin and acid fuchsin. These dyes
are repelled by the negatively charged proteins of the cell wall of the bacteria by ionic repulsion enabling
contrast of the cell surface.

Exercise 5 Gram Staining

1. Did your results agree with the information in your textbook? If not, why not?

Answer:

Yes, the combination of crystal violet dye and iodine produced violet-iodine complex causing their
molecules to be trapped inside the cell walls of the gram positive microorganisms producing the violet or
purple color because of their three layers of peptidoglycan. For instance, Staphylococcus epidermidis
and Bacillus subtilis stained purple. On the other hand the gram negative Escherichia coli that turned
colorless, clear, with the application of ethyl alcohol of In addition the colorless gram negative retained
the color of the counterstain safranin (red) when applied.

2. Why will gram-positive cells more than 24 hours old stain gram-negative?

Answer:

Because the cells after 24 hours could have died and their cell walls have degraded prohibiting it from
retaining the primary stain such as purple from cystal violet. This would cause the appearrance of the
cell clear when the decolorizing agent Ethyl alcohol is applied. As a result, it would retain the red color of
the counterstain safranin when applied thereby causing an inaccurate result.

3. Can iodine be added before the primary stain in a Gram stain?

Answer:

No, the iodine cannot be added before the primary stain in a gram stain since the iodine bonds with the
primary stain chemically producing a cystal violet iodine (CV-I) complex preventing them from escaping
the thick three layered peptidoglycan of gram positive cells. If it was added first and then wash with a
distilled water, the iodines could washed out.
4. Lit the steps of the Gram-staining procedure in order (omit washings), and fill in the color of
gram-positive cells and gram-negaive cells afer each step.

Step Chemical Gram-Postive Cells Gram-Negative Cells

Add Crystal violet Violet/purple Violet/purple

Add Gram iodine (mordant) Violet/purple Violet/purple

Add Ethyl alcohol Purple Clear

Add Safranin (counterstain) Purple Red/pink

5. Which step can be omitted without affecting determination of the Gram reaction?

Answer:

All the steps in question number 4 above are important for the gram staining. However, if one had to
choose which one to omit without affecting gram reaction determination, the last step safranin can be
omitted. The reason is that this counterstain would just let us see the gram negative cells. Without this
step we could determine if the cell is gram positive or negative after applying the alcohol. If the color
does not wash out it is gram positive. If it washed out it is gram negative.

Exercise Acid-Fast Staining

1. What are the large stained areas on the sputum slide?

Answer:

This was not performed in the class.

2. What is the decolorizing agent in the Gram stain? In the acid-fast stain?

Answer:

In the gram stain the decolorizing agent is Ethyl-alcohol whereas in the acid-fast stain the decolorizing
agent is Acid-alcohol.

3. What diseases are diagnosed using the acid-fast procedure?

Answer:

Using acid-fast procedure one can diagnose pathogenic species that have cell walls that are difficult to
penetrate with other stains because of mycolic acid, such as the specific epithets of Mycobacterium and
Nocardia. For example, Mycobacterium tuberculosis which is the causative agent of tuberculosis and
Mycobacterium leprae which is the causative agent of leprosy.

4. What is phenol (carbolic acid), and what is its usual application?

Answer:

It is an alcohol with a phenyl group, benzene. It is used to control surgical infections and odor of sewage.
However, it has disagreeable odor and can irritate the skin. Other use is for throat lozenges because of its
anesthetic effect.

Exercise 9 Microbes in the Environment

Data

Area sampled Diameter Appearance Margin Elevation Pigment Number of this

Type

Palm Pinpoint-1.5 mm Circular Entire Convex, umbonate Yellow NA

Table Pinpoint-1.5 mm Circular Entire Convex, umbonate No pigment NA

Top of hand Pinpoint-1.0 mm Circular Undulate Umbonat Yellow NA

Blockboard Clean Clean Clean Clean Clean NA

1. How can you tell whether there is bacterial growth in the nutrient broth?

Answer:

The broth will become torbid or cloudy in the present of bacterial growth.

2. What is the minimum number of different bacteria present on one of your plates? How do you
know?

Answer:

There minimum was one colony. This is based on a single pigment and morphology.

3. What is the value of Petri plates in microbiology?

Answer:

Petri plates containing solid media provides a large surface area for streaking bacterium and the
observation of bacterial colonies.

4. What are bacteria using for nutrients in nutrient agar? What is the purpose of agar?

Answer:

The agar serves as a gelling agent for solidifiying culture. The others mixed with the agar provide the
bacterial nutrients. For example the beef extract mixed with the agar provides amino acids as nutrients.
5. Which environment has the most total bacteria? The least? Provide a reason for the differences
in total bacteria in theses two places.

Answer:

The area on the top of the table that was swiped has the most total bacteria. This is due to its horizontal
flat position causing it to be more receptive to microbes especially when not disinfected or cleaned
often. On the other hand, the blockboard has no signs of bacteria. This is due to its vertical surface
position on the wall causing it not to be receptive to bacteria and it is often cleaned after class.

6. Which environment has the most different bacteria? The least? Provide a reason for the
differences in bacteria in these two places?

Answer:

The environment that has the most different bacteria is the floor. This is due to it high foot traffic
location and bacterial accumulation and for not being thoroughly cleaned. The least is the blackboard
since it is cleaned often and it vertical surface area is not conducive for bacterial attractions.