HES 032 (Microbiology and

STUDENT ACTIVITY SHEET Parasitology) BS NURSING /SECOND

YEAR Session # 4

LESSON TITLE: The Domain Materials:

Bacteria LEARNING OUTCOMES: Book, pen, notebook, and paper

Upon completion of this lesson, the nursing student can: References:

1. List the various ways in which bacteria can be classified Engelkirk, P., & Engelkirk, J. (2015). Burton's
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correctly; Microbiology for the Health and Sciences. 10
2. Outline precisely the (3) three basic shapes of bacteria; ed., Philadelphia: Lipincott Williams & Wilkins
3. Classify effectively the bacteria based on its morphological
arrangements;
4. Enumerate correctly the different staining procedures.
5. Explain the principles of Gram Staining and Acid Fast
Staining efficiently

Daily Productivity Tip:

Learn, Write, Rewrite, Relearn. Believe in the power of writing because it might help you organize your
ideas. Make your best notes and mnemonics today!

LESSON PREVIEW/REVIEW (5 minutes)

Recall from the last session the cell structures found in a eukaryote and a prokaryote.

EUKARYOTIC CELL BOTH PROKARYOTIC CELL

Nucleus Cell Membrane Ribosomes
Endoplasmic Reticulum Chromosomes Glycocalyx
Ribosomes Cytoplasm Pili
Golgi Complex Cell Wall Spores
Lysosomes Flagella Plasmid

Lastly, what are the two domains of prokaryotic microorganisms?

1. Bacteria
2. Archaea
That’s great! Now let’s begin with today’s session.

MAIN LESSON (40 minutes)

Many characteristics of bacteria are examined to provide data for identification and classification.Today’s session, you will
get to know the major categories of bacteria based on their cell morphology and staining characteristics.
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Before we start, please open chapter 4 of Burton’s Microbiology for the Health Sciences, 10 ed.

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 Lesson 1: Cell Morphology

With the compound light microscope, the size, shape, and morphologic arrangement of various bacteria are easily
observed. Bacteria vary greatly in size, usually ranging from spheres measuring about 0.2 µm in diameter to 10.0-µm-
long spiral-shaped bacteria, to even longer filamentous bacteria.

I. Three general shapes of bacteria are:

bacillus (plural: bacilli) coccus (plural: cocci) spirilla

Spiral-shaped bacteria are referred to as spirochetes:

Tightly-coiled : Treponema pallidum
Loosely-coiled : BorreLia spp.
Curved or comma-shaped bacilli : Vibrio spp.
Gull-wing : Campylobacter spp.

II. Morphological arrangement of bacteria are:

cocci
diplococci – cocci in pairs
streptococci – cocci in chains
staphylococci – cocci in clusters
tetrad – cocci in packets of four
octad – cocci in packets of eight
bacilli
diplobacilli – bacilli in pairs
streptobacilli – long, filamentous bacilli in chain
coccobacilli – resembling short, elongated cocci
diptheroids – bacilli stack up next to each other.

Think and Learn: Based on you readings, can you cite examples of bacteria belonging to each
morphological arrangement?

Did you know?

The shape of a bacterium is determined by heredity. Though, most bacteria are monomorphic; that is, they
maintain a single shape, there are bacteria that can alter its shape because of a number of environmental
conditions. They are described as being pleomorphic. Bacteria from the genus Mycoplasma do not have cell
walls; thus, when examine microscopically, they appear in various shapes.

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Lesson 2: Staining Procedure

As they exist in nature, most bacteria are colorless, transparent, and difficult to see. Therefore, various staining methods
have been devised to enable scientists to examine bacteria.

Staining Techniques
Simple staining a single stain is used; directed towards coloring the forms
and shapes present
Example: Malachite green, methylene blue, crystal
violet

Diagnostic Antibody/DNA probe-mediated staining directed specifically on identification of organisms such as

Chlamydiatrachomatis,Legionellapneumophilia,
Bordetella pertussis, etc.
Negative staining utilized to demonstrate the presence of diffuse capsule
surrounding some bacteria. It is excellent technique to
study bacterial gas vacuoles and viral morphology.
Example: India Ink or Nigrosin dye (acidic stain)
Differential staining divides the bacteria into separate groups; directed
towards coloring components of those elements present.
Example: Gram staining, Acid Fast Bacilli staining

Steps in Differential Staining:

Gram Stain
Most commonly used in differential stain in the clinical microbiology laboratory.

Reaction Theories:
Theory Gram-Positive Bacteria Gram –Negtive Bacteria
Benian Less permeable to decolorizing agent Permeable to decolorizing agent
Stern and Stearn (Law of Magnetism) Low isoelectric point High isoelectric point

Principle:
Bacteria with thick cell walls containing teichoic acid retain the crystal violet-iodine
complex dye after decolorization and appear PURPLE, thus they are Gram-
Positive. Other bacteria with thinner cell walls containing lipopolysaccharides do
NOT retain the dye complex and appear PINK and they are Gram-Negative. Gram positive – PURPLE or
BLUE
.
Some strains of bacteria are consistently neither blue to purple nor pink to red Gram negative – PINK
after Gram staining; they are referred to as Gram-variable bacteria. Examples of
Gram-variable bacteria are members of the genus Mycobacterium. Remember there is always
that boy who has a negative
Mycobacterium species are more often identified using a staining procedure call feeling for pink colours
the acid-fast stain.

Think and Learn: What are the chemical stains used in the Gram staining technique?
 Crystal violet (primary stain)
 Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall)
 Decolorizer (e.g. ethanol)
 Safranin (secondary stain)
 Water (preferably in a squirt bottle)

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Acid Fast staining
used to stain bacteria have high lipid content in their cell walls.

Methods:
1. Ziehl-Neelsen Method – Hot staining Method
2. Kinyoun’s Method - Cold staining Method

Principle
The primary stain binds to mycolic acid in the cell walls of the Mycobacteria and is retained after decolorization with acid
alcohol. Acid-fast bacilli retain the primary stain and colored red while non-AFB are either blue or green (methylene blue
or malachite green counterstains) color.

Mycolic acid renders the cells resistant to decolorization, thus termed as “acid fast”.

Think and Learn: How do you prepare a bacterial smear for staining?
The preparation of a smear is the first step in most bacterial staining methods. Cells from a culture are
distributed in a thin layer over a small area of a microscope slide in a smear preparation, dried, and then fixed to the slide
by heating or other chemical fixatives.

Morphologic and Staining Differences among the Bacteria

Morphology Gram-Positive Gram-Negative
Coccus Staphylococcus Neisseria
Streptococcus
Bacillus Spore-forming: Enterics: Escherichia coli, Proteus
Clostridium spp., Enterobacter spp., Klebsiella
Bacillus spp., Salmonella spp., Shigella spp.,
Non spore-forming: Pseudomonas spp., Helicobacter
Listeria spp., Yersinia spp., Campylobacter
Corynebacterium spp., Vibrio spp., Bordetella spp.,
Francisella spp.
Mycobacterium (weakly Gram-positive)
Spirals Spirochetes: Treponema spp.,
Borrelia spp., Leptospira spp. (weakly
Gram-negative)
Pleomorphic Chlamydia spp., Ricketssia spp.,
Mycoplasma spp.

All cocci are Gram-positive except Neisseria spp., Branhamella spp., Moraxella spp., Veilonella spp. All
bacilli are Gram-negative except the spore-forming bacilli, Bacillus spp. and Clostridium spp., Mycobacteria
spp., and Corynebacterium spp.

TAKE A QUICK BREAK. BREATHE SLOWLY. WRITE FREELY.

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CHECK FOR UNDERSTANDING (15 minutes)
After studying this module, answer the following questions. You will answer and rationalize this by yourself. This will
be recorded as your quiz. One (1) point will be given to correct answer and another one (1) point for the correct ratio.
(Total: 10 points)

Multiple Choice

1. Gram Staining Procedure: Arrange the following dyes in correct order.

a. Gram’s Iodine
b. Crystal Violet
c. Safranin
d. Alcohol
A. 1,2,4,3
B. 2,3,1,4
C. 4,3,1,2
D. 2,1,4,3
ANSWER: D
RATIO: The steps of the Gram Staining Procedure are as follows: primary stain, mordant, decolorizer, and finally
secondary stain or counter stain.

2. Statement 1: Ziehl-Neelsen Method: Hot Method

Statement 2: Kinyoun Method: Cold Method
A. Statement 1 is true; statement 2 is false
B. Statement 2 is true; statement 1 is false
C. Both of the statements are true
D. Both of the statements are false
ANSWER: C
RATIO: Both statements are correct. The Ziehl-Neelsen Method is for hot staining, whereas the Kinyoun Method is for
cold staining.

3. The group of bacteria that lack rigid cell walls and take on irregular shapes is:
a. chlamydia.
b. mycobacteria.
c. mycoplasmas.
d. rickettsia.
ANSWER: C
RATIO: Mycoplasmas are essentially bacteria that lack a solid cell wall during their entire life cycle, although being
much smaller than bacteria.

4. This staining is especially useful in the tuberculosis laboratory (“TB lab”) where the mycobacteria are readily seen
as red bacilli against a blue or green background in a sputum specimen from a tuberculosis patient.
a. gram staining
b. simple staining
c. acid-fast staining
d. structural staining
ANSWER: C
RATIO: Acid-fast staining used to stain bacteria has high lipid content in their cell walls. It is the most often used to
diagnose an active tuberculosis infection.

5. The cell wall of many gram-positive bacteria can be selectively removed by:
a. Spermine
b. Lipoteichoic acid
c. Lysozyme
d. Amylase
e. para-amino benzoic acid

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 ANSWER: C
RATIO: Lysosomes can degrade a bacteria's cell wall.

RATIONALIZATION ACTIVITY
The instructor will now rationalize the answers to the students. You can now ask questions and debate among yourselves.
Write the correct answer and correct/additional ratio in the space provided.

1. ANSWER:
RATIO:

2. ANSWER:
RATIO:

3. ANSWER:
RATIO:

4. ANSWER:
RATIO:

5. ANSWER:
RATIO:

LESSON WRAP-UP (5 minutes)

You will now mark (encircle) the session you have finished today in the tracker below. This is simply a visual to
help you track how much work you have accomplished and how much work there is left to do.

You are done with the session! Let’s track your progress.

Exit Ticket: One-minute paper

a. How do you feel about today’s session? __ Happy __ Satisfied __ Sad __ Confused
I feel happy upon reading the manual guide because it has many information’s that cater some knowledge.

b. What question(s) do you have as we end this session?

I don’t have any questions anymore
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Reading Assignment: For the next session, read chapter 7 of Burton’s Microbiology for the Health Siences, 10 ed.

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