INTRODUCTION TO MICROBIOLOGY AND STRUCTURE OF BACTERIA

Learning Objectives
At the end of this lesson student will be able to:
1. Explain the difference between prokaryotic and eukaryotic cells and be able to
distinguish each type.
2. Explain the difference between flagella and pili.
3. Identify the following structures on the slides and explain the functions of each: the
plasma membrane, cytoplasm, nucleus, nucleolus, cell wall, and plastids.
4. Describe methods used to classify bacteria
5. Describe bacteria based on shape and arrangements.

Microbiology is a subject which deals with living organisms that are individually too small to be
seen with the naked eye. It considers the microscopic forms of life and deals about their
reproduction, physiology, and participation in the process of nature, helpful and harmful
relationship with other living things, and significance in science and industry.
Subdivision of microbiology
Bacteriology deals with bacteria.
Mycology deals with fungi.
Virology deals with viruses.
Bacterial species
- A bacterial species is defined by the similarities found among its members. Properties such as
biochemical reactions, chemical composition, cellular structures, genetic characteristics, and
immunological features are used in defining a bacterial species. Identifying a species and
determining its limits presents the most challenging aspects of biological classification for any
type of organism.
Bacterial Cell
General property:
• Typical prokaryotic cell
• Contain both DNA and RNA
• Most grow in artificial media
• Replicate by binary fission
• Almost all contain rigid cell wall
• Sensitive to antimicrobial agent

The distinguishing features between Eukaryotic cell and Prokaryotic cell

Features Prokaryotic cell Eukaryotic cell

. Size 1μm 10μm
. Nuclear membrane Absent Present
. Chromosome Single Multiple
. Nucleolus Absent Present
. Histones Absent Present
. Sexual reproduction Absent Present
. Cytoplasmic ribosomes 70s 80s
. Mitochondria Absent Present
. Endoplasmic reticulum Absent Present
. Lysosomes Absent Present.

STRUCTURE OF BACTERIA
The bacterial structure is considered at three levels.
1. Cell envelope proper: Cell wall and cell membrane.
2. Cellular element enclosed within the cell envelope: Mesosomes, ribosomes, nuclear apparatus,
polyamides, and cytoplasmic granules.
3. Cellular element external to the cell envelope: Flagellum, Pilus and Glycocalyx.
Cell envelope proper
A. Cell wall
Multi layered structure and constitutes about 20% of the bacterial dry weight.
Average thickness is 0.15-0.5 μm. Young and rapidly growing bacteria have a thin cell wall but
old and slowly dividing bacteria have a thick cell wall. It is composed of N-acetyl Muramic acid
and N-acetyl Glucosamine backbones cross-linked with peptide chain and pentaglycine bridge.
Components of the cell wall of Gram-negative bacteria
1. Peptidoglycan
2. Lipoprotein
3. Phospholipid
4. Lipopolysaccharide
Components of the cell wall of Gram-positive bacteria
1. Peptidoglycan
2. Teichoic acid
Functions of cell wall
1. Provides shape to the bacterium
2. Gives rigidity to the organism
3. Protects from environment
4. Provides staining characteristics to the bacterium
5. Contains receptor sites for phages/complements
6. Site of action of antibody and colicin
7. Contains toxic components to host
Cell membrane
Also named as the cell membrane or cytoplasmic membrane It is a delicate trilaminar unit
membrane.
It accounts for 30% of the dry weight of the bacterial cells. It is composed of 60% protein, 20-
30% lipids and 10-20% carbohydrate.
Function of cell membrane
1. Regulates the transport of nutrients and waste products into and out of the cell.
2. Synthesis of cell wall components
3. Assists DNA replication
4. Secrets proteins
5. Carries on the electron transport system
6. Captures energy in the form of ATP
Glycocalyx (capsule and slime layer)
Capsule is gel firmly adherent to the cell envelope. Slime is gel easily washed off from the cell
envelope.
All bacteria have at least a thin slime layer. The capsule is composed of polysaccharide and
protein (D-Glutamate of Bacillus anthracis)
Features of capsule
1. Usually weakly antigenic.
2. Not necessary for viability.
3. Endows virulence.
4. Protects from phagocytosis.
5. Capsulated strains are invariably non-motile.
6. Visualized by negative staining and capsule staining.
7. Detected by quellung phenomenon
Flagellum
It is the organ of locomotion in bacterial cells and consists of three parts. These are. The filament
. The hook
. The basal body
The basal body and hook are embedded in the cell surface while the filament is free on the
surface of a bacterial cells.
Flagellar arrangements
1. Atrichous: Bacteria with no flagellum.
2. Monotrichous: Bacteria with single polar flagellum.
3. Lophotrichous: Bacteria with a bunch of flagella at one pole.
4. Amphitrichous: Bacteria with flagella at both poles.
5. Peritrichous: Bacteria with flagella all over their surface.
Pili (fimbriae)
It is a hair-like structure composed of protein (pilin)
Two types (Based on function)
. Common pili: The structure for adherence to the cell surface.
. Sex pili: The structure for transfer of genetic material from the donor to the recipient during the
process of conjugation.
Comparison between flagella and pili

Character Flagella Pili

. Size Large small
. Thickness +++ +
. Origin Cell membrane Cell wall
. Organ of locomotion + _
. Organ of adhesion _ +
. Required for conjugation _ +

Spores
Resting cells which are capable of surviving under adverse environmental conditions like heat,
drying, freezing, action of toxic chemicals and radiation.
Bacterial spore is smooth-walled and oval or spherical in shape. It does not take up ordinary
stains.
It looks like areas of high refractivity under a light microscope. It is significant in the spread of
disease and indicators of sterility of materials.
Spores are detected by
. Simple staining methods
. Special staining methods

Arrangements of spores
1. No bulging of cell wall
. Oval central
. Oval subterminal
- Spherical central

2. Bulging of cell wall

. Oval subterminal
. Oval terminal
. Spherical terminal

CLASSIFICATION OF BACTERIA
The bacterial classification depends on the following characteristics.
1. Morphology and arrangement
2. Staining
3. Cultural characteristics
4. Biochemical reactions
5. Antigenic structure
6. The base composition of bacterial DNA

Morphology and staining of bacteria are the commonly used characteristics to classify bacteria.
1. Morphology of bacteria
When bacteria are visualized under light microscope, the following morphology are seen.
 1. Cocci (singular coccus): Round or oval bacteria measuring about 0.5-1.0μmb in
diameter. They are found in single, pairs, chains, or clusters.
2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tempered, square or
swollen ends; with a size measuring 1-10μm in length by 0.3-1.0μm in width.
3. Coccobacilli (singular coccobacillus): Short rods.
4. Spiral: Spiral shaped bacteria with a regular or irregular distance between twisting.
Eg. Spirilla and spirochetes

2. Staining of bacteria
Bacterial staining is the process of the coloring of colorless bacterial structural components using
stains (dyes). The principle of staining is to identify microorganisms selectively by using dyes,
fluorescence, and radioisotope emission. Staining reactions are made possible because of the
physical phenomena of capillary osmosis, solubility, adsorption, and absorption of stains or dyes
by cells of microorganisms.
Individual variation in the cell wall constituents among different groups of bacteria will
consequently produce variations in colors during microscopic examination.
The nucleus is acidic in character and hence, it has a greater affinity for basic dyes. Whereas, the
cytoplasm is basic in character and has a greater affinity for acidic dyes.
e.g.
Gram staining method
Developed by Christian Gram.
Most bacteria are differentiated by their gram reaction due to differences in their cell wall
structure.
Gram-positive bacteria are bacteria that stain purple with crystal violet after decolorizing with
acetone-alcohol.
Gram-negative bacteria are bacteria that stain pink with the counterstain (safranin) after losing
the primary stain (crystal violet) when treated with acetone-alcohol.

Procedure:
1. Prepare the smear from the culture or from the specimen.
2. Allow the smear to air-dry completely.
3. Rapidly pass the slide (smear uppermost) three times through the flame.
4. Cover the fixed smear with crystal violet for 1 minute and wash with distilled water.
5. Tip-off the water and cover the smear with gram’s iodine for 1 minute.
6. Wash off the iodine with clean water.
7. Decolorize rapidly with acetone-alcohol for 30 seconds.
8. Wash off the acetone-alcohol with clean water.
9. Cover the smear with safranin for 1 minute.
10. Wash off the stain wipe the back of the slide. Let the smear to air-dry.
11. Examine the smear with oil immersion objective to look for bacteria.
Interpretation:
. Gram-positive bacterium ……………Purple
. Gram-negative bacterium …………..Pink

3. CULTIVATION OF BACTERIA IN CULTURE MEDIA

Culture media
It is the media containing the required nutrients for bacterial growth.
Uses:
- Isolation and identification of micro-organisms
- Performing anti-microbial sensitivity tests

Common ingredients of culture media: Peptone, Meat extract, Yeast extract, Mineral salts,
Carbohydrates & Agar

5. COMMON BIOCHEMICAL TESTS

CATALASE TEST
This test is used to differentiate those bacteria that produce the enzyme catalase, such as
staphylococci, from non-catalase producing bacteria such as streptococci.

Method
• Pour 2-3ml of the hydrogen peroxide solution into a test tube.
• Using a sterile wooden stick or a glass rod, remove a good growth of the test organism and
immerse it in the hydrogen peroxide solution.
Note: A nichrome wire loop must not be used because this may give a false-positive reaction.
• Look for immediate bubbling.

Results
Active bubbling ----------------- A positive test (Catalase produced)
No release of bubbles ---------- A negative test (No catalase produced)

6. BACTERIAL GENETICS

Genetics is the study of inheritance. Bacterial inherited characteristics are encoded in DNA.
Bacteria have two types of DNA that contain their genes. These are:
. Chromosome
. Extra chromosome: Plasmid
The bacterial chromosome is circular, double-stranded DNA attached to the bacterial cell
membrane. DNA replication in bacteria is semi-conservative i.e. each strand of DNA is
conserved intact during replication and becomes one of the two strands of the new daughter
molecules.
Plasmids are self-replicating extrachromosomal DNA molecules. It multiplies independent of
the host cell. Multiple copies of the same plasmid may be present in each bacterial cell. Different
plasmids are also often present in the same bacterial cell.
Plasmid types
There are many types of plasmid types. The following are examples.
a. R factors: Plasmids that contain genes that code for antibiotic resistance.
b. Col factors: Plasmids that contain genes that code for extracellular toxin (colicines)
production that inhibit strains of the same and different species of bacteria.
c. F(fertility) factors: Plasmids that can recombine itself with the bacterial chromosome.
It promotes transfer of the chromosome at a high frequency of recombination into the
chromosome of a second (recipient) bacterial cell during mating.
Genetic variation in Bacteria
Mechanisms: Mutation and Gene transfer
1. Mutation: It is due to a chemical alteration in DNA. It could be spontaneous or
induced by chemical and physical means Mutants are variants in which one or more bases
in their DNA are altered; which are heritable and irreversible
 Types of mutation
1. Substitution: Change of a single base.
2. Deletion: Loss of a base.
3. Insertion: Addition of a base.
2. Gene transfer
There are three types of gene transfer that alters the DNA gene content of bacteria.
These are:
- Transformation
- Transduction
- Conjugation

1. Transformation occurs when fragments of exogenous bacterial DNA is taken up and

absorbed into recipient bacterial cells.
Transformation of genes from one bacterium to another results in
. Change in pathogenicity of the bacterium.
. Change in antibiotic sensitivity pattern of a bacterium.
Competence: The recipient bacterium must be competent to absorb the exogenous fragments of
bacterial DNA.
Frequency: The frequency of transformation is low
2. Transduction occurs when fragments of chromosomal DNA is transferred or transduced into
a second bacterium by phage. During phage replication, the bacterial DNA may be accidentally
enclosed instead of the normal phage DNA, and when this particle which enclosed the bacterial
DNA infects a second bacterial cell, the
DNA from the first bacterium is released and incorporated into the chromosome of the second
bacterium.
3. Conjugation occurs when plasmid DNA is transferred from donor to recipient bacterium by
direct contact via a sex pilus.
Quiz
1. State the components of the cell wall of gram-positive and negative bacteria 6mks
2. Describe flagella arrangements 5mks
3. Describe the arrangements of spores 3mks
4. Describe plasmid types 3mks
5. Describe types of mutation 3mks
REFERENCES
1. Richardson, J. H., Tulis, J. J., & Vesley, D. (1995). Laboratory safety: principles and
practices (Vol. 2). D. O. Fleming (Ed.). Washington, DC: ASM Press.
2. Lennette, E. H., & Balows, A. (1985). Manual of clinical microbiology 4 Ed.
3. Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., Yolken, R. H., & Morgan,
D. R. (1995). Manual of Clinical Microbiology (6th edn). Trends in microbiology, 3(11),
449-449.
4. http://www.columbia.edu/itc/hs/medical/pathophys/id/2009/introNotes.pdf. Accessed
on 10. April 2020.