Medical Microbiology

for year II Optometry Students

December, 2024

1
 Introduction to Microbiology….
KeyTerminologies
➢Pathogens: is infectious agent that can produce disease.
e.g bacteria, virus..

Infection:

➢The invasion and multiplication of microorganisms, that

are not normally present within the body and cause harm
or illness to host.
2
 Contd…
Sepsis

➢ The condition or syndrome caused by the presence of microorganisms

or their toxins in the tissue or the bloodstream.

Septicemia

➢ A wide spread destruction of tissue due to adsorption of disease

causing bacteria or their toxins from blood stream.

➢ Bacteremia

The presence of bacteria in the blood 3

 Contd…
➢ Disease: is a particular abnormal condition that negatively affects the
structure or function of all or part of an organism and differing from
physical injure.

➢ Diseases are often known to be medical conditions that are associated

with specific signs and symptoms.

➢4
 Contd..
❖Nosocomial infection
➢ (healthcare facility acquired infection or healthcare
associated infections)
➢ An infection acquired in healthcare facility by a patient who
was admitted for a reason other than that infection.
❖Acute infection
 A disease or condition which occur/develops rapidly and can
be dangerous.

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 1.Introduction to Microbiology
❑1.1.Scope of Microbiology
❑Medical Microbiology: - a branches of medical science that deal with
microorganisms that cause infectious diseases of human beings.

❑ It Involves the study of

✓Pathogens

✓Disease caused by them and

✓The body defences against disease

6
 Contd,
➢ Medical microbiology is also concerned with

❖Epidemiology

❖Transmission of pathogens

❖Disease prevention measures

❖Aseptic techniques

❖Laboratory diagnosis

❖Treatment of infectious diseases

❖The production of vaccines to protect against infectious disease

7
 Contd,
❑Sub-division of Medical Microbiology
➢Bacteriology

➢Virology

➢Mycology

➢Immunology

➢Parasitology

8
 Contd,
1.2.History of Microbiology
➢ In some ancient civilization, disease was believed to be a punishment
sent from the God for human wrong doing.

➢ Many philosophers during early period believe that disease was

transmitted by invisible animals.

❖Hippocratus (father of medicine)observed that ill health resulted due

to changes in air, winds, water, climate, food, nature of soil and habits of
people. 9
❑Antonio Van Leeuwenhoek (1632-1723 G.C.)
▪ Microbiology emerged as a science after the discovery of
microscope.
▪ Father of Microbiology, observed “animalcules” using simple
microscope with one lens.
▪ He was the first who properly described the different shapes of
bacteria.
▪ He examined pond water and discovered an invisible creature called
“Animalclues” small animals.
▪ They were every where in water droplets, particle of soil etc.
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 Contd,

❑ The golden age of Microbiology (1870-1890)

▪ Louis Pasteur (1822-1895 GC)

▪ Major contribution of Louis Pasteur

➢ Microbial theory of fermentation.

➢ disproved the theory of abiogenesis.

➢ Principles and practice of sterilization and pasteurization.

➢ Development of vaccines against anthrax and rabies.

➢ Discovery of streptococci bacteria

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 Louis Pasteur….
➢ In classic series of experiment Pasteur clarified the role of yeast in
fermentation and showed bacteria were responsible for sour wine.

 Experiment 1 Grape juice ➔No fermentation

Without yeast

 Experiment 2 Grape juice + Yeast ➔Fermentation only

 Experiment 3 Grape juice+ Yeast + Bacteria ➔ Fermentation

& sour
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 Louis Pasteur….
 Louse Pasture after performing the above experiment he concluded the
following:-
1) Yeast cells are very important for fermentation to take place.
2) Bacteria's can cause souring of wine due to formation of lactic acid.
3) Different microorganisms cause different fermentation products.
4) Bacteria which is present in fresh grape juice can be eliminated by
gentle heating or boiling (heating at 65oc for 30 min i.e. pasteurization)
5) Fermentation occurs anaerobically.
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 Contd,
❑ Robert Koch(1870s)
▪ Known as the father of bacteriology
▪ Introduced
✓ Staining techniques
✓ Pure culture medium.
✓ Isolation of bacterial colonies using of agar.
▪ Developed Koch's Postulates
✓ which are a sequence of experimental steps for directly relating a
specific microbe to a specific disease.
▪ Koch discovered:
✓ Bacillus anthracis
✓ Mycobacterium tuberculosis
✓ Vibrio cholera
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 Contd,
❑Alexander Fleming (1928s)

▪ Alexander Fleming a Scottish biologist and pharmacologist, observed

bacterial staphylococci colonies disappearing on plates contaminated
with fungus mold.

▪ Fleming discovered that a mold accidentally growing

on one of his Petri dishes had anti-bacterial activity.

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 Contd,
➢ Fleming extracted the compound from the mold responsible for
destruction of the bacterial colonies.

▪ The mold was producing penicillin.

▪ This was the first antibiotic discovered.
➢ The product of the mold was named penicillin, after the
Penicillium mold from which it was derived.
➢ Nobel Prize in Physiology of Medicine in 1945.

16
 Contd,
❑Joseph Lister
➢used a chemical disinfectant to prevent surgical wound
infections
➢ after looking at Pasteur’s work showing microbes are in the air,
can spoil food, and cause animal diseases

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 Theories of origin of microorganisms
1.3.Spontaneousgeneration (Abiogenesis )

▪ This was the theory of Aristotle and his followers.

▪ This is a theory that says life comes from non living nature.

➔Snake emerged from horse hair.

➔ Maggots emerged from rotten meat

➔ Flies from fresh and rotting fruit

➔Frogs emerged from slime mud.

➢ Key to developing the germ theory of disease was a refutation of the concept of
spontaneous generation.
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2. Biogenesis
Contd,

▪ This theory says life comes from pre-existing cells.

▪ Different individuals did a lot to prove this theory

❑ Francisco Redi-1665

o He did an experiment in 1665, with covered and uncovered meat

showing that maggots developed only in meat that flies could flesh to lay
eggs

o He proved that living things are comes from pre-existing living things.

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 The Redi experiment

Jar-1 Jar-2 Jar-3

❑Jar-1
• Left open
• Maggots developed
• Flies were observed laying eggs on the meat in the open jar
❑Jar-2
•Covered with netting
• Maggots appeared on the netting
• Flies were observed laying eggs on the netting
❑Jar-3
•Sealed
• No maggots developed 20
 Contd,

❑ Louis Pasture – 1858

➢He proved the controversy between the two theories in 1859.

➢Louis pasture devised swanked flask in order to prove micro

organisms do not generate spontaneously in sterilized broth
exposed in to air.

21
 Contd,

1.4. Germ theory of disease

Germ theory of disease explains about microbes can spread through the
population and causes a specific disease.
▪ L. Pasture discovered that microbes can cause disease.
▪ Joseph Lister a surgeon who explained sepsis and discovered antiseptics
so called the father of antiseptic surgeon.
▪ Robert Koch Explained & proved microorganisms can be isolated and can
cause a specific disease. He studied anthrax in his experiment.

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 Germ theory of disease…
 A definite proof of the germ theory of disease was offered by Robert
Koch.

 Foundation of modern medicine.

 Germ theory of disease is the single most important contribution to

medical science and practice ever.

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Germ theory of disease…

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25
From this experiment Koch formulated the following postulates:

1) Diseased animal contains the causative organism of that disease.

2) The organism can be isolated and grown on pure culture media.

3) Pure culture can produce disease when inoculated into susceptible animal.

4) It is possible to obtain the organism on culture from experimental infected

animal.

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❑ Exception/ Limitation to Koch’s postulates
a) Many healthy people carry pathogens, but do not exhibit symptoms of the disease.
▪ E.g. HIV, Typhoid fever
b) Some microbe are very difficult or impossible to grow in the laboratory on artificial
media.
▪ E.g. Virus, M. leprae , T. palladium etc...

c) Certain disease develops only when an opportunistic pathogen invades a weak

(susceptible) host.

d) Many species of microbes are species specific e.g. Brucella aborutus cause abortion
in animals but not in humans.

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 WHY MICROBIOLOGY IS IMPORTANT?

➢ Microbes immune defense: live on and inside of us and protect us from

pathogens simply by taking up space (normal flora).

➢ Microbes boost the immune system: bind to immune system cells and

stimulate them to divide and reproduce. T cells and B cells.

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 Contd…
Microbes keep us slim. Microbes play an important role in our body
shape by helping us digest and ferment foods,

➢ produce chemicals that shape our metabolic rates.

➢ involved in photosynthesis and accounts for >50% of earth’s oxygen.

➢ Also involved in decomposition and nutrient recycling.

➢ Used to synthesis drugs, hormones and enzymes.

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 Modern Uses of Microbes

▪ Biotechnology, the use of microbes as miniature biochemical factories to produce

food and chemicals is centuries old.

▪ Genetic engineering makes use of molecular biology and recombinant DNA

techniques as new tools for biotechnology.

▪ Gene therapy replaces missing or defective genes in human cells through genetic
engineering.

▪ Genetically modified bacteria are used to protect crops from pests and freezing.

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31
 General bacteriology….
❑Bacteriology
▪ the major sub-division of medical microbiology which study
about medically important bacteria.

❑Bacterial Cell
➢General property
▪ Typical prokaryotic cell
▪ Contain both DNA and RNA
▪ Most grow in artificial media
▪ Replication is by binary fission
▪ Contain rigid cell wall
▪ Sensitive to antimicrobial agent
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2.1 Eukaryotic and prokaryotic cells
➢ Based on the cellular complexity or organization among unicellular
and Multicelluar organisms.
➢ by Electron microscope
➢ Microorganisms can be classified as
1) Prokaryotic
▪Bacteria
2) Eukaryotic
▪Fungi
▪Protozoa
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Eukaryotic and Prokaryotic cells....
❑Prokaryotic
oPro➔ primitive
oKaryon➔ nucleus/ Nut shell
 Cells of lower life forms
 Having DNA which is not enclosed by membrane
 nuclear material is distributed in mass through the cytoplasm.
 Example: Bacteria
 They divide by binary fission.

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A typical prokaryotic cell

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Eukaryotic and Prokaryotic cells....
❑Eukaryotic cells
▪ EU➔ True/ real
▪ Karyon ➔ nucleus/ Nut shell
➢ More advanced, larger, contain membrane bounded organelles.
▪ Fungi
▪ Protozoa
▪ Cells of plants and animals
➢ They divide /multiply/ by a process called mitosis

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A typical eukaryotic animal cell
2.2 Morphology and structure of bacterial cells

❑Morphology: appearance of bacteria when visualized under light microscope, which

includes

➢Size

➢Shape

➢Arrangement

❑ Size: -

▪ Bacteria have different size.

▪ Most bacteria range from 0.5-10 μm in length and 0.2 – 2.0 μm in diameter.
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 Morphology...........
❑Shape and arrangement
1. Cocci
▪ Round or oval bacteria
▪ measuring about 0.5-1.0 μm in diameter.

❑ Arrangement
✓Micrococcus: - single
✓Diplococcic: - arranged in pair
✓Streptococci: - chain forming cocci
✓Staphylococci: -Cocci in irregular groups (
in cluster). 39
 Morphology
2. Rod shaped (Bacilli)
▪ stick- like bacteria
▪ with a size measuring 1-10μm in length by 0.25 - 1.0 μm in
width.
❑Arrangement
o Singly eg S. typhi
o Mass together, e.g. Mycobacterium leprae.
o Forming angle (Chinese letter) eg Corynebacterium
diphtheria

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 Morphology............
i) Cocco bacillus
▪ They are short bacilli.
▪ Their size is smaller than bacilli and bigger than coccus
e.g., Haemophilus influenza
Bordetella pertussis

ii)Comma shaped
▪They assume rod shape and are curved like comma
▪ e.g., Vibrio cholera
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 Morphology.......
3. Spiral shaped bacteria
• It is a twisted ,flexible, coiled, motile organisms.
a) Treponemes
▪ thin delicate with regular tight coils.
▪ 6 –15 μm by 0.2 μm in width
▪ Examples Treponema pallidum
b) Borreliae
▪ large spirochaetes with irregular open coils.
▪ 10–20 μm in length by about 0.5μm in width
▪ Examples Borrelia recurrentis 42
Morphology.......
c) Leptospira
▪ Thin spirochaetes with many tightly
packed coils that are difficult to
distinguish.

▪ 6–20μm in length by 0.1μm in width and

have hooked ends.

▪Leptospira interrogans

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Morphologic arrangements of cocci and examples of bacteria
having these arrangements.
Structure of bacterial cells

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 Structure of bacterial cells...........

❑ Based on the position structure occupy

I) The envelope structure:

➢ The cell membrane
o Encloses the cytoplasm
o Proteins (60 %) and lipid (40 %) (bilipid layer).
o Unlike eukaryotic cell not contain sterols, except Mycoplasma species.
➢ Cell wall
✓ Multi layered structure
✓ Constitutes about 20% of the bacterial dry weight
✓ Average thickness is 0.15 - 0.5μm .
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 Structure of bacterial cells...........

❑ Comparison of cell walls of Gram-positive and Gram-negative

bacterial cells.

Component Gram-Positive Gram-Negative

Bacterial Cell Bacterial Cell
• Peptidoglycan Thicker(85%) Thinner, single layered (3-12%)
• Teichoic acid Present Absent
• Lipopolysaccharide
(Outer membrane) Absent Present
• Periplasmic space Absent present

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 Structure of bacterial cells...........
❑Functions of Cell Wall
❖Provides shape to the bacterium

❖Gives rigidity to the organism

❖Protects from environment

❖Provides staining characteristics to the bacterium

❖Contains receptor sites for phages

❖Site of action of antibody

❖Contains components toxic to host

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 Structure of bacterial cells...........
II) Cytoplasmic structures

• Enclosed with in the cell envelope

• Contains different Cytoplasmic organelles such as

➢Mesosomes

➢Ribosome

➢Nuclear apparatus

➢Plasmid

➢Cytoplasmic granules 49
 Structure of bacterial cells...........
❑ Mesosomes

▪ Convoluted invagination of cytoplasmic membrane

▪ It is involved in DNA segregation during cell division

▪ Aiding cell in cellular respiration and analogous to mitochondrion

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 Contd…
❑ Nuclear apparatus
▪ No nuclear membrane.
▪ DNA is concentrated in the cytoplasm as a nucleotide (chromosomal DNA).
▪ Bacterial genome consists of double stranded DNA arranged in a circular form.

❑ Ribosome
➢ ribosomes are 70S in size, and
➢ composed of 30S and 50S subunits.
➢ the sites of protein synthesis.
➢ targets for antibiotics that inhibit protein synthesis
51
 Structure of bacterial cells...........
❑ Plasmid
▪ Extra chromosomal DNA.
▪ Smaller than chromosomal DNA.
▪ Code for variable numbers of genes e.g. virulent property, antibiotic resistance.
❑ Cytoplasmic granules
▪ are distinct granules that may occupy a substantial part of the cytoplasm.
▪ Their presence vary with bacteria and metabolic activity.
▪ Represent accumulated food reserves.
▪ For example,
– Glycogen granules (polysaccharides)
– Poly-beta hydroxy butyrate granules (lipid)
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 Structure of bacterial cells...........

III) External structures

➢ External to the cell envelope
❑ Pilli
▪ Hair like filaments structure
▪ Found mainly on gram negative bacteria.
▪ Function of pilli
• Common Pili: adherence to host surface.
• Sex (F) pili: involved in bacterial conjugation transfer
of genetic material (DNA)

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 Structure of bacterial cells...........

❑Flagella
➢ organ of locomotion

➢ It is composed of protein named as flagellin.

➢ The Flagellar antigen in motile bacterium is

named as H antigen.

➢ mainly seen in bacilli bacteria.

➢ The number and arrangement of flagella on

the cell are diagnostically useful.

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 Structure of bacterial cells...........
❑ Flagellar Arrangements
✓ Atrichous →Bacteria with no flagellum
✓ Monotrichous → with single polar flagellum
✓ Lophotrichous → with bunch of flagella at one pole
✓ Amphitrichous → with flagella at both poles
✓ Peritrichous → Bacteria with flagella all over their surface.

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 Structure of bacterial cells...........
http://science.nhmccd.edu/biol/wellmeyer/bacteria/capsules3.jpg

❑ Capsule
➢ Hardest part of a bacterium which is non-living.
➢ Composed of complex polysaccharides.
➢ Important in protection of bacteria
➢ resistance to Phagocytosis.
E.g
❑Haemophilus influenzae
❑Streptococcus pnemoniae
❑Klebsiella species
❑Bacillus anthracis
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 Structure of bacterial cells...........
❑ Spores
➢ Metabolically inert forms triggered by adverse env’tal conditions.
➢ allows re-growth when conditions are favorable.
➢ smooth walled and oval or spherical in shape.
➢ Uses to surviving under adverse environmental conditions
o Heat - Drying -Radiation
o Freezing - Toxic chemicals

➢ It is significant in spread of disease and sterility of materials.

➢ Pathogenic bacteria that produce spores include
❑ Clostridium species
❑ Bacillus species
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Endospores formation within a vegetative
bacterium

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59
 2.3 Classification and Identification of bacteria

❑Classification of bacteria based on

➢ Morphology

➢ Staining reaction

➢ Growth requirement

➢ Cultural characteristics

➢ Biochemical tests

➢ Etc……………..

60
 Classification &Identification.....
A) Morphology
➔ Cocci
▪ Streptococci species (S. pyogen, S. pneumonia)
▪ Staphylococci species ( S. aureus)
▪ Neisseria species ( N. gonorrhea)
➔ Rod
▪ B. anthracis
▪ Salmonella species , E. coli etc……

➔ Spiral
▪ Borrelia
▪ Treponema
▪ Leptospira
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 Classification & Identification.....

B)Staining
▪ is the process of coloring of colorless object using stains (dyes).

➢ Uses of staining
▪ To observe the morphology of bacteria.
▪ To differentiate one group of bacteria from the other group.

➢ Stain in Microbiology:
▪ Staining is possible due to the difference in the composition of the
dyes and the cellular components.

62
 Classification &Identification.....

➢ Basic principle of staining

▪ Acidic part of the cell negatively charged (nucleic acid,

nucleoproteins of the nucleus) stains with basic dye (methylene
blue), and

▪ Basic component of the cell positively charged (cytoplasm) stains

with acidic dyes (eosin).

▪ Other structure stained by a combination of this two (neutral stain).

63
 4.2 Types of stain
➢Type of staining methods
1. Simple staining method

2. Differential staining method

3. Special staining method

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 Types of stain….
1) Simple staining method

▪ Only a single dye is used

➢ Simple stain used for determining shape and arrangement of

bacteria

➢ Simple stain can be applied

a) For urgent cases

b) If differential staining reagents are not available.

65
 Types of stain….
➢ Two kinds of simple stains
i) Positive staining
▪ The bacteria or its parts are stained by the dye
▪ E.g. 1% methylene blue, 1% Crystal violent.
ii) Negative staining
▪ The dye stains the background and the bacteria
remain unstained.
▪ E.g. India ink , Negrosin

66
 Staining techniques
❑Procedure
1) Make a smear and label it.
2) Allow the smear to dry in air.
3) Fix the smear over a flame.
4) Apply a few drops of positive simple stain like (1%
methylene blue, 1% Crystal violet for 1 minute).
5) Wash off the stain with water.
6) Air-dry and examine under the oil immersion objective.

67
 Types of stain….
Classification &Identification.....
2) Differential staining method
▪ Multiple stains (dye) are used to distinguish different group of
bacteria.

▪ Common example of differential staining technique

➢Gram staining

➢Ziehl-Neelsen (acid fast staining technique)

68
 Types of stain….
A) Gram stain
▪ Originally developed by Christian Gram in 1884
▪ A critical test for a rapid, presumptive diagnosis of infectious agents
▪ Used to classify bacteria on the basis of their Gram reaction,
morphology (shape & arrangement).
▪ Most bacteria are differentiated by their gram reaction (Gram
positive and Gram negative bacteria)due to difference on their cell
wall structure.
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 Advantage of gram stain
• Rapid procedure
• Cost effective
• Accessible
• Effective screening technique
• Semi-quantitative
• Provides culture clues

70
 Gram stain…….
❑Purpose of Gram staining techniques
➢ to identify
1) Gram reaction (Gram pos. or Gram neg.)

2) Shape ( cocci, rod, spiral)

3) Arrangement ( chain, cluster, pair, mass)

4) Location (intercellular or extracellular)

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 Specimen for Gram staining
❑Clinical specimens (direct smears):
▪ Wounds
▪ Eye lesions
▪ Body fluids (Sterile)
▪ Body tissues
▪ Discharges

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 Materials and reagents ....
i) For smear preparation iii) For staining processes
▪ Slides • Staining rack
▪ applicator stick • Forceps
▪ Sprit lamp/bunsen burner • Cotton
▪ Labeling materials • Washing bottle
ii) For safety iv) For examination
▪ PPE • Microscope
• Oil immersion

73
 Gram stain…….
❑Required reagents:
1) Crystal violet (Gentian violet).....Primary stain
2) Gram's iodine ............................Mordant
3) Acetone- Alcohol........................Decolourizer
4) Safranin........................................Counter stain
▪ Gram staining technique includes the following
five major process sequentially.
o Smear ➔ Fix ➔ Staining ➔ Examining ➔ Report

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Gram staining

75
 Gram staining technique….
❑ Fixing smears
➢ The purpose of fixation is
▪ to preserve microorganisms and
▪ to prevent smears being washed from slides
during staining.
➢ Method of fixation
▪ Heat and alcohol, or occasionally by other
chemicals.
76
 Fixation
A) Heat fixation
▪ This is widely used but can damage organisms and alter their staining
reactions especially when excessive heat is used.
▪ Heat fixation also damages leucocytes and is therefore unsuitable for
fixing smears which may contain intracellular organisms such as N.
gonorrhoeae and N. meningitidis.
▪ When used, heat fixation must be carried out with care.

77
 Fixation……
❑ Technique for Heat fixation

1) Allow the smear to air-dry completely.

2) Rapidly pass the slide, smear uppermost, three times through the
flame of a spirit lamp or pilot flame of a Bunsen burner.

3) Allow the smear to cool before staining it.

78
 Fixation…..
B) Alcohol fixation
➢Less damaging to morphology of microorganisms than heat.
➢Cells, especially pus cells, are also well preserved.
➢Recommended for fixing intracellular bacteria.
➢Alcohol fixation is more bactericidal than heat and reduce
contamination.

79
 Fixation….
❑A method of alcohol fixing smears
1) Allow the smear to air-dry completely.

2) Depending on the type of smear, alcohol-fix as follows:

3) Leave the alcohol on the smear for a minimum of 2

minutes or until the alcohol evaporates.

80
 Gram Staining Procedure
1) Cover the fixed smear with crystal violet stain for 30–60 seconds.
3) Rapidly wash off the stain with clean water.
4) Tip off all the water, and cover the smear with Lugol’s iodine for 30–60
seconds.
5) Wash off the iodine with clean water.
6) Decolorize rapidly (few seconds) with acetone–alcohol. Wash
immediately with clean water.
7) Cover the smear with Safranin (neutral red) stain for 2 minutes. 81
 Gram Staining techniques…….

8) Wash off the stain with clean water.

9) Wipe the back of the slide clean, and place it in a draining rack for
the smear to air-dry.

10) Examine the smear microscopically, first with the 40 objective to

check the staining and to see the distribution of material, and then
with the oil immersion objective to report the bacteria and cells.

82
Summary of Gram Staining technique

83
Summary of Gram Staining technique

84
Staining techniques……
❑Interpretation
• Gram-positive bacterium …Purple (Dark purple)
• Gram-negative bacterium …Pink (pale to dark red)
• Yeast cells . . . . . . . . . . . . . . .Dark purple
• Nuclei of pus cells . . . . . . . . Red
• Epithelial cells . . . . . . . . . . . .Pale red

85
86
87
Gram Staining techniques…….

➢Gram positive ➢Gram negatives

▪ Staphylococcus ▪ Neisseria spp.
▪ Streptococcus ▪ Haemophilus spp.
▪ Clostridium ▪ Vibrio
▪ Salmonella
▪ Bacillus ▪ Shigella spp
▪ Corynebacterium ▪ Proteus
▪ Etc….. ▪ Klebsiella
▪ Brucella
▪ Yersinia
▪ Coliforms
▪ etc…..

88
 Differential staining method
B) Ziehl- Neelsen staining Method
▪ The Ziehl-Neelsen (Zn) technique is used to stain Mycobacterium
species.
▪ Mycobacterium, unlike most other bacteria, do not stain well by the
Gram technique
▪ Can stain other acid fast organisms which cannot be stained with gram
stain.
➢Acid fast bacilli (AFB)
▪ Mycobacterium tuberculosis
▪ Mycobacterium leprae
▪ Mycobacterium. ulcerans
89
 Ziehl- Neelsen staining Method….
➢Principle
▪ Mycobacterium species stained with carbol fuchsin combined with
phenol.
▪ The stain binds to the mycolic acid in the mycobacterial cell wall.
▪ After staining, an acid decolorizing solution is applied.
▪ This removes the red dye from the background cells, tissue fibres,
and any organisms in the smear except mycobacteria which retain
(hold fast to) the dye and are therefore referred to as acid fast
bacilli (AFB).
▪ Counterstained with malachite green or methylene blue which
stains the background material, providing a contrast color against
which the red AFB can be seen.
90
Ziehl- Neelsen staining Method….

❑Reagents required
1) Carbol-fuchsin

2) Acid-Alcohol

3) Methylene blue/Malachite green

91
 Procedure for Ziehl-Neelson staining method
1)Prepare the smear from the primary specimen and fix it by passing
through the flame and label clearly
2)Place fixed slide on a staining rack and cover each slide with
concentrated carbol fuchsin solution.
3)Heat the slide from underneath with sprit lamp until vapor rises (do not
boil it) and wait for 5 minutes.
4)Wash off the stain with clean water.
5)Cover the smear with 3% acid-alcohol solution until all color is removed
(2-5minutes).
6)Wash off the stain and cover the slide with 1% methylene blue. for 1-
2minute.
7)Wash off the stain with clean water and let it air-dry.
8)Examine the smear under the oil immersion objective to look for acid fast
bacilli.
92
 AFB ( Acid Fast Bacilli)
❑Interpretation
▪ Acid fast bacilli(AFB)…………............Red Rods
– Straight or slightly curved rods, occurring singly or in small groups
▪ Back ground material …………….....Blue/ Green
▪ Cells ………………………………………….. Blue/ Green

93
 Ziehl- Neelsen staining Method….
❑Reporting of sputum smears
➢When any definite red bacilli are seen, report the smear as
‘AFB positive’, and give an indication of the number of bacteria
present as follows:
▪ More than 10 AFB/field………………….+3
▪ 1–10 AFB/field ………………………………+2
▪ 10–100 AFB/100 fields…………………..+1
▪ 1–9 AFB/100 fields ……………report exact number

94
 Acid fast stain using Sodium hypochlorite
centrifugation technique
❑Materials • new and clean slides
• burning spirit • timer
• diamond pencil or pencil • 250 ml staining bottles with
• little plastic bag for the spout
waste disposal • Naocl,
• wooden sticks or wire loops • Microscope
• staining reagents • Centrifuge
• sand jar • Test tubes, Alcohol
• Bunsen burner or spirit • Forceps
lamp • funnels
❑Specimens
•Pulmonary: sputum
•Extra-pulmonary
•laryngeal swabs … etc 95
96
Types of stain.............
3. Special Stains
a) Capsule staining method

b) Spore staining method

c) Flagella staining

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 Classification &Identification.....

❑Cultivation of Bacteria in Culture Media

➢ Culture Media
▪ It is the medium which contain the required nutrient for bacterial
growth.

➢ Use of culture media

▪ for isolation and identification of microorganism.
▪ to perform anti microbial sensitivity test.

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 Classification &Identification.....
❑Types of culture media
➢Basic
▪ nutrient agar & nutrient broth
➢Enriched
▪ Blood agar, Chocolate agar
➢Enrichment
▪ Refers to liquid media that inhibitis the growth other of
bacteria
▪ Tryptosoya broth, Selenite F broth

99
 Types of culture media….

➢Selective
▪ Salmonella Shigella agar MacConkey agar, Thiosulfate Citrate Bile
Salts Sucrose
➢Differential/ Indicator
▪ MacConkey agar
▪ TCBS agar (Thiosulphate Citrate Bile salt Sucrose)
➢Transport
▪ Amies transport media
▪ Cary Blair transport media

100
LF vs NLF

101
Swarming growth Proteus

102
P. areuginosa

103
N. gonorrhea

104
S. aureus

105
Salmonella species

106
 Classification &Identification.....
C) Based on growth requirement

1) Air requirement
➢ Obligatory (strict) aerobes: - need free oxygen for their growth.
▪ E.g. Pseudomonas aeruginosa
➢ Facultative anaerobes:- can grow either in the presence or absence of O2.
▪ E.g. S. aureus, E. coli, salmonella, Shigella, Vibrio, Haemophilus, Proteus spp etc..
➢ Obligate or strict anaerobes: -
▪ only grow in absence of free oxygen.
▪ E.g. Clostridium tetani, Borrelia, Treponema, etc….

➢ Microaerophilic: - need small amount of oxygen for growth.

▪ E.g. Campylobacter species.
➢ Carboxyphilic: - bacteria require an atmosphere which contains CO2.
▪ E.g. Neisseria meningitides
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108
 Classification &Identification.....
2) PH requirement
▪ Most bacteria grow or reproduce at a pH range of 6 -8.
▪ Classification of bacteria based on PH
a) Neutrophilic
• Bacteria's which grow at a pH between 6-8.

b) Acidophilic
• bacteria which can grow at a pH less than 7.0.
• E.g Lactobacillus
c) Alkalophilic
• Few bacteria's grow at a pH higher than 8.0.
• E.g. Vibrio cholera. 109
 Classification &Identification.....

3) Temperature
▪ wide range of temperature
▪ This range can influence enzymatic activity.
➢ Mesophilic
▪ bacteria which can grow at a temperature B/n 30 - 37 oC.
➢ Psycrophilic
▪ Grow at a temperature between 15oC – 20 oC.
➢ Thermophilic
▪ Those bacteria which grow between 50oC – 60oC.

❖ Note:-
▪ Most pathogenic bacteria's are mesophils.
▪ Spore form bacteria can survive in any of the above temperature conditions

110
 Classification &Identification.....

4) Moisture
▪ Most bacteria need optimum amount of moisture.
▪ Some bacteria needs high amount of moisture. Example
➢ T. palladium
➢N. gonorrhoea

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 Classification &Identification.....
5) Nutritional requirement
• Bacteria need nutrition for synthesis of protoplasm and energy.
➢ Simple: - All bacteria need source of
• Carbon - Energy
• Nitrogen - Water and mineral salts
➢ Complex
• some bacteria need growth factors (vitamins).
• Fastidious bacteria.
• E.g. Neisseria gonorrhea, Haemophilus influenza
➢ Halophilic
• Those bacteria which live (grow) on high salt concentration.
• E.g. Staphylococcus aureus
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 Classification &Identification.....
d)Biochemical reaction
• Biochemical can be used for classifying bacteria in to different groups.
i) Enzymes
▪ Coagulase positive. Eg S. aureus
▪ Catalase positive. Eg Staphylococci species
▪ Urease. Eg Protus species
ii) Metabolic end product
▪ Gas producer
▪ acid, or H2S from carbohydrates.
iii) Carbohydrate utilization
▪ Lactose fermenter
▪ Glucose fermenter
▪ Sucrose fermenter
113
Biochemical test

114
Citrate test

115
Coagulase test

116
Indole test

117
118
Urease test

119
KIA

120
 2.4 Nomenclature and taxonomy of bacterial cells
❑Taxonomy
 Science of systemic classification, identification and naming living
things (Microbes).
 Taxon:-a group or category of related organisms
 It is dynamic.
▪ Domain
▪ Kingdom
▪ Phylum
▪ Class
▪ Order
▪ Family
▪ Genus
▪ Species
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Classification &Identification.....
Taxonomic rank Example: Escherichia coli
▪ Domain………………. Prokaryotes (Bacteria)
▪ Kingdom……………...Monera ( the prokaryotes)
▪ Phylum……………......Gracilicutes
▪ Class……………..……Scotobacteria (bacteria that do not require light for
metabolism
▪ Order……………..…...Eubacteria
▪ Family……………...….Enterobacteriaceae
▪ Genus……………...…..Escherichia
▪ Species…………….….coli

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 2.4 Nomenclature and taxonomy of bacterial cells

❑Classification

➢is the orderly arrangement of bacteria into groups( taxa).

➢Based on similarity or relation ship.

➢different groups of scientists may classify the same organisms differently.

123
 Taxonomy.....

❑Identification
▪ is the practical use of classification criteria
➢To distinguish certain organisms from others.

➢To isolate and identify the causative agent of disease.

▪ Using a stain or other techniques.

124
 Nomenclature and taxonomy.....
❑ Species

➢ A bacterial species is defined by the similarities found among its

members.
➢ Properties used for defining a bacterial species:-
▪ Biochemical reactions
▪ Chemical composition(capsule, cell wall)
▪ Cellular structures,
▪ Genetic characteristics
▪ Immunological features
125
 Nomenclature and taxonomy.....
❑Scientific name (systematic name)

➢is also called binomial system of nomenclature

➢genus name + species name

▪ italicized or underlined
▪ genus name is capitalized and may be abbreviated
▪ species name is never abbreviated
▪ a genus name may be used alone to indicate a genus group
▪ a species name is never used alone,
➢Bacillus subtilis or
➢B. subtilis 126
 Nomenclature and taxonomy.....

❑Common or descriptive names (trivial names)

➢ names for organisms that may be in common usage, but are not
taxonomic names.

➢ Example:-

▪ Tubercle bacillus →(Mycobacterium tuberculosis)

▪ Meningococcus → (Neiserria meningitidis)

▪ Group A streptococcus →(Streptococcus pyogenes)

127
 2.5 Nutrition, growth and multiplication

❑Bacterial Nutrition
• Nutrient must be provided for optimal bacterial growth.
❑ Carbon source
➢ Autotrophs
▪ free-living
▪ non-parasitic bacteria
▪ use carbon dioxide as carbon source.
➢ Heterotrophs
▪ Parasitic bacteria
▪ require more complex organic compounds as their source of carbon and
energy.

128
 Nutrition, growth and multiplication.....
❑ Nitrogen source
▪ proteins and inorganic molecules
▪ ammonium salts
▪ nitrates

❑ Mineral
▪ Sulfur and phosphorus, Sodium, potassium, magnesium, calcium, iron,
chlorine, zinc, copper, iodine etc
▪ essential for physiological activities of bacteria.

❑ Growth factor
▪ are organic compounds that are required in small amounts
▪ cell can not synthesize it from other carbon source.
▪ These are
▪ amino acids
▪ Purines & pyrimidines
▪ vitamins
129
 Nutrition, growth and multiplication.....

❑Growth
▪ It is an orderly increase in all the components of an organism
or
▪ the increase in the number of cells.

• The bacteria reproduce asexually (binary fission) in which

one parent cell divides to form two progeny cells.

• The division processes continue in a geometric way, that is

after cell division in to two cell the population double.
➢ Number of cell 1, 2, 4, 8, 16, 32, 64... ………
➢ Exponential 20, 21, 22, 23, 24, 25, 26…………2n,
130
 Nutrition, growth and multiplication.....

❑Generation time
➢ The time needed for a cell population to double in number.

➢ the time required for a reproduction cycle.

➢ vary greatly from species to species.

➢ Fast-growing bacteria cultivated in vitro have a generation time of 15–30 minutes.

➢ e.g. E. coli

➢ Obligate anaerobes grow much more slowly than aerobes.

➢ Mycobacterium tuberculosis have an in-vitro generation time of 12–24 hours.

131
 Nutrition, growth and multiplication.....

❑Bacterial growth phases

➢ The normal bacterial growth curve has four phases.
1. Lag phase
▪ The period of adaptation with active macro molecular synthesis like DNA,
RNA, various enzymes and other structural components.
▪ It is the preparation time for reproduction
▪ no increase in cell number.
2. Exponential (log) phase
▪ The period of active multiplication of cells.
▪ Cell division precedes at a logarithmic rate, and determined by the
medium and condition of the culture.
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 Nutrition, growth and multiplication.....

3. Maximal stationary phase

▪ maximal cell density or yield.
▪ no further increase in viable bacterial cell number.
▪ The growth rate is exactly equal to the death rate.
▪ A bacterial population may reach stationary growth when one of the
following conditions occur:
✓The required nutrients are exhausted
✓Inhibitory end products are accumulated
✓Physical conditions do not permit a further increase in population size

133
 Nutrition, growth and multiplication.....

4. Decline phase
▪ The period at which the rate of death of bacterial cells exceeds the rate of new cell
formation.

▪ There is drastic decline in viable cells.

134
Nutrition, growth and multiplication.....

135
 Nutrition, growth and multiplication.....

❑Factors Influencing Bacterial Growth in Vitro

▪ Each bacterial species has a specific tolerance range for specific environmental
parameters.
▪ Rate of bacterial growth are greatly influenced by the following environmental
parameters.
➢Nutrition
➢Salinity (salt)
➢Temperature
➢Pressure (Osmotolerant)
➢Oxygen
➢Light radiation
➢PH
➢Moisture, etc…

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