Microbiology

• Microbiology deals with living organisms that are

too small to be seen with the naked eye.

• Clinical microbiology studies the pathogenic

agents of infectious diseases.

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Eukaryotes:
ᴑ A true membrane bound nucleus
ᴑ usually containing multiple chromosomes
ᴑ a mitotic apparatus
ᴑ a well defined endoplasmic reticulum, and
ᴑ mitochondria.

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• Prokaryotes:
ᴑ Naked DNA.
ᴑ without associated basic proteins.
ᴑ divides amitotically by binary fission.
ᴑ bounded by a semi rigid cell wall.

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Microbiology
• Bacteria ---- Bacteriology
• Viruses ---- Virology
• Fungi ---- Mycology
• Parasites ---- Parasitology
- Protozoa
- Helminths

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BACTERIOLOGY

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 Nomenclature
• For bacteria, fungi , protozoa and helinths

- Binomial nomenclature system

Genus and species
eg; Escherichia coli

genus species

• In case of Viruses this rule is not followed. 6

 Bacterial Cell:
General properties
• Typical prokaryotic cell
• Contain both DNA and RNA
• Most grow in artificial media
• Replicate by binary fission
• Almost all cotain rigid cell wall
• Sensitive to antimicrobial agents

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 STRUCTURE OF BACTERIA:

Bacterial structure is described at three levels.

1. Cell envelope proper: Cell wall and cell membrane

2. Cellular elements enclosd within the cell envelop.

3. Structures external to the cell envelop:

Flagellum, Pili , Capsule , Glycocalyx.

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Ultrastructure of Bacterial Cell:

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Ultrastructure of Bacterial Cell:

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 Bacterial Cell Wall :

1. Cell envelope proper ( Cell Wall + Cell membrane)

A. Cell wall
• Multi layered structure , constitutes about 20%
of bacterial dry weight.
• Young and rapidly growing bacteria have thin cell
wall.
• Old and slowly growing bacteria have thick cell wall.
• Average thickness 0.15 – 0.5 μm.

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• Components of cell wall of Gram neg; bacteria.
--- Peptidoglycans

– Lipoproteins
– Phospholipids
– Lipopolysaccharides
• Components of cell wall of Gram Postive bacteria.
-- Peptidoglycans
-- Tiechoic acids

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B. Cell membrane
• Also named as cytoplasmic membrane.
• It is a delicate trilaminar unit membrane .
• It is composed of 60% protein, 20-30% lipids and
10 – 20 % carbohyderates.

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• The plasma membrane or bacterial cytoplasmic
membrane -- phospholipid bilayer
• Has all the functions of a cell membrane
- permeability barrier for most molecules
- serving as the location for the transport of
molecules into the cell.

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 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/complements
• Site of action of antibody.
• Contains toxic components to the host

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• Active Transport –
Three main types :
The Sodium-Potassium pump,
Exocytosis, and
Endocytosis.

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• Passive Transport:
-- Simple diffusion – movement of small or
lipophilic molecules (e.g. O2, CO2, etc.)
-- Osmosis – movement of water molecules
(dependent on solute concentrations)
-- Facilitated diffusion – movement of large or
charged molecules via membrane proteins
(e.g. ions, glucose, etc.)

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• Function of cell membrane
– Regulates the transport of nutrients and waste
products into and out of the cell.
– Synthesis of cell wall components.
– Assists in DNA replication.
– Secretes proteins.
– Carries on electron transport system.
– Captures energy in the form of ATP.

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Nuclear apparatus of Bacteria :

• Well defined nucleus with nuclear membrane ,

discrete chromosome and mitotic apparatus are
NOT present in bacteria.

• Bacterial genome consists of single molecule of

double stranded DNA arranged in a circular form
and named as nucleoid.

• Bacteria may have extra genetic material named

plasmids.
• 21
• Plasmids –
• Besides nuclear apparatus, bacteria may have
extra chromosomal genetic material named as
plasmids.

• No role in normal function.

- May confer certain additional properties
like - Virulence, drug resistance etc.

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Cellular Elements Internal To The Cell Envelope
A. Mesosomes
• Convoluted invagination of cytoplasmic
membrane often at sites of septum formation.
• It is involved in DNA segregation during cell
division and respiratory enzyme activity.

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 B. Ribosomes
• Cytoplasmic particles which are the sites of
protein synthesis.
• It is composed of RNA(70%) and proteins(30%).

C. Polyamines
• They are found in association with bacterial DNA,
ribosomes and cell membrane.
• Antimutagenic.
• Increase resistance of protoplast to lysis.

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 D. Cytoplasmic granules
• They represent accumulated food reserves.
- Glycogen, Poly-beta hydroxy butyrate
amd Volutin.

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 3. Cellular elements external to the
cell envelope

i. Glycocalyx (capsule and slime layer)

ᴑ Capsule is composed of polysaccharides and
proteins.
ᴑ It is in the form of gel firmly adherent to cell
envelope.
ᴑ Slime is gel easily washed off from cell envelope.
All bacteria have at least a thin slime layer.

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• Features of capsules
– Usually weakly antigenic.
– Not necessary for viability.
– Endows virulence.
– Protects from phagocytosis.
– Capsulated strains are invariably non-motile.
– Visualized by negative staining and capsule staining.

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ii. Flagellum
-- Organ of locomotion.
-- Consists of thee parts.
filament ,
hook,
basal body

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iii. Pili (fimbriae)
• Hair like structure composed of protein (pilin).
• Two types (Based on function)
- Common pili: The structure for adherence to
cell surface.
- Sex pili: The structure for transfer of genetic
material from the donor to the
recipient during the process of
conjugation.

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 Spores

• Spores (endospores) are the dormant form of

vegetative bacteria and are highly resistant to
physical and chemical influences.

• Nutrient starvation, Temp or pH extremes , cell

crowding or antibiotic exposure.
• Survive harsh conditions.

Bacteria such as the Bacillus and

Clostridium species are able to form spores.
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iv. Spores
• Forms capable of surviving under adverse
environmental conditions like heat, drying,
freezing, action of toxic chemicals and radiation.
• It is significant in spread of disease and indicator
of sterility of materials.

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 CLASSIFICATION OF BACTERIA:

• Bacterial classification depends on the following

characteristics.
- Morphology and arrangement
- Staining
- Cultural characteristics
- Biochemical reactions
- Antigenic structure
- Base composition of bacterial DNA

• Morphology and staining are the characteristics

which are used mostly, in classification. 33
A. Morphology of Bacteria:
□ Bacteria classified by shape into -
1. Cocci : Round or oval bacteria measuring
about 0.5-1.0 μm in diameter.They are found as
single, pairs, chains or clusters.

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 2. Bacilli (singular bacillus): Stick-like bacteria with
rounded, tepered, square or swollen ends; with a
size measuring 1-10 μm in length by 0.3-1.0 μm in
width.

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3. Coccobacilli (singular coccobacillus): Short rods.

4. Spiral: Spiral shaped bacteria with regular or

irregular distance between twisting eg; spirila,
Spirochaetes.

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B. Staining of bacteria
• The principle of staining is to identify
microorganisms selectively by using dyes,
fluorescence or radioisotope emission.

• Individual variation in the cell wall constituents

variations in colors during microscopic exam.

• Nucleus is acidic in character and hence, it has

greater affinity for basic dyes.
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• Cytoplasm is basic in character and has greater
affinity for acidic dyes.
General methods of staining
• Direct staining
Microorganisms are stained with simple dyes.
E.g., methylene blue
• Indirect staining – Needs mordants.
A mordant is the substance which, when
taken up by the microbial cells, serves as a link
or bridge to make the staining possible (cell-
mordant-dye- complex); eg. Gram iodine. 40
• Accentuator :
- Not essential for chemical union of the
microbial cells and the dye.
- Does not participate in the staining reaction.
- Merely accelerates or hasten the speed of
staining.
• Decolorization (Differentiation):
- Selective removal of excess stain.

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• Types of staining methods
ᴑ Simple staining method
- only single dye is used
- eg. Methylene blue, gram safrnin

ᴑ Differential staining method

- uses more than one chemical stain
- differentiates between two groups
- eg. Gram staining - G+ve , G-ve
ᴑ Special staining method
- stain used to give colour to a special part
- eg. Melachite green (spores) / Sefranin 42
 GRAM STAINING
• Developed by Christian Gram.
• Most bacteria are differentiated by their gram
reaction due to differences in their cell wall
structure.
• Gram-positive bacteria stain purple with crystal
violet after decolorizing with acetone-alcohol.
• Gram-negative bacteria stain pink with the
counter stain (safranin) after losing the primary
stain (crystal violet) when treated with acetone-
alcohol.
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• Required reagents:
. Crystal violet
. Gram’s Iodine
. Acetone-Alcohol
. Safranin

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• Procedure:
• Prepare the smear from the culture or from
the specimen.
• Allow the smear to air-dry completely.
• Rapidly pass the slide (smear upper most)
three times through the flame.
• Cover the fixed smear with crystal violet for 1
minute and wash with distilled water.
• Tip off the water and cover the smear with
gram’s iodine for 1 minute.

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• Wash off the iodine with clean water.
• Decolorize rapidly with acetone-alcohol for 30
seconds.
• Wash off the acetone-alcohol with clean
water.
• Cover the smear with safranin for 1 minute.
• Wash off the stain wipe the back of the slide.
Let the smear to air-dry.
• Examine the smear with oil immersion
objective to look for bacteria.

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• Interpretation:
. Gram-positive bacterium ………… Purple
. Gram-negative bacterium ……….. Pink

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 Cultivation of Bacteria
• Culture medium:
It is the medium containing the required
nutrients for bacterial growth.

Culture media are ued for

1. Isolation and identification of micro-
organisms.
2. Performing anti-microbial sensitivity
test.

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Common ingredients of culture media
. Peptone
. Meat extract
. Yeast extract
. Mineral salts
. Carbohydrates
. Agar
. Water

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• Peptone: Hydrolyzed product of animal and plant
proteins, Free amino acids, peptides and
proteoses(large sized peptides).
It provides nitrogen; as well as carbohydrates,
nucleic acid fractions, minerals and vitamins.
• Yeast extract: It is bacterial growth stimulants.
• Mineral salts:
. Sulfates as a source of sulfur.
. Phosphates as a source of phosphorus.
. Sodium chloride

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• Carbohydrates:
. Simple and complex sugars are a source of
carbon and energy.
. Assist in the differentiation of bacteria.
- Sucrose in TCBS agar differentiates vibro
species.
. Lactose in MacConkey agar differentiates
enterobacteria.

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• Agar: It is an inert polysaccharide from seaweeds.
. Not metabolized by the micro-organism.
. high gelling strength.
. high melting temperature(90-95 oc)
. low gelling temperature
. It forms firm gel at 1.5% W/V concentration.
. It forms semisolid gel at 0.4-0.5% W/V conc.
. May provide calcium and organic ions.
. used to solidify culture media.
• Water
Deionized or distilled water must be used in the
preparation of culture media 52
 Types of culture media
1. Basic /Simple / All purpose media
ᴑ Supports the growth of micro-organisms that do not
require special nutrients.
ᴑ Uses :
. To prepare enriched media
. To maintain stock cultures of control bacterial
strains.
. To subcuture pathogenic bacteria from
selective /differential medium prior to performing
biochemical or serological tests.
. eg. Nutrient Broth, Nutrient Agar. 53
2. Enriched media
Fortified / enriched with
whole blood,
lyzed blood,
serum,
special extracts
vitamins
to support the growth of pathogenic bacteria.
eg. Blood Agar, Chocolate Agar

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3. Enrichment media
Fluid medium that increases the numbers of a
pathogen by containing enrichments and /or
substances that discourage the multiplication of
unwanted bacteria.
eg. Selenite F broth
Akaline peptone water

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4. Selective media
Contain substances (eg. Antibiotics) that prevent
or slow down the growth of bacteria other than
pathogens for which the medium is intended.

eg. Modified Thayer –Martin Agar

Salmonella-Shigella( SS) agar

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5. Differential media
Indicator substances are added to differentiate
bacteria.
eg. TCBS Agar differentiates sucrose fermenting
(acid producing) yellow colonies of
Vibrio cholerae from other vibrio species
showing blue colonies ( non-fermnting ).

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 6. Transport media
• Media containing ingredients to prevent the
overgrowth of commensals and ensure the
survival of pathogenic bacteria when
specimens can not be cultured soon after
collection.
eg. Amies transport medium
Stuart medium
Kelly-Blair medium

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 Physical Forms of Culture Media
1. Solid culture media
. Plate cultures in petri dishes
. Stab/slope cultures in tubes and bottles
. Used for description of bacterial colonies
- Size : diameter in mm
- Out line : circular, entire, wavy, indented
- Elevation: flat, raised, low convex and dome
shape.
-Transparency: transparent, opaque, and translucent.
- Surface: smooth (mucoid) and shiny, rough, dull.
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 - Color: colorless, white, pink, pigmented.
- Changes in medium: hemolysis in blood agar,
blackening due to H2S production.
2. Semisolid culture media
- Medium to demonstrate motility in bacteria.
eg. Lysol indol motity medium.

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3. Fluid culture media
Bacterial growth in fluid media is shown by a
turbidity in the medium.
Uses :
. as an enrichment media
. as biochemical testing media
. as blood culture media
. as transport media

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 Preparation of culture media
• Culture media contains essential ingredients for
microbial growth requirements.
• For successful isolation of pathogens, culture media
must be prepared carefully.
• The major processes in preparation of culture media
-Weighing and dissolving of ingrediants.
- Sterilization and sterility testing.
- Addition of heat-sensitive ingredients.
- Dispensing of culture media.
- pH testing .
- Quality assurance.
- Storage . 62
 BACTERIAL NUTRITION
• Bacteria, like all cells, require nutrients for the
maintenance of their metabolism and for cell
division.
• The four most important elements of bacteria are
carbon, hydrogen, oxygen and nitrogen.

●. Carbon
. required for the synthesis of numerous organic
compounds that comprise living things.

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• Depending on their requirements, bacteria can be
classified as
• Autotrophs: Free-living, non-parasitic bacteria ,
- use carbondioxide as carbon source.
• Heterotrophs: Parasitic bacteria,
- require organic compounds as source
of carbon and energy.
Human pathogenic bacteria are heterotrophs.
• Principal source is carbohydrates
oxidation, in the presence of O2.
ATP fermentation, in the absence of O2
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● Hydrogen and oxygen
– Obtained from water.
– Essential for the growth and maintenance of cell.
● Nitrogen
– Constitutes 10% of dry weight of bacterial cell.
-- Main source of nitrogen is NH3 (from NH4 salts)
● Growth factors
-- organic compounds.
-- required in very small amounts for growth.

-- cannot be synthsized.
-- may be amino acids, purines, pyrimidine, vits.
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 Bacterial growth
• It is an orderly increase in all the components of an
organism.
• It is an increment in biomass.
• It is synchronous with bacterial cell reproduction.
Generation time
• “It is the time taken for the size of a bacterial
population to become double”.
• Bacteria grow by taking nutrients and incorporate
them into bacterial components; then bacteria
divide into two equal daughter cells and double the
number. 66
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 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.

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 2. Exponential (log) phase:
• Period of active multiplication of cells.
• Bacterial division proceedes at a logarithmic rate,
and determined by the medium and condition of
the culture.

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3. Maximal stationary phase:
• Period when the bacteria have achieved their
maximal cell density or yield.
• No further increase in viable bacterial cell number.
• Growth rate is exactly equal to the death rate.

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• 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.

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 4. Decline phase:
• Rate of death of bacteria exceeds the rate of new
bacterial production.
• There is drastic decline in viable cells.
• Few organisms may persist for so long time to this
period, at the expense of nutrients released from
dying micro-organisms.

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 Quantitative measurement of bacterial growth
• Bacterial growth is measured by determining number
of bacteria. The common measuring methods are -
1. Viable plate count
• The most common method.
• Involves counting the number of bacterial colonies
after 18 - 24 hours incubation.
• Procedure -
1. Sample is serially diluted.
2.The suspension is inoculated on solid media by
surface spread technique i.e. the suspension is
spread. 73
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 3. The concentration of bacteria in the original sample can be
determined by counting the visible colonies multiplied by the
dilution factor.

• Number of colonies = Number of colonies X dilution factor

Volume of sample

• NB: The statistically significant plate count is

between 30 and 300 colonies.
- Less than 30 colonies on a plate are not
accepted, for statistical reasons.
- Greater than 300 colonies on a plate are too
numerous to be counted.
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 2. Direct count:
• Direct microscopic counting of bacteria in the
sample, using counting chamber.
• It is relatively quick and does not need the sample
to be incubated.
• Procedure
. Serial dilution of the sample is made.
. Counting area of the chamber is filled with the
sample.
. Total number of bacteria in the sample per unit
volume is calculated.
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Serial Dilution

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 3. Turbidimetric method:
• It is the method of determination of bacterial
growth in liquid media. Bacterial growth
increases the turbidity of liquid to absorb light.
• The turbidity of the suspension is determined by
spectrophotometer.

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Factors influencing bacterial growth in vitro
• Not all bacterial species grow under identical
environmental conditions.
• Each bacterial species has a specific tolerance
range for specific enviromental parameters.
• Rates of bacterial growth are greatly influenced by
the following enviromental parameters.
. Nutrition . Oxygen
. Temperature . pH
. Salinity . Pressure
. Light radiation 79
 1. Nutrition:
• Following nutrients must be provided for optimal
bacterial growth.
– Hydrogen donors and acceptors
– Carbon source
– Nitrogen source
– Minerals: sulfur and phosphorus, trace
elements
– Growth factors: amino acids, purines,
pyrimidines and vitamins.

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2. Temperature:
• Temperature tolerance range:
- Mminimum and maximum temperature at
which a micro-organism can grow; - different in
different species of bacteria.
• Optimal growth range of temperature:
- The temperature at which the maximum growth
rate occurs; and results in the shortest
generation time of bacteria.

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• Based on different optimal growth temperature
requirements, bacteria are divided into:

. Psychrophilic bacteria = 15-20 o c; grow best at low T0 range

. Mesophilic bacteria = 30-370c; grow best at middle T0 range
. Thermophilic bacteria= 50-60oc; grow best at high T0 range

• Most human pathogens and many of the normal

flora of human body are mesophilic in nature.

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3. Oxygen:
• Base on oxygen requirements and tolerance,
bacteria are classified as:

. Obligate aerobes
. Obligate anaerobes
. Facultative anaerobes
. Microaerophiles

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 Obligate aerobic bacteria.
• They grow only when free oxygen is available to
support their respiratory metabolism.
• They obtain ATP by using oxygen in respiration.

Obligate anaerobic bacteria.

• Grow in the absence of oxygen.
• Exposure to oxygen kills anaerobes.

Facultative anaerobic bacteria.

• They grow in the presence or absence of oxygen.
• They obtain ATP by fermentation or anaerobic
respiration
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 Microaerophilic bacteria:
• They grow best at reduced oxygen tension;
high oxygen tension is toxic to them.

4. Hydrogen ion concentration

• Neutrophilic - grow best at near neutral PH value.
• Acidicophilic - prefer to grow at low PH value.
• Alkalinophilic - prefer to grow at high PH value.
• Most pathogenic bacteria grow best at PH 6-8.

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 5. Salinity :
• Most bacteia - can not tolerate high salt conc.
• High salt concentration disrupts membrane
transport systems and denatures proteins.
• Some bacteria (halophiles) have adaptive
mechanisms to tolerate high salt concentration.

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 BACTERIAL GENETICS
• Genetics - study of inheritance.
• Inherited characteristics - encoded in DNA .
• Two types of DNA in bacteria -
. A circular Chromosome
. Extra chromosomal : Plasmid
• The bacterial chromosome
. Chromosome + several proteins + RNA
= nucleoid

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• DNA replication in bacteria - semi-conservative.
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Plasmids:
• Self-replicating extra chromosomal DNA molecules.
• Multiply independent of the host cell.
• Multiple copies of the same plasmid may be
present in each bacterial cell.
• Different plasmids – in the same bacterial cell.
• Many plasmid types -
a. R factors: genes code for antibiotic resistance.
B. Col factors: genes code for extracellular toxin

(colicines) production .
. Inhibit strains of the same and different species.
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C. Fertility Factors: . Can recombine itself with the
bacterial chromosome.
. Promote transfer of the chromosome at a high
frequency of recombination into the chromosome
of a second (recipient) bacterial cell during mating.

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91
B
A
C
T
E
R
I
A
l C
ON
J j
G
A
T
I
O
nBacterial conjugation

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Genetic variation in Bacteria:
• Mechanisms: Mutation and Gene transfer.
a) Mutation. Due to a chemical alteration in DNA.
. Could be spontaneous or induced by chemical /
physical means.
. Mutants are variants in which one or more bases
in their DNA are altered.
. Change is irreversible and heritable.
Types of mutation
- Substitution: Change of a single base.
- Deletion: Loss of a base.
- Insertion: Addition of a base 93
 b) Gene transfer:
• Three types of gene transfer - alter the DNA gene content
of bacteria - Transformation , Transduction , Conjugation &
Transposition.
1. Transformation –
- Fragments of exogenous bacterial DNA are taken
up and absorbed into recipient bacterial cells.
- Transformation from one bacterium to another
results in
. Change in pathogenicity of the bacterium.
. Change in antibiotic sensitivity pattern of
bacterium.
. Frequency: The frequency of transformation is low
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95
 2. Transduction-
• Fragment of chromosomal DNA transferred or
transduced into a second bacterium by phage.

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 3. Conjugation:
• DNA is transferred from donor to recipient
bacterium by direct contact via a sex pilus.

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 4. Transposition:
• Transposons(Jumping genes) -fragments of DNA
that can move extremely readily (transpose),
from plasmid to plasmid or from plasmid to
chromosome(and viceversa).
• Plasmid genes become part of the chromosomal
component of genes.
• Transferring transposon is a copy of original one.
• Transposons code for toxin production, resistance
to antibiotics as wellas other fuctions.

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• Transposition (horizontal gene transfer), the
transfer of genetic material between organisms
other than by vertical gene transfer.
• Transposons, or genetic transposition, a mutation
in which a chromosomal segment is transferred to
a new position on the same or another
chromosome.

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 NORMAL FLORA
• Normal flora are the microorganisms that live on
another living organism (human or animal) or
inanimate object without causing disease. ... We
are covered with, and contain within our
intestines, approximately one hundred trillion
bacteria that form the normal flora of our bodies.

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