General Characteristics of microbes.pptx

GENERAL
CHARACTERISTICS OF
MICROBES
UNIT 2

A)Classification of
Microorganisms

● Classification - orderly arrangement of units under
study into groups of larger units.
● Carolus Linnaeus (1707-1778) - Established
classification in Biology.
● Microorganisms are classified according to 9 different
characteristics.

Classification on the basis of ;-
1. Body & nuclear organisation
2. Morphological characteristics
3. Nutritional requirements
4. Oxygen requirements
5. Temperature requirements
6. Osmotic condition requirements
7. Distribution
8. Economical importance
9. Host resistance or Pathogenicity

1.Classification
on the Basis of
Body & Nuclear
Organisation

On the basis of body organisation:-
1. Microbes beyond cellular organisation - These
organisms completely lack cell structure in their body.
Eg :- Viruses
2. Microbes with cellular organisation - these organisms
have their body made up of cells. Eg:- Bacteria,
microalgae, microfungi, protozoans etc.

On the basis of nuclear organisation :-
1. Prokaryotes - Microorganisms that possess incipient
nucleus & lacks nuclear membrane, nucleoplasm &
nucleolus. Eg:- Archaebacteria, Cyanobacteria &
Eubacteria
2. Eukaryotes - Microorganisms that possess a well
developed nucleus with nuclear membrane,
nucleoplasm & nucleolus. Eg:- Microalgae, protozoans,
slime moulds, microfungi

2. Classification
on the Basis of
Morphological
Characteristics

I According to Shape of Bacteria
1) COCCI (Spherical or Oval)
a. Cocci in cluster. Eg:- Staphylococcus
b. Cocci in chain Eg:- Streptococci
c. Cocci in pair Eg:- Diplococcus pneumonia
d. Cocci in groups of four -tetrad Eg:- Gaffkya tetragena
e. Cocci in groups of eight Eg:- Sarcina lutea

Diplococcus
pneumonia
Gaffkya tetragena
Sarcina lutea

2. BACILLUS (Cylindrical or Rod shaped)
a) Coccobacilli :- length of the cell greater than its breadth. Eg:-
Brucella
b) Cuneiform. Eg:- Corynebacterium
c) Vibrio:- comma shaped, curved rods. Eg:-Vibrio cholerae
d) Spirilla :- Rigid spiral shaped bacteria
e) Spirochetes :- longer, thinner, flexible & coil shaped bacteria
f) Actinomycetes :- branching type filamentous bacteria

COCCOBACILLI CUNEIFORM VIBRIO
SPIRILLA SPIROCHETES ACTINOMYCETES

3. Bacteria without stable shape
● Lacks cell wall
● Does not have stable morphology
● Called as MYCOPLASMA
● They occur as round & oval
bodies & as interlacing filaments

II According to their Size
1. Higher bacteria :- they are branching filamentous bacteria.
Eg:- Actinomycetes
2. Lower bacteria :- they are single, small prokaryotic cells.
Eg:- Cocci, Bacilli, Vibrio, Spirochetes etc.

III According to their Flagella
1. Monotrichous :- bacteria with single polar flagellum. Eg:-
Vibrio cholerae
2. Amphitrichous :- bacteria with single polar flagella or tuft of
flagella at both poles. Eg:- Alcaligenes faecalis
3. Lophotrichous :- Bacteria with tuft of flagella at one pole. Eg:-
Spirilla
4. Peritrichous :- Bacteria with flagella all around the cell. Eg:-
Salmonella typhi.

IV According to their Staining Reaction
A) GRAM STAIN
1. GRAM POSITIVE BACTERIA:- The bacteria which appear violet after
gram staining. Eg:- Staphylococcus aureus
2. GRAM NEGATIVE BACTERIA :- The bacteria which appear pink or red
after gram staining. Eg:- E.coli
) ACID-FAST or ZIEHL-NEELSEN STAIN
1. ACID FAST ORGANISM :- The bacteria which appear pink in colour after
staining. Eg:- Mycobacterium group
2. NON ACID FAST ORGANISM :- The bacteria which appear blue in
colour after staining. Eg:- E.coli

V Based on Biochemical Reaction
Basic classification:-
1. AEROBIC & FACULTATIVE AEROBIC BACTERIA
2. ANAEROBIC BACTERIA
Aerobic & facultative aerobic bacteria are further classified into:-
Cocci : Gram Positive
Catalase positive : Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus saprophyticus
Catalase negative : Staphylococcus pyogenes, Staphylococcus pneumoniae,
Viridans group Streptococci
Cocci : Gram Negative :- Neisseria gonorrhoeae, Neisseria meningitidis,
Moraxella catarrhalis

Bacilli : Gram Positive : Bacillus anthracis, Bacillus cereus,
Corynebacterium, Mycobacterium
Bacilli : Gram Negative
a) Enterobacteriaceae :- E.coli, Klebsiella, Salmonella, Shigella
b) Non-Enterobacteriaceae - Fermentive Bacilli :- Aeromonas,
Vibrio, Pasteurella
c) Non-Enterobacteriaceae -Non Fermentive Bacilli :-
Pseudomonas, Moraxella, Acineobacter, Eikenella
Coccobacilli : Gram Negative :- Actinobacillus, Bartonella,
Brucella, Haemophilus, Chlamydia

Mycoplasma :- Mycoplasma hominis, Mycoplasma pneumonia,
Ureaplasma urealyticum
Treponema :- Treponema pallidium, Leptospira, Borelia
burgdorferi, Borelia recurrentis

3. Classification
on the basis of
Nutritional
Requirements

1. PHOTOAUTOTROPHS :- Use light energy to produce food.
Eg:- photosynthetic microalgae, cyanobacteria
2. CHEMOAUTOTROPHS :- Use chemical energy to produce
food. Eg:- sulphur bacteria, iron bacteria, nitrifying bacteria
3. PHOTOHETEROTROPHS :- These are microbes that use light
energy in obtainment of their food from external environment.
Eg:- purple non-sulphur bacteria
4. CHEMOHETEROTROPHS :- These are microbes that use
chemical energy in obtainment of their food from external
environment. Eg:- Saprophytic bacteria, symbiotic bacteria,
microfungi, protozoa, colorless microalgae.

4. Classification
on the basis of
Oxygen
Requirements

1. OBLIGATE / TRUE AEROBES :- Grow in presence of Oxygen
only.
2. OBLIGATE / TRUE ANAEROBES :- Grow in absence of Oxygen
only.
3. FACULTATIVE AEROBES :- Grow even in absence of Oxygen.
4. FACULTATIVE ANAEROBES :- Grow even in presence of
Oxygen.

5. Classification
on the basis of
Temperature
Requirements

1. PSYCHROPHILIC / CRYOPHILIC :- Grow at sufficiently low
temperature. Best growth occurs at 0 deg as the minimum,
10-15 deg as optimum & below 20 deg as maximum.
2. MESOPHILIC :- Grow on moderate temperature. Best growth
occurs at 10-15 deg as the minimum, 30-40 deg as optimum
& below 45 deg as maximum.
3. THERMOPHILIC :- Grow at higher a temperature lethal to
many other microbes . Best growth occurs at 45 deg as the
minimum, 50-85 deg as optimum & above 100 deg as
maximum.

6. Classification
on the basis of
Osmotic
Condition
Requirements

1. OSMOPHOBIC :- These microbes die if subjected to substrate
of high osmotic concentrations.
2. OSMOPHILIC :- These microbes grow best on substrates of
high osmotic concentrations./
3. HALOPHILIC :- These microbes preferably grow in high
osmotic concentrations produced by dissolved salts.
4. OSMODURIC :- These microbes grow normally on substrates
of moderate osmotic concentrations & are resistant to wide
osmotic changes in the substrate on which they grow.

7.
Classification
on the basis of
Distribution

1. HYDROSPHERIC / AQUATIC :- These microbes grow in water.
2. LITHOSPHERIC / TERRESTRIAL :- These microbes grow on or
in soil or rocky substances.
3. ATMOSPHERIC / AERIAL :-These microbes are found in
atmosphere.

8. Classification
on the basis of
Economical
Importance

1. USEFUL MICROBES
2. HARMFUL MICROBES

9. Classification
on the basis of
Host Resistance
or Pathogenicity

1. PATHOGENS :- These microbes causes disease in the
host. Eg:- Vibrio cholerae
2. NON-PATHOGENS :- These microbes do not cause any
disease in the host. Eg:- Lactobacilli.

SIZE OF BACTERIA
● Size of bacterial cell is 1/10th size of eukaryotic cells.
● 0.5-5.0 micrometers in length
● Size of medically important bacteria :- 0.2-1.5 micrometers in
diameter & 3-5 micrometers in length
● Smallest bacteria :- Mycoplasma (0.3 micrometers)

SHAPE OF BACTERIA /
MORPHOLOGICAL TYPES
1. COCCI
a) Micrococci
b) Diplococci
c) Streptococci
d) Staphylococci
e) Tetrad
f) Sarcina (Octad)
2. BACILLI
a) Micro Bacilli
b) Diplobacilli
c) Streptobacilli
d) Palisade arrangement (bacilli
lined side by side like
matchsticks & at angles to
one another. Eg:-
Corynebacterium) diphtheria
e) Comma shaped
f) Spirillum - bacilli

3. COCCOBACILLI
4. SPIROCHETES

STRUCTURE OF A TYPICAL
BACTERIAL CELL

Structures External to Cell Wall
1. FLAGELLA
● They are long, hollow, helical filamentous organs of
locomotion.
● Arise from cytoplasmic membrane & pass out through cell
wall.
● Size:- 10-20 nm in diameter & 3-20 micrometer in length.
● Found in both gram positive & negative bacteria.

3 Parts of flagellum are :-
1. Basal body :- It represents a border & thicker basal region of
flagellum attached with plasma membrane.
2. Hook :- It represents a border & thicker basal region of
flagellum & passes out through the cell wall.
3. Filament :- It is the thinner, elongated & terminal part of the
flagellum.
● The basal body comprises of a rod & two or more sets of
encircling innermost (M & S) & outer most (P & L) rings.
● Gram negative bacteria consists of all these 4 rings.

● Flagella is attached to cell membrane through M ring.
● Ring S is located just above the cell membrane.
● Flagella is attached to peptidoglycan & outer
lipopolysaccharide membranes through P & L rings
respectively.
● Gram positive bacteria lacks outermost ring (P & L).
● Flagellum consists of protein called FLAGELLIN.

2. PILI (FIMBRIAE)
● They are hollow, non-helical, filamentous appendages that
are thinner, shorter & more numerous than flagella.
● It occurs in both flagellated & non flagellated bacteria.
● Each bacteria possess 100-200 peritrichously borne pili.
● They are 1-1.5 micrometers in length & 4-8 nm in diameter.
● They are made up of protein PILIN.

FUNCTIONS
1. F-pilus (fertility pilus) or sex pilus which involved in bacterial
conjugation helps in transfer of genetic material between
bacterial cells.
2. Some pili allow the pathogenic bacteria to attach to
epithelial cells lining the respiratory, intestinal or
genitourinary tract & thus establish an infection. Eg:-
Neisseria gonorrhoeae

3. CAPSULE / SLIME LAYER
● The high molecular substances produced by bacteria during
their active growth gets collected on surface of cells & forms a
gelatinous covering around these cells & when these covering
does not form a persistent layer but is present more diffusely
forming a loose mass or matrix around the bacterial cell, it is
called SLIME LAYER or BIOFILM.
● When this gelatinous layer forms a well developed persistent
layer, it is called CAPSULE.

● Bacterial capsules are composed of either polysaccharide
(Eg:- Klebsiella pneumonia) or polypeptide (Eg:- Bacillus
anthracis)
FUNCTIONS
● Provides protection against temporary drying by binding
water molecules.
● It may block attachment of bacteriophages

● It may inhibit engulfment of pathogenic bacteria by
phagocytes & thugs contribute to virulence of bacteria.
● It may promote attachment of bacteria to smooth surfaces.
Eg:- Streptococcus mutans.
● It may protect bacteria from antibacterial agents such as lytic
enzymes found in nature.
● Bacteria over produces capsular material when overfed with
sugars, become reserves of carbohydrates for subsequent
metabolism.

4. CELL WALL
● It is the rigid structure surrounding the plasma membrane.
● Present in all prokaryotes except Mycoplasma &
Methanoplasma.
● Peptidoglycan (Murein, Muramic acid, Mucopeptide) is the
main constituent of bacterial cell wall.
Peptidoglycan
● It is an insoluble , porous, cross linked polymer of enormous
strength & rigidity.
● It is found only in prokaryotes & founds as a “bag shaped
macromolecule” surrounding the cytoplasmic membrane.

Peptidoglycan consists of 2 parts:-
1. A peptide portion : composed of 4 amino acids (L-alanine, D-
glutamine, either L-lysine or diaminopimelic acid & D-alanine).
2. Glycan or sugar portion : Made up of two alternating units of
N-acetyl glucosamine (NAG) & N-acetyl muramic acid (NAM).
Peptidoglycan is present in gram positive & negative bacteria.

Cell wall of gram positive bacteria
● Cell wall appears as a thick homogeneous layer.
● Consists of peptidoglycan.
● Remaining is made up of proteins, polysaccharides & teichoic
acid.
● Teichoic acid affect the passage of ions thus maintaining low
pH of cell wall so that autolysins don't degrade the cell wall.

Cell wall of gram negative bacteria
● Cell wall is more complex than gram positive.
● Contains an outer membrane that surrounds a thin underlying
layer of peptidoglycan.
● Rich in lipids.
● Outer membrane is attached to peptidoglycan by means of “
Braun’s lipoprotein”.
● This membrane is bilayered mainly of phospholipids, proteins
& lipopolysaccharides (LPS).
● LPS has toxic properties & known as Endotoxins which is
released only after lysis of cell wall.

● Outer membrane serves as impermeable barrier to prevent
escape of important enzymes.
● It also prevents the entry of various external chemical &
enzymes that could damage the cell wall.
● Its impermeable to larger molecules (protein) but permeable
to smaller molecules (amino acids, peptides) to pass across
by means of channels in special proteins called “porins”.

Functions of cell wall
● The cell wall being rigid structure, gives shape to the cell.
● Cell wall prevents the cell from expanding & bursting,
when osmotic pressures are exerted on it.

Structures internal to cell wall
1. CYTOPLASMIC MEMBRANE / PLASMA MEMBRANE
● Present just beneath the cell wall
● Thickness : 7.5 nm
● Composed of phospholipids & proteins.
● Ultra structure of cell membrane has been explained by Fluid
Mosaic Model proposed by Singer & Nicolson in 1974.
● The phospholipids form a bilayer in which most of the
proteins are firmly held called INTEGRAL or INTRINSIC
PROTEINS which can be removed only by destruction of
membrane.

● Other membranes called PERIPHERAL or EXTRINSIC
PROTEINS are loosely attached & can be removed by mild
treatments such as osmotic shock.

Functions of plasma membrane
● It act as a differentially permeable barrier, regulating the flow
of materials in & out of the cell. Transport proteins in the
membrane facilitates the passage of small molecules
(nutrients & wastes) across the membrane.
● It contains various enzymes involved in respiratory
metabolism & in synthesis of cell wall, septum formation,
membrane synthesis & DNA replication.
● It is the site of proton motive force.
● It contains specific attachment sites for chromosomes & for
plasmid that plays vital role at the time of cell division.

1. Difference
between flagella &
fimbriae.
2. Difference
between gram
positive &
negative bacterial
cell wall.

2. CYTOPLASM
It is a homogeneous aqueous solution bounded by cell
membrane & is divided into 3 distinct areas.
● Cytoplasmic area :- Granular in appearance & rich in
ribosomes in which proteins are synthesized.
● Chromatin area :- Rich in DNA
● Fluid portion :- Consisting of dissolved substance ( cell
solutes, metabolites)
● It lacks endoplasmic reticulum, golgi apparatus, mitochondria
& a true bound nucleus.

3. INCLUSIONS
● Present in prokaryotic cells
● Theory are distinct granules that may occupy a substantial
part of the cytoplasm.

4. MESOSOMES
● They are characteristic infoldings in the form of system of
convoluted tubules & vesicles present especially in gram +
bacteria.
● On the basis of their location they may be:-
1. Central - Central mesosomes penetrate deeply into the
cytoplasm & are located near the middle of cell & appear to
be attached with nuclear membrane. Involved in DNA
replication & septa formation.
2. Peripheral - They do not penetrate into cytoplasm & are
located near periphery. They are involved in export of
exocellular enzymes like penicillinase.

5. NUCLEAR MATERIAL
● Does not contain membrane bound nucleus or mitotic
apparatus.
● Known as Nucleoid, the chromatin body, the nuclear
equivalent & bacterial chromosome.
● Its a large circular molecule of DNA , more or less free in
cytoplasm although coiled & super coiled anchored by
proteins.
● The total DNA content of a prokaryote is referred to as Cell
Genome.
● The DNA can be visible under light microscope by Feulgen
staining which is specific to DNA.

6. ENDOSPORES
● Spores are highly resistant, metabolically dormant structures
produced by certain bacteria during their unfavourable
environmental conditions.
● Since these spores are formed within the parent cell, they are
called Endospores.
● Endospores are thick walled, highly refractile bodies that are
usually produced by cells growing rich in culture media when
approaching the end of active growth.
● Eg:- Bacillus ( B. anthracis, B. subtilis), Clostridium (Cl. tetani,
Cl. botulinum), Sporosarcina, Thermoactinomyces

Formation of spore (Sporulation)

● Resistance of spore :- endospores are resistant to ordinary
boiling, heating & disinfectants. They can withstand boiling
upto 3 hrs, dry heat at 150 deg for 1 hr. They can be destroyed
by autoclaving at 121 deg C for 15-20 minutes.
● Germination of spore :- The conversion of spore into
vegetative cell under favourable environmental conditions is
called germination & it may occur less than 2 hrs.

Germination of bacterial spore
3 stages:-
1. ACTIVATION :- Activated by heat (60 deg C for 1 hr), low pH
(acidic), abrasion that can damage the spore coat.
2. INITIATION :-Binding of effector substances to spore coat,
which activates autolysins that destroy peptidoglycan of
cortex, allowing uptake of water & release of calcium
dipicolinate.
3. OUTGROWTH :- With disintegration of cortex & swelling of
spore, a single germ cell emerges after breaking open the
spore coat. The new vegetative cell contains spore
protoplasts which is followed by a period of biosynthesis
producing an outgrowth that transfers to new vegetative cell.

C) Growth &
Nutrition of
Microbes

Bacterial growth involves:-
● Increase in cell mass
● Duplication of DNA
● Synthesis of new cell wall & cellular membrane
● Cell division by BINARY FISSION

Nutritional types of bacteria
On the basis of Carbon & Energy sources for growth, bacteria are
classified into 2 groups :-
1) Autotrophic bacteria
● Photoautotrophic bacteria
● Chemoautotrophs or Chemosynthetic bacteria
1) Heterotrophic bacteria
● Photoheterotrophs
● Chemoheterotrophs

Physical / Environmental Factors Affecting
Growth Of Bacteria
1. TEMPERATURE
● Psychrophiles / Cryophiles
a. Obligate / Strict Psychrophiles :- can't grow above 20-22 deg
C of temperature & dies at room temperature.
b. Facultative Psychrophiles :- grow at 0 deg C, but growth is
best at a range of 20-30 deg C.

● Mesophiles
a. Thermotolerants / Thermoduric :- they can withstand with
high temperature but can’t multiply.
b. Psychro / Cryotolerants :- also called psychroduric or
cryoduric. They can survive at very low temperature but don’t
grow & multiply.
● Thermophiles
a. Obligate thermophiles :- they grow only at temperature above
50 deg C, & can’t grow in mesophilic range.
b. Facultative thermophiles :- they can grow both at high
temperature & in mesophilic range. (37-50 deg C).

2. OXYGEN
1. Aerobes
2. Anaerobes
● Obligate aerobes
● Obligate anaerobes
● Facultative aerobes
● Facultative anaerobes
Capnophilic :- these are organisms which can grow best in an
atmosphere of 5-10 % of carbon dioxide.
Microaerophilic :- they require low levels of oxygen but can’t
tolerate the level of oxygen in the atmosphere.

3. HYDROGEN ION CONCENTRATION (pH)
● Acidophiles :- microbes which grow at an optimum pH well
below neutrality (7.0).
● Neutrophiles :- Microbes which grow best at neutral pH.
● Alkaliphiles :- Microbes that grow best under alkaline
conditions.

4. OSMOTIC PRESSURE
● Osmophobic
● Osmophilic
● Halophilic
● Osmoduric
5. LIGHT
Bacteria except phototrophs grow in darkness.

6. WATER
● Water activity of pure water is 1.0
● Bacteria live over a range of water activity from 1.0 - 0.7
7. CARBON DIOXIDE

BACTERIAL GROWTH CURVE
A characteristic growth curve obtained in batch culture ( culture
in which no fresh nutrients are added) consists of four phases.
1. LAG PHASE
2. LOG / EXPONENTIAL PHASE
3. STATIONARY PHASE
4. DEATH / DECLINE PHASE

GROWTH CURVE OF BACTERIA IN BATCH CULTURE

1. LAG PHASE
● Bacteria adapts themselves to growth conditions.
● Phase of intense metabolic activity (prepare for reproduction,
DNA synthesis, cell division)
● Increase in size (volume) of bacterial cell but no increase in
cell number.
● Length of this bacteria depends upon the bacteria & quality of
cell culture media.

2. LOG / EXPONENTIAL PHASE
● Bacteria multiply in its maximum rate & their number
increases exponentially or by geometric progression.
● Plotting the natural logarithm of cell number against time
produces a straight line.
● Duration of this phase is limited since the bacteria is growing
in a constant medium of batch culture.

3. STATIONARY PHASE
● Growth rate slows down as a result of nutrient depletion &
accumulation of inhibitory end (toxic) products.
● Growth rate becomes equal to death rate rate.

4. DEATH / DECLINE PHASE
● Death rate exceeds reproduction rate & thus no. of viable
bacterial cells start declining.
● Entire bacterial population dies after viable period & culture
becomes sterile.

Composition of Blood
The two main constituents of blood are liquid plasma or blood
serum & the various types of cells which floats in it.
Composition of blood serum
● Water : Main constituent. 90% of blood is water.
● Dissolved gases : Oxygen, CO2, Nitrogen
● Dissolved solids : plasma proteins like fibrinogen, globulins,
albumins, antibodies, enzymes, hormones & waste materials.
Inorganic substances like Fe, Ca, Mg, Na, K, Chlorides,
phosphates, carbonates, bicarbonates, sulphates.

● Nutrients : fatty acids, glucose, amino acids, cholesterol &
fats.
● Excretory materials : Urea, Ammonia, Uric acid & Creatinine
● Blood corpuscles (formed elements) : RBC, WBC,
Thrombocytes or Platelets.

Functions of Blood
● Transport of nutrients
● Transport of excretory products
● Transport of respiratory gases
● Transport of hormones
● Protection against infection
● Clotting of blood
● Maintenance of temperature
● Regulation of pH & water balance
● Maintenance of physiological state of body
● Transport of organic & inorganic substances

Disorders of Blood
● Anemia
● Hemophilia
● Polycythemia
● Leukemia

Body Fluids
● They are dilute watery solutions containing dissolved
chemicals that are found inside cells (Intracellular fluid or ICF)
as well as surrounding them (Extracellular fluid or ECF).
● The ECF that fills the narrow spaces between cells of tissues
is called Interstitial fluid.
● ECF within blood vessels - Plasma
● ECF within lymphatic vessels - Lymph

● ECF in & around brain & spinal cord - Cerebrospinal fluid(CSF)
● ECF within joints - Synovial fluid
● ECF within eyes - Aqueous humour & Vitreous body
● The proper functioning of the body cells depends on well
regulation of composition of interstitial fluid often called
internal environment.
● Serous fluids - Pleural, Pericardial & Peritoneal fluids.

E) Laboratory
Methods For
Identification Of
Microbes

Steps for isolation & identification of pathogenic
bacteria
● Collection of specimen
● Direct microscope
examination
● Dry mount lamination
(preparation of smear,
fixing of smear & staining)
● Culture
● Observation of microbes
● Motility test
● Biochemical reactions
● Serological diagnosis
● Animal pathogenicity
● Antibiotic sensitivity test
● Phage typing

Specimen Collection
Precautions in specimen collection
● Specimen should be collected under aseptic technique.
● Specimen should contain only those organisms from the site
where it was collected.
● Avoid contaminating discharge or ulcer material with skin
commensals.
● Specimen should be collected in dry sterile, leak proof
container free from all traces of disinfectants.

● Every specimen should accompanied by request for which
include the information like patient’s name, date & time of
collection, ward details if it is collected in hospital,
investigation required, clinical notes.
● High risks specimens like HIV, Hepatitis B must be handled
with extra care & should be mentioned as HIGH RISK
SPECIMEN.

Sl.No: Specimen Container Patient
preparation
Transport
to lab
Storage
1 Urine
(midstream)
Sterile screw cap
with wide mouthed
bottle
Male : clean glans
& collect midstream
urine
Female : clean area
around urethral
opening & collect
midstream urine
Within 2 hrs
at 2-8 deg C
24 hrs at 2-8 deg C
2 Stool Clean or leak proof
screw cap with wide
mouthed bottle
No specific
preparation
Within 24
hrs at 2-8
deg C
72 hrs at 2-8 deg C
3 Rectal
swab
Swab placed in
enteric transport
medium
Insert 2.5 cm past
anal sphincter
Sterile
screw cap
with wide
mouthed
bottle
72 hrs at 2-8 deg C

4 Blood &
bone
marrow
Vacutainer tube with
polyanethol sulphate
Draw blood
during sterile
episode &
collect two sets
from left &
right arm.
Within 2 hrs at
room
temperature
Should be
incubated at 37
deg C
5 Sputum Sterile screw cap with wide
mouthed bottle
Collected after
deep coughing
Within 2 hrs at
room
temperature
24 hrs at room
temperature
6 CSF Sterile screw cap tube or
bottle
Disinfect skin
before
aspirating
Keep
immediately at
temperature
6 hours at 37
deg C
7 Body fluids Sterile screw or anaerobic
transport
Disinfect skin
before
aspirating
Keep
immediately at
room
temperature
Prepare plate
as soon as
received
8 Skin
scrapings
Anaerobic transporter Disinfect skin,
do not allow to
dry
Within 24 hrs at
room
temperature
24 hrs at room
temperature

Direct microscopic examination
● First step in laboratory diagnosis of pathogenic organism.
● It is useful when organism cannot be cultured i the lab.this
may be done with a wet unstained specimen or with a dry
fixed stained specimen.
● Two methods are :-
1. Wet mount method
2. Hanging drop method

1. WET MOUNT METHOD
● It is done without fixing the specimen.
● It helps in the identification of motile organisms, fragile
structures & sporulating bodies.
● Stained smears are also used to study staining characters.
● Specimens that are used are:- urine sediment, fecal
suspension, skin scrapings.
METHOD
● Transfer a loopful of the specimen to a microscopic slide.
● Put a drop of saline & place the cover slip.
● Avoid air bubbles.
● Examine slide under microscope with low power objective
(100 X magnification).

2. HANGING DROP METHOD
● It is a method in which a drop of bacterial suspension is
enclosed in an airtight chamber prepared in a special
depression / concavity slide.
● It is used to examine the motility of bacteria in a given
culture.
● Most frequently used to examine the stool specimen of
suspected Cholera patients.

● Observe the slide through the eyepiece.
● Focus the edge of the drop carefully. The cells will look either
dark or slightly green, very small rods or spheres.
● Observation :- BROWNIAN movement should be visible on
slide indicating motility of given organism.

STAINING TECHNIQUES
Stains are dyes or reagents used for differential colouring of
microbes to observe their structures with much clarity under
microscope. They may be either:-
● ACIDIC DYES
● BASIC DYES
● NEUTRAL STAINS
DRY MOUNT LAMINATION :- It is the examination of the
stained smear. Morphological features are studied better
through this method.

Purpose
of
staining
1. To view the
organism with
much clarity.
2. To determine
peculiar
structures.
3. To differentiate
one organism
from another.

Types of Stains
DIRECT /
GENERAL
STAIN
INDIRECT
STAIN
SELECTIVE
STAINS
DIFFERENTIAL
STAINS
They are able
to stain
bacteria with
exceptions of
bacterial spore
& bacteria with
waxy coating
on their cell
wall.
They stain
only the
background.
Eg:- Nigrosin
or India ink
They are
used to stain
particular
part of the
organism like
spores,
flagella ,
nuclei etc.
They differentiate
two groups of
bacteria in a
mixture. (gram
positive &
negative )

Staining Techniques / Procedures
There are two staining procedures for light microscopy:-
a. SIMPLE STAINING
b. DIFFERENTIAL STAINING
SIMPLE STAINING
● Single staining reagent is used (methylene blue, crystal
violets, carbol fuchsin).
● All cells & structures stain in the same manner.
● Its of two types. (Positive staining & Negative staining)

i) Positive staining :- Here the stain (methylene blue) is basic
(cationic) & attaches to the surface of the object that are
negatively charged.
ii) Negative staining :- The stain(India ink, Nigrosin) is acidic
(anionic). Bacterial cell appears transparent & only background is
stained.

Differential staining
● More than one staining reagents are used.
● Specific objects exhibit different staining reactions which are
distinguishable.
● Main two differential staining procedures are Gram staging &
Acid fast staining.

Simple Staining Of Bacteria
Principles
1. Bacterial smear is stained with single dye or staining reagent.
2. Purpose is to demonstrate cell size, shape & arrangement of
bacterial cells.
3. Bacterial cell surface is negatively charged, so it tends to bind
strongly to the cationic chromogen of basic dyes.
Reagent :- Loeffler’s methylene blue solution

Procedure ;-
● Prepare a smear of a given culture by spreading a thin film on
a clean glass slide.
● Dry it by waving in air & then heat fix by passing the slide 2-3
times through the flame wit the smeared side facing upwards.
● Stain the smear by flooding it with one of the staining
solutions & allowing it to remain covered with the stain for the
time designated below;-
METHYLENE BLUE (blue) - 1 minute
CRYSTAL VIOLET (purple) - 30 seconds
CARBOL FUCHSIN (red) - 20 seconds

● Wash the slide gently with running water to remove excess
stain.
● Air dry the slides or blot dry with blotting paper.
● Apply oil directly to the smear & focus the smear under low
power objective & then under oil immersion objective.
Results
The bacteria will appear blue or red depending upon the stain
used with characteristic morphology.

Gram Staining Of Bcateria
● The technique was developed by Danish physician, Dr. Hanes
Christian Gram (1884).
● Gram staining determines gross morphology, differentiate
bacteria into two major distinct groups, ie; Gram Positive &
Negative.
● It involves 6 basic steps;- Smear preparation, heat fixing of
smear, staining with crystal violet (primary stain), use of
Iodine/Lugol’s iodine (mordant), treatment with acetone
alcohol mixture (decolourizing agent) & use of Safranin
(counterstain).

Principles
● In gram negative bacteria the cell wall is thin, multilayered
containing high lipids which are readily dissolved by alcohol,
resulting in pore formation of the cell wall facilitating the
leakage of the crystal violet-iodine complex & resulting in
discoloration of gram negative bacteria & takes up safranin &
appears red.

● Cell walls of grampostive bacteria is thick, composed mainly
of proteins & cross linked mucopeptides.
● On the application of decolourizing agent, dehydration is
caused resulting in closure of pores of cell wall thereby
retaining the primary stain colour & appears as blue or purple.
Reagents
1. Crystal violet stain
2. Gram iodine solution
3. Ethyl alcohol (95%) or alcohol-acetone (1:1) solution
4. Safranin solution

Procedure
● Make smear of a given culture on a clear glass slide.
● Air dry the smear & heat fix it.
● Cover the smear completely with crystal violet & leave the
stain on the slide for 1 minute.
● Wash the slide gently in distilled water or tap water.
● Flood the smear with Gram or Lugol’s iodine solution & wait
for 1 minute.
● Wash with tap water gently & drain carefully.
● Add ethyl alcohol or alcohol-acetone solution drop by drop,
until the smears becomes free from any colorization.

● Wash the slide gently under tap water & drain.
● Now counterstain with safranin & wait for 30 seconds.
● Wash again & blot dry with blotting paper or simply air dry &
observe under oil immersion objective.
Results
Bacteria that appear BLUE/VIOLET/PURPLE are assigned as
Gram Positive & those appearing as RED/PINK as Gram
Negative.

ACID FAST STAINING OF BACTERIA
● Technique was developed by Paul Ehrlich (1882) & was
modified by Ziehl-Neelsen.
● Hence known as Ziehl- Neelsen stain.
● It's a differential stain & is used to identify mainly the
Mycobacterium especially Mycobacterium tuberculosis &
Mycobacterium leprae.
● Bacteria are classified into :- Acid fast & Non acid fast
organisms.

Reagents used:-
1. Carbol fuchsin solution (Primary stain)
2. Acid-alcohol solution (3% HCl in alcohol)
3. Methylene blue (counter stain)
Results:-
Acid fast organisms will appease bright red on a blue
background while non acid fast organism will appear dark blue in
colour.

CULTURE MEDIA
It is a solid or liquid preparation for cultivating microbes by
letting them to reproduce in a predetermined laboratory
conditions (giving adequate nutrients & meeting all growth
requirements).

Classification of Culture Media
A) Classification based on nature of ingredients
1. The media which require living cells or tissues which are
parasitized by microbes to be cultured. Eg: Rickettsiae
2. The media that doesn't require living cells or tissues.
This media can be divided into two:-
i) Synthetic media /Defined media
ii) Non-synthetic media /Complex media

Synthetic media /Defined media
● These are the media in which only pure chemicals in definite
concentrations are used.
● These are useful for nutritional & metabolic studies.
Non-synthetic media / Complex media
● These are the media in which exact chemical compositions of
each of the constituent is not known with certainty.

B) Classification based on consistency (physical state) of
medium
1. Liquid Media :- These are used in liquid form. Eg:- Nutrient
broth, Brucella broth, Nitrate broth.
2. Semisolid Media :- These media contain 0.5% or less of agar
imparting custard consistency to the medium. Eg:- Cysteine
Trypticase agar medium.

3. Liquefiable Solid Media :- This media is prepared by adding
suitable amount of gelatin or agar to the liquid medium to
remain solid when cool but become liquid when warm or vice
versa. Hence its also called Solid reversible to Liquid media.
Eg:- Salmonella - Shigella Agar, Nutrient Gelatin Medium, Blue-
Esculin Agar Medium.
4. Solid Media :- These media always remain olid. Eg:- Nutrient
Agar Medium, Bile Salt Agar Medium.

C) Classification based on application or
function
1. Cultivation media
2. Storage media
3. Enrichment media
4. Differential media
5. Selective & selective
differential media
6. Assay media
7. Maintenance media
8. Transport media
9. Enumeration Media
10. Anaerobic media
11. Sugar media

1. CULTIVATION MEDIA :- Used for general cultivation of
bacteria. Eg:- Nutrient Broth/Agar
2. STORAGE MEDIA :- Bacterial cultures are preserved & stored
for longer periods of time by Lyophilization (deep - dry
method).
3. ENRICHMENT MEDIA :- These are the media in which
nutritional environment is adjusted in such a way that the
growth of certain bacterial types is enhanced selectively
within a mixed population. Eg:- GN Broth, Salenite F, Loefller’s
serum slope.

4. DIFFERENTIAL / INDICATOR MEDIA :- These are the media
which differentiate two organisms by their characteristic
reactions towards the medium like haemolysis or biochemical
changes.
Eg:- Blood -Agar medium which differentiates haemolytic species
from non-haemolytic species, MacConkey Agar or EMB Agar
differentiates lactose fermenters from non lactose fermenters.

5. SELECTIVE & SELECTIVE DIFFERENTIAL MEDIA
i) Selective media :- this media allows the growth of selective
groups of organisms while inhibiting others by various inhibitors
like antibiotics, dyes, bile salts.
Eg: MacConkey Agar for isolation of E.coli, Deoxycholate citrate
agar for Salmonella & Shigella, Lowenstein-Jensen medium for
M.tuberculosis.

ii) Selective differential media :- This media allows the selective
growth of certain organisms & also differentiation of various
groups of organisms on the basis of their biochemical reactions
in a particular media.
Eg:- Blood Agar for isolation of bacterial pathogen (S.pyogenes &
S.pneumoniae)

6. ASSAY MEDIA :- Also called Media for Special Purpose. This
has got profound influence on the bacterial cells with respect to
formation of enzymes, toxins, antibiotics & other bioactive
products. Its used to test the effectiveness of antimicrobial drugs
& to assess the effect of disinfectants, antiseptics, cosmetics &
preservatives on the growth of microbes. Eg:- Pyridoxine deficient
growth medium for Streptococcus faecalis.
7. MAINTENANCE MEDIA :- its used to maintain physiological
characteristics & hence viability of bacterial cultures.

8. ENUMERATION MEDIA :- They are used primarily to count the
number of microbes in milk, water, food & other samples & find
their broad application in concerned industries.
9. ANAEROBIC MEDIA :- These media are used for the growth of
anaerobes. Eg:- Robertson’s cooked meat medium or
Thioglycolate broth for isolation of Clostridium species.
10. SUGAR MEDIA :- This media carries out sugar fermentation,
which is important for the identification of most of the microbes.

11. TRANSPORT MEDIA :- These media inhibit self destruction & multiplication of
microbes & hence maintains the specimen in a “status quo”. Used for Neisseria
species.
Different transport media for transporting different specimens
a) Cary-Blair & Amies transport media :- used for transporting fecal material
on rectal swab & are effective in preserving Salmonella, Shigella & Vibrio.
b) Stuart transport media :- used for CSF & cervical specimens. Preserves both
Neisseria species & Trichomonas vaginalis for 18-24 hrs.

c) Venkataraman - Ramakrishnan (V.R) media :- used to transport
stool specimen of suspected cholera patient.
d) Bile Peptone transport medium :- used to transport stool
specimen of suspected cholera patient. Useful medium for field
works when more delay is expected.
e) Brucella Broth :- Used to transport biopsy specimen from
duodenal ulcer patients. The medium preserves the etiological
agent under refrigeration for 24-48 hrs.

General Characteristics of microbes.pptx

General Characteristics of microbes.pptx

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