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Bacteriology
Bacteriology
Definition:
• A branch of microbiology dealing with the
identification, study, and cultivation of bacteria and with
their applications in medicine, agriculture, industry, and
biotechnology
OR
• The branch of science concerned with the study of
bacteria
• Bacteria: Microscopic living organisms normally are
unicellular with no distinct nuclei , reproduce by fission
or forming spore found everywhere and can exist
independently(Free living) or dependently(parasitic) can
be dangerous as cause disease or beneficial as process
of fermentation(Wine production)
Morphology and Classification
• Bacteria are Prokaryotes as they lack nuclei and other organelles
which characterize the true cell(Eukaryotes ie : Algae ,Fungi ,
Animal and plant cells).
• Size: Bacteria cells range from 0.2µm-3 µm in diameter
•
Compared to RBC which is 8µm in diameter
• Major classes: Are based on staining properties some are GRAM
stains other are ACID - FAST stains
• Those staining Gram positive show Purple colour
• Gram negative show Red Colour
• Gram negative have thin cell wall
• However Gram resistant bacteria stain poorly or no stain at all. ie:
Mycobacterium
Morphology and Classification
• Ultrastructure:
• Generally Gram +ve and –ve have similar internal structures
but dissimilar cell envelopes
A. Internal Structure:
 Nucleoid: Central region not surrounded by nuclear
membrane but contains DNA
 Ribosome: Differ in size and shape with eukaryotic ribosomemajor target for antibiotic action
 Plasmids: Small and circular fragments of extrachromosomal
DNA may carry antibiotic resistance genes
Morphology and Classification
• B. Cell Envelope(Cytoplasmic membrane+Cell
wall)
Cytoplasmic(cell,plasma)membrane Structurally similar
to eukaryotic membrane but lacks sterols.
Associated with enzymes and other proteins that carry
out energy production , transportation of nutrients and
synthesis of structural components
Cell wall of Gram +ve bacteria:
Has thick peptidoglycan layer forming meshlike
exoskeleton -This is porous for Antibiotics
Morphology and Classification cont:
Teichoic and lipoteichoic acids : Associated with
peptidoglycan are antigenic may promote adhesion to host
tissue.
Cell wall of Gram +ve bacteria has:
 Peptidoglycan layer: adjacent to cytoplasmic membrane
 Outer membrane :maintains bacterial structure acts as
permeability barrier protects against adverse environment
conditions
 Periplasmic space: Is btn outer membrane and cytoplasmic
membrane has degradative enzymes and nutrient binding
proteins
Morphology and Classification cont:
Lipoproteins: Covalently linked to peptidoglycan
layer are inserted into the outer membrane
connecting the two structures.
Cell wall of Mycobacterium:
Structure of peptidoglycan differs slightly from other
bacteria
Waxlike lipid coat containing mycolic acid surrounds the
peptidoglycan –like layer(This is responsible with
virulence and antiphagocytic activity of mycobacteria).
Mycobacteria and other mycolic acid producing bacteria
can be identified acid-fast stains.
Morphology and Classification cont:
• Other external structures:
Bacterial capsule: Loose layers of polysacharide or
protein surrounds cell wall of some bacteria
Pili(fimbriae) :Short , hairlike appendages contains
protein subunits anchored in plasma membrane of some
bacteria – promotes adhesion of bacteria to host cells
Fagella long, ropelike appendages –useful in motility
Difference btn Prokaryotic and Eukaryotic cells
Charecteristics
Size:
Cell wall:
Prokaryotic cells
0.5 - 3µm
Plasma Membrane:
Has no sterols except
Mycoplasma
Has protein,peptidoglycan Only Plant and fungal
& lipids
differ
Nuclear membrane: Absent
Single,Circular DNA
Genome:
molecule in nucleoid
Organelles:
Cell division:
Eukaryotic cells
>5µm
Absent
Binary fission
Has sterols
Present
Multiple,linear DNA
molecules in
nucleus
Present
Mitosis and
Meiosis
Comparison of Gram +ve and Gram –ve Bacteria
Characteristics
Gram +ve
Gram-ve
Absent
Thick
Absent
Present
P/nt in some sp
Present
Thin
Present
Absent
P/nt in some spp
Very Sensitive
Very permiable
Some species
Some spp.
Largely resistant
Structure:
•Outer membrane
•Peptidogycan layer
•Lipopolysaccharides
•Teichoic Acids
•Capsule,pili,flagella
Functional :
•Lysozyme sensitivity
•Antibiotic permiable
•Sporulation
•Exotoxin production
Impermeable to many
None
Some spp.
Bacterial Growth
• Bacterial growth curve:
When placed in a new media show a pattern of growth and
multiplication in four phases(Lag,Long
(Exponential),Stationary and Death)
• Lang phase: Bacteria adapt themselves to growth
condition,they mature but unable to divide,synthesis of
RNA,enzymes and other molecules takes place.
• Long phase(Exponential): There is cell doubling ,if
growth not limited doubling continues at constant rate
growth condition determine growth.
Bacterial Growth Cont..
• Stationary phase: Growth rate and death rate are equal,
The number of new cells created is limited by the growth
factor
• Due to a growth-limiting factor such as the depletion of
an essential nutrient, and/or the formation of an
inhibitory product such as an organic acid.
• Death(Decline) phase: Bacteria die this can be to lack of
nutrients , environmental factors ie: high or low
temperature below the tolerance band for the species,
or other injurious conditions.
Growth Requirements
• Different bacteria show variation in the condition
conducive to their growth.
Temperature: Low temperatures tend to reduce growth
rates which has led to refrigeration hence useful in food
preservation(pathogenic bacteria grow at 35
˚ᶜ-37˚ᶜ(near
normal body temperature)
Acidity: Optimal acidity for bacteria tends to be around pH
6.5 to 7.0 with the exception of acidophiles.
Oxygen requirement: Obligate aerobes require O2 for
growth ie: Mycobacteria tuberculosis,Neisseria etc.
Anaerobes do not require O2 for growth
Growth Requirements
• Obligate anaerobes are killed by O2
ie:Treponema,Actinomyces
• Aerotolerant anaerobes: Can survive in the
presence of O2 but grow best in its absence (ie:
Clostridium spp)
• Facultative anaerobes: Can grow under anaerobic or
aerobic conditions( E.coli,Staphylococcus aureus)
Growth Requirements
Nutrient Requirements
• Undemanding eaters: Can be cultured on simple
media(e.g.E.coli,Salmonella)
• Demanding eaters: Require complex media with
numerous growth factors(e.g Haemophilus,Neisseria)
Cell Division
• Most bacteria divide by binary fission
 Chromosome duplication is initiated at specific sequence
(replication origin) in the DNA
 Synthesis of new membrane and cell wall in the centre
of the cell forms a septum that eventually divides the
cytoplasm into two daughter cells with complete
chromosome.
Bacterial Spores(Endospores)
• Spores are formed by some Gram – positive bacteria
representing a dormant state that is resistant to heat ,
dry and chemicals.
• Spore formation : a variant type of cell division is
induced by depletion of essential nutrients needed for
normal growth-Antibiotics and Toxins are produced
during sporogenesis prior to release of the spore from
the bacterial cell.
• Germination of spores: Cell germinated into vegetative
form starts by demage on the spore coat by trauma ,
water or aging and requires special nutrients once spore
demaged the cell becomes susceptible to agents.
Normal Flora
Microorganisms that normally reside at a given site and
under normal circumstances do not cause disease. , even
beneficial, in their usual sites, and they inhibit the growth
of pathogens, but can cause infection if introduced into
unusual sites.
Non sterile sites in the body(e.g: lumen of GIT, UGT, Mouth ,
Saliva , Pharynx and Skin) harbor certain organisms
Skin: Staphylococcus epidermis,Proprionibacterium acnes
Upper respiratory tract:Staphylococcus aureus,
Staphylococcus pneumoniae,Haemophilus influenzae
Colon:Escherichia coli (E.Coli),Bacteroides fragilis
Normal Flora cont…
Urogenital Flora: Candida spp
Conjunctival Flora: Haemophilus and
Staphylococcus
Oral and Upper Respiratory Tract Flora:Neisseria,
Bordetella, Corynebacterium, and Streptococcus
spp.
Sterilization
• The removal of all microorganisms and other pathogens
from an object or surface by treating it with chemicals or
subjecting it to high heat or radiation.
Uses of Sterilization:
Sterilization for Surgical Procedures: Gloves, aprons,
surgical instruments, syringes etc.
Sterilization in Microbiological works like preparation of
culture media, reagents and equipments where a sterile
condition is to be maintained.
Sterilization
• Classification of Sterilization methods:
Physical and Chemical Agents
Physical Agents:
1. Heat
2. Radiation
3. Filtration
Chemical Agents:
Physical Agents:
• STERILIZATION BY HEAT:
This is cheaper and rapid method of sterilization achieved
by heating above 100˚ᶜ
Types of Heat :
A. Sterilization by moist heat
B. Sterilization by dry heat
Sterilization by Moist Heat:
Acts by denaturation and coagulation of protein,
breakage of DNA strands, and loss of functional integrity
of cell membrane.
Sterilization by Moist Heat:
• Sterilization at 100°C:
Boiling: Boiling at 100°C for 30 minutes in a water bath(
Syringes, rubber goods & surgical instruments)
Steaming: Steam (100°C) more effective than dry heat at
the same temperature(Steam has more penetrating
power, more heat is given up during condensation).
Sterilization Cont..
• Sterilization above 100°C
Autoclaving: Sterilization is by steam under pressure.
temperature higher than 100°C, higher temperature is
obtained by employing a higher pressure .
Autoclave is closed and made air-tight, and water starts
boiling.
Inside pressures increases and now the water boils above
100°C.
At 15 ib per sq. inch pressure, 121°C temperatures is
obtained kept for 15 minutes to kill spores. -like a
pressure cooker.
Sterilization Cont..
Sterilization below 100°C
 Pasteurization:Heating of milk to temperature & for a period
of time to kill pathogens presenting in milk without changing
colour, flavour & nutrities (Mycobacterium bovis, Salmonella
species, E.coli and Brucella species be present in milk present
in milk)-Spore forming are not destroyed.
 Methods of Pasteurization:
(i) Flash Method(Heating is done at 72°C for 15 seconds-short
time method).
(ii) Holding Method(Heating is done between 63°C and 66°C for
30 minutes).
Sterilization Cont..
Inspissation: Stiffening of protein without coagulation as
the temperature is below coagulation temperature.
Sterilization by dry heat:
How it works? (1) Protein denaturation, (2) Oxidative
damage, (3) Toxic effect of elevated electrolyte (in
absence of water).
Dry heat at 160°C (holding temperature for one hour is
required to kill the most resistant spores) unsuitable for
clothing –can be spoiled.
Red Heat(Wire loops used in microbiology laboratory
sterilized by heating to red on bunsen burner or spirit
lamp flame ).
Sterilization Cont..
Flaming: Involves passing instrument without allowing to
turn red-incomplete sterilization-scarpel
Sterilization by Hot Air:
Hot Air Oven (Sterilizer) Most common method used for
sterilization(Glass wares, swab sticks, all-glass syringes,
powder and oily substances) holding temperature of
160°C for one hour is recommended to kill spores.
Sterilization holding time: Is the time for which the
entire load in the autoclave requires to be exposed.
Disinfection
Disinfection: A procedure of treatment that eliminates
many or all pathogenic microorganisms with the
exception of bacterial spores.
Disinfectants: A germicide that inactivates virtually all
recognized pathogenic microorganisms but not
necessarily all microbial forms. They may not be effective
against bacterial spores.
Antiseptics: Mild forms of disinfectants that are used
externally on living tissues to kill microorganisms, e.g. on
the surface of skin and mucous membranes.
Disinfection cont..
• Factors for successful disinfectants:
•
•
•
•
•
•
Type and level of microbial contamination
Concentration of active ingredients
pH of disinfectant
Temperature
Humidity
Duration of contact btn disinfectant and item to be
disinfected
• Pressure of organic matter or soil load
Disinfection cont…
• Chemical Disinfectant Groups:
a. Aldehydes(Formaldehyde,Paraformaldehyde &
Glutaraldehyde):
 Formaldehyde & Paraformaldehyde: Have broad
spectrum biocidal activity effective for both surface and
space contamination-implicated to cancer.
 Paraformaldehyde: Generates formaldehyde when
heated –useful in space contaminations.
 Glutaraldehyde: Colourless liquid with sharp pungent
smell useful when exposed for many hours-effective to
virus,Spore and vegetative bacteria
Disinfection cont…
b. Chlorine and Idophores:
 Chlorine: Good on clean surface but inactivated
by organic matter they are broad spectrum,
inexpensive and fast acting ie: Calcium
hypochlorite
 Idophores: An Iodine and solubilizing agent –For
both antiseptic and disinfection
c. Quaternary Ammonium Compounds:
They are odorless,colourless they have detergent
action
Disinfection cont…
d. Heavy Metals:
 Soluble salts of mercury,Silver lactate and Mercuric
compouds,they are good bactericidal but they are
hazardous should be used with care.
e. Alcohols:
They disrupt cellular membrane, solubilization of lipids
and denaturation of proteins. Effective against lipid
cotaining virus and broad spectrum bacterial
spp.,innactivate spore forming bacteria but they
evaporate rapidly.
References:
1. Microbiology and Immunology
By : Rosenthal K.S
2. Introduction to Diagnostic Microbiology
By: Delost M.D
3. Review of Medical Microbiology(15th edition)
By:E.Jawetz,J.L.Melnick,E.A.Adelberg
4. District Laboratory Practice in Tropical Countries(Part 1&2)
By : Monica Cheesbrough
5. Biology(4thEdition)
6. By: Sylvia S.Mader
7 . Wikipedia
8. Medical Microbiology(Updated 3rd Edition)
By:Mims,Dockrell,Goering,Roitt,Wakelin,Zuckerman)
.
THE END
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