GENRAL MICROBIO

1. Comparison of various types of microscopy.

• Light microscopy: forms a dark image against a brighter
• Dark-field microscopy: object appears bright against a dark back ground uses for detecting
unstained causative agents of syphilis, recurrent fever, leptospirosis and motility of the
organism.
• Bright-field microscopy similar to light microscopy
• Phase-contrast microscopy
• Luminescent microscopy: exposure to UV rays , most commomly accepted to distinguish
between primary and secondry fluorecence
• Electron microscopy (EM) types: uses microbiology for investigation of the struture of
microorganims. Apply in virology Studing sections of tissue cell and microorganisms.
Transmission - internal structure Scannin EM- ( examine the surface)
• Scanning electron microscopy: uses a fine beam or spot of electrons thta is focused rapidly
back and forth over the specimen.

2. Methods of examination of living microorganisms.

• Light microscopy:
• Dark-field microscopy
• Bright-field microscopy
 • Phase-contrast microscopy
• Luminescent microscopy
• Electron microscopy
• Scanning electron microscopy
3. Main differences between prokaryotic and eukaryotic cells.
Answer:
Eukaryotic
Presence of Nucleus. A well-
defined nucleus is present
and enclosed within nuclear
membrane
Multicellular
Usually cell wall is absent, if
present (plant cells and
fungus), comprises of
cellulose (polysaccharide).
Linear, double-stranded
DNA.
80S ribosome
Mode of reproduction is
commonly sexual
Cell division is by mitosis
Transcription occurs in
nucleus and translation in
cytosol.
Organelles are membrane
bound and are specific in
function
Prokaryotic
Well-defined nucleus is
absent, rather 'nucleoid' is
present which is an open
region containing DNA
Single-celled (unicellular)
Cell wall present, comprise
of peptidoglycan or
mucopeptide
(polysaccharide).
Circular, double-stranded
DNA.
70S ribosome
Mode of Reproduction is
asexual.
Cell division is by Binary
Fission, (conjugation,
transformation,
transduction)
Transcription and
Translation Occurs together
Organelles are not
membrane bound, if
present.
 Multiple origins of Single origin of replication
replication

Microtubules, Endoplasmic Microtubules,Endoplasmic

reticulum, lysosomes and reticulum , Lysosomes and
Peroxisomes present Peroxisomes absent

Examples: Plants and Examples: Archaea,

Animals. Bacteria

4. Simple methods of staining, their practical value.

Answer:-
• Procedure
1. Clean and dry microscope slides thoroughly.
2. Flame the surface in which the smear is to be spread.
3. Flame the inoculating loop.
4. Transfer a loop full of tap water to the flamed slide surface.
5. Reflame the loop making sure the entire length of the wire that will enter the tube
has been heated to redness
6. Remove the tube cap with the fingers of the hand holding the loop.
7. Flame the tube mouth.
8. Touch the inoculating loop to the inside of the tube to make sure it is not so hot that
it will distort the bacterial cells; then pick up a pinhead size sample of the bacterial
growth without digging into the agar.
9. Reflame the tube mouth, replace the can, and put the tube back in the holder.
10.Disperse the bacteria on the loop in the drop of water on the slide and spread the
drop over an area the size of a dime. It should be a thin, even smear.
11.Reflame the inoculating loop to redness including the entire length that entered the
tube.
12.Allow the smear to dry thoroughly.
13.Heat-fix the smear cautiously by passing the underside of the slide through the
burner flame two or three times. Test the temperature of the slide after each pass
against the back of the hand. It has been heated sufficiently when it feels hot but
can still be held against the skin for several seconds. Overheating will distort the
cells.
 14.Stain the smear by flooding it with one of the staining solutions and allowing it to
remain covered with the stain for the time designated beloW
− Methylene blue - 1 minute
− Crystal violet - 30 seconds
− Carbol fuchsin - 20 seconds
During the staining the slide may be placed on the rack or held in the fingers.
15.At the end of the designated time rinse off the excess stain with gently running tap
water. Rinse thoroughly.
16.Wipe the back of the slide and blot the stained surface with bibulous paper or with a
paper towel.
17.Place the stained smear on the microscope stage smear side up and focus the smear
using the 10X objective.
18.Choose an area of the smear in which the cells are well spread in a monolayer. Center
the area to be studied apply oil directly to the smear, and focus the smear under oil
with the 100X objective.
19.Draw the cells observed.

5. Practical value of Gram staining of bacteria. Algorithm and mechanism of

staining. Examples of
• Gram - positive and Gram - negative microorganisms.
• Answers : Gram staining is a common technique used to differentiate two large groups of
bacteria based on their different cell wall constituents. The Gram stain procedure
distinguishes between Gram positive and Gram negative groups by coloring these cells red
or violet.

• The gram-staining
1) The crystal violet dye stains all cells blue/purple.
2) The iodine solution (a mordant) is added to form a crystal violet–iodine
complex; all cells continue to appear blue.
3) The organic solvent, such as acetone or ethanol, extracts the blue dye
complex from the lipid-rich, thin-walled gram-negative bacteria to a greater
degree than from the lipid-poor, thick-walled gram-positive bacteria. The gram-
negative organisms appear colorless; the gram-positive bacteria remain blue.
4) The red dye safranin stains the decolorized gram negative cells red/pink;
the gram-positive bacteria remain blue.
 • gram-negative cells are completely decolorized by alcohol. As a last step, a counter stain
such as the red dye safranin) is applied so that the decolorized gram-negative (cells will take
on a contrasting color; the gram-positive cells now appear purple.
• Gram-positive microorganisms:
− Corynebacterium
− Clostridium
− Listeria
− Bacillus
− Staphylococcus aureus
− Staphylococcus epidermis
− Staphylococcus saprophyticus
− Streptococcus pyogenes
− Streptococcus pneumoniae
− Enterococcus
• Gram-negative microorganisms:
− Escherichia coli
− Neisseria gonorrhoeae
− Neisseria meningitidis
− Chlamydia trachomatis
− Yersinia pestis
− Salmonella
− Shigella
− Helicobacter pylori

6. Procedure and principle of Ziehl-Neelsen technique with results, its diagnostic

value.
Answer:

• Ziehl-Neelsen ver-sion can be used. For rapid screening purposes, auramine stain, which can
be visualized by fluorescence microscopy, is used.
 • Neisser's staining
− Stain with Neisser's methylene blue for 1 min.
− Dye is poured off and smear is washed with water
− Pour in Lugol's solution to act for 20-30 sec
− Without washing with water, stain preparation with vesuvin for 1-3min
− Then wash it with water and dry.Results:
− Cytoplasm appears pink and granules deep blue

7. Structure of bacterial cell.

Answer:
• The cell wall is the outermost component common to all bacteria (except Mycoplasma
species, which are bounded by a cell membrane, not a cell wall). Some bacteria have
surface features external to the cell wall, such as a capsule,flagella, and pili, which are less
common components and are discussed next.
• The cell wall is located external to the cytoplasmic membrane and is composed of
peptidoglycan
• The peptidoglycan provides structural support and maintains the characteristic shape of the
cell.
8. A cell wall: its functions and methods of the examination. The cell wall structure
of Gram-positive and Gram-negative bacteria.
Answers:
• The peptidoglycan layer is much thicker in gram-positive than in gram-negative bacteria.
Many gram-posi-tive bacteria also have fibers of teichoic acid that protrude outside the
peptidoglycan, whereas gram-negative bacteria do not have teichoic acids
• the gram-negative bacteria have a complex outer layer consisting of lipopolysaccharide,
lipoprotein,and phospholipid. Lying between the outer-membrane layer and the
cytoplasmic membrane in gram-negative bacteria is the periplasmic space, which is the site,
in some species, of enzymes called β-lactamases that degrade peni-cillins and other β-
lactam drugs.
• Gram positive bacteria have cell walls composed of thick layers of peptidoglycan.
• Gram positive cells stain purple when subjected to a Gram stain procedure.
• Gram negative bacteria have cell walls with a thin layer of peptidoglycan. The cell wall also
includes an outer membrane with lipopolysaccharide (LPS) molecules attached.
• Gram negative bacteria stain pink when subjected to a Gram stain procedure.Value of cell
wall:
• osmosis – movemoent of water through semipermeable membrane from areas of low
concentration solution to areas of high concentration solution
• involved in the transport of materials and metabolites into and out of the cell
• defined shape of cell
• protection from external environment
• protection from invasion by viral, bacterial and fungal pathogens
• withstand the turgor pressure which develops within the cells due to high osmotic pressure

9. Principles and diagnostic value of complex staining methods: Aujesky staining,

Loeffler staining, and Neisser staining.
➢ Loeffler's staining
• on a fixed smear pour alkaline methylene blue to act for 3-5min
• wash with water
• dry with filter paper and examine under microscope
• Loefflers Methylene Blue stain is used for the identification of diphtheria bacilli,since it
differentiates the deeply staining metachromatic granules from the pale blue-staining
cytoplasm .
➢ Results:
 • metachromatic granules - deep blue, cytoplasm – pale blue
➢ Neisser's staining
• Stain with Neisser's methylene blue for 1 min.
• Dye is poured off and smear is washed with water
• Pour in Lugol's solution to act for 20-30 sec
• Without washing with water, stain preparation with vesuvin for 1-3min
• then wash it with water and dry.
➢ Results:
• cytoplasm appears pink and granules deep blue
➢ Aujesky staining
• 0.5% sulphuric acid plus heat
• Stained by ziehl neerson technique
➢ Results :
• Spore (red color ) and bacterial cell (blue color )

10. Bacterial capsules: a value, methods of their examination. Diagnostic value of

capsule formation phenomenon. Examples of pathogenic bacteria, which form
capsules.
➢ Capsules are important for:
• adhesion (associated with virulence in bacteria)
• avoidance of immune response (protects bacteria from phagocytic cells)
• protection from dehydratation
• composition of capsule: 98% water and 2% polysaccharide or glycoprotein or both
• most important virulence factor for bacteria
• very delicate structure

➢ Example: Bacillus anthracis – capsule is made up of polypeptide (D glutamic acid)

Streptococcus – alpha-aminoacids
Klebsiella pneumoniae – capsule is made up of glucose, galactose, rhamnose

➢ Diagnostic value:
• used in diagnosis
• used in formation of vaccines
• used in serotyping
 ➢ Capsule stain:
• use negative staining technique
• Acid dye (negatively charged)
• Basic dye (positively charged)
• Capsule (unstained, chemical)

11. Bacterial spores: structure, function. Examples of sporogenic microorganisms.

Sporulation. It is unicellular structure.

➢ Spores – form life cycle in many plants, algae, bacteria, fungi.

• There are 2 types of spore:

1) Endospores – are formed within vegetative cell. Bacteria producing endospores:

Bacillus, Sporosarcina,...
Has following layers: exosporium, spore coat, cortex, core

2) Exospores – are formed in either one of ends of vegetative cell (on surface of cell)
Structures: coat,outer membrane, core, cortex, germ cell wall, inner membrane

➢ The spore stain

• Spores are most simply observed as intracellular refractive bodies in unstained cell
suspensions or as colorless areas in cell stained by conventional methods.
• The spore wall is impermeable, but dyes can be made to penetrate it by making the
preparation.
• Prevent decolorisation of spore by period of alcohol treatment sufficient to decolorize
vegetative cells.
• Later can finally be counterstained.
• Spores are commonly stained with Malachite green or Carbofuchsin.

12. Classification of bacteria according to the spore location. Methods of spore

examination.

13. Classification of bacteria according to the localization of flagella.

Answer:

➢ Monotrichous bacteria have a single flagellum (e.g., Vibrio cholerae).

➢ Lophotrichous bacteria have multiple flagella located at the same spot on the bacterial
surfaces which act in concert to drive the bacteria in a single direction. In many cases, the
bases of multiple flagella are surrounded by a specialized region of the cell membrane,
called the polar organelle
➢ Amphitrichous bacteria have a single flagellum on each of two opposite ends (only one
flagellum operates at a time, allowing the bacterium to reverse course rapidly by switching
which flagellum is active).
➢ Peritrichous bacteria have flagella projecting in all directions (e.g., E. coli).
➢ Atrichous bacteria are lacking flagella
➢ According to location of flagella .
• Monotrichous :-pseudomona auregenasa
• Amphitrichous:-aquasspirillum serpens
• Lopotrichous:-pseudomona fluorescence
• Peritrichous:- salmonella typhi
• Atrichous:-spirochates.

14. Morphological classification of cocci. Their role in human pathology

➢ Bacteria can be classified into 6 major groups.
Cocci – spherical or oral cells.
• Monococci (single) – Monococcus
• Diplococci (in pairs) – Streptococcus pneumoniae
• Staphylococci (in grape-like clusters) – Staphylococcus aureus
• Streptococci (in chains) – Streptococcus pyogenes
 • Tetrad (in group of four) – Micrococcus
• Sarcinoma (in group of eight)
− Produce catalase, coagulase.
− Mannitol fermentation.
− Hemolysin, leukocidin.
− Exfoliate toxin
− Toxin shock syndrome
− Enterotoxins
• These organism are parasite in mouth, intestine, and respiratory tract of humans and
other animals.

15. Morphological classification of rod-shaped bacteria. Their role in human

pathology
Answer:
• Bacteria include those microorganisms which, as a rule, donot produce spores
(colibacillus, and organisms responsible for enteric fever, paratyphoids, dysentery,
diphtheria, tuberculosis, etc.). Do not produce spore.
• Bacilli and clostridia include organisms the majority of which produce spores (hay
bacillus, bacilli responsible for anthrax, tetanus, anaerobic infections, etc.) Bacilli ( fever,
paratyphoid’s, dysentery )

16. Morphological classification of spiral-shaped bacteria. Their role in human

pathology.
Answer:
• Vibriones are cells which resemble a comman in appearance. Typical representatives of
this group are Vibrio cholerae, the causative agent of cholera, and aquatic vibriones which
are widely distributed in fresh water reservoirs.
• Vibrocholerae- cause of cholera
• Spirilla- (L. spira coil) are coiled forms of bacteria exhibiting twists with one or more turns.
Only one pathogenic species is known {Spirillum minus} which is responsible for a disease in
human transmited through the bite of the rats or another rodents
• Spirochaetes – Borrelia. Their cells have large, obtuse-angled, irregular spirals, the number
of which varies from 3 to 10. Pathogenic for man are the causative agents of relapsing fever
 transmitted by lice (Borrelia hispanica), and by ticks (Borrelia persica, etc.). These stain blue-
violet with the Romanowsky-Giemsa stain
• Ileptospira -are characterized byvery thin cell structure. The leptospirae form 12 to 18 coils
wound close to each other, shaping small primary spirals. The organisms have two paired
axial filaments attached atopposite ends (basal bodies) of the cell and directed toward each
other.cause leptospirosis from animals )
• treponema ( syphilis, treponema pallidum) exhibits thin, flexible cells with 6-14 twists. The
micro-organisms do not appear to have a visible axial filament or an axial crest when viewed
under the microscope

17. Morphology and ultrastructure of viruses. Antigenic structure. Methods of

cultivation.
➢ Contain 2 following components:
1) a nucleic acid genome
2) a protein capsid that covers the genome
3) lipid envelope
• 1 & 2 – together nucleocapsid.
➢ Viruses structure:
− Icosahedral (cubical)
− appear spherical in shape, but closer looks actually reveals they are icosahedral
− genetic material is fully enclosed inside the capsid
− examples: Poliovirus, Rhinovirus and Adenovirus
➢ Envelope (enveloped icosahedral and helical)
− structure is conventional icosahedral or helical structure that is surrounded by a lipid
bilayer membrane
− virus is enveloped
− infectivity of these viruses is mostly dependent on enelope
− examples: Influenza virus, Hepatits C and HIV
➢ Complex
- structures have a combination of icosahedral and helical shape
- head of virus has an icosahedral shape with helical shape tail
- bacteriophage uses its tail to attach to bacteria, creates a hole in the cell wall and then
inserts its DNA into the cell using the tail as a channel.
➢ Helical (spiral)
 − shaped like hollow tubes with protein walls
− promotes self assemble
− size of capsid is a function of nuclei acid
− example: Bacteriophage M13
➢ 4 morphotypes predominance:
• naked icosahedral
• naked helical
• enveloped icosahedral
• enveloped helical
• Viruses' properties:
• obligate intracellular parasites
• contain a single type of nucleic acid, either DNA or RNA
• contain a protein coat (capsid) consisting of individual protein units (capsomeres)
• multiply inside living cells
➢ Methods of viruses examining:
• filtration through membranes of gaded porality
• sedimentation in the ultracentrifuge
• direct observation in electron microscope
• ionizing radiation
• X-ray crystallography
➢ Main methods of cultivation:
• inoculation of virus into animals
• inoculation of virus into embryonated eggs
• tissue culture
➢ Inoculation:
1. media – artificially prepared nutrients
2. culture growth on the media (for example: colony on Agar)
3. culturing
4. contamination (unwanted microbes)
5. pure culture (only 1 type of microbe)Nature of viruses:
➢ Viruses are infective microorganisms
• are much smaller than bacteria
• no independent metabolism
• simple structure
 • absence of cellular structure
• nucleic acids (have only 1 nucleic acid, either DNA or RNA)
• do not growth and no division

18. What are differences between viruses and bacteria. Classifications of viruses.
Answer:
➢ Viruses are even smaller than bacteria and require living hosts — such as people, plants or
animals — to multiply. Otherwise, they can't survive. When a virus enters your body, it
invades some of your cells and takes over the cell machinery, redirecting it to produce the
virus.

, Classification
19. CPE (cytopathic effect) of viruses. Types. Examples.
Answer:
➢ (CPE) is the term used to describe the damage, both morphologic and functional, inflicted
on the cell by the virus. In the clinical laboratory, the presence of a virus in the patient’s
specimen is often detected by seeing a CPE in cell culture.
➢ Technique

(1) Hemadsorption (i.e., attachment of erythrocytes to the surface of virus-

infected cells). This technique is limited to viruses with a hemagglutinin protein
on their envelope, such as mumps, parainfluenza, and influenza viruses.
(2) Interference with the formation of a CPE by a second virus. For example,
rubella virus, which does not cause a CPE, can be detected by interference
with the formation of a CPE by certain enteroviruses, such as echovirus or
Coxsackie virus.
(3) A decrease in acid production by infected, dying cells. This can be detected
visually by a color change in the phenol red (a pH indicator) in the culture
medium. The indicator remains red (alkaline) in the presence of virus-infected
 cells but turns yellow in the presence of metabolizing normal cells as a result
of the acid produced. This technique can be used to detect certain
enteroviruse
Total destruction Enteroviruses Monolayer shrink,
become
dense(pyknosis)

Subtotal destruction Togaviruses . Detachment or death of

alphaviruses some
picornaviruses , cells in the monolayer
paramyviruses

Focal degeneration Herpes viruses , pox Localized areas on

viruses infection
cells enlarged rouded
Swelling and clumping Adenoviruses Cell like grape( enlarge
and
clump)
Foamy degeneration Retroviruses , Large and numerous
(vacuolization paramyxoviruses cytoplasmic vacuoles
,flaviviruses
Cell fusion (syncitium or Paramyxoviruses Fusion of plasma
polykaryon) membrane

20. Types and stages of viruses with cells interaction.

➢ There are 6 phages in the varius replication cycle:
1. attachment
2. penetration/ entry
3. uncoating
4. replication and expression
5. assembly
6.release
− Virus attachment protein (VAP) attaches to the receptor of the host cell.Receptor on
the host cell enable virus to enter into the host cell.
➢ Virus can enter through 3 different routes:
 • direct penetration
• fusion
• endocytosis
− The viral DNA enters the host cell.Uncoating – then viral contents are released.Viral
RNA enters the nucleus, where is replicated by the viral RNA polymerase.New phage
particles are assembled.New viral particles are made and released into extracellular
fluid.The cell, which is not killed during this process, continues to make new virus.

21. Types of virus reproduction according to Baltimore classification.

Answer:-
➢ Baltimore classification also closely corresponds to the manner of replicating the
genome, so Baltimore classification is useful for grouping viruses together for both
transcription and replication. Certain subjects pertaining to viruses are associated with
multiple, specific Baltimore groups, such as specific forms of translation of mRNA and
the host range of different types of viruses. Structural characteristics such as the shape
of the viral capsid, which stores the viral genome, and the evolutionary history of viruses
are not necessarily related to Baltimore groups.

I. double stranded DNA viruses

II. single stranded DNA viruses
III. double stranded RNA viruses
IV. IV-positive sense single stranded RNA viruses
V. Negative sense single stranded RNA
VI. single stranded RNA viruses with a DNA intermediate in their life cycle

22. Structure of bacteriophage. Concept about virulent and temperate phages.

Lysogenic bacteria.
➢ Phage conversion and its practical value. Usage of bacteriophage phenomenon in
medicine. Structure: head, neck, tail Bacteriophages carry only genetic information
needed for replication of their nucleic acid and synthesis of their protein coats.
➢ 6 morphological types:
1. head with rigid tail having contractile apparatus and may have appendages
2. head with flexible tail without contractile sheath, it may have terminal appendages
3. head with short tail
 4. head with large capsomers at each apex of hexagon, it has no tail
5. simple head without capsomers and tail
6. long flexible tail without head
➢ Isolation of bacteriophages:
1. plague assay technique
2. steps:
− serial dilutions
− add in host to phage dilution
− incubation 37C, 20min
− add in agar
− pour on solidified agar
− incubate 37C, 18-24 hours
− observation of plague formation
Phage titer is expressed as plague forming units (PFU) per mililiter.

➢ Use in medicine:
• use in treatment of bacterial infections, diagnosis of certain infections diseases
• use for identification of pathogenic bacteria
• use in molecular biology
• use of antibiotics made it possible to prevent bacterial contamination

23. Types of bacterial respiration. Examples of bacteria according to the types of

respiration.

Obligate aerobes Need 02 to growth Nocardia

Bacillus
Mycobacterium
tuberculosis

Obligate anaerobes Not 02 Clostridium

Bacteroids
Actinomyces
Peptostreptococcus

Facultative anaerobes Use O2 but have anaebic Staphylococcus spp.

 methods of Streptococcus
energy production Escherichia coli
Yersinia pestis
Lesteria spp.

Microanaerophiles Need O2 but are harmed Campylobacter jejuni

by Helicobacter pylori
atmospheric Campylobacter fetus
concentrations of O2 Mycrobacterium
ulcerans

Aerotolerant Not need O2 but not Lactobacilli

anaerobes harmed by it Streptococci
Capnophites Need nigh concentration Capnophiles
of carbon Campylobacter

24. The mechanism of bacterial replication. The main growth phases of the
microbial population in liquid media. Practical value.

➢ Replication: DNA by uncoiling o helix , strand by breaking of the hydrogen bonds between
the complementary strands and new strands by complementary base pairing
• Phases :
1. LAG: bacteria are metabolically active by no dividing( synthesizing DAN, ribosome’s
and enzymes )
2. EXPONENTIAL (LOG): exponential growth ( cells are most susceptible to inhibitors)
3. STATIONRY : growth reached a plateaus as the number of dying cells equals the
number of dividing cells
4. DEATH: exponential decreased of living cells
Practical value :Important when the aim is use to inoculate and know the number of bacterial
isolate

25. Enzymes of bacteria: classifications, their feature.

Answer:
➢ The first number represents enzymes that are classified according to the mechanism of
enzymatic reaction. According to the type of reactions that the enzymes catalyze, enzymes
 are classified into seven categories, which are oxidoreductases, transferases, hydrolases,
lyases, isomerases, ligases, and translocases.

Hydrolyses Breakdown of carbon and nitrogen

atoms /
oxygen and sulphurs atoms

Transferases Catalyse by transferring certain

radicals from one
molecule to another

Oxidative enzyme Catalyse the oxidation reduction

process
Isomerases and racemases Important in carbohydrate metabolis

26. The methods and principles of isolation of pure culture.

Methods :

Streak plate Colony forming units , on the surface

of the agar plate using wire loop
Serial dilution Inoculation a series tubes with
microbial suspension.

Spread plate Number of bacteria per unit volume

of an sample is reduced by serial
dilution before spread in agar plate

Single cell isolation Individual cell of the required kind is

picked ou

Enrichment culture Use to isolated small number of

microorganism slow growth
Poor plate Involves plating of diluted samples
mixed with melted agar medium
 ➢ Principles:
1. isolation of bacteria
2. separation from other bacteria to obtain pure culture
3. study of pathogen sensitivity
27. Classifcation of nutrient media.
Bacteriological culture media can be classified in two ways.
A. Based on consistency, culture media are grouped into:
− Liquid media (or broth)
− Semisolid media
− Solid media
B. Based on die growth requirements, culture media are classified as
− Routine laboratory media: They are prepared from nutrients, such as aqueous extract of
meat, peptone, etc. they can further be classified into various types based on functional
use or application, as follows-
o Simple/ basal media
o Enriched media
o Enrichment broth
o Selective media
o Differential media
o Transport media
o Anaerobic media
− Defined or synthetic media: lliey are prepared from pure chemical substances and the
exact composition of die media is known.
o Simple syndietic media
o Complex synthetic media

28.The mechanisms of microbial nutrition.

29. Stages of isolation of pure cultures of aerobic microorganisms.
1) Macro- and microscopic examination of researched stuff and showing an agar media for
obtaining separate colonies..
2) Macro- and microscopic examination of colonies and reinoculation on agar media with
oblique surface
3) Checking of the purity of the isolated culture and its identification
4) Output (conclusion) about isolated culture
Fortner's method
Gram's method
 Agar media method of strokes
Drygalsky method
− Prepare smears to study under microscope
− Use spatula to put material to solid medium in Petri dish.
• Mechanical separation = growing in colonies. Put material to liquid enrichment medium
and to Petri dishes with solid nutrient media 37 degreeC for 18-24 hours.
• Following 24h incubation. Microscopic examination.

30. The methods of creation of anaerobic conditions.

• Fortner's medthod
i. Petry dish with blood agar is diluted on two halves
ii. Anaerobic and aerobic bacteria are cultivated on different parts
iii. Petry dish is closed hermetic
iv. At first aerobic bacteria
v. Anaerobic bacteria begin growth when aerobic bacteria have used all oxygen.
• Kitt-Tarozzi
o 1 st day – inoculate material into Kitt-Tarozzi media.
Meat peptone broth. To add oxygen.
o 2 nd day – chnages in enrichment media.
take broth culture with Pasteur pipete, transfere. Microscopic examination.
• Kitt-Tarozzi = we should put patological material, obtain separated colonies. We have two
methods:
i. Zeissler's method (Drygalski) :- We need incubate Petri dish, use spatula and
separate colonies in Petri dish. We should make incubation for 3 days. Use
microscopic examination.
ii. Veinberg's method

31. Methods of the isolation of pure cultures of anaerobic bacteria.

• To isolate anaerobic bacteria- which grow only in the absence of oxygen but grow In the
presence of carbon dioxide ( 20%)
• Creating anaerobic conditions called anaerobiosis established by various methods.
➢ In one way anaerobic conditions can be created in an airtight closed container-
anaerobic jar (McIntosh and Fylde’s jar made up of either stainless steel or
polycarbonate/ polypropylene
 ➢ In another way anaerobic conditions can be created by using anaerobic media in test
tubes ( containing reducing substance) provide anaerobic condition.

32. Identification of pure culture: morphological, tinktorial, cultural, biochemical,

serological, biological.
• pure culture are the presence of single,isolate colonies of the same type.
➢ Surface plating: Screaking with intermittent healing is a routinely used method in the
laboltory.
➢ Selective media and enrichment broch: it is employed for isolating pathogens from
specimens containing normal fora, e.g. feces.
➢ Pre-treatment of specimens: Suitable bactericidal substances are used for pre-
treatment of specimens co isolate a particular bacterium, e.g. concentration and
decontamination of sputum sample with 4% NaOH before culturing for
Mycobacterium tuberculosis.
➢ Anaerobiosis: Obligate aerobes and anaerobes may be separated by incubating the
places under aerobic and anaerobic con die ions
➢ Heating: Mixture of bacteria in liquid medium wich different optimum growth
temperatures can be separaced by heating the medium at different temperatures.
For example, heating at 60' C would allow only the thermophilic bacteria co grow.
➢ Fillers: Filters of different pore diameters are widely used for separating bacteria of
different sizes and also for separating viruses from bacteria.
➢ Based on motility: Motile bacteria can be separated from non-motile bacteria by sub
culturing chem on co Craigie cube.
➢ Animal inoculation: Pathogenic bacteria can be separated from non-pathogenic
bacteria by inoculating the mixture into susceptible animals followed by their
isolation from die lesions which would be produced only by the pathogenic bacteria.
For example, Bacillus anthracis can be separated from other aerobic spore bearing
bacilli by inoculation into guinea pigs

33. How are studied peptolytic, proteolytic and haemolytic properties of bacteria?
➢ pectolytic isolates were characterized using physiological and biochemical tests.
− which were selected according to keys of Buchanan and Gibbons Bradbury .
− properties: Gram reaction – staining, KOH reaction, presence of L-alanine
aminopeptydase, spore production, cell morphology, fluorescent pigment production ,
presence of oxidase,catalase , oxidative/fermentative test (O/F) ,levan production from
 sucrose , hydrolysis of: gelatin, starch and aesculin, nitrate reduction,presence of
arginine dihydrolase, acid production from D-glucose, lactose,Dmannitol, D-sorbitol, D-
raffinose and D-xylose.
➢ Proteolytic bacteria are a type of bacteria that can produce protease enzymes, which are
enzymes that can break down peptide bonds in protein molecules.
Proteolytic enzyme, also called protease, proteinase, or peptidase, any of a group
of enzymes that break the long chainlike molecules of proteins into shorter fragments
(peptides) and eventually into their components, amino acids.
➢ Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to
induce hemolysis when grown on blood agar is used to classify certain microorganisms. This
is particularly useful in classifying streptococcal species.
➢ There are three types of hemolysis, designated alpha, beta and gamma. Alpha hemolysis is
a greenish discoloration that surrounds a bacterial colony growing on the agar. This type of
hemolysis represents a partial decomposition of the hemoglobin of the red blood cells.

34.Microbial antagonism. Mechanisms and method of its examination.

• Microbial antagonism refers to the presence of normal micro biota.
− that protects the body by competing with pathogens in a variety of ways to prevent
pathogens from invading the body.
− Antibiotics are medicines that help stop infections caused by bacteria.
• They can be divided into 2 classes based on their mechanism of action.
➢ Bactericidal antibiotics: They kill the bacteria by inhibiting cell wall synthesis
− Examples: Beta-lactam antibiotics ( penicillin derivatives), cephalosporins and
vancomycin. Also bactericidal are daptomycin, fluoroquinolones, metronidazole,
etc.
➢ Bacteriostatic antibiotics: They limit the growth of bacteria by interfering with bacteria
protein production, DNA replication< or other aspects of other bacterial cellular
metabolism. They include tetracyclines, sulfonamides, spectinomycin, trimethoprim, etc

35.Types of symbiosis of microorganisms: commensalism, mutualism, parasitism.

• symbiosis: A close and often long-term interaction between two or more different biological
species
 ➢ Commensalism is a relationship between species in which one benefits and the other is
unaffected. Humans are host to a variety of commensal bacteria in their bodies that do
not harm them but rely on them for survival (e.g. bacteria that consume dead skin).
➢ Mutualism, a relationship in which both species benefit, is common in nature. In
microbiology, there are many examples of mutualistic bacteria in the gut that aid
digestion in both humans and animals.
➢ Parasitic relationships, in which one species benefits and the other suffers, are very
common in nature. Most of the microorganisms studied in medical microbiology are
parasitic and feed on human tissue. For example, cholera, leshmaniasis, and Giardia are
all parasitic microbe

36.Antibiotics. Classifications of antibiotics according to their origin.

− Antibiotics are medicines that help stop infections caused by bacteria
• Classification according to origin
1. Microbial origin:
i. Bacterial origin:
− Bacillus polymyxa: PolymyxiN
− Chromobacter violaceum: Bacitracin
− Micromonospora spp: Gentamycin
ii. Fungal origin:
− Penicillium notatum: Penicillin
− Cephalosporin spp: Cephalosporin
iii. Actimomycetes origin:
− Streptomyces griseus: Streptomycin
− S. venezuelue: Chloramphenicol
− S. erythreus: Erythromycin
− S. mediterranae: Rifampicin
− S. venezuelae: Chloramphenicol

2. Semi-synthetic antibiotics:
− Examples: Amoxycillin, Ampicillin, Doxycycline, Tigecycline, Sulfonamide etc

3. Synthetic antibiotics:FIND
− Examples: Chloramphenicol (* it was extracted from Streptomyces
venzuelae but now produced synthetically), 4-quinolones, Sulfonamide
• Classification according to chemical structure
− Carbohydrate containing Antibiotics:
− Pure saccharides antibiotics: examples; Streptozotocin
− Aminoglycosides: examples; Streptomycin
− N/O glycosides: eg. Chromomycin
− Other: eg; Lincomycin
− Macrocyclic lactone antibiotics: eg. Erythromycin
− Quinolones antibiotics; eg. Fluroquinolone
− N-containing heterocyclic antibiotics: eg. Beta-lactum
− O-containing heterocyclic antibiotics: eg. Cycloserine
− Alicyclic antibiotics: eg. Cycloheximide
− Aromatic antibiotics (Nitrobenzene): eg. Chloramphenicol
− Aliphatic amine antibiotics: eg. Spermidine
− Peptide antibiotics: eg. Polymyxin, Bacitracin, Gramicidin

37.Antibiotics. Classifications of antibiotics according to their mechanism of action.

➢ According to mechanism of action
• They can be divided into 2 classes based on their mechanism of action.
o Bactericidal antibiotics: They kill the bacteria by inhibiting cell wall synthesis
− Examples: Beta-lactam antibiotics ( penicillin derivatives), cephalosporins and
vancomycin. Also bactericidal are daptomycin, fluoroquinolones, metronidazole, etc.
o Bacteriostatic antibiotics: They limit the growth of bacteria by interfering with
bacteria protein production, DNA replication< or other aspects of other bacterial
cellular metabolism. They include tetracyclines, sulfonamides, spectinomycin,
trimethoprim, etc.

38.Antibiotics. Classifications of antibiotics according to their action spectrum.

➢ According to action spectrum

• Spectrum means the number of the organisms affected by the same drugs.

o Broad spectrum antibiotics: They affect several types of bacteria and fungi and it is
usually used where the specific type of micro organism is unknown.
 o Narrow spectrum antibiotics: They are used only when we know the specific type of
micro organism. These are more effective on specific micro organisms but less
effective on others

39.Determination of bacteria susceptibility to antibiotics by the serial dilution

method in liquid media. Concept about minimal inhibitory concentration, minimal
bactericidal, and minimal bacteriostatic concentrations.
➢ Susceptible :- bacteria are those that belong to the most susceptible sub-population and
lack mechanisms of resistance. Serial dilution method in fluid media allows to determine
minimal inhibitory concentration (MIC) and minimal bactericidal concentration of the drug
for the isolated micro organisms.
− A series of double dilution of the drug is prepared in the test tubes on the fluid growth
medium (1ml each) .
− Concentration decreases correspondingly from 128 to 0.06 mcg/ml( basic
concentration can vary depending on the drug activity).
− 0.05 ml of the physiological saline is introduced in each tube ,
− that contain 106 /ml of studied microbial cells.
− Tubes are inoculated during 18-20 h at 37 degrees .
− After this term has ended, results are accounted by the medium turbidity visually or
nephelometrically , comparing results with control
➢ Serial dilution in fluid solid media is mostly similar to the dilution method in fluid media.
− Double serial dilution method is prepared 1:10000 to 1:320000,
− and then 1 ml of the each tube content is introduced in 9ml melted and cooled to 45
degrees nutritive agar.
− Then investigated bacterial test culture is inoculated in this agar.
− Obtained agar mixture is poured is poured in petri dishes and incubated for 18-20 h at
37 degrees.
➢ Minimal inhibitory concentration This is the lowest concentration of a chemical, usually a
drug, which prevents visible growth of a bacterium or bacteria. MIC depends on the micro
organism, the affected human being, and the antibiotic itself
➢ Minimal bactericidal and bacteriostatic concentration
o The minimal bactericidal concentration is the lowest concentration of an antibacterial
agent required to kill a particular bacterium. It can be determined from broth dilution
minimum inhibitory concentration tests by sub culturing to agar plates that do not
contain the test agent.
 o The minimal bacteriostatic concentration is the lowest concentration of the
antibacterial agent required to stop the activities of a certain kind of bacteria

40.Methods of the susceptibility determination of bacteria to antibiotics. Standard

disk diffusion method in detail
➢ Disk Diffusion Method
o Disk diffusion tests are the most widely used method. They are suitable for rapidly
growing pathogenic bacteria; however, they are not suitable for slow growing bacteria.
o The disk diffusion method is so named because:
− It uses filter paper disks impregnated with appropriate concentration of the
antibiotic solution
− The test bacterium is inoculated (as lawn culture) on the solid medium and then
the antibiotic disks are applied The antibiotic in the disks diffuses through the
solid medium, so that the concentration is highest near the site of application of
the antibiotic disk and decreases gradually away from it
− Sensitivity to the drug is determined by the zone of inhibition of bacterial
growth around the disk.

❖ Kirby-Bauer Disk Diffusion Method

− A sterile cotton swab is dipped into inoculum and squeezed to drain out the excess
fluid. Then the swab is inoculated on to the Mueller-Hinton agar plate by streaking
the syub three times over the entire agar surface.
− After drying the surface of agar plate for 3-5 minutes the antibiotic disks are applied
using either sterile forceps or multi disk dispenser
− Disks should not be placed closer than 20 mm (center to center) on the MHA plate
− Ordinarily, maximum up to 6 disks can be applied on a 100 mm plate (five in the
periphery and one in the center)
− The plates are then incubated at 37 C for 16-18 hours.
− The zones of complete growth of inhibition around each Var of the disks are
measured using a ruler or Vernier caliper. The diameter of the disk (6 mm) is also
included in this measurement
− The interpretation of zone size into sensitive, intermediate or resistant is based on
the standard zone size interpretation chart, Clinical and Laboratory Standards
Institute(CLSI) guideline
− Control strains should be tested each time when a new batch of disks or Mueller-
Hinton agar (MHA) is used,
 ❖ Stokes Disk Diffusion Method
− The MHA plate is divided into two halves. The test strain and control strain are
inoculated separately in each half.
− An uninoculated gap of 2-3 mm wide should separate
− the test and the control area on which the antibiotic disks are applied
− The plates are then incubated at 37°C for 16-18 hours
− the sensitivity report is prepared by comparing the zones of inhibition of control and
test strain

❖ Primary or Direct Disk Diffusion Test

− The primary or direct disk diffusion test may be performed when results are required
urgently and single pathogenic bacterium is suspected in the specimen (for sterile
fluid, urine or positively flagged blood culture bottle). Here, the specimen is directly
inoculated uniformly on the surface of an agar plate and the antibiotic disks are
applied. The results of the primary test should be verified by testing the isolates
subsequently. This test is of no use when mixed growth of different bacteria is
suspected to be there in the specimen, e.g. pus, stool, sputum, etc.

41.The resistance of bacteria to antibiotics. Mechanisms of its development and

methods of warning.
• Antibiotic resistance is the ability of bacteria or other microbes to resist the effect of
antibiotics.
• The major resistance mechanisms of microbes are decreased drug uptake, efflux pumps,
enzymes that inactivate an anti microbial chemical and target alteration by mutation.
They are also biofilms
1) Decreased uptake: The outer layer of gram negative bacteria makes it much more
difficult for certain anti-biotic to penetrate. Gram positive have a cell wall composed of
mostly peptidoglycan, a very rigid substance. This is a prime target of beta lactams
antibiotic such as penicillin and cephalosporins. The anti biotic locks on the beta
lactams structure in the cell wall, preventing expansion, and the cell ruptures as it
grows. Gram negative bacteria have a much thinner cell wall itself and this is protected
by a lipopolysaccharide molecule in the capsule, an outer membrane and what is
known as a periplasmic space, in short it is a much more heavily armoured vehicle.
 Porins are openings in the cytoplasmic membrane through which antibiotic agents can
gain entry. A reduced number of these porins is one means of resistance.
2) Efflux pumps: Some bacteria, eg pseudomonas have a system called efflux pump. As its
name suggests, this is a system whereby the bacterium has a pump to expel ingested
chemicals. Antibiotic efflux pumps are believed to contribute significantly to acquired
bacterial resistance because of the very broad variety of substances they recognise,
they are expression in important pathogens and their cooperation with other
mechanisms of resistance, such as decrease uptake.
3) Enzyme inactivation: Some microorganisms have developed the ability to produce
enzymes that are able to inactivate certain antibiotics. The most notable example is
penicillinase that can inactivate penicillin, but there are others.

42.Main principles of rational antibiotic therapy.

• Presence of substantiated indications for prescription of an antibiotic
• Choosing the most effective and the least toxic drug, in time administration
• Introduction of optimal doses with optimal frequency, taking into consideration the
complexity of the disease
• Choosing of the optimal way of introduction
• Estimation of duration of treatment
• Control after treatment
• Monitoring and prophylaxis of negative side effects
• Decision on expediency of combined antibiotic treatments

43.Side effects of an antibiotic therapy.

➢ Side effects
• Nausea
• Development of the resistant to antibiotics bacterial strain
• Allergic reactions
• Toxic reactions
• Endotoxic reactions
• Dysbiosis

44.Disinfection and sterilization. Difference between them.

➢ Sterilization
 − Sterilization is a process by which all living microorganisms, including viable spores,
are either destroyed or removed from an article, body surface or medium
− It results in reduction of at least 10 log colony-forming
− units of microorganisms and their spores It can be achieved by a physical agent or a
chemical agent (called chemical sterilant).

➢ Disinfection
− It refers to a process that destroys or removes most if not all pathogenic organisms
but not bacterial spores.
− It results in reduction of at least 10 log colony-forming units of microorganism, but
not spores
− The primary goal in disinfection is to destroy potential pathogens, but it also
substantially reduces the total microbial population
− Agents: Disinfection can also be achieved by a physical agent or a chemical agent
(called disinfectant) and they are normally used only on inanimate objects, not on
body surfaces.

45.Methods of sterilization. The objects for sterilization. Control of sterilization.

➢ Methods of sterilization;
• Through torch flame
o Boiling
o Dry heat
o Fluid steam
o Steam with pressure
o Tyndallisation
o Filtrating
• Object’s for sterilization:
o Surgical toolkit
o Medical dressing gowns
o Pathogenic cultures
o Petri dishes
o Physiological solutions
o Bacteriological loops ‘
• Control of sterilization;
o Chemical control: Sulphur, benzoic acid, betta naphthol
 o Biological control: spores culture of microorganism

46.Value of genetics for development of general and medical microbiology, virology,

molecular biology. Microbiologic bases of gene engineering. Achievements of gene
engineering, usage gene engineering drugs in medicine.
➢ Value of genetics;
• To understand the gene function of microorganisms
• Microbes provide relatively simple system for studying genetic phenomenon and thus
useful to other higher organisms.
• Microorganisms are used for isolation and multiplication of specific genes of higher
organisms which is referred as gene cloning.
• Microbes provide many value added products like antibiotics, growth harmones etc.
Microbial genetics will be helpful to increase these products productivity by microbial
technology
• Understanding the genetics of disease causing microorganisms especially virus, will be
useful to control diseases.
• Gene transfer among the prokaryotes play major role in the spread of the genes in a
particular environment. Microbial genetics will be useful to study the gene transfer from
one organism to another
➢ Genetic engineering is the deliberate manipulation of DNA, using techniques in the
laboratory to alter genes in organisms.
• Microbial genetics is a subject area within microbiology and genetic engineering. It
studies the genetics of very small (micro) organisms; bacteria, archaea, viruses and some
protozoa and fungi. This involves the study of the genotype of microbial species and also
the expression system in the form of phenotypes.
➢ Achievements of gene engineering; Genetic engineering will have important effects on
agriculture, notably in the development of new vaccines and diagnostic reagents, in the
production of transgenic animals, and in the application of enzyme technology in food
production and processing.
➢ Gene engineering drugs in medicine: vaccines, Antivenoms, bacteria derived toxins,
Immunoglobulins, monoclonal antibodies, Allergens, blood products and clotting factors,
hormones such as insulin, growth hormone they are made from gene engineering.
47.Value of genetics for development of general and medical microbiology, virology,
molecular biology. Microbiologic bases of gene engineering. The scheme
recombinant DNA technology. Achievements of gene engineering, usage gene
engineering drugs in medicine.

➢ Value of genetics;
• To understand the gene function of microorganisms
• Microbes provide relatively simple system for studying genetic phenomenon and thus
useful to other higher organisms.
• Microorganisms are used for isolation and multiplication of specific genes of higher
organisms which is referred as gene cloning.
• Microbes provide many value added products like antibiotics, growth harmones etc.
Microbial genetics will be helpful to increase these products productivity by microbial
technology
• Understanding the genetics of disease causing microorganisms especially virus, will be
useful to control diseases.
• Gene transfer among the prokaryotes play major role in the spread of the genes in a
particular environment. Microbial genetics will be useful to study the gene transfer from
one organism to another
➢ Genetic engineering is the deliberate manipulation of DNA, using techniques in the
laboratory to alter genes in organisms.
• Microbial genetics is a subject area within microbiology and genetic engineering. It
studies the genetics of very small (micro) organisms; bacteria, archaea, viruses and some
protozoa and fungi. This involves the study of the genotype of microbial species and also
the expression system in the form of phenotypes.
➢ Achievements of gene engineering; Genetic engineering will have important effects on
agriculture, notably in the development of new vaccines and diagnostic reagents, in the
production of transgenic animals, and in the application of enzyme technology in food
production and processing.
➢ Gene engineering drugs in medicine: vaccines, Antivenoms, bacteria derived toxins,
Immunoglobulins, monoclonal antibodies, Allergens, blood products and clotting factors,
hormones such as insulin, growth hormone they are made from gene engineering.
➢ The scheme recombinant DNA technology
• The procedure of recombinant DNA technology involves the following steps:
 1. M. Treatment with restriction enzyme: The DNA from the microorganism is
extracted and then is cleaved by enzymes called restriction endonucleases to
produce mixture of DNA fragments.

2. Southern blot: The fragment containing the desired gene is isolated from the
mixture of DNA fragments. This is done by:
− Electrophoresis DNA fragments are electrophoretically separated by subjecting
to agar gel electrophoresis .
− Transfer to nitrocellulose membrane. The separated DNA fragments are
transferred from the gel to a nitrocellulose membrane
− Detection of desired gene: The DNA fragment containing the desired gene is
detected adding a specific DNA probe, complementary to the gene of interest
− Isolation: The band containing the desired gene is isolated by DNA extraction
and then, is subjected to electrophoresis in a different gel.
3. Recombination with a vector: The isolated DNA fragment is annealed with a
vector by DNA ligase enzyme
4. Introduction of the vector into bacteria: The vector is introduced into bacteria
usually by transformation (injecting by electroporation) and rarely by phage vector
by transduction.
5. Cloning: Culture of the bacteria containing the desired gene followed by
expression of the gene products, yields a large quantity of desired protein.

48.Hereditary variation: mutations, their types

➢ Mutation is a change that occurs in our DNA sequence, either due to mistakes when the
DNA is copied or as the result of environmental factors such as UV light and cigarette
smoke.
• Mutations can be of many types, such as substitution, deletion, insertion, and
translocation.
➢ Mutations occur in one of the two ways:
1) Spontaneous mutations: Mutations that occur naturally in any dividing cells that
arise occasionally without adding any mutagen.
2) Induced mutations: These mutations on the other hand, are as a result of exposure
of the organism to a mutagen, an agent capable of inducing mutagenesis. Examples
of mutagens include
49.The types of mutagens (physical, chemical, biological). Methods of isolation of
mutants.
➢ Types of mutagens;
• Physical:
− Radiation
− Heat
• Chemical:
− Base analogs
− Alkylating agents
− intercalating agents
− Metal ions
• Biological
− Viruses
− Bacteria
− Transposons
➢ There are 4 Methods for isolation of mutants;
a. Replica Plating Technique
b. Resistance Selection Method
c. Substrate Utilization Method
d. Carcinogenicity Test
50.Reparations: photoreactivation and dark reparation. The mechanisms of
reparations and their importance
➢ Photoreactivation is a type of DNA repair mechanism present in prokaryotes, archaea and
in many eukaryotes. It is the recovery of ultraviolet irradiated damages of DNA by visible
light.
➢ Dark repair of DNA by a mechanism that does not require light. There are two general
classes of DNA repair;
• The direct reversal of the chemical process generating the damage.
• The replacement of damaged nucleotide bases
➢ The mechanisms of reparations and their importance
• They ensures the survival of a species by enabling parental DNA to be inherited as
faithfully as possible by offspring. It also preserves the health of an individual. Mutations
in the genetic code can lead to cancer and other genetic diseases.

51.Genetic recombination: transformation.

➢ TRANSFORMATION
• Definition
− Transformation is a process of random uptake of free or naked DNA fragment from
the surrounding medium by a bacterial cell and incorporation of this DNA fragment
into its chromosome in a heritable form.
− Natural transformation has been studied so far only in certain bacteria-
Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter and Pseudomonas

• Mechanism of Transformation
− When bacteria lyse, they release large amounts of dsDNA into the surrounding
environment. Their uptake depends up on the competency of the bacteria present
in the surroundings.

• Competency for Transformation

− Competent bacteria refers to the cells multiplying in log phase of cell division and
expressing certain transformation promoting factors called competence factors O
Bacteria expressing competence factors (e.g. S. pneumo niae) can uptake any DNA
fragment irrespective of source.
 − But competence factors are not expressed by all bacteria that mediate
transformation e.g. Haemophilus influenzae. In such case, the uptake of DNA occurs
only from the closely related species.
− The transformation [Frequency of very competent cells is around 10 for most
genera. Steps involved in transformation are as follows (Fig. 6.7):
i. A long dsDNA fragment comes in contact with a competent bacterium and
binds to DNA-binding protein present on its surface and then it is nicked by a
nuclease.
ii. One strand is degraded by the recipient cell exonuclease
iii. The other strand associates with a competence specific protein and is
internalized, which requires energy expenditure
iv. The single strand enters into the cell and is integrated into the host
chromosome in place of the homologous region of the host DNA.

52.Genetic recombination: transduction

➢ TRANSDUCTION
• Definition
− Transduction is defined as transmission of a portion of DNA from one bacterium to
another by a bacteriophage (bacteriophage is a virus that infects and multiplies
inside the bacterium).
• Mechanism of Transduction
− During the transmission of bacteriophages from one bacterium to other, a part of
the host DNA may accidentally get incorporated into the bacteriophage and then
gets transferred to the recipient bacterium. This leads to acquisition of new
characters by the recipient bacterium coded by the donor DNA. Bacteriophages can
perform two types of life cycle inside the host bacteria.
1) Lytic or virulent cycle: Bacteriophage multiplies in host cytoplasm, produces
large number of progeny phages, which subsequently, are released causing
death and lysis of the host cell.
2) Lysogenic or temperate cycle: In contrast to virulent cycle, here the host
bacterium is unharmed. The phage DNA remains integrated with the bacterial
chromosome as the prophage, which multiplies synchronously with bacterial
DNA. However, when the phage DNA tries to come out, it is disintegrated from
host chromosome, comes out into the cytoplasm, and behaves as a lytic phage.
 It replicates to produce daughter phages, which are subsequently released by
host cell lysis.

• Types of Transduction
− Transduction is of two types, either generalized or restricted

• Generalized Transduction
− It involves transfer of any part of the donor bacterial genome into the recipient
bacteria. Generalized transduction usually occurs as a result of defective assembly
during the lytic cycle of virulent and some temperate phages.
− Packaging errors may happen occasionally due to defective assembly of the
daughter phages. Instead of their own DNA, a part of host DNA may accidentally be
incorporated into the daughter bacteriophages
− The resulting bacteriophage (called transducing phage) often injects the donor DNA
into another bacterial cell but does not initiate a lytic cycle as the original phage
DNA is lost
− The donor DNA may have three fates inside the recipient bacterium

• Abortive transduction: About 70-90% of the transferred DNA is not integrated with the
recipient bacterial chromosome, but often is able to survive and express itself. Such
bacteria containing this non-integrated, transduced DNA are called abortive
transductants

• Stable gene transfer: The donor DNA gets integrated with recipient bacterial
chromosome

• Unstable gene transfer: In some cases, the donor DNA gets disintegrated by the host
cell enzymes.

• Restricted or Specialized Transduction

− In contrast to generalized transduction, the restricted transduction is capable of
transducing only a particular genetic segment of the bacterial chromosome that is
present adjacent to the phage DNA.
− It occurs as a result of defect in the disintegration of the lysogenic phage DNA from
the bacterial chromosome. Restricted transduction has been studied intensively in
the 'lambda' phage of E. coll
 − When a prophage (le lysogenic bacteriophage is integrated with the bacterial
chromosome) leaves the host chromosome, portions of the bacterial chromosome
present adjacent to the phage DNA may get wrongly excised along with it
− Such transducing phages carrying a part of bacterial DNA in addition to their own
DNA, when infect another bacterium, the transfer of the donor DNA takes place in
two ways
1) 1.The entire transducing genome (L.e. phage DNA + donor DNA) acts as a
prophage and gets integrated to the recipient chromosome. This occurs if
the recipient bacterium is already infected by another helper bacteriophage
2) Crossover between the donor DNA and a part of recipient DNA-
leads to integration of the donor DNA into the recipient
chromosome and a part of recipient DNA into the phage DNA.

• Role of Transduction
− In addition to chromosomal DNA, transduction is also a method of transfer of
episomes and plasmids.

o Drug resistance: Transduction may be a mechanism for the transfer of bacterial

genes coding for drug resistance; for example, plasmid coded penicillin
resistance in staphylococci
o Treatment: Transduction has also been proposed as a method of genetic
engineering in the treatment of some inborn metabolic defects.
53. Genetic recombination: conjugation.
Answer :
• It occurs between two different but related viruses of the same family infecting a host cell
simultaneously.
• The two viruses exchange segments of nucleic acids between them so that a hybrid
(recombinant virus) results.
• Such hybrids possess new genes not found in both die parent viruses, are genetically stable
and able to replicate.
• Conjugation refers to the transfer of genetic material from one bacterium (donor or male)
to another bacterium (recipient or female) by mating or contact with each other and
forming the conjugation tube and it is also called conjugation bridge.
• Types : F+ X F- matting , HRF conjugation , F’ conjugation , colicinojenic
 54.Pathways of penetration of causative agents into the organism. Concept about routs of
diseases transmission, portals of entry and their connection with the duration of the
incubation period.
Answer:

• Pathways of penetration of causative agents into organism are: transmission by Contact (direct and/or
indirect), Droplet, Airborne, Vector and Common Vehicle, fomites ,oral
• The portal of entry is the means by which the infectious microorganisms gains access into the new host.
This can occur, for example, through ingestion, breathing, or skin puncture, mucus membrabe ,
gastrointestinal tract
• The specific incubation period for a disease process is the result of multiple factors, including:
o Dose or inoculum of an infectious agent
o Route of inoculation
o Rate of replication of infectious agent
o Host susceptibility
o Immune response

55.Pathogenicity of microorganisms. Factors of microbial virulence, their characteristics

Answer :
 • Pathogenicity is the ability to produce disease in a host organism. Microbes express their pathogenicity by
means of their virulence, a term which refers to the degree of pathogenicity of the microbe.
• Factors that are produced by a microorganism and are called virulence factors. Virulence factors are
molecules produced by bacteria, viruses, fungi, and protozoa that add to their effectiveness and enable
them to achieve the following:
o Colonization of a niche in the host (this includes attachment to cells)
o Immunoevasion, evasion of the host's immune response
o Immunosuppression, inhibition of the host's immune response
o Entry into and exit out of cells (if the pathogen is an intracellular one)
o Obtain nutrition from the host

56.Methods of virulence determination and units its activity

Answer :

• There are three methods :

• Method 1 is based on homology searches of known virulence factor-encoding genes in the same or other
species
• Method 2 identifies genes that are likely to result from lateral gene transfer (such genes are often localized
in pathogenicity islands)
• Method 3 identifies virulence genes by comparing the genomes of strains with different pathogenicity
profiles.

57.Bacterial protein exotoxins. Properties, chemical structure. Determination of potency of

exotoxin. Their role in the pathogenesis of diseases.
Answer :

• exotoxin is a toxin secreted by bacteria and can cause damage to the host by destroying cells or disrupting
normal cellular metabolism.
• Type • Mostly Gram +
• Relation to microbe • By-products of growing cell
• Chemistry • Protein
• Fever? As symptoms • No
• Neutralized by antitoxin? • Yes
• There are three main types of exotoxins
1. superantigens (Type I toxins)
2. exotoxins that damage host cell membranes (Type II toxins); and.
3. A-B toxins and other toxin that interfere with host cell function (Type III toxins
• Chemical structure : Exotoxins are protein and nonprotein bacterial metabolites released from their
cytoplasm to the extracellular medium during their normal cell cycle function. Exotoxins are part of a
defensive system of bacteria to avoid capture and killing by leucocytes.
• ROLE : The capsule is considered a virulence factor because it enhances the ability of bacteria to cause disease
(e.g. prevents phagocytosis). The capsule can protect cells from engulfment by eukaryotic cells, such as
macrophages. A capsule-specific antibody may be required for phagocytosis to occur.
 58.Bacterial endotoxins. Properties, chemical structure, classification. Role in the pathogenesis
of infectious diseases.
Answer :
• Gram (-) cell wall have lipopolysaccharides (LPS) on their outer membrane. The lipid part of the LPS is called
endotoxin or lipid A. Lipid A is released upon bacterial cell death often causing fever and inflammation. At
high levels, endotoxins can cause hemorrhaging and septic shock.
• Type • Gram -
• Relation to Microbe • Outer membrane
• Chemistry • Lipid A
• Fever? As symptoms • Yes
• Neutralized by Antitoxin? • No
• Chemical structure : Endotoxin is a lipid-polisaccharide heteropolymer consisting of three elements: lipid A,
core oligosaccharide, and O-specific polysaccharide, also called antigen-O.
• Role in the pathogenesis of infectious diseases.

1. Coronary artery disease: Conditions in the coronary arteries are associated to the lipopolysaccharides
mainly from the bacteria called Helicobacter pylori and Chlamydia pneumoniae.
2. Crohn's Disease and Ulcerative Colitis: Although circulating endotoxin levels that are higher than
normal have been recorded in people suffering from Crohn's disease and in ulcerative colitis cases,
the role of endotoxemia in these diseases is unclear. High levels of lipopolysaccharides may result
from damage to the intestinal lining caused by the disease itself or they may be caused by
endotoxemia.
3. Cystic Fibrosis: Cystic fibrosis is a disease of the digestive system related to bacterial endotoxins,
which results from genetic defects in calcium channels that cause changes in the viscosity of the
intestinal lining and the lungs.

**VS**

59.The dynamics of development of infectious diseases.

Answer :

• The five periods of disease (sometimes referred to as stages or phases) include the
• Incubation : is the time from exposure to the causative agent until the first symptoms develop and is
characteristic for each disease agent.
• Prodromal : The prodromal period occurs after the incubation period. During this phase, the pathogen
continues to multiply and the host begins to experience general signs and symptoms of illness, which typically
result from activation of the immune system, such as fever, pain, soreness, swelling, or inflammation.
• Illness :
• Decline
• convalescence periods