UDC 579.

61(07)
Approved by: YSMU Foreign Students’ Educational and Methodological
Council by protocol N6,16.03. 2018; YSMU Educational and Methodological
Council of Theoretical Subjects by protocol N25, 21.06.2018; Recommended for
publishing by YSMU Academic Council by protocol N17, 26.12.2018

Authors: Marine S. Hovhannisyan, Gayane M. Poghosyan,

Susanna Yu. Zalinyan, Kristina H. Abgaryan,
Dianna M. Muradyan
Reviewers: Hasmik S. Hovhannisyan, DSC,
Professor of Epidemiology Department
Vigen A. Asoyan, PhD,
Associate Professor of Infectious Diseases
Department
Language
Editor: Meline N. Bisharyan, Department of Foreign
Languages
Computer Design: Avetisyan H. Manyak

Marine S. Hovhannisyan, Gayane M. Poghosyan, Susanna Yu. Zalinyan, Kristine

H. Abgaryan, Dianna M. Muradyan. Laboratory Manual on Medical
Microbiology (General part)/-Yerevan-YSMU, 2019, 158 pages

Knowledge and best practice in the field of Medical Microbiology are constantly
changing and this manual includes contemporary information about treatment, diagnosis
and prophylaxis of infectious diseases.
The Laboratory Manual on Medical Microbiology is intended for English speaking
students for higher education in the YSMU faculties of General Medicine, Stomatology
and Pharmacy.
This manual contains the following chapters: Morphology and Physiology of
Microorganisms, Microbial Ecology, Microbial Genetics, Infection, Antimicrobial
Medications, Immunodiagnostics, also tests with answers and list of abbreviations.

ISBN 978-9939-65-214-6 UDC 579.61(07)

©YSMU, Marine S. Hovhannisyan, 2019
©YSMU, Gayane M. Poghosyan, 2019
©YSMU, Susanna Yu. Zalinyan, 2019
©YSMU, Kristina H. Abgaryan, 2019
©YSMU, Dianna M. Muradyan, 2019
2
 Medical Microbiology, virology and immunology belong to
biomedical sciences, the knowledge of which is necessary to every
doctor and medworker, because it insures general biological
knowlege and at the same time as a propaedeutics direction it is used
in the practical sphere of medicine.
The important problems of General microbiology are: the
classification and nomenclature of microbes, the studying of
ultrastructure, morphology, physiology, genetics of pathogenic
microbes, their standards and peculiarities, their habitat in living
environment (ecology of microbes) and relationship with host
organism in norm and pathological conditions the role of distinct
species in etiology and pathogenesis of different infectious diseases,
the study of infectious processes and development of immune
mechanisms, laboratory diagnosis, prophylaxis, treatment methods
elaboration for infectious diseases.
There is well known saying, prevention is better than cure,
therefore, methods of prevention of infectious diseases by vaccines
have been described. However, if disease has established than an
early and accurate diagnosis is essential, and the the latist
diagnostic methods have been described in proper details.

3
anti-HGG - anti-human-gamma-globulin
anti-HGG - anti-human-gamma-globulin
atm. - atmosphere
CD - cluster of differentiation
CFT - complement fixation test
cm – centimeter
col-plasmid - plasmid for synthesis of colicins
CPE - cytopathic effects
CV - crystal violet
DFA - direct fluorescent antibody test
DLM - dosis letalis minima
DL50 - dosis letalis 50
DMT - dosis maxima tolerance
DMC - dosis minima curativa
E-test - epsilometer test
e.g. – for example
ELISA - enzyme-linked immunosorbent assay
EMB agar - eosin methylene blue
etc. - and so on
f-plasmid - plasmid for synthesis of conjugative (fertility) pili
FACS - fluorescence activated cell sorting
FITC - fluorescein isothiocyanate
(G+) - Gram positive
(G-) - Gram-negative
GK - Greek
h - Hours
HAV - hepatitis A virus
Hep-2 - human epithelioma of larynx cell line
4
HeLa – an immortal cell line derived from cervical
cancer in 1951 from Henrietta Lacks
Hfr - high frequency recombination
HIV – human immunodeficiency virus
I - iodine
IFA - indirect fluorescent antibody test
Ig - immunoglobulin
LPS - lipopolysaccharide
m 3 -
cubic meter
MHD - minimum hemolytic dose
MIC - minimum inhibitory concentration
min - minute
ml - milliliter
mm – millimeter
MPB - meat-peptone broth
MPA - meat-peptone agar
MPN - most probable number
MRVP test - methyl red, Voges-Proskauer test
MR - methyl red test
MSA - mannitol salt agar
MUG agar - 4-methylumbelliferone glucuronide agar
NAG - N-acetyl glucosamine
NAM - N-acetylmuramic acid
nm - nanometer
R-plasmid - resistance plasmid
R type colony - rough colony
RBC – red blood cell
rhu-Mabs - recombinant human monoclonal antibodies
Rh – Rhesus
S - for Svedberg ultracentrifuge
S type - smooth
5
sec. - second
TA - teichoic acid
TSI - triple sugar iron agar
tox-plasmid – plasmid for production of toxin
USMLE – United States Medical License Examination
UVL – ultraviolet ligth
UVR - ultraviolet radiation
V-P test - Voges-Proskauer
VDRL – Veneral Disease Research Laboratory
WI-38 - human embryonic lung cell strain
μm - micrometer
μg – microgram
μl - microliter

6
1.Bacteriological, virological, serological laboratories and organization of
laboratory work.
2.Microscopic techniques.
3.Demonstration of slides.
THE AIM OF THE LESSON:
1.to know the structure of microbiological laboratory.
2.to know the technique of immersion microscopy, phase-contrast and
dark-field microscopy, examination of microorganisms (on the slides).
METHODICAL INSTRUCTIONS:
1. Microbiological laboratory
Microbiological laboratories are included in the structure of each
big hospital and they routinely work with microbial cultures and
consequently must use rigorous methods of controlling microorganisms.
To work with pure cultures, all media and instruments that contact the
culture must first be rendered sterile to avoid contaminating the culture
with environmental bacteria. All materials used to grow microorganisms
must again be treated before disposal to the avoid contamination of
workers and the environment.
For cultivation of microorganisms, sterilization of culture media
and different tools, examination of specimen each laboratory has
different apparatus, tools, equipment (thermostat, microscopes, Pasteur’s
stove, autoclave, refrigerator, scales, distillatory vessel and so on).

7
 Laboratory facilities:
• Interior surfaces of the walls, floors and ceilings are water
resistant so that they can be easily cleaned.
• Bench tops are impervious to water and resistant to acids, alkalis,
organic solvents, and moderate heat.
• Windows in laboratory are closed and sealed.
• An autoclave for decontaminating laboratory wastes is available,
preferably within the laboratory.
Safety in microbiological laboratory
During the microbiology course the student should learn how to
handle safely microorganism containing fluids; during the practice be
able to perform experiments so that bacteria and viruses remain in the
desired containers. The use of specific methods to exclude
contaminating microorganisms from the environment is called aseptic
technique, which is the vital part of the work.
The rules of work in the microbiological laboratory:
1. wear white gown, tie long hair back;
2. each student must have his/her constant place;
3. do not eat, drink, smoke, store food or apply cosmetics in the
laboratory;
4. don not perform unauthorized experiments;
5. do not use equipment without instruction;
6. the duty student delivers material for work to each student and
then collects material back in the presence of the teacher;
7. sterilize every instrument contaminated with microbes;
8. disinfect spilled microbial culture on the table, floor with 2% of
chloramines solution;
9. wash hands after every laboratory work.
2. Microscopes and technique of microscopy
Virtually all organisms studied in microbiology cannot be seen
with the naked eye, but require the use of optical systems for
magnification. The morphological study of bacteria requires the use of
microscopes. Microscopy has come a long way since Leeuwenhoek first

8
observed bacteria three hundred years ago using hand ground lens. The
following types of microscopy are being employed now:
1)Light microscopy In light microscopy, light typically passes through a
specimen and then through a series of magnifying lenses. The most
common type of light microscope, and the easiest to use, is the bright-
field microscope, which evenly illuminates the field of view. Bacteria
may be examined in the living state or after fixation and staining.
Immersion system of microscopy is used in the microbiological
laboratory. Oil immersion lens has the highest magnification (97 X to
100X) and must be used with immersion oil. When immersion oil is
placed between the slide and the oil immersion lens, the light ray
continues without refraction because immersion oil has the same
refractive index (N=1.52) as glass (N=1.52). The result of using oil is that
light loss is minimized, and the lens focuses very close to the slide.
2)Phase-contrast microscopy exploits the difference between the
refractive index of cells and surrounding medium, resulting in a darker
appearance of the denser material. As light passes through cells, it is
refracted slightly differently than when it passes through its
surroundings. Special optical devices amplify those differences, thereby
increasing the contrast.
3)Dark field microscopy Organisms viewed through a dark-field
microscope stand out as bright objects against a dark background. A
special mechanism directs light toward the specimen at an angle, so that
only light scattered by the specimen enters the objective lens. Dark-field
microscopy can detect members of the genus Treponema. These thin,
spiral-shaped organisms stain poorly and are difficult to see via bright-
field microscopy.
4)Fluorescence microscopy takes advantage of the fluorescence of
substances. Fluorescence substances absorb short wavelengths of light
(ultraviolet) and give off light at a longer wavelength that can be seen by
the use of special light filters. Some organisms fluoresce naturally under
special lighting; if the specimen to be viewed does not naturally
fluoresce, it is stained with one of a group of fluorescent dyes called
fluorochromes. When microorganisms stained with a fluorochrome are
9
examined under a florescence microscope with an ultraviolet or near-
ultraviolet light source, they appear as luminescent, bright objects
against a dark background. The principal use of fluorescence microscopy
is a diagnostic technique called the fluorescent - antibody technique, or
immunofluorescence.
5) Electron microscopy In the electron microscope a beam of electrons is
employed instead of the beam of light used in the light microscope. The
object which is held in the path of the beam scatters the electrons and
produces an image which is focused on a fluorescent viewing screen.
Resolving power is about 0.1nm. Microscope is a very important tool for
a microbiologist to examine the shape, structure, size of microorganisms.
6)Ocular-micrometer and object-micrometer for measure the size of
microorganisms.
Preparation of microscope for work:
• raise the condenser up to the stage, open the diaphragm until
the light just fills the field;
• settle the light with a mirror or a lamp;
• place a drop of immersion oil in the center of the slide, put it on
the stage. Focus with 90O objective;
• under the eye control move the body tube with macro knob for
moving the oil immersion lens into position;
• find an image, then focus with the fine adjustment knob;
• when observation is completed move the turret to bring a low-
power objective into position;
• clean the oil of the objective lens with paper saturated with
alcohol.

10
1.Technique and stages of smear preparation.
2.Simple staining.
3.Differential (complex) staining.
THE AIM OF THE LESSON:
1.to prepare smears from bacterial culture (St. aureus and E. coli), stain
them with simple staining (fuchsine and methylene blue), microscope
them and draw.
2.to have some notion about aniline dyes.
METHODICAL INSTRUCTIONS:
1.Smear preparation
1. Clean the glass microscope slide with abrasive soap or cleanser and
remove it by cotton (slide must be grease-free).
2. Place 1 or 2 drops of physiological solution in the center of the slide,
using the inoculating loop.
3. Sterilize the inoculating loop by holding it in the hottest part of the
flame until it is red hot. The entire wire should get red. Allow the
loop be cooled, so microbes won't be killed. The loop that is too hot
will spatter the medium and move bacteria into the air.
4. Using the cooled loop scrape a small amount of the bacterial culture.
Emulsify the cells in the drop of physiological solution, and spread
the suspension. The smear should look like diluted milk. Flame the
loop again in order to kill bacteria. In case of broth culture do not use
physiological solution, because the bacteria are already suspended in
water. Place 2 or 3 drops of the culture on the slide. Spread the
culture. Flame the loop.
5. Let the smear dry.
6. Fix the smear by one of the following methods:
a)pass the slide quickly through the blue flame two or three
times (thermal fixation)
11
 b)cover the smear with 95% methyl alcohol for 1 min (chemical
fixation).
7. Stain the smear (Dye is applied and washed off with water).

2.Simple staining
It can be difficult to observe living microorganisms with the
bright-field microscope. Most are nearly transparent. Consequently,
cells are frequently immobilized and stained with dyes.
Staining procedures that use only one stain are called simple
stains. It is easy way to increase the contrast between otherwise
colorless cells and a transparent background. Simple staining can be used
to determine cell morphology, size and arrangement and can't be used
for detail examination of the structure of bacteria. Stained smear must
be washed with water, dried, examined microscopically.
Most stains in microbiology are synthetic aniline dyes derived
from benzene. The dyes are usually salts, although a few are acids and
bases, composed of charged colored ions. Basic dyes, which carry a
positive charge, are more commonly used for staining than the
negatively charged acidic dyes. Because opposite charges attract, basic
dyes stain the many negatively charged components of cells, including
nucleic acid and many proteins, whereas acidic dyes are repelled.
Common basic dyes include methylene blue, crystal violet, safranin and
malachite green. Acidic dyes are sometimes used to stain backgrounds,
against which colorless cells can be seen, a technique called negative
staining.
3.Differential (complex) staining
Differential staining techniques are used to distinguish one
group of bacteria from another. They take advantage of the fact that
certain bacteria have distinctly different chemical structures in some of
their components. These lead to differences in their staining properties.
The two most frequently used differential staining techniques are the
Gram stain and the acid-fast stain.

12
1.Spherical, rod shaped and spiral bacteria.
2.Microscopic examination of stained smears of cocci, rods, spiral
bacteria.
THE AIM OF THE LESSON:
1.to know the basic shapes of bacteria and their arrangement.
2.to examine microscopically spherical, rod shaped and spiral bacteria
and to draw them.
METHODICAL INSTRUCTIONS:
1.Shapes of bacteria
Depending on the shape, bacteria are classified into several
varieties:
1.Cocci (from kokkos, meaning berry) are spherical or oval cells.
2.Rods (bacilli - from bacillus, meaning rod ) are cylindrical cells.
3.Spiral shape, in which 3groups are described –vibrio, spirilla,
spirochaete (figure 1.1).
Cocci – (GK-coccus-berry) - cocci are subdivided into six groups accor-
ding to cell arrangement and cell division:
1.Micrococci - are arranged singly or irregularly
2.Diplococci - are arranged in pairs
3.Streptococci (GK–streptos-chain)- are arranged in chains
4.Tetracocci (GK–tetra–four) - form groups of fours
5.Sarcinae (GK-cartio-to tie) - resemble packets of 8, 16 or more cells
6.Staphylococci (GK–staphyle–cluster of grapes) - resemble clusters of
grapes
Rods - rod-shaped or cylindrical forms are subdivided into:
Bacteria - include those micro-organisms which as a rule do not produce
spores - (e.g. organisms responsible for enteric fever, dysentery,
diphtheria, etc.)
Bacilli and Clostridia - include organisms the majority of which produce
spores. Bacilli possess aerobic type of respiration (anthrax bacilli);
13
clostridia possess anaerobic type of respiration (Cl. tetani, Cl. botulinum
etc.).
Rods exhibit differences in the form of ends, size, arrangement and
spore-formation.
Spiral shape bacteria – There are three groups:
Vibrios which possess one coil, which is ¼ part of the whole twist
(Vibrio cholerae)
Spirilla (GK-speira-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 humans,
transmitted through the bite of rats and other rodents (Sodoku).
Spirochaetes (GK-speira-coil, chaite- filament) which are flexible
organisms with different quantity of twists.

Figure 1.1 Bacterial shapes

Cells have a variety of other shapes. Bacteria that

characteristically vary in their shape are called pleomorphic or
polymorphic (pleo-meaning many and morphic referring to shape).
14
1.Structure of bacterial cell.
2.Structure, chemical composition and functions of capsule.
3.Methods of capsule revealing.
THE AIM OF THE LESSON:
1.to know chemical composition, structure, basic functions of capsule.
2.to know methods of capsule revealing: Ionne staining method, Burry-
Hinss staining method, Quellung reaction.
METHODICAL INSTRUCTIONS:
1.Structure of bacterial cell
Bacteria differ essentially from plant and animal cells in structure. They
consist of cell membrane, cytoplasm, nucleoid and supplementary
structures – flagella, pili (fimbriae), spores, and capsule.
The cell membrane consists of three components:
The external layer – glycocalyx which may form a capsule
The middle layer – cell wall
The internal layer – cytoplasmic membrane
2.Structure, chemical composition and functions of capsule
Capsules vary in their chemical composition depending on the
species of bacteria. It is generally composed of polysaccharides (S.
pneumoniae, Cl. perfringens, N. meningitidis, Klebsiella) and in some
microorganisms it consists of polypeptides (Bacillus anthracis, Yersinia
pestis). Some bacteria can form capsule both in vivo and in vitro
conditions (K. pneumoniae, K. ozenae, K. rhinoscleromatis), which are
called capsulated bacteria, others form capsule only in the organism.
Capsule has several functions:
1.Protective - protection of microorganism from biological, physical,
chemical agents (phagocytosis, antibodies, antibacterial drugs, drying).
2.It is determinant of virulence of many bacteria. Loss of the capsule
may render the bacterium avirulent (St. pneumoniae).

15
3.Capsular polysaccharides ensure the antigenicity (superficial K-
antigen).
4.The capsule may play a role in the adherence of bacteria to human
tissues, which is an important initial step in causing infection.
3.Methods of capsule revealing
Capsule cannot be stained with ordinary stains like Gram
staining. It can be visualized by Ionne (figure 1.2), Burry-Hinss staining
methods and Quellung reaction (figure 1.3). The latter is a reaction of
capsular antigen with specific antibody which causes a characteristic
swelling of the capsule and allows rapid identification of capsular
serotypes of Streptococcus pneumoniae, Neisseria meningitidis etc.

Figure 1.2. Ionne staining method

Figure 1.3. Quellung reaction

16
Ionne staining method
1.Cover the smear with 2% acetic acid - 2min.
2.Wash with water.
3.Stain with 2% gentian-violet - 2min.
4.Wash with water, dry.
Microscopic view: violet bacteria are surrounded by pink capsule.

Burry-Hinss staining method

1.Place 2-3 loopful of microbial culture and black India ink on the slide
and mix.
2.Dry and fix by thermal method.
3.Stain by red dye (e.g. water fuchsine or safranin).
4.Wash with water, dry.
Microscopic view: Bacterial cell is stained red and capsule stands out
against the India ink-stained background as a halo around the organism.
Quellung reaction
Specific identification of an organism can be made by using antiserum
against the capsular polysaccharide. In the presence of the homologous
antibody, the capsule will swell greatly.

17
1.Structure, chemical composition and functions of the cell wall.
2.Gram staining of mixed smears prepared from gram-positive and
gram-negative bacterial culture.
THE AIM OF THE LESSON:
1.to know the chemical composition, structure, main functions of the
cell wall in gram positive and gram negative bacteria.
2.to know Gram staining and differentiate G(+) and G(-) bacteria.
METHODICAL INSTRUCTIONS:
1.Structure, chemical composition and functions of the cell wall
The cell wall is a multilayer rigid structure located external to
the cytoplasmic membrane. It is composed of a macromolecular network
called peptidoglycan (murein) which consists of N-acetyl glucosamine
(NAG) and N-acetylmuramic acid (NAM) which are combined with
glycosyl bonds. The enzyme lysozyme, which is present in human tears,
mucus and saliva, can cleave the peptidoglycan backbone by breaking its
glycosyl bonds, thereby contributing to the natural resistance of the host
to microbial infection.
NAM includes tetrapeptide side chains, which consist of four
amino acids: D-alanine, L-alanine, D-glutamine and diaminopimelic acid
which is found only in bacterial cell. These tetrapeptides are combined
with peptide bonds. Since peptidoglycan is present in bacteria but not in
human cells, it is a good target for antibacterial drugs (β-lactamates)
which suppress synthesis of peptide bonds.
The structure, chemical composition and thickness of the cell
wall differ in gram-positive and gram-negative bacteria (table 1.1).
Cell wall has several functions: it
1.Determines the shape of bacteria
2.Has protective meaning
3.Ensures the tinctorial properties (staining by Gram method)

18
4.Ensures virulence
5.Contains receptors for bacteriophages, bacteriocines and different
chemical structures (ensures attachment of bacteriophage to the host
cells)
6.Provides antigenicity (somatic O antigen)
7.Has taxonomic meaning
8.Participates in metabolism.

Gram-positive Gram-negative
Color of Gram-stained cell purple reddish-pink
Peptidoglycan thick layer thin layer
Teichoic acids present Absent
Periplasm absent Present
Outer membrane absent Present
Teichoic acids present absent
Endotoxin absent present
(lipopolysaccharide)
Porin proteins absent present
Sensitivity to penicillin more susceptible less susceptible
Table 1.1 Comparison of features of Gram-positive and Gram-negative
bacteria

2.Gram staining
The Gram stain is by far the most widely used procedure for
staining bacteria. That was discovered by Dr. Hans Christian Gram in
1884. It is a differential stain that allows to classify bacteria either Gram
positive (G+) or Gram-negative (G-), which reflects a fundamental
difference in the chemical structure of their cell wall: Gram-positive
bacteria are stained purple, and Gram-negative bacteria – reddish-pink.
The Gram stain procedure involves the following steps:
1.Prepare mixed smear from culture of St. aureus and E. coli. Dry. Fix.
2.Gram staining:

19
a)Cover the smear with the sheet of paper saturated with Gentian-violet
(crystal-violet). Moisten it with water - 1 min.
b)Take off the paper. Apply mordant Lugol’s solution (iodine) - 1 min.
c)Pour off stain. Apply ethyl alcohol (70%) - 0,5-1 min.
d)Rinse the smear with water.
e)Apply secondary stain fuchsine - 1 min.
f)Wash the smear with water, gently dry with a paper towel or
adsorbent paper.
Microscopic view: gram-positive bacteria (St. aureus) are stained purple,
gram-negative bacteria (E. coli) are stained reddish-pink (figure 1.4).

(a) (b)
Figure 1.4 Gram stain
(a) Steps in Gram stain procedure.
(b) Results of a Gram stain. G(+) cells (purple) are Staphylococcus
aureus, G(-) cells (reddish-pink) are E.coli.

Essence of Gram staining

Bacteria are stained differently because of chemical and physical
differences in the cell wall: G(+) cell wall consists of thick and
multilayer peptidoglycan. Gentian violet and iodine molecules enter the
peptidoglycan. The iodine (I) combines with the crystal violet (CV) and
teichoic acid (TA) of the cell wall to form a CV-I-TA complex, which is
saved in the cell wall and is not washed out during cultivation by
alcohol and these bacteria are stained purple (figure 1.5).

20
Figure 1.5 Gram positive cell wall

In Gram(-) cell wall peptidoglycan is thin and monolayer,

teichoic acid is absent and during cultivation by alcohol cell wall is
decolorized because CV-I complex is washed out through the thin
layer of peptidoglycan and red dye fuchsine stains the decolorized gram
negative cells red (figure 1.6).

(a) (b)
Figure 1.6 Gram negative cell wall
(a) Membrane of Gram negative bacteria.
(b) Components of an outer membrane of Gram negative cell wall.

21
1.Structure, chemical composition of the cytoplasm, cytoplasmic
membrane, nucleoid, ribosome, inclusions (volutin granules).
2.Methods of revealing of inclusions.
THE AIM OF THE LESSON:
1.to know the structure, chemical composition, main functions of
cytoplasm, cytoplasmic membrane, nucleoid, ribosome, inclusions
(volutin granules).
2.to know methods of revealing of inclusions: Neisser’s and Loffler’s
staining methods
METHODICAL INSTRUCTIONS:
1.Structure, chemical composition of the cytoplasm, cytoplasmic
membrane
Prokaryotic cell has some differences compare with eukaryotic
cell which is described in table 1.2.
Prokaryotic cell Eukaryotic cell
DNA within a nuclear No Yes
membrane
Mitotic division No Yes
Histone proteins No Yes
Chromosome number Single, circular DNA Multiple, linear DNA
Membrane bound organelles No Yes
(nucleus, mitochondria,
chloroplasts, endoplasmic
reticulum, Golgi apparatus,
lysosomes, peroxisomes)
Size of ribosome 70S 80S
Peptidoglycan containing cell Yes No
wall

Table 1.2. Comparison of prokaryotic and eukaryotic cell structures

22
 Cytoplasmic membrane is a thin structure lying inside the cell
wall and enclosing the cytoplasm of the cell. It consists of three layers:
bilayer phospholipids (15-30%) and the protein layer (50-70%).
Cytoplasmic membrane plays a vitally important role and the affection
of this structure can cause death of the cell (figure 1.7).

Figure 1.7 Cytoplasmic membrane

The cytoplasmic (plasma) membrane has several functions: it

1.participates in active transport of molecules into the cell
2.synthesizes precursors of the cell wall
3.secrets enzymes and toxins
4.provides osmotic pressure
5.participates in spore-formation
6.participates in cell division
7.participates in replication of DNA

Mesosome is the invagination of cytoplasmic membrane into the

cytoplasm which is connected with nucleoid and participates in the cell
division. They are the principal sites of respiratory enzymes in bacteria
and are analogous to mitochondria of eukaryotes in which energy
generation by oxidative phosphorylation occurs. They participate in
sporulation.
Cytoplasm consists of about 80% of water and contains primarily
proteins (enzymes), carbohydrates, lipids; inorganic ions are present in

23
cytoplasm. The major structures in the cytoplasm are DNA, ribosomes,
inclusions, plasmids.
Nucleoid (bacterial chromosome) of bacteria is a single circular
molecule of double-stranded DNA that contains all the genetic
information required by a cell.
Plasmids are extrachromosomal, double-stranded circular DNA
molecules that are capable of replicating independently of the bacterial
chromosome. Although plasmids are usually extra-chromosomal, they
can be integrated into the bacterial chromosome. Plasmids may be
gained or lost without harming the cell. Under certain conditions,
however, plasmids are an advantage to cells. Plasmids may carry genes
for antibiotic resistance (R-plasmid), tolerance to toxic metals,
production of toxins (tox-plasmid), synthesis of conjugative pili (f-
plasmid), synthesis of colicins (col-plasmid) etc. Plasmids can be
transferred from one bacterium to another. In fact, plasmid DNA is used
for gene manipulation in biotechnology.
Ribosomes are intimately involved in protein synthesis.
Prokaryotic ribosomes differ from Eukaryotic ribosomes in size and
chemical composition which is expressed as a distinct unit, S (for
Svedberg), that reflects how fast they move when they are spun at very
high speeds in an ultracentrifuge. The faster they move toward the
bottom, the higher the S value and the greater the density. Bacterial
ribosomes are 70S in size (with 50S and 30S subunits), whereas
eukaryotic ribosomes are 80S in size (with 60S and 40S subunits).
Several antibiotics work by inhibiting protein synthesis in the
ribosomes. Because of differences in prokaryotic and eukaryotic
ribosomes, the microbial cell can be killed by an antibiotic, while the
eukaryotic host cell remains unaffected.
Inclusions – The cytoplasm contains several different types of
granules (inclusions) that serve as storage areas for nutrients and energy:
glycogen, starch, sulfur, wax, polyphosphates (volutin granules). Some
inclusions serve as a basis for differentiation of the microorganisms; e.g.
volutin granules or Babesh-Ernst granules. For Corynebacterium
diphtheriae they have differential-diagnostic significance as they are
24
arranged strictly at poles of pathogenic Corynebacteria whereas in non-
pathogenic Corynebacteria they are either absent or are arranged on the
whole surface of the cell. Besides, volutin granules possess the property
of metachromazia (metachromatic granules) and they are stained
differently compared with the color of dye. The staining methods for
volutin granules are Neisser’s and Loffler’s methods.
2.Methods of revealing of inclusions
Neisser’s staining method:
1.Fixe smear and stain with Nasser’s vinegar sour blue - 1 min.
2.Wash with water.
3.Cover the smear with Lugol’s solution - 30 sec.
4.Stain with vesuvin or chrizoidin- 15 sec.
5.Wash, dry.
Microscopic view: cytoplasm of bacteria is stained light-brown, volutin
granules - dark-brown or black (figure 1.8).

Figure 1.8 Neisser’s staining method

Leoffler’s staining method is simple staining - Loffler’s blue is

used.
Microscopic view: cytoplasm of bacteria is stained light blue,
volutin granules - dark-blue or red.

25
1.Flagella. Examination of living microorganisms in "wet mount" and
"hanging drop" by phase-contrast microscopy. Microvilli.
2.Acid-fast staining. Ziehl-Neelsen procedure.
THE AIM OF THE LESSON:
1.to know the structure, chemical composition and functions of flagella
and pili.
2.to prepare "wet mount" and "hanging drop" and microscopy.
3.to know the chemical composition of acid-fast bacteria. Acid-fast
staining.
METHODICAL INSTRUCTIONS:
1.Flagellum is a long protein structure responsible for most types of
bacterial motility. It is composed of three basic parts. The filament is
composed of identical subunits of a protein called flagellin, which
extends into the exterior environment. The basal body anchors the
flagellum to the cell wall and cytoplasmic membrane. The hook is a
curved structure that connects the filament to the cell surface (figure
1.9).
Flagella have two important functions:
1.motility
2.antigenicity (flagellar H antigen)
By the number and location of flagella bacteria are classified into
the following groups:
monotrichous - bacteria having a single flagellum at one pole of the cell
(e.g. Vibrio cholerae);
amphitrichous - bacteria with two polar flagella or with a tuft of flagella
at both poles (e.g. Spirillum volutans);
lophotrichous - bacteria with a tuft of flagella at one pole (blue green
milk bacillus);

26
peritrichous - bacteria having flagella distributed over the whole surface
of their bodies (e.g. E. coli, Salmonella) (figure 1.9).

(a) (b)
Figure 1.9 Flagella (a) structure (b) location

Motility may be determined by observing bacteria by phase-

contrast microscopy, by "hanging drop" and "wet mount" preparations of
unstained bacteria.
Microvilli are hair like microfibrils, from1 to 1.5 μm in length and 4 to 8
nm in diameter, that extend from the cell surface. They are shorter and
straighter than flagella and are composed of subunits of a protein called
pilin, arranged in helical strands.
Microvilli are subdivided in two types:
1)Common which ensure enlargement of the bacterial surface and
participate in metabolism and gives antigenicity to bacteria
2)Specialized microvilli:
• Fimbria are those pili which enable attachment of bacteria to
specific receptors on the human cell surface, which is a
necessary step in the initiation of an infectious process for many
microorganisms. They are found mainly on gram negative
microorganisms.
• F-pili (sex pili, conjugative pili) form the attachment between
the male (donor) and the female (recipient) bacteria during
conjugation, a mechanism of DNA transfer from one bacterial
27
 cell to another. Typically, sex pili are somewhat longer than the
types of pili that mediate other characteristics.
2.Acid-fast staining. Ziehl-Neelsen procedure.
The acid-fast stain is a procedure used to stain a small group of
organisms that do not readily take up stains. Among these are
Mycobacterium tuberculosis and Mycobacterium leprae that are said to
be acid-fast, because they resist decolorization with acid-alcohol after
being stained with carbolic fuchsine. This property is related to the high
concentration of lipids in the cell wall: mycolic acids, wax (D-wax),
glycolipids, trehalose dimycolate (cord factor), sulfatides.
Acid-fast staining: Ziehl-Neelsen procedure:
a)Prepare and fix a smear.
b)Cover the smear with carbolic fuchsine. Heat it until vapors appear.
Repeat it 2-3 times.
c)Wash the slide.
d)Immerse smear into the 5% H2 SO4 for decolorizing.
e)Wash with water.
f)Counterstain with methylene blue for about 3-5 min.
g)Wash with water and blot dry.
Microscopic view: acid-fast bacteria are stained red and the surrounding
flora is stained blue (figure 1.10).

Streptococci
Staphylococci
M. tuberculosis

Figure 1.10 Acid-fast stain M. tuberculosis is acid-fast bacteria and is

stained red.

28
1.Spore.
2.Ozheshko procedure.
THE AIM OF THE LESSON:
1.to know the structure, chemical composition and function of spores.
2.to know the method of spore revealing: Ozheshko procedure.
METHODICAL INSTRUCTIONS:
1.Spore is an anabiotic form of the bacteria and is highly resistant
structure formed in response to adverse conditions by two medically
important Gram positive genera: Clostridium and Bacillus.
Sporulation in bacteria is not a means of reproduction: each
bacterium forms one spore, which on germination gives rise to one
vegetative cell; this is, therefore, preservation of the species (resting
stage).
Sporulation is a highly ordered sequence of changes that initiates
when cells are grown in low amounts of carbon or nitrogen. The stages
of sporulation:
1.formation of sporogene zone inside the bacterial cell. The process is
characterized by thickening of the cytoplasm in a certain region and
formation of a fore-spore.
2.formation of membranes: invagination of cytoplasmic membrane
which surrounds the sporogene zone by two layers. Between these two
layers peptidoglycan-containing material is laid down, forming the
cortex which includes dipicolinic acid, that is absent in vegetative cell.
Dipicolinic acid combines with calcium ions and this complex appears to
play a role in thermoresistance. This region externally is covered with
proteins, lipids, polysaccharides which are absent in vegetative cells and
form spore coat.
3.maturation of spores: the rest of the cell gradually disappears. Instead
of a vegetative cell, a mature spore, one-tenth of the size of parental cell,

29
is produced. Sporulation is completed within 18 to 20 hours (figure
1.11).
When conditions become favorable, the spore germinates: it
takes on water and swells. The spore coat and cortex then crack open
and a vegetative cell grows out within 4 to 5 hours.

Figure 1.11 The process of sporulation

Spore is stable to physical and chemical agents due to:

• multilayer membrane
• dipicolinic acid
• calcium compounds
• inactivation of enzymes
• absence of free water
Location of spore inside the bacteria has differential-diagnostic
meaning. Spores may locate:
1.centrally - in the centre of the cell (the causative agent of anthrax:
Bacillus anthracis);
30
2.terminally - at the end of the rod and hence the drum–stick
appearance (the causative agent of tetanus: Clostridium tetani);
3.subterminally - towards the ends and microbes take on the form of a
spindle (the causative agents of botulism, gas gangrene: Clostridium
botulinum, Clostridium perfringens).
2.Ozheshko procedure.
Spores are impermeable to most stains, they are thermo- and
acid resistant and for revealing of spores Ozheshko staining method is
used. Although these structures do not stain with a Gram stain, they can
often be seen as a clear smooth object within an otherwise purple-
stained cell (figure 1.12).
Spore stain by Ozheshko:
1.Prepare a thick smear from spore-forming bacteria. Dry, do not fix.
Add 0,5% hydrochloric acid solution (HCl) and heat it until vapors
appear (2min). HCl is for softening of spore membranes.
2.Wash with water, dry, fix.
3.Stain by Ziehl-Neelsen method (see at the end of Lesson 1.7).
Microscopic view: spores are stained red, vegetative cells - blue.

Figure 1.12 Spore stain by Ozheshko and Gram

31
1.Spirochetes. Observation of Treponema, Borrelia and Leptospira in
stained smears.
2.Rickettsia, Chlamydia, Mycoplasma, Actinomycete.
THE AIM OF THE LESSON:
1.to know the taxonomy, morphology and ultrastructure of spirochetes,
rickettsia, mycoplasma, chlamydia, actinomycete.
2.to define the spirochetes in stained smears.
METHODICAL INSTRUCTIONS:
1.Spirochetes– belong to the order Spirochaetales and family Spiro-
chaetaceae. Spirochetes vary in size from 5 to 500 μm in length. They
are elongated, motile, flexible bacteria. The body of Spirochete consists
of the hollow coil which is enclosed by the cytoplasmic membrane; the
outer membrane is the thin cell wall. All spirochetes are actively motile
and achieve their motility by means of two or more axial filaments. The
axial filaments are enclosed in the space between the outer sheath and
the body of the cell. One end of each axial filament is attached near a
pole of the cell on the disks - blepharoplasts. By rotating its axial fila-
ment the cell rotates in the opposite direction to move through liquids
like a corkscrew.
Generally spirochetes are free living organisms. They are found
in contaminated water, in swage, soil, and decaying organic material,
and within the bodies of humans and animals (conditionally pathogenic
- T. orale, T. buccalis, T. denticola, T. refringens).
Three pathogenic genera belong to this family: genus
Treponema, which includes Treponema pallidum, the cause of syphilis;
genus Borrelia, the cause of relapsing fever and Lyme disease; genus
Leptospira, the cause of leptospirosis.
These three genera are differentiated by the staining properties,
the number of twists, amplitude of twists (figure 1.13).

32
 Figure 1.13 Spirochetes

Borreliae are large spirochetes with irregular, wide, open coils,

the number of which varies from 3 to 10 (distance between them is 2 to
4 μm) and they stain blue-violet with the Romanowsky-Giemsa stain.
Borreliae are readily stained by ordinary methods and are Gram
negative. They have secondary spirals. Borreliae of medical importance
are Borrelia recurrentis- causative agent of relapsing fever, Borrelia
burgdorferi - causative agent of Lyme disease.
Treponemes (Greek: trepin- turn, nema-thread) exhibit thin, de-
licate, flexible cells with 8-12 twists (distance between turn 1 μm) and
tapering ends. They don't form secondary spirals. These spirochetes
stain pale-pink with the Romanowsky-Giemsa stain. A typical represen-
tative is the causative agent of syphilis - Treponema pallidum. Morozov
staining method also is used (silver impregnation method).
Leptospira (GK – leptos thin, speira -coil) are characterized by
20-30 coils set so close together that they can be distinguished only
under dark ground illumination in the living state or by electron
microscopy. They have secondary spirals which give them an S or C-like
appearance. They stain pinkish with the Romanowsky-Giemsa stain.
Spirochetes can be observed by the following methods:
• in native preparations in dark-field or phase contrast microscopy.
• staining by silver impregnation method (Morozov method).
• staining by Romanowsky-Giemsa method.
Microscopic view: by Romanowsky-Giemsa method Treponema stain
pale pink; Borrelia – blue-violet; Leptospira - pink-red.
33
2.Rickettsia, Chlamydia, Mycoplasma, Actinomycete.
Rickettsiae are polymorphic prokaryotic microorganisms. By shape they
can be:
Coccal shape (0,3-0,6μm)
Rod shape (1-2μm)
Filamentous (40μm)
Bacillar (3-4μm)
Rickettsiae are obligate intracellular parasites, because they are
unable to produce sufficient energy to replicate extracellularly.
Therefore, rickettsiae must be grown in cell culture, embryonated eggs,
or experimental animals. But, unlike viruses they have cellular structure
that is DNA, RNA, cell membrane, G (-) cell wall. Rickettsiae are non-
motile; do not produce spores and capsules. Rickettsiae divide by bynary
fission within the host cell. They stain well by the Giemsa stain (they
appear blue) and Zdradowski stain (they appear red). The diseases caused
by rickettsiae are known as rickettsiosis: epidemic and endemic typhus
fever, Q-fever, etc. (figure 1.14).

Nucleus

Rickettsia
Figure 1.14 Rickettsia

Chlamydiae are obligate intracellular parasites. They lack the

ability to produce sufficient energy to grow independently and therefore
can grow only inside host cells and cannot grow on artificial nutrient
media. They have prokaryotic cell structure. Chlamydiae are small, non-
motile, non-capsule forming bacteria. Their cell walls resemble those of
gram-negative bacteria but lack muramic acid. Chlamydia has a
replicative cycle different from that of all other bacteria (figure 1.15).
The cycle begins when extracellular, metabolically inert, “spore-like”
34
elementary body enters the cell and reorganizes into a larger,
metabolically active reticulate body (formerly also called the initial
body).

Figure 1.15 Replicative cycle of chlamydia

The latter undergoes repeated binary fission to form daughter

elementary bodies, which are released from the cell. Within cells, the
site of replication appears as an inclusion body, which can be stained
and visualized microscopically (figure 1.16). These inclusions are useful
in the identification of these organisms in the clinical laboratory.
Growth, reproduction and maturation of Chlamydia organisms are
completed in 40-72 hours. Chlamydiae are agents of psittacosis,
trachoma, lymphogranuloma venereum and other infections. They are
stained by Romanowsky-Giemsa method.

35
Figure 1.16 Chlamidiae inside the cell

Actinomycetes are true bacteria (related to Corynebacteria and

Mycobacteria), but they form long, branching filaments that resemble
the hyphae of fungi. They are gram-positive, but the peptidoglycan of
Actinomycetes contain arabinose, galactose, which are absent in
bacteria, but some are also acid-fast. There are two medically important
genera Streptomyces and Nocardia. Nocardia are common in soil. Some
species, such as Nocardia asterioides, occasionally cause chronic,
difficult-to-treat pulmonary Nocardiosis. Species of Streptomyces are
valuable because they produce most of our commercial antibiotics
(figure 1.17).

Figure 1.7 Actinomycetes

36
 Mycoplasmas are pathogens of the respiratory and genitourinary
tracts and joints. They are the smallest free-living organisms. The
smallest mycoplasmas are 125-250 nm in size. Their most striking
feature is the absence of a cell wall. Consequently, mycoplasma stain
poorly with Gram stain, and antibiotics that inhibit cell wall synthesis,
e.g., penicillin and cephalosporin, are ineffective, but they are inhibited
by tetracycline or erythromycin. Their outer surface is a flexible three-
layer cell membrane; hence, these organisms can assume a variety of
shapes. They can be grown in the laboratory on artificial media, but
they have complex nutritional requirements, including several lipids.
They grow slowly and require at least 1 week forming a visible colony.
The colony frequently has a characteristic “fried-egg” shape, with a
raised center and thinner outer edge (figure 1.18).

Figure 1.18 Colonies of mycoplasma

37
1.Definition of sterilization. Methods:
a) physical
b) mechanical
c) chemical
2.definition of disinfection.
3.definition of asepsis, antisepsis.
THE AIM OF THE LESSON:
1.to know the basic methods of sterilization.
2.to prepare the utensils and dishes for sterilization.
METHODICAL INSTRUCTIONS:
Sterilization is the killing or removal of all microorganisms, including
bacterial spores, which are high resistant.
Physical methods of sterilization
• Flaming is probably the most common method by which
microbes are killed in heat. It is used to sterilize inoculating
loop, forceps. Inoculating loop is held in the hottest part of the
flame (at the edge of the inner blue area) until it is red hot.
• Boiling - 100OC in 10 minutes kills vegetative forms of bacterial
pathogens, many viruses and fungi. Endospores and some
viruses, however, are not destroyed so quickly. One type of
hepatitis virus (e.g. HAV), can survive up to 30 minutes of
boiling, and some bacterial endospores resist at boiling
temperature for more than 20 hours. Boiling is therefore not
always a reliable sterilization procedure. However, boiling
makes food or water safe to eat or drink. Boiling method is used
for sterilization of syringes, surgical not sharp instruments, slides
and cover slips. If water supply is hard distilled water should be
38
 used, otherwise instruments become covered with film of
calcium salts.
• Hot-air sterilization (dry air) is performed in Pasteur's oven (hot
air oven). Generally, a temperature of about 165OC maintained
for nearly one hour ensures sterilization; at 180OC - 40 minutes.
This method of sterilization is used to sterilize different
glassware, forceps, scissors, scalpels, all glass syringes, Petri
dishes, tubes and pipettes, some pharmaceutical products such as
liquid paraffin, dusting powder, fats and grease). All glass ware
must be clean, perfectly dry and properly plugged and mouth
wrapped before being placed in the oven. The oven must be
allowed to cool slowly for about two hours before the door is
opened, since the glassware may crack due to sudden or uneven
cooling.
• Moist heat sterilization is performed under the pressure in
autoclave. Increased pressure raises the boiling point of water
and produces steam with higher temperature. Sterilization in an
autoclave is most effective when the organisms either contact
the steam directly or are contained in a small volume of aqueous
(primarily water) liquid. This is usually sufficient to kill all
organisms and their spores and render materials sterile.
Autoclaving is used to sterilize culture media, instruments,
dressings, intravenous equipments, applicators, solutions,
syringes, transfusion equipment, and numerous other items that
can withstand high temperature and pressure (table 2.1).

Pressure Temperature Sterilization

(atm.)
0.5 110oC 180min
1.0 120 C
o 45min
2.0 130 C
o 20min
Table 2.1 Relationship between the pressure and the temperature of
steam.
39
 • Sterilization with air-steam is performed under 100OC in Koch's
apparatus or in autoclave with open valve. For effective
sterilization fraction sterilization is used – at 100OC for 20-30min
during 3 days.
• Thindalization is the fraction sterilization under 58 - 60OC for 1
hour, during 5-6 days (for sera, vitamins and other thermolabile
products).
• Pasteurization. In pasteurization the temperature is maintained
at 50-63OC for 15-30min or 70-80OC for 5-10 min to kill
designated organisms that are pathogenic or cause spoilage. It is
used for food sterilization (milk, juice, alcoholic beverages-wine,
beer). Vaccines prepared from non-spore-forming bacteria may
be inactivated in a water bath at 60OC for an hour.
• Sterilization with ultraviolet radiation (UVR) is used for
sterilization of air in microbiological laboratories, boxes,
operation rooms, other closed environments (germicidal lamps).
Mechanical sterilization
• Filtration is the passage of a liquid or gas through a screen-like
material with pores small enough to retain microorganisms.
Filtration is used to sterilize heat-sensitive materials, such as
culture media, enzymes, vaccines and antibiotic solutions.
In recent years, membrane filters, composed of such substances
as cellulose esters or plastic polymers, have become popular for
an industrial and laboratory use.
• Desiccation. To grow and multiply, microbes require water. In
the absence of water - a condition known as desiccation -
microbes are not capable of growth or reproduction but can
remain viable for years. Then, when water is made available to
them, they can resume their growth and division. This ability is
used in the laboratory when microbes are preserved by
lyophilization. During lyophilization (freeze-drying), a
suspension of microbes is quickly frozen at temperature ranging
from 54OC to 72OC, and the water is removed by high vacuum
(sublimation). While under vacuum, the container is sealed by a
40
 high-temperature torch. The remaining powder like residue that
contains the surviving microbes can be stored for years. The
microbes can be revived at anytime by hydration with suitable
liquid nutrient medium.
Chemical methods of microbial control
• Disinfection is the killing of many but not all microorganisms on
the surfaces of objects. For adequate disinfection, pathogens
must be killed but some microorganisms and bacterial spores
may survive. Disinfectants vary in their tissue-damaging
properties from the corrosive phenol-containing compounds,
which should be used only on inanimate objects, to less toxic
materials such as ethanol and iodine, which can be used on skin
surfaces. Chemicals used to kill microorganisms on the surface of
skin and mucous membranes are called antiseptics.
• Antisepsis is chemical disinfection of the skin, mucous
membranes or other living tissues.
Asepsis is a complex of procedures that provide the absence of pathogens
on an object or area. Aseptic techniques are designed to prevent the
entry of pathogens into the body. Whereas surgical asepsis is designed to
exclude all microbes, medical asepsis is designed to exclude microbes
associated with communicable diseases.
Several chemical agents are used as antiseptics and disinfectants.
Chemicals vary greatly in their ability to kill microorganisms and are
class. Chemical agents act primarily by one of three mechanisms:
1.disruption of cell membranes: alcohols, detergents, phenols.
2.modification of proteins: chlorine, iodine, heavy metals, hydrogen
peroxide, formaldehyde, ethylene oxide, acids and alkalis.
3.modification of DNA: a variety of dyes not only stain microorganisms
but also inhibit their growth - gentian violet (crystal violet), malachite
green.
Some of the chemicals act by more than one mechanism.

41
1.Types and mechanisms of nutrition.
2.Culture media. Classification.
3.Bacterial growth in culture media.
4.Definition of inoculation and re-inoculation.
THE AIM OF THE LESSON:
1.to know different types of culture media (complex and simple media,
differential-diagnostic media, elective media, enrichment media).
2.to pour melted agar into the tube.
3.to prepare blood agar.
4.to inoculate and re-inoculate culture into the solid and liquid nutrient
agar.
METHODICAL INSTRUCTIONS:
1.Types and mechanisms of nutrition.
A constant exchange of compounds with the surrounding
environment is inherent in all organisms. To carry out the processes of
nutrition and reproduction certain conditions are necessary: presence of
food materials from which microbes synthesize the component parts of
their cell, and, by oxidation of different substances, receive the required
energy.
Bacterial metabolism is closely similar to the metabolism of the
higher organisms, but there are some differences:
1. Microorganisms haven’t digestive organs.
2. Assimilation and dissimilation are very intensive.
3. The whole surface of the bacterium cell participates in the
nutrition process.
4. Bacteria have an expressed adaptive ability to the nutritive
conditions of the environment.

42
 All organisms, including microbes, can be classified
metabolically according to their nutritional pattern – that is, their
source of energy and their source of carbon and nitrogen.
Bacteria are subdivided into 2 groups by their principal carbon
and nitrogen source (figure 2.1):

Figure 2.1 Classification of microorganism based on the

types of nutrition

43
 1. Autotrophs (self-feeders) - use inorganic carbon (carbon
dioxide).
2. Heterotrophs (feeders on others; GK-heteros-another) - require
an organic carbon source.
Heterotrophs can be:
a) saprophytes - feeders of death organic compounds
b)parasites -depend on hosts and classified into:
• obligate intracellular parasites (rickettsia, chlamydia, viruses)
• facultative parasites (conditionally pathogenic and pathogenic
microorganisms that can live without host and multiply on
artificial nutrient media).
Considering the energy source microorganisms can generally be
classified into 2 groups:
1. Phototrophs - use light as their primary energy source.
2. Chemotrophs - depend on oxidation-reduction reactions of
inorganic or organic compounds for energy.
Chemotrophs are classified into:
a)lithotrophs, which use inorganic donors of electrons (H2, NH3, H2S,
Fe2+)
b)organotrophs, which use organic compounds as donors of electrons.
Heterochemoorganotrophs are the most important
microorganisms in medical microbiology.
Besides peptones, carbohydrates, fatty acids and inorganic ele-
ments bacteria require the special substances - growth factors:
• vitamines
• amino acides
• purine, pyrimidine
• lipides - sterols (for mycoplasma - cholesterol)
• Fe-porphyrins (haem)
Microorganisms, which are able to synthesize growth factors by
themselves, are named prototroph; and microorganisms which are not -
able to synthesize growth factors by themselves are named auxotroph.
Entrance of nutrients into bacterial cell is a complex of physical-
chemical processes and several factors promote these processes: different
44
nutrient concentrations, molecular size, their dissolution in water and
lipids, pH of environment, penetration through membranes, etc.
There are four mechanisms (figure 2.2) of penetration of nutrients into
the cell:
1. Passive diffusion
2. Light diffusion (facile)
3. Active transport
4. Translocation of radicals

Figure 2.2 Mechanisms of nutrition

2.Culture media. Classification.

A nutrient material prepared for the growth of microorganisms
in a laboratory is called a culture medium. They are used in
microbiological laboratories to isolate bacteria and identify them, to
obtain toxins and antibiotics. The microbes that grow and multiply in or
on a culture medium are referred to as a microbial culture.
45
 Culture media should have:
• sufficient nutrients for the particular microorganism
• growth factors
• sufficient moisture
• optimal pH
• isotonic conditions
Culture media should be:
• absolutely sterile
• transparent
• at the proper temperature
The presence of the growth factors is very important.
Classification of nutrient media is carried out:
1.According to the consistence:
a)liquid
b)semisolid
c)solid (plate agar, slant agar, column agar)
To create solid and semisolid media it is necessary to add agar-
agar (a polysaccharide extracted from marine red algae) as a solidifying
agent (2-2,5% for solid media and 0,5-0,75% for semisolid media).
Agar is an ideal solidifying agent as it is bacteriologically inert (no
influence on bacterial growth), it remains solid at 37°C, and
is transparent.
In solid media bacteria may be identified by studying the colony
character, mixed bacteria can be separated and it is used for the isolation
of bacterial pure culture.
Agar slants are test tubes containing solid culture media that
were left at an angle while the agar solidified. Agar slants, like agar
plates (in Petri dish), provide a solid growth surface, but slants are easier
to store and transport than Petri plates. Agar is allowed to solidify in the
bottom of a test tube to make an agar deep or column agar is used to
grow bacteria that prefer less oxygen than is present on the surface of
the medium. Semisolid agar deeps can be used to determine whether a
bacterium is motile. Motile bacteria will move away from the point of
inoculation giving an inverted “fir tree” appearance.
46
2.According to its origin:
a)natural (vegetables, milk, egg, blood, serum are added )
b)artificial (meat-peptone broth – MPB, meat-peptone agar - MPA)
c)synthetic (includes amino acids, mineral salts, vitamins)
3.According to its composition:
a)simple - meat-peptone broth (MPB), meat-peptone agar (MPA)
b)complex - blood agar, sugar agar
4.According to the meaning:
a)common - MPB, MPA
b)special, elective or selective media are designed to suppress the growth
of unwanted bacteria and encourage the growth of desired microbes.
Selective media are the following:
• Roux’s media (coagulated serum), Loffler’s media (three parts of
serum and one part of sugar broth) are elective for C. diphtheria.
• Bile broth is elective for Salmonella.
• Selenite media is elective for Enterobacteria.
• Basic peptone broth (1% peptone water) is elective for V.
cholerae.
• Yolk salt agar, milk salt agar, MSA- mannitol salt agar, are
elective for Staphylococci.
c)differential-diagnostic media contain carbohydrates, proteins and
indicators which make easier to identify bacteria based on their
fermentative activities: proteolytic and sugarlytic.
Differential-diagnostic media are the following:
• Endo’s media consists of MPA, 1% lactose and Andrade’s
indicator (basic fuchsine decolorized by sodium hyposulfite).
• Levin’s media contains MPA, 1% lactose and decolorized methyl
blue.
• Ploskirev’s media contains MPA, 1% lactose, iodine, brilliant
green, salts of bile acids, indicator (neutral red dye).
• Ressel’s media is slant agar which contains glucose, lactose and
indicator.
• TSI - triple sugar iron agar contains glucose (0,1%), lactose (1%),
sucrose (1%), ferrous sulfate and indicator. TSI tube
47
 contains butt (poorly oxygenated area on the bottom) and
slant (angled well oxygenated area on the top). Indicator is
phenol-red. It is yellow in acidic condition and red under
alkaline condition. Ferrous sulfate is indicator of H2S formation
(figure 2.3).

Figure 2.3 Triple sugar iron agar

If lactose (or sucrose) is fermented, a large amount of acid is

produced, which turns the phenol red indicator yellow both in butt and
in the slant. Some organisms generate gas, which
produces bubbles/cracks on the medium.
If only glucose is fermented (1/10 of carbohydrates), small amount
of acid is formed, and the oxygen deficient butt turns yellow, but on the
slant the acid is oxidized to carbon dioxide and water by the
microorganism and the slant will be red (alkaline or neutral pH).
If neither lactose/sucrose nor glucose is fermented, both the butt and
the slant will be red. The slant can become a deeper red-purple (more
alkaline) as a result of production of ammonia from the oxidative
deamination of amino acids.

48
 If H2S is produced, the black color of ferrous sulfide is seen (table
2.2).
slant butt gas H2S microb
acid acig + - Escherichia, Klebsiella,
Enterobacter
alkaline acid - - Shigella, Serratia,
alkaline acid + + Salmonella, Proteus
alkaline alkaline - - Ps. aeruginosa
Table 2.2 Biochemical activity of different bacteria in triple sugar iron
agar
• MacConkey agar is a selective and differential media used for the
isolation and differentiation of non-fastidious gram-negative
rods, particularly members of the family Enterobacteriaceae and
the genus Pseudomonas. It contains MPA, 1% lactose, crystal
violet, bile salts, indicator (neutral red dye).
• EMB agar (eosin methylene blue) is a selective and differential
medium. It contains MPA, lactose, sucrose, eosin and methylene
blue. It is selective because the EMB dyes inhibit the growth of
gram-positive organisms, allowing the growth of gram-negative
bacteria. EMB is differential in that the colonies of lactose
fermenting organisms have different colors.
Above mentioned media are used for the differentiation of enteric
bacteria.
• Hiss’ row is universal differential-diagnostic media.
d)enrichment culture media enhance the growth of microorganisms. It
is helpful in isolating organisms from sources when the bacterium is
present in relatively small numbers (e.g.- sugar broth is used as an
enrichment media in staphylococcal sepsis).
3.Bacterial growth in culture media.
A single bacterium, supplied with the right nutrients and conditions,
will multiply on the solid medium in a limited area to form a colony, a
mass of cells descended from the original one. In other words, colony is
a visible accumulation of generation of one mother cell on solid nutrient
49
media. About 1 million cells are required for a colony to be easily visible
to the naked eye.

Figure 2.4 S and R types of colonies

The colony morphology can give initial clues to the identity of

the organism. Colony differ by their edges (smooth-S type, rough-R
type, jagged, wavy, curly), surface (concave, convex, smooth, rough),
size (small - 1-2mm, middle - 2-4mm, large - 5mm and more), consis-
tency (dry, mucous, homogenous), transparency and color (figure 2.4).
Color of colonies is provided by pigments which are lipid-
soluble or water-soluble. For example, colonies of Serratia marcescens
are red when incubated at 22OC due to
a production of lipid-soluble pigment.
Pseudomonas aeruginosa often
produces a water soluble greenish
pigment, which discolors the growth
medium. Pigment has protective
meaning for some microorganisms.
Microbes, depending on their
type of respiration, form diffuse
Figure 2.5 Types of growth in turbidity, pellicle or sediment when are
liquid nutrient media grown in liquid nutrient media (figure
2.5).
50
4.Transfer of bacteria: definition of inoculation and re-inoculation.
Aseptic technique.
In the laboratory bacteria must be cultured in order to facilitate
identification and to examine their growth and metabolism. Bacteria are
inoculated, or introduced into various forms of culture media in order to
keep them alive and to study their growth. Inoculation must be done
without introducing unwanted microbes, or contaminants, into the
media. Aseptic technique is used in microbiology to exclude
contaminants.
All culture media are sterilized,
or rendered free of all life, prior to use.
Sterilization is usually accomplished
using an autoclave. Containers of
culture media, such as test tubes or Petri
plates should not be opened before the
work.
Transfer and inoculation are
usually performed with a sterile (figure
2.6), heat-resistant, non-corroding, no-
chrome wire attached to an insulated
handle. When the end of the wire is
Figure 2.6 Sterilization of bent into a loop, it is called an
inoculating loop holding the inoculating loop; when it is straight, is
wire in a burner flame called inoculating needle. Re-
inoculation is a transfer of microbes
from one culture medium to another.

51
1.Isolation of pure culture of aerobic bacteria:
a)mechanical dilution method
b)biological method
THE AIM OF THE LESSON:
1.to isolate the pure culture of aerobic bacteria from a mixed sample.
2.to observe and describe the morphological, tinctorial, cultural
properties of microbes:
a)preparation of smears from the suspicious colonies, staining by Gram
method, microscopy.
b)re-inoculation on the surface of curve agar from suspicious colonies
for isolation of pure culture.
c)final identification of isolated culture.
METHODICAL INSTRUCTIONS:
In nature many different organisms including bacteria live
together as a mixed population, jointly contributing to numerous
activities and processes in their surroundings. In the laboratory bacteria
are isolated and grown in pure culture in order to study the functions of
a particular species: growth characteristics, pathogenicity, metabolism
and antibiotic susceptibility. Since bacteria are too small to separate
directly without sophisticated micromanipulation equipment, indirect
methods of separating must be used.
In 1880 Robert Koch prepared solid media, after which
microbiologists could separate bacteria by dilution and trap them on the
solid media. An isolated bacterium grows into a visible colony that is
generation of one mother cell.
There are 2 methods of pure culture isolation:
I. Method, based on the principles of mechanical dilution of
microorganisms in nutrient media:

52
 a) Pasteur’s method - cereal dilution of investigating material in
liquid nutrient media.
b) Koch’s method - cereal dilution of investigating material in
melted agar and followed cooling of agar in Petri dishes.
c) Drigalsky’s method - inoculation of investigating material on
the surface of solid nutrient media with the help of the spatula or
inoculating loop.
d) Clonal culture method - transferring of one bacterial cell with
micromanipulator into the nutrient media and obtaining of the
generation (clone).
II. Biological method - infecting of laboratory animals susceptible to
certain species of microorganisms.
The pure culture can be obtained:
• by using selective media
• by heating the investigating material at 80-100OC to destroy
vegetative bacterial cells. Only spores can survive extreme heat,
so this method is used to isolate the pure culture of spore-
forming bacteria.
• by treating the investigating material with 5% H2SO4 before
seeding. This procedure destroys all microbes, except acid-fast
bacteria and it is used to isolate the pure culture of them, for
example, M. tuberculosis.

Isolation of pure culture by Drigalsky’s mechanical dilution method

1st step - inoculation of the investigating material on the surface of solid
medium in a Petri plate by mechanical dilution for obtaining isolated
colonies. The streak plate is the most common isolation technique in use
today (figure 2.7). In the streak plate technique a loop is used to streak
the sample many times over the surface of a solid culture medium in a
Petri plate. By streaking the loop repeatedly over the agar surface the
bacteria fall of the loop one by one and are ultimately distributed over
the agar surface, where each cell develops into a colony.

53
Figure 2.7 Streak plate technique

2nd step –
• macroscopic observation of colonies (figure 2.8)
• microscopic investigation of microbes of suspicious colony
• re-inoculation of microbes from suspicious colony on the surface
of curved agar for isolating pure culture.

Figure 2.8 Colonies on the surface of Endo media

54
3rd step –
• identification of the isolated pure culture (figure 2.9) by
morphological, tinctorial (staining), cultural properties
• re-inoculation of the isolated pure culture into differential-
diagnostic media (Hiss’ variegated row) for pure culture
identification by biochemical properties

Figure 2.9 Pure culture

4th step – identification of the pure culture by biochemical

properties: sugar lysing and protein lysing activities (figure 2.10).
So, during the isolation of pure culture the isolated
microorganism can be distinguished by the following properties:
morphological, tinctorial, cultural, biochemical, antigenic, toxigenic etc.
Revealing of sensitivity to antibacterial drugs and phagotyping
are important steps after isolation of pure culture.

55
Figure 2.10 Steps of pure culture isolation of aerobe and facultative
anaerobe bacteria

56
1.The 3-rd and the 4-th days of isolation of pure culture of aerobic
bacteria. Biochemical activity of microorganisms:
a) sugarlytic activity.
b) proteolytic activity.
THE AIM OF THE LESSON:
1.to check the purity of isolated culture (preparation of smear and
staining by Gram method).
2.to identify isolated pure culture (inoculation in Hiss’ variegated row).
METHODICAL INSTRUCTIONS:
Enzymatic activity is widely used to differentiate bacteria. Some
bacteria catabolize sugars oxidatively, producing carbon dioxide and
water. Oxidative catabolism requires molecular oxygen (O2). Most
bacteria, however, ferment glucose without using oxygen. Fermentative
catabolism does not require oxygen but may occur in the presence of
oxygen. The metabolic end-products of fermentation are small organic
molecules, usually organic acids. Some bacteria are both oxidative and
fermentative; others are neither but obtain their carbon and energy by
other means.
Fermentation processes can produce a variety of end-products,
depending on the substrate, the incubation, and the organism. In some
instances large amounts of acid may be produced, while in others a
majority of neutral products may result. The MRVP test is used to
distinguish between microbes that produce large amounts of acid from
glucose and those that produce the neutral product acetoin. MRVP
medium is a glucose-supplemented nutrient broth used for the methyl
red (MR) test and the Voges-Proskauer (V-P) test. If an organism
produces a large amount of organic acid from glucose, the medium will
remain red when methyl red is added, indicating the pH is below 4,4. If
neutral products are produced, methyl red will turn yellow, indicating a
pH above 6.0. The production of acetoin is detected by the addition of
57
potassium hydroxide (KOH) and α-naphthol. If acetoin is present, the
upper part of the medium will turn red; a negative V-P reaction will
turn the medium light brown (figure 2.11).

Figure 2.11 MRVP test a) methyl red test b) Voges-Proskauer test

Biochemical activity of microorganisms is defined in a Hiss’

“variegated’’ row. It is row of tubes which are filled with MPB along
with 1% of different carbohydrates: glucose, lactose, saccharose,
maltose, mannit, to define sugarlytic activity. Andrade’s indicator (pH
indicator – neutralized fuchsine) is added in each tube. Microorganisms
taken from pure culture are inoculated into each tube which is
incubated in the thermostat for 24 hours at 37OC.
Acid and gas may be produced when microorganism metabolizes
carbohydrates. Acid formation changes the pH of media into acidic
(pH<4.5) and the pH indicator changes its color from yellow to pink. To
reveal gas formation an inverted tube (Durham’s tube) is used (figure
2.12). In the presence of gas the inverted tube is filled with gas and floats
on the surface of media.
Hiss’ serum sugars are used for certain fastidious organisms like
pneumococci, gonococci and Meningococci that require serum for
growth (it contains 3% of serum).
Instead of Andrade’s indicator phenol red indicator may be used
for revealing the fermentation of sugars, which is red (neutral) in an

58
uninoculated fermentation tube; fermentation that results in acid
production will turn the indicator yellow (pH of 6,8 or below).
Hiss’ media is largely used for identification of bacterial species
(figure 2.13).

Figure 2.12 Revealing of acid and gas in Hiss’ media

Figure 2.13 Differentiation of bacteria in Hiss’ media

59
 To define proteolytic activity a tube with MPB is used.
Fermentation of proteins is expressed by H2S, indole, ammonia
formation. For detection of H2S production an indicator is used which is
blotting paper saturated with plumbum acetate. A black precipitate
forms (PbS) due to the reaction of H2S with plumbum acetate.
Production of indole is the result of enzymatic removal of amino
group from tryptophan. Indole reacts with a chemical reagent turning it
to red color (indicator is a paper saturated with 12% of oxalic acid).
Ammonia production is revealed by lacmus, which is red and
turns to blue when pH changes to alkaline.
Gelatinase activity and peptonation of milk are also expressions
of proteolytic activity. To reveal this property tubes with gelatin and
milk are added in Hiss’ row. Liquefying of gelatin is detected as a funnel,
inverted fir-tree, layered liquefaction.
Isolated microorganism can be identified by the following
properties: morphological, tinctorial, cultural, biochemical, antigenic,
toxigenic etc.

60
1.Types of respiration.
2.Methods of the cultivation of obligate anaerobes.
3.Isolation of pure culture of obligate anaerobes.
THE AIM OF THE LESSON:
1.to demonstrate tools and culture media for anaerobes.
2.to know methods of cultivation of obligate anaerobes.
3.to prepare smear from the soil and stain it by Gram method.
4.to isolate pure culture of anaerobic bacteria from the soil by Weinberg
method.
METHODICAL INSTRUCTIONS:
1.Types of respiration.
Respiration in bacteria is a complex process which is
accompanied with the liberation of energy required by the
microorganism for the synthesis of different organic compounds.
According to the type of respiration all microbes can be
subdivided into:
• Obligate aerobes, which grow in the presence of oxygen, develop
well in an atmosphere containing 21 % of oxygen. They require
oxygen to grow because their ATP-generating system depends
on oxygen as the hydrogen acceptor (brucellae, tubercle bacilli,
Pseudomonas, etc). They grow on the surface of liquid media
and form pellicle.
• Facultative anaerobes, which utilize oxygen to generate energy
in the presence of it, but they also use the fermentation pathway
to synthesize ATP in the absence of sufficient oxygen (E. coli).
Growth is more rapid when oxygen is present because aerobic
respiration yields the most ATP of all these processes.

61
 • Obligate anaerobes, for which the presence of molecular
oxygen is a harmful growth-inhibiting factor, because they lack
either superoxide dismutase or catalase, or both. Obligate
anaerobes vary in their response to oxygen exposure; some can
survive but are not able to grow, whereas others are killed
rapidly, because of hydrogen peroxide accumulation
(Bacteroides, Cl. tetani, Cl. botulinum).
• Microaerophiles, which can develop well in the presence of 1%
(2-10%) of oxygen. Higher concentrations are inhibitory
(Helicobacter pylori).
• Aerotolerant organisms such as Clostridium histolyticum, can
survive and grow in presence of oxygen, but do not multiply.
• Capnophiles which metabolize well in the presence of 5-15%
CO2 (Helicobacter pylori) (figure 2.14).

Figure 2.14 Oxygen requirements of bacteria and their growth in liquid

nutrient media

62
2.Methods of the cultivation of obligate anaerobes.
For cultivation of obligate anaerobes special anaerobic
conditions are necessary, which are created by the following methods:
1)physical method - Anaerostat is used, which is an apparatus that is
equipped with air locks and is filled with inert gases (figure 2.15).
2)mechanical method - inoculation in the deep of solid media (in
column agar or in Vinial-Veon tubes).
3)chemical method - special substances or chemicals are used (pirogalol)
that adsorb oxygen.
4)biological method of Fortner - aerobes and anaerobes are inoculated
on the surface of one, hermetically, tightly capped Petri plate. The
aerobes grow at first, then - anaerobes.
For cultivation of anaerobic bacteria
special media, called reducing media
are used. These media contain
ingredients that chemically combine
with dissolved oxygen and deplete it:
a)Kitt-Tarozzi media – contains
MPB, 0.5-1% glucose and pieces of
organs of animals 0.5gram (liver,
brain, minced meat), that connect
Figure 2.15 Anaerostat oxygen. Before using, the tubes with
media are heated to remove the
solved oxygen, and then are covered with the sterile Vaseline.
b)Ceisler’s media – is sugar-blood agar
c)Wilson – Bler’s media – contains MPA, 1% glucose, Na2SO4 and FeCl3.
3.Isolation of pure culture of obligate anaerobes.
Pure culture isolation for anaerobes is performed by two methods
(Ceisler’s and Weinberg’s) and includes the following steps (figure 2.16):
1st step - inoculation of investigating material in Kitt-Tarozzi media
which is heated before inoculation
2nd step - re-inoculation of material from Kitt-Tarozzi media into
Ceisler’s sugar-blood agar (Ceisler’s mechanical dilution method) or in

63
Vinial-Veon tubes (Weinberg’s method-serial dilutions by physiological
solution) in order to have separate colonies

Figure 2.16 Isolation of pure culture of anaerobes by Weinberg’s and

Ceisler’s methods
3rd step –
• macroscopic observation of colonies
• microscopic investigation of microbes of suspicious colony
• re-inoculation of microbes from suspicious colony into Kitt-
Tarozzi media for getting pure culture.
4th step –
• identification of isolated pure culture by morphological,
tinctorial (staining), cultural properties
• re-inoculation of isolated pure culture into differential-
diagnostic media (Hiss’ variegated row) for pure culture
identification by biochemical properties
5th step – identification of pure culture by biochemical properties: sugar
lysing and protein lysing activities.
So, during the pure culture isolation the isolated microorganism
can be distinguished by the following properties: morphological,
tinctorial, cultural, biochemical, antigenic, toxigenic etc.
Revealing of sensitivity to antibacterial drugs and phagotyping are
important steps after the isolation of pure culture.
64
1.Cultivation of intracellular parasites.
2.Cell cultures. Methods of viral growth indication.
THE AIM OF THE LESSON:
1.to know methods of cultivation of viruses and other intracellular
parasites in cell cultures, in the organism of living animals and in
embryonated egg.
2.to know the methods of viral growth indication.
3.to determine viral reproduction by hemagglutination reaction.
METHODICAL INSTRUCTIONS:
1.Cultivation of intracellular parasites.
Viruses, rickettsia and chlamydia are obligate intracellular
parasites and cannot reproduce in artificial nutrient media.
Intracellular parasites can be cultured:
1. in the organism of laboratory animals
2. in embryonated eggs
3. in cell cultures (figure 2.17)

Cell cultures Embrionated egg

Laboratory animal

Figure 2.17 Methods of cultivation of intracellular parasites

65
Infection of animals - It is the earliest method of cultivation of viruses
causing human diseases. For this method different animals are used
(monkeys, rats, rabbits, mice, guinea pigs). It depends on the
susceptibility of the animal to different microbes. New-born animals are
chosen which are more susceptible to infectious diseases. Depending on
the type of the infectious disease animals may be infected by several
routes - intracerebral, subcutaneous, intravenous, intraperitoneal or
intranasal.
This method is used as a diagnostic procedure for identifying and
isolating a virus from a clinical specimen. Infection of animals is also
used to study pathogenesis of infectious diseases, immune response,
epidemiology and oncogenesis.
Embryonated eggs - The embryonated chicken egg was first used for the
cultivation of viruses by Goodpasture in 1931 (figure 2.18).

Figure 2.18 Cultivation of viruses in embryonated egg

The embryonated egg offers several sites for cultivation:

chorioallantoic membrane, amniotic cavity, allantoic cavity, etc. The
growth of the viruses in infected embryonated eggs may be indicated by
the death of embryo and presence of visible lesions (pocks) on the
membranes caused by hemagglutinin of virus.

66
2.Cell cultures. Methods of viral growth indication.
Human and animal tissues are used as tissue cultures. The idea of
cell culture dates back to the end of the nineteenth century, but it was
not a particular laboratory technique until the development of
antibiotics in the years following World War II. To prepare cells for
growth outside the body a tissue is removed from an animal and minced
into small pieces. The cells are separated from one another by treating
them with a protease enzyme, such as trypsin. The suspension of cells is
then placed in a screw-capped flask in a medium containing a mixture of
amino acids, minerals, vitamins, and sugars as a source of energy
(Hencs’, Hotinger’s, Eagle’s media, “199” medium) (figure 2.19). The cells
bathed in the proper nutrients attach to the bottom of the flask and
divide every several days, eventually covering the surface of the dish
with a single layer of cells, a monolayer.

a) b)
Figure 2.19 a) Eagle’s media b) Screw-capped flasks for cell culture

Types of cell cultures:

• Primary cell cultures – these are normal cells freshly taken from
the body and cultured. They are capable of only limited growth
in culture and cannot be maintained in serial culture. Common
examples of primary cell cultures are Rhesus monkey kidney cell
67
 culture, human amnion cell culture, chick embryo fibroblast.
Primary cell cultures are used for the isolation of viruses and
their cultivation for vaccine production.
• Diploid cell lines – human embryonic lung cell strain /WI-38/;
Rhesus embryo cell attain/HL-8/. These are cells of a single type
that retain the original diploid chromosome number and
karyotype during serial subcultivations for a limited number of
times. After about fifty serial passages, they undergo
“senescence”. These are widely used for culturing viruses that
require a human host.
• Continuous cell lines - these are cells of a single type, usually
derived from cancer cells that are capable of continuous serial
cultivation indefinitely. Examples are standard cell lines derived
from human cancer /HeLa/; human epitheloma of larynx cell
line /Hep-2/; etc. These cell lines may be maintained by serial
subcultivation or stored in the cold /-70oC/ for being used when
necessary. They are sometimes called “immortal” cell lines. One
of these, the HeLa cell line was isolated from the cancer of a
woman who died in 1951.
Detection of viral growth in cell culture is performed by the
following methods.
1.Cytopathic effect - many viruses cause morphological changes in cell
culture in which they grow. These changes can be readily observed by
the microscopic examination of cell cultures. These changes are known
as “cytopathic effects” (CPE) and the viruses causing CPE are called
“cytopathogenic viruses’’. Cytopathic effect may be manifested by the
degeneration of the cells (enteroviruses). Some viruses can form
syncytium (figure 2.20), simplast – giant multinucleated cells (measles,
mumps viruses etc.), herpes virus causes discrete focal degeneration, etc.

68
Figure 2.20. Syncytia

2.Metabolic inhibition (color test). In normal cell culture the pH of

medium turns acid due to cellular metabolism. If the investigating
material contains viruses, they grow in the cells of culture, cellular
metabolism is inhibited and acid is not produced. This can be made out
by the color of the indicator (phenol
red) incorporated in the medium.
3.Hemadsorption. When
hemagglutinin containing viruses
grow in cell cultures, their presence
can be indicated by the addition of
erythrocytes to the culture.
Erythrocytes will adsorb onto the
surface of the cells. This is known as
Figure 2.21 Hemadsorption “hemadsorption” (figure 2.21).
4.Patches or plaque formation. This is
the most common method for detecting and quantifying the amount of
virus present in any sample by adding a known volume of virus
containing solution to actively metabolizing stained cells.
The infection, lysis, and subsequent infection of surrounding
cells lead to a clear zone or plaque surrounded by the uninfected cells
(figure 2.22).

69
 Figure 2.22 Patches or plaque formation

Each plaque represents one virion, initially infecting one cell,

and so the number of virions in the original solution can be readily
determined. Plaque assay can be used with any virus that lyses their host
cell, including bacteriophage.
5.Hemagglutination. Some animal viruses clamp or agglutinate red blood
cells because they interact with the surfaces of the cells. This
phenomenon is called hemagglutination (figure 2.23). Hemagglutination
can be measured by mixing serial dilutions of the viral suspension with a
standard amount of red blood cells. The highest dilution showing
maximum agglutination is the titer of the virus.

Figure 2.23 Hemagglutination

70
6. Transformation. Tumor forming viruses (oncogenic) induce cell
“transformation” and loss of contact inhibition, so that growth appears
in a piled-up fusion producing “microtumors”.
7. Immunofluorescence. Fluorescent dyes can be attached to known
specific antibodies, which are then used to detect the presence of viral
antigen. This gives positive results earlier than other methods and,
therefore is applied in diagnostic virology.
8. Interference. The growth of a noncytopathogenic virus in cell culture
can be tested by subsequent challenge with a known cytopathogenic
virus. The growth of the first will inhibit the infection by the second
virus due to interference.
9. Formation of inclusion bodies. These are discrete areas of cells
containing viral proteins or viral particles. They have either
intranuclear or intracytoplasmic location (figure 2.24). Viral inclusions
are formed by rabies virus (Babesh-Negri bodies), smallpox virus
(Guarnieri’s inclusions), herpes viruses, and adenoviruses. Inclusion
bodies are revealed by methods of Romanowsky-Giemsa, Muromcev and
Turevich.

Figure 2.24 Intracytoplasmic inclusions

71
1.Structure of bacteriophage.
2.Interaction between bacteriophages and sensitive cells. Virulent and
temperate phages. Lysogeny.
3.The usage of phages. Isolation and titration of phages.
THE AIM OF THE LESSON:
1.to know the basic properties of phages.
2.to examine Petri plates with negative colonies of virulent and
temperate phages, to distinguish them.
3.to know the method of titration of phages.
METHODICAL INSTRUCTIONS:
1.Structure of bacteriophage.
Bacteriophages are viruses that infect bacteria. Most phages have
very complicated structure; are tadpole or spermatozoid shaped. They
consist of head, neck, tail and basal plate from each angle of which six
teeth and filaments are going out by which phage is adsorbed on the
bacterial cell (figure 2.25).

a) b)
Figure 2.25 (a) bacteriophage by electron microscope
(b) structural components of bacteriophage

72
 The nucleic acid DNA or RNA is contained in the hexagonal
head. The head is surrounded with protein membrane or capsid. The
neck is free of this membrane. The tail is composed of a hollow core
surrounded with protein membrane and forms the cylindrical symmetry
and the free filament of nucleic acid is hanged in this cylinder. The
lysozyme enzyme is contained in the basal plate. Phage breaks down a
portion of the bacterial cell wall due to lysozyme and penetrates
through it into the bacteria.
2.Virulent and temperate phages. Lysogeny. Interaction between
bacteriophages and sensitive cells.
How phages affect the cells they infect depends primarily on the
phage and based on it they are classified into:
• virulent or lytic phages, which go through productive life cycle
and exit by lysing the bacterium. An example is phage T4, which
infects E. coli.
In all lytic phage infections the phage nucleic acid enters the
bacterium while its protein coat remains on the outside. The interaction
of phage and bacteria is manifested in the following phases occurring in
succession (figure 2.26):
1. Attachment 2. Penetration 3.Reproduction: biosynthesis,
maturation 4. Release

Figure 2.26 Interaction of phage and bacteria

73
 • temperate phages (temperatus means controlled), which in
contrast to virulent bacteriophage do not cause lyses and death
of host cell. These ones penetrate into the bacterial cell and
integrate their DNA into the genetic material of the bacteria or
DNA replicates as a plasmid. The integrated phage is known as a
prophage (figure 2.27).
The prophage behaves like a segment of the host chromosome and
replicates with it. This phenomenon is called lysogeny, and a bacterium
that carries a prophage is called lysogenic bacterium. The phage DNA
often codes for proteins that modify the properties of the host and
lysogenic bacterium gets new properties: toxigenicity, capsule
formation, biochemical activity. This phenomenon is known as
lysogenic conversion or phage conversion. An example is lambda phage
(λ), which infects E. coli.

Figure 2.27 Interaction of temperate phage and bacteria

3.The usage of phages. Isolation and titration of phages.

Phages are used in prophylaxis, treatment and diagnosis of
certain infectious diseases based on the specificity between phage-
bacteria interaction. They are also used in genetic investigations as they
perform transduction.
Bacteriophage can be cultivated in liquid or solid cultures of
bacteria. The use of solid media makes possible to define the location of
bacteriophage by plaque-forming method. Host bacteria and
bacteriophages are mixed together in melted agar, which is then poured
into a Petri plate, containing hardened nutrient agar. Each
74
bacteriophage that infects a bacterium multiplies, and releases several
hundred new viruses. The new viruses infect other bacteria and more
new viruses are produced. Bacteria in the area surrounding the original
virus are destroyed, leaving a clear area, or plaque, against a confluent
"lawn" of bacteria (figure 2.28 a)). The lawn of bacteria is produced by
the growth of uninfected bacterial cells.

a) b)
Figure 2.28 Cultivation of bacteriophage (a) plaque-forming method
(b) clearing of broth

In liquid media the growth of phage is expressed by clearing of

broth (figure 2.28 b)).
Since the number of phages in a natural source is low, the
desired host bacteria and additional nutrient are added as an enrichment
procedure. After incubation bacteriophages can be isolated by
centrifugation of the enrichment media and by membrane filtration.
Filtration has been used for removing microbes from liquids for
sterilization since 1884, when Pasteur's associate Chamberland, designed
the first filter candle. Today filtration of viruses is done using membrane
filters with pore size usually 0,45 μm, that physically exclude bacteria
from the filtrate.

75
 The viral activity can be measured in the sample by performing
sequential dilutions of the viral preparation and assaying for the
presence of viruses (Appelman’s titration method). In the broth-clearing
assay, the endpoint is the highest dilution of virus (smallest amount of
virus) producing lyses of bacteria and clearing of the broth. The titer, or
concentration, that results in a recognizable effect is the reciprocal of
the endpoint. In the plaque-forming method (Gracia’s method) the titer
is determined by counting plaques. Each plaque theoretically
corresponds to a single infective virus in the initial suspension. Some
plaques may arise from more than one viral particle, and some virus
particles may not be infectious. Therefore, the titer is determined by
counting the number of plaques and multiplying by the reciprocal of the
dilution. For example 32 plaques in a 1:10² dilution is equal to 3.2 x 10³
p.f.u. (plaque-forming units).

76
1.Ecology and microbial ecology.
2.Normal microbiota.
THE AIM OF THE LESSON:
1.to know the types of relationships between the microorganism and
host organism.
METHODICAL INSTRUCTIONS:
1.Ecology and microbial ecology.
Ecology is the study of the relationships of organisms (plant and
animal, large and small) to each other and to their environment. Living
organisms interact with one another in symbiotic relationships, such as
commensalisms, mutualism, and parasitism. Organisms in a given area,
e.g. the community, interact with each other and non-living
environment, forming an ecological system, or ecosystem (major
ecosystems include oceans, rivers and lakes, forests, etc.).
The region of the earth inhabited by living organisms is called
biosphere. Within the biosphere, ecosystems vary both in biodiversity
(the number of species present and their evenness of distribution) and
biomass (the weight of all organisms present).
Microorganisms play a major role in most ecosystems, and many
ecosystems host microbes unique to themselves. The environment
immediately surrounding an individual microorganism, the
microenvironment, is most relevant to that cell, but because microbes
are so small the microenvironment is difficult to identify and measure.
Microbial ecology is the study of numerous interrelationships
between microorganisms and the world around them; how microbes
interact with organisms other than microbes, and how microbes interact
with the non living world around them. Interaction between

77
microorganisms and animals, plants, other microbes, soil, and our
atmosphere have far-reaching effects on our lives. We are all aware of
the diseases caused by pathogens, but this is only one example of the
many ways that microbes interact with humans. Most relationships
between humans and microbes are beneficial rather than harmful.
2.Normal microbiota.
A diverse microbial flora is associated with the skin and mucous
membranes of every human being from shortly after birth until death.
The human body, which contains about 1013 cells, routinely harbors
about 1014 bacteria (figure 3.1). The microorganisms that establish more
or less permanent residence without producing diseases are known as
normal microbiota. The normal microbial flora is relatively stable, with
specific genera populating various body regions during particular periods
in an individual's life. Microorganisms that may be present for a few
days or months are called transient microbiota. Human microflora is the
result of a mutual adaptation of micro and macro-organisms in the
process of evolution. Most bacteria of the normal and constant
microflora of the human body have adapted themselves to life in certain
parts of the body.
Microorganisms of the normal flora may aid the host (by
competing for microenvironments more effectively than such pathogens
as Salmonella or by producing nutrients the host can use), may harm the
host (by causing dental caries, abscesses, or other infectious diseases), or
may exist as commensals (inhabiting the host for long periods without
causing detectable harm or benefit). Even though most elements of the
normal microbial flora inhabiting the human skin, nails, eyes,
oropharynx, genitalia, and gastrointestinal tract are harmless in healthy
individuals, these organisms frequently cause disease in compromised
hosts. By most investigators viruses and parasites are not considered
members of the normal microbial flora because they are not commensals
and do not aid the host.

78
 Figure 3.1 Numbers of bacteria that colonize different parts of the body

Thus, the relationship between normal microbiota and a healthy

person may be classified as one of two types of symbiosis (the close
association between two organisms): commensalism or mutualism. In a
commensal relationship one organism is benefited and the other is
unharmed. For example, many of the bacteria living in the large
intestine are supplied with continual food and a constant temperature,
while the host is neither benefited nor harmed. In a mutualistic
relationship both organisms are benefited. Some bacteria that live on
and in our bodies receive a constant supply of nutrients, while they, in
exchange, supply something of benefit to us. For example, E.coli
synthesizes vitamin K and certain B vitamins for its human host.
A third kind of relationship is parasitism, when one organism
gains while the other is harmed. In a disease state the microorganism is a
parasite and is harming its human host. An organism like this is called a
pathogen.

79
 Some members of the normal microbiota become parasites under
certain conditions. These organisms are called opportunists.
Quantity of microorganisms for adults is approximately 1014
organisms and obligate anaerobes are predominating. Microorganisms of
normal microflora enfold in high hydrated exo-polysaccharide-
mucinous matrix forming a biological membrane, which is stable to
different influence.
Quantitative and qualitative composition of microbiota is
changed during the life and depends on sex, age, nutrition, etc. Besides,
hesitation of microbiota of human body depends on diseases and the use
of chemotherapeutic and immunologic remedies, antibiotics. Estimation
of quantitative and qualitative composition of microbiota gives a
possibility to find out disorders and conditions associated with it:
disbiosis or disbacteriosis (quantitative and qualitative changes in normal
microbiota).

80
1.Microflora of water. Safety of drinking water. Detection techniques of
coliforms.
2.Microflora of soil.
3.Microflora of air.
THE AIM OF THE LESSON:
1.to know the method of water testing.
2.to know the indicators of fecal contamination of soil.
3.to know the sanitary-demonstrating microbes of air.
METHODICAL INSTRUCTIONS:
1.Microflora of water. Safety of drinking water. Detection techniques of
coliforms.
Large numbers of microorganisms in water generally indicate
high nutrient levels in the water. Water contaminated by inflows from
sewage systems from biodegradable industrial organic wastes is
relatively high in bacterial counts. Some diseases can be transmitted
through water: Typhoid fever, cholera, leptospirosis, which are caused
by bacteria, hepatitis A which is caused by a virus. Water is unfavorable
environment for pathogenic and conditionally pathogenic bacteria and
survival period depends on their type, dose, temperature of the water,
presence of organic substances and the composition of saprophytes.
Endospores of Bacillus anthracis can survive for years; Salmonella,
Leptospira, Hepatitis A virus – for months; Shigella (causative agent of
dysentery), Vibrio cholera (causative agent of cholera), Brucella
(causative agent of brucellosis) – for days, weeks.
A primary concern regarding the safety of drinking water is the
possibility that it might be contaminated with any of wide variety of
intestinal pathogens. It is not feasible to test for all the pathogens,
because they occur in such small numbers that they might be missed by
sampling, that is why indicator organisms are checked to detect fecal
contamination of water. These are microbes that routinely found in
feces, survive longer than intestinal pathogens, and are relatively easy to
81
detect and enumerate. The most common group of bacteria used as
indicator organisms is total coliforms – lactose fermenting members of
the family Enterobacteriaceae, including E.coli. These are facultative
anaerobic, Gram-negative, rod shaped, non-spore-forming bacteria that
ferment lactose, forming acid and gas within 48 hours at 35°C. Although
total coliforms are routinely present in the intestinal contents of warm-
blooded animals, certain species can also thrive in soil and on plant
material. Thus, the presence of these organisms does not necessarily
imply fecal pollution. The most common fecal coliform is E.coli.
Established public health standards specify the maximum
number of coliforms allowable in each 100ml of water, depending on
the intended use of the water (for example, drinking, water-contact
sports, river etc.).
Coliforms can be detected and enumerated in the multiple-tube
technique and membrane filter technique.
In multiple-tube technique coliforms are detected in three stages
(figure 3.2). In the presumptive test, dilutions from a water sample are
added to lactose fermentation tubes, which can be made selective for
gram-negative bacteria by the addition of different substances.
Fermentation of lactose to gas is a positive reaction.

Figure 3.2 Principle of multiple-tube technique

82
 Samples from the positive presumptive tube at the highest
dilution are examined for coliforms by inoculating differential medium
in the confirmed test. A confirmed test can be done on MUG agar (con-
tains 4-methylumbelliferone glucuronide). Almost all strains of E.coli
produce the enzyme ß-glucuronidase which converts MUG to a fluores-
cent compound that is visible with an ultraviolet lamp (figure 3.3).

Figure 3.3 Fluorescence of E.coli colonies on MUG agar

The number of coliforms is determined by a statistical

estimation called the most probable number method. In the presumptive
test, tubes of lactose broth are inoculated with samples of the water
being tested. A count of the number of tubes showing acid and gas is
then taken, and the figure compared to statistical tables. The number is
the most probable number of coliforms per 100 ml of water (figure 3.4).

83
 Figure 3.4 Table of determination of most probable number

In the membrane filter technique water is drawn through a filter

with pore size of 0,45μm (figure 3.5).
Bacteria are retained on the filter,
which is then placed on a pad of
suitable nutrient medium. Nutrients
that diffuse through the filter can be
metabolized by bacteria trapped on the
filter. Each bacterium that is trapped on
the filter will develop into a colony
(figure 3.6). Bacterial colonies growing
on the medium can be counted for
100ml of water:
100 x number of coliform colonies
volume of sample filtered
Figure 3.5 Bacterial filter

84
 Sanitary microbiological condition of the drinking water is also
appreciated by:
Microbial number: quantity of mesophilic chemoorganotrophes in 1ml
of water (till 100).
Coli-titer: the minimum quantity of water which contains one E. coli
(>300ml).
Coli index: quantity of E. coli in one liter of water (<3).

Figure 3.6 Membrane filter technique

2.Microflora of soil.
The soil is one of the main reservoirs of microbial life. The most
numerous organisms in soil are bacteria. Typical garden soil has millions
of bacteria in each gram. The population is the highest in the top few
centimeters of the soil and declines rapidly with depth.
For vegetative forms of many bacteria soil is an unfavorable
environment. Human pathogens, which are mostly parasites, generally
find the soil an alien, hostile environment. Even relatively resistant
enteric pathogens, such as Salmonella species, have been observed to
survive for only a few weeks or months when introduced into the soil.
Most human pathogens that can survive in soil are endospore-forming
bacteria. For example, endospores of Bacillus anthracis, which causes
anthrax in animals, can survive in certain soils for decades before finally
85
germinating when ingested by grazing animals. Disposing of the body of
animal infected with anthrax requires care so that the soil is not seeded
with the endospores from the dead animal.
Clostridium tetani (the causative agent of tetanus), Clostridium
botulinum (the causative agent of botulism), Clostridium perfringens
(the causative agent of gas gangrene) are all endospore-forming
pathogens whose normal habitat is the soil. From soil they are
introduced into foods or wounds, where they grow and produce toxins.
Soil is the source of infections: wound infections, enteric infections, and
food-toxinfections by contaminating vegetables, fruits.
Presence of E. coli, Clostridium perfringens, Enterococcus
feacalis, Enterobacter and Citrobacter in the soil indicate the fecal
contamination of the soil. E. coli and Enterococcus fecalis (S. feacalis )
are indicators of fresh contamination; Enterobacter and Citrobacter are
indicators of non-fresh contamination. C.perfringens is the indicator of
old contamination.
Indicators of exact valuation (appreciation) of soil microbial
contamination are Coli (perfringens) titer, Coli (perfringens) index and
microbial number.
Coli (perfringens) index is the amount of microorganisms in 1
gram of soil.
Coli (perfringens) titer is the smallest mass of soil in which one
microorganism is revealed.
Microbial number is the total amount of microbes (quantity of
saprophytes, thermophiles and nitrifying bacteria) in 1 gram of soil.
“Clear” soils contain no more then 1-1,5 millions of bacteria in 1 gram.
The results always must be correlated according to the type of
the soil (structure of the soil, concentration of mineral and organic
substances, temperature, pH, humidity, concentration of carbon dioxide,
osmotic pressure, physical-chemical condition).
3.Microflora of air.
Air is not a favorable environment for microorganisms as it does
not contain enough moisture and nutrients to support their growth and
reproduction, and it is often exposed to sunlight which is harmful for
86
microbes. They occur in relatively small numbers in air when compared
with soil or water. There are vegetative cells and spores of bacteria,
fungi and algae, viruses and protozoan cysts in air. The sources of these
microbes may be soil, water, plant and animal surfaces. These organisms
may be either commensals or plant or animal pathogens.
The main source of airborne microorganisms is human beings.
Their surface flora may be shed at times and may be disseminated into
the air. Similarly, the commensal as well as pathogenic flora of the upper
respiratory tract and the mouth are constantly discharged into the air by
activities like coughing, sneezing, talking and laughing and may be the
reason of transmitting of many viral and bacterial airborne droplet
infections. The microorganisms are discharged out in three different
forms which are grouped on the basis of their relative size and moisture
content. They are droplets, droplet nuclei and infectious dust.
Droplets are usually formed by sneezing, coughing and talking.
Each droplet consists of saliva and mucus and each may contain
thousands of microbes. It has been estimated that the number of bacteria
in a single sneeze may be between 10,000 and 100,000. The size of the
droplet determines the time period during which they can remain
suspended Small droplets in a warm, dry atmosphere are dry before they
reach the floor and thus quickly become droplet nuclei.
Large aerosol droplets settle out rapidly from air onto various
surfaces, get dried and become infectious dust.
The microflora of air can be studied under two headings:
outdoor and indoor microflora.
Although most of the microorganisms present in air are harmless
saprophytes and commensals, less than I % of the airborne bacteria are
pathogens. Even though the contamination level is very low, the
probability of a person becoming infected will be greatest if he is
exposed to a high concentration of airborne pathogens. Outdoor air
doesn't contain disease causing pathogen in a significant number to
cause any infection. Dispersion and dilution by large volume of air is an
inherent mechanism of air sanitation in outside air.

87
 In the case of indoor air chance for the spread of infectious
disease is more, especially in areas where people gather in large
numbers. For example, in theatres, schools etc.
Air within the hospital may act as a reservoir of pathogenic
microorganisms which are transmitted by the patients. Infection
acquired during the hospitalization is called nosocomial infection which
may arise in a hospital unit or may be brought in by the staff or patients
admitted to the hospital. Carriers, either with the manifestation of
corresponding symptoms or without any apparent symptoms, may
continuously release respiratory pathogens in the exhaled air.
Staphylococcus aureus is the most commonly found pathogen in
air since the carriers are commonly present.
Sanitary microbiological condition of the air is appreciated by:
• microbial number: quantity of microorganisms in 1m3 of
air .
• revealing of sanitary demonstrating microorganisms of the
air: α hemolytic (S. pneumonia) and β hemolytic (S.
pyogenes) streptococcus, Staphylococcus aureus.
Koch’s sedimentation method, Krotov’s aspiration method and
filtration methods are used for microbiological study of air.

88
1.Modification. Mutation.
2.Plasmids.
3.Genetic recombinations: transformation, transduction, conjugation.
THE AIM OF THE LESSON:
1.to reveal motility of E.coli under the influence of phenol to make a
conclusion about the mechanism of discovered changes.
2.to perform the procedure of the experiment of modification,
transformation and transduction.
METHODICAL INSTRUCTIONS:
1.Modification. Mutation.
Microbial genetics studies the laws of heredity and variability.
There are hereditary (mutation and recombination) and non hereditary
(modification) types of variability.
Modifications are changes of phenotype due to the action of
environmental factors. The good example of this is the loss of motility of
E.coli under the action of phenol solution.
Experiment: to identify the character of loss of motility of E.coli
the following experiment should be done: culture of E.coli is inoculated
into two tubes with nutrient broth: in one tube phenol solution-1:100 is
previously added. After incubation in thermostat 37ºC during 18-20
hours we define motility of bacterial cells, in "hanging" drop, prepared
from two tubes.
To decide the mechanism of loss of motility the culture treated
with phenol is re-inoculated in a new tube and incubated. After 20h

89
"hanging drop" is prepared and motility is checked. Reversion of lost
feature tells about the non-hereditary character of variability.
Mutation is a change in the DNA base sequence of the wild type
organism.
2.Plasmids are extrachromosomal, double-stranded, circular DNA
molecules that are capable of replicating independently of the bacterial
chromosome. Although plasmids are usually extrachromosomal, they
can be integrated into the bacterial chromosome. They control different
characteristics of bacteria:
Col-plasmids (colicinogenic factor) lead to synthesis of colicines
(antibiotic-like substances that are specifically and selectively lethal to
other enterobacteria).
R-plasmids (antibioticoresistance) lead to the spread of multiple
drug resistance among bacteria.
F-plasmids (fertility factor) – are responsible for conjugation.
Tox-plasmids - are responsible for toxigenicity.
2.Genetic recombinations: transformation, transduction, conjugation.
Genetic recombination refers to the exchange of genes between
two DNA molecules to form a new combination of genes on a
chromosome. Genetic material can be transferred between bacteria in
several ways. In all the mechanisms the transfer involves a donor cell
that gives a portion of its total DNA to a recipient cell. The recipient cell
that incorporates donor DNA into its own DNA is called recombinant.
The transfer of genetic information from one cell to another can
occur by three methods:
• Conjugation
• Transduction
• Transformation
Conjugation is the mating of two bacterial cells during which DNA is
transferred from the donor to the recipient cell (figure 4.1).
The mating process is controlled by F (fertility) plasmids (F-
factor), which carries the genes for the proteins required for
conjugation.

90
Figure 4.1 Conjugation

91
 One of the most important proteins is pilin, which form the sex
pilus (conjugation tube). Mating begins when the pilus of the donor
“male” bacterium carrying the F factor (F+) attaches to a recipient on the
surface of the recipient “female” bacterium, which does not contain an F
factor (F-). The cells are then drawn into direct contact by “reeling in”
the pilus. After an enzymatic cleavage of the F factor DNA, one strand is
transferred across the conjugal bridge into the recipient cell. The process
is completed by synthesis of the complementary strand to form a double
- stranded F factor plasmid in both the donor and recipient cells. The
recipient is now an F+ male cell that is capable of transmitting the
plasmid further. In this instance only F factor, and not chromosome, has
been transferred.
Some F+ cells have their F plasmid integrated into the bacterial
DNA and thereby acquire the capability of transferring the chromosome
into another cell. These cells are called Hfr (high – frequency
recombination) cells (figure 4.2). During this transfer, the single strand
of DNA that enters the recipient F-cell contains a piece of the F factor at
the leading end followed by the bacterial chromosome and then by the
remainder of the F factor.
The time required for complete transfer of the bacterial DNA is
approximately 100 minutes. Most mating results in the transfer of only a
portion of the donor chromosome, because the attachment between the
two cells can break. The donor cell genes that are transferred vary, since
the F plasmid can integrate at several different sites in the bacterial
DNA. The bacterial genes adjacent to the leading piece of the F factor
are the first and therefore the most frequently transferred. The newly
acquired DNA can recombine into the recipient’s DNA and become a
stable component of its genetic material. Plasmids other than F are also
transferred by conjugation.
Transformation is the process, where genes are transferred from
one bacterium to another as "naked" DNA in solution (figure 4.3).

92
Figure 4.2 Hfr strain formation

93
Figure 4.3 Transformation

94
Direct uptake of donor DNA by recipient cells depends on heir com-
petence for transformation. Natural occurrence of this property is
unusual among bacteria, and some of these strains are transformable
only in the presence of competence factors, produced only at a specific
point in the growth cycle. The competent condition is in the logarithmic
phase of growth. Transformation falls into three phases:
1. adsorption of donor DNA on the surface of recipient cell
2. entry of DNA into the recipient cell
3. integration of DNA into the chromosome and
recombination.Experiment of transformation:
The auxotroph strain of B.subtilis that requires tryptophan for
growth may be cultivated without it with the help of DNA of
prototroph culture of B.subtilis. Auxotroph strain of B.subtilis lacks
enzyme tryptophan-synthetase. DNA of prototroph culture transferred
into the auxotroph cells restores synthesis of tryptophan-synthetase
enzyme.
Materials:
1. DNA of prototroph culture.
2. B.subtilis - auxotroph culture.
3. Petri plates with minimal nutrient media (Spetsayzen media).
Prepare suspension from B.subtilis and pour it into 2 tubes (0,5
ml): add 0,5ml DNA in the first tube, physiological solution - into the
another. The tubes incubate in thermostat for 30-60 min after which the
content of the tubes inoculate on the Petri plates with Spetsayzen media
for 2 days.
Many small colonies are revealed in the Petri plates, inoculated
with bacterial culture mixed with DNA.
There is not bacterial growth on the plates inoculated with
bacterial culture mixed with physiological solution.
Transduction is the transfer of the cell DNA by means of
bacterial virus (bacteriophage or phage) (figure 4.4). There are three
types of transduction:
• Generalized

95
 • Specialized
• Abortive
The generalized type occurs when the virus carries a segment
from any part of the bacterial chromosome. The specialized transduction
involves the transfer of only a few specific genes. Abortive type, when
phage carries a segment from the bacterial chromosome but it does not
integrate into the recipient DNA, it remains in the recipient’s cell
cytoplasm.
Transducing phages are termed defective because they lack the
DNA necessary to form complete phage and lyse the recipient cell.
Experiment of transduction:
With the help of transducing phage, discharged from E.coli
lactose (+) strain lactase activity can be transferred to E.coli lactose (-)
strain.
Materials:
1. E.coli lactose (-) strain
2. transducing phage discharged from E. coli lactose (+) strain.
3. Endo media.
Place a drop of the suspension of lactose (-) strain of E.coli into
the tube with transducing phage and incubate it in the thermostat for 30
min after which inoculate the mixture on ½ of the surface of Endo
media. The second half of the surface is inoculated by lactose (-) strain
of E. coli. Endo media is placed into the thermostat for 24 h. The next
day on the first half of Endo media surface red and uncolored colonies
are revealed, on the second half – only uncolored colonies.

96
Figure 4.4 Transduction

97
1.Pathogenicity. Virulence.
2.Infectious disease. Infectious dose. Routs of transmission of
communicable diseases. Types of infectious diseases.
THE AIM OF THE LESSON:
1.to know important components for establishment of infection: the role
of microorganism, macroorganism and environment in the development
of infectious process.
2.to show the role DLM and DL50 for evaluation of virulence and
toxigenicity.
METHODICAL INSTRUCTIONS:
1.Pathogenicity. Virulence.
Pathogenicity is a potential ability of certain species of microbes
to cause an infectious process.
Virulence is the degree of pathogenicity. Virulence is measured by the
number of organisms required for causing disease and toxigenicity
measured with the following units:
DLM (Dosis letalis minima) is a minimal number of microbes or minimal
dose of toxin which may cause death of 85-95% of susceptible
experimental animals.
DL50 (Dosis letalis 50) is the number of administered microbes or dose
of toxin that results in death of susceptible experimental animals in 50%
of cases.
2.Infectious disease. Infectious dose. Routs of transmission of
communicable diseases. Types of infectious diseases.

98
 Infectious process may develop when there are pathogenic
microbe, susceptible organism and proper environmental factors (figure
5.1). An infection that results in disease, a noticeable impairment of
body function, is called an infectious disease.

Figure 5.1 Chain of infection

Infectious dose is also important for spread of a contagious

disease. It is the minimum number of microbes necessary to establish an
infection. For example, the intestinal disease shigellosis is quite
contagious in humans because only 10 to 100 cells of a Shigella species
need to be ingested to establish infection. In case of salmonellosis
infectious dose is 106. The difference in infectious doses reflects the
ability of Shigella species to survive in acidic conditions during passage
through the stomach.
Communicable disease can be spread either directly or indirectly
from one host to another. Droplet infection, when microorganisms are
carried on liquid drops from a cough or sneeze, is a method of direct

99
contact (influenza, tuberculosis, smallpox, measles, mumps). Some
diseases can be transmitted by sexual intercourse (syphilis, gonorrhea).
Disease can also be transmitted by contact with contaminated
inanimate objects, or fomites. Drinking glasses, bedding, and towels are
examples of fomites that can be contaminated with pathogens from
feces, sputum, or pus. Fecally contaminated food and water may be the
source of infection – fecal-oral or alimentary route (Hepatitis A and
Hepatitis E, cholera).
Some diseases are transmitted from one host to another by
vectors. Vectors are insects and other arthropods that carry pathogens.
In mechanical transmission, insects carry a pathogen on their feet and
may transfer the pathogen to a person’s food. For example, houseflies
may transmit the causative agents of typhoid fever from the feces of an
infected person to food. Transmission of a disease by an arthropod’s bite
is called biological transmission. An arthropod ingests a pathogen while
biting an infected host. The pathogen can multiply or mature in the
arthropod and then be transferred to a healthy person in the arthropod’s
feces or saliva. The course of infectious disease is shown in table 5.1.

Table 5.1 The course of infectious diseases

Disease may also be transmitted from mother to fetus – vertical

route of transmission (syphilis, rubella, toxoplasmosis,
cytomegaloviruses).
100
 The continual source of an infection is called the reservoir.
Humans who harbor pathogens but who do not exhibit any signs of
disease are called carriers.
There are many types of infectious diseases, which are described
in the table 5.2.

Classification principle Types of infection

Nature of pathogenic agent Bacterial, viral, fungal, protozoal
The origin Exogenous, endogenous
(autoinfection)
Distribution of the pathogen in the Focal, generalized: bacteremia,
body viremia, sepsis (septicemia),
septicopyemia, toxicoseptic shock
Number of the types of the Monoinfection, mixed infection
causative agents
Repeated manifestations of the Reinfection, super infection, relapse
diseases caused by the same agent
Duration of the interaction Acute, chronic, carrier state
between the causative agent and
host
Manifestation Manifest, asymptomatic
Source of infection:
human Anthroponose
animal Zoonose
environment Sopronose
Table 5.2 Types of infectious diseases

101
1.Antimicrobial chemotherapy. Antibiotics. Criteria for evaluation of
antimicrobial drugs.
2.The mechanism of action of antimicrobials.
3.Resistance to antimicrobial drugs. Methods of revealing of
susceptibility to an antimicrobial drug.
THE AIM OF THE LESSON:
1.to know the mechanisms of action of antimicrobial drugs.
2.to know the methods of antimicrobial susceptibility testing.
METHODICAL INSTRUCTIONS:
1.Antimicrobial chemotherapy. Antibiotics. Criteria for evaluation of
antimicrobial drugs.
Antimicrobial chemotherapy is the treatment of infectious
diseases by chemical drugs. Antimicrobial chemicals absorbed or used
internally, whether natural (antibiotics) or synthetic, are called
chemotherapeutic agents.
Antibiotic is a compound naturally produced by certain molds
and bacteria that inhibits the growth or kills other microorganism
because of antagonistic interrelations between microorganisms of
various species. Antibiotic is now defined as a substance produced by the
microorganism or a similar substance produced wholly or partially by
chemical synthesis that is capable of inhibiting the growth or causing
death of other microorganisms in low concentrations.
Criteria for evaluation of antimicrobial drugs are the following:
1. Absence of toxic action on the human organism or selective toxicity,
causing great harm to the microorganism. They do this by interfering
with essential biological structure or biochemical processes that are
common in microorganism but not in human cells.
102
 The toxicity of a given drug is expressed as the chemotherapeutic
index, which is the lowest dose toxic to the patient divided by the dose
typically used for therapy. In other words, it is the ratio of the maximal
tolerable dose of the drug to the minimal chemotherapeutic dose.

DMT (Dosis maxima tolerance)

DMC (Dosis minima curativa)

Antimicrobials that have a high therapeutic index are less toxic

to the patient. For example, penicillin G. Antimicrobials, that are too
toxic for systemic use can sometimes be used for topical applications,
such as first aid antibiotic skin ointments.
2. Antimicrobial action, which may be either bactericidal or
bacteriostatic. Bactericidal drug kills bacteria. Bacteriostatic drug
inhibits growth of bacteria but does not kill them.
3. Spectrum of activity, which is defined by the range of
microorganism they kill or inhibit. Antimicrobials that affect a wide
range of bacteria are called broad-spectrum antimicrobials. These are
very important in the treatment of acute life-threatening diseases
when immediate antimicrobial therapy is essential and there is no
time to culture and identify the disease causing agent. The
disadvantage of broad-spectrum antimicrobials is that they disrupt
the normal flora that plays an important role in excluding
pathogens. Antimicrobials that affect a limited range of bacteria are
narrow-spectrum antimicrobials. Their use requires identification of
pathogen and testing sensitivity of isolated strain to antibiotics.
4. Prolonged use of these drugs should not form antibiotic-resistant
strains and L-forms.
5. The drug should not have adverse effects (allergic reaction, toxic
effect, suppression of normal flora)

2.The mechanism of action of antimicrobials. A number of bacterial

processes utilize enzymes or structures that are either different, absent,
or not commonly found in eukaryotic cells. Main targets of most
103
antimicrobial drugs are several microbial processes, including the
synthesis of bacterial cell wall, proteins, and nucleic acids, metabolic
pathways, and the integrity of the cytoplasmic membrane (figure 6.1).

Figure 6.1 The mechanism of action of antimicrobials

3.Resistance to antimicrobial drugs. After the introduction of sulfa drugs

and penicillin there was great hope that they would soon eliminate most
bacterial diseases. Nowadays drug resistance limits the usefulness of all
known antimicrobials. As these drugs are increasingly used and misused,
the bacterial strains that are resistant to their effects have a selective
advantage over their sensitive counterparts.
Bacteria may be intrinsically resistant to one or more classes of
antimicrobial agent (e.g. mycoplasma) or may acquire resistance by
mutation or acquisition of resistance genes from other organism.
Acquired resistance genes may enable a bacterium to
• produce enzymes that destroy the antibacterial drug

104
 • express efflux system that prevent the drug from reaching its
intracellular target
• modify the drug’s target site
• produce an alternative metabolic pathway that evades the action
of the drug.
Acquisition of new genetic material may be through:
1.Vertical gene transfer – resistance genes are transferred directly to all
bacterial progeny during DNA replication (figure 6.2).
2.Horizontal gene transfer – small molecules of DNA (R plasmids) can be
transferred between individual bacteria of the same species or even
between different species.
In many cases susceptibility of a pathogenic microorganism to a
specific antimicrobial drug is unpredictable. Unfortunately it has often
been the practice to try one drug after another until a favorable response
is observed or, if the infection is very serious, to give several together.
Both approaches are undesirable. A better approach is to determine the
susceptibility of the specific pathogen to various antimicrobial drugs and
then choose the drug that acts against the offending pathogen but
against as few other bacteria as possible.
Antimicrobials for susceptibility test should be chosen with
discrimination. Only those clinically relevant should be tested. Thus
chloramphenicol need not be tested against urinary pathogens as the
drug is excreted in urine mostly in inactive form. Nitrofurantoin needs
to be tested only against urinary pathogens.

105
Figure 6.2 The selective advantage of drug resistance
106
 Sensitivity tests to antimicrobial drugs are the following:
• dilution test
• diffusion test (Kirby-Bauer disk diffusion method)
• diffusion and dilution test (E-test)
Dilution test is too laborious for routine use. Serial dilutions of
the drug are prepared and inoculated with the test bacterium. This test
is generally employed when the therapeutic dose should be regulated
accurately as in the treatment of bacterial endocarditis, for tests on slow
growing bacteria such as M.tuberculosis. Dilution tests may be done by
the broth dilution or agar dilution methods. In broth dilution method
serial dilutions of the drug in broth are taken in tubes and a standardized
suspension of the test bacterium is inoculated. After overnight
incubation, the minimum inhibitory concentration (MIC) is determined
by the lowest concentration of drug that prevents visible growth of the
organism. This provides the physician with a precise concentration of
drug to guide the choice of both the drug and the dose (figure 6.3).

Figure 6.3 Revealing of MIC

107
 Kirby-Bauer disk diffusion method uses filter paper discs, 6.0mm
in diameter, charged with appropriate concentrations of the drugs
(figure 6.4 a)). They may be prepared in the laboratory or purchased
commercially. Disc diffusion method is in common use, in this method a
suitable dilution (turbidity matching 0.5 MacFarland standard) of a
broth culture or a broth suspension of the test bacterium is inoculated
on the surface of a solid medium (Mueller-Hinton agar or nutrient agar)
as a lawn by spreading with a cotton swab. Mueller Hinton agar allows
the chemotherapeutic agent to diffuse freely. After drying the plate
(37°C for 30 minutes), antibiotic discs (4-6 per 9cm plate) are applied
with sterile forceps. After overnight incubation, the degree of sensitivity
is determined by measuring the zones of inhibition of growth around
the disc (figure 6.4 b)). Growth will be inhibited around discs containing
antibiotics to which the bacterium is susceptible but not around those to
which it is resistant.

a) b)
Figure 6.4 Kirby-Bauer disk diffusion method (a) filter paper discs
charged with appropriate concentrations of the drugs (b) measuring the
zones of inhibition of growth around the disc

108
 The diameter of the zone of inhibition is influenced by a variety
of factors, such as diffusibility of the drug, disc concentration, nature
and composition of the medium, its thickness, presence of inhibitory or
stimulatory substances, pH and time of incubation.
Based on the zone of inhibition around the disc the results are
recorded and interpreted according to the zone diameters available in
the tables of the guidelines (figure 6.5).

Figure 6.5 Inhibition zone diameters of some antimicrobial drugs

The disc diffusion test, as described above, is done after the

pathogenic bacteria are isolated from clinical specimens. When results
are required in a hurry, the “primary disc diffusion test” may be
performed. Here the swab or other clinical specimen is directly
inoculated uniformly on the surface of a plate and discs are applied. The
results of a primary test should be verified by testing the isolates
subsequently.
E-test (diffusion and dilution test) or Epsilometer test is a recent
modification of the agar diffusion susceptibility test employing a
quantitative diffusion gradient. It uses an absorbent strip with a known
gradient of drug concentrations along its length. When the strip is

109
placed on the agar plate seeded with the test bacterium, the antibiotic
diffuses into the medium. The MIC is obtained by noting the lowest
concentration of the gradient which inhibits bacterial growth (figure
6.6).

Figure 6.6 E-test

110
1.Serological reactions. Principles of immunological testing.
2.Quantifying antigen-antibody reactions.
3.Zone of equivalence.
THE AIM OF THE LESSON:
1.to know the principles of immunological testing.
2.to know the methods of obtaining of antibodies.
3.to know the dilution method of serum.
METHODICAL INSTRUCTIONS:
1.Serological reactions. Principles of immunological testing.
Reactions of antigens and antibodies are highly specific. An
antigen will react only with antibodies elicited by itself or by a closely
related antigen. Because of the great specificity, reactions between
antigens and antibodies are suitable for identifying one by using the
other. This is the basis of serological reactions or immunoassays: antigen-
antibody interactions in vitro for diagnosis.
A person who has not been exposed to a given pathogen has no
specific antibodies against it in their serum, and is referred to as
seronegative. Once infected, that person will produce specific antibodies
about a week to 10 days later, becoming seropositive. This change from
seronegative to seropositive is referred to as seroconversion. As the
infection progresses, increasing amounts of specific antibodies are
produced, causing the amount of those antibodies in the blood to
increase. A rise in the amount of specific antibodies, or titer, is
characteristic of an active infection. Small, but steady amounts of
antibodies indicate a previous infection or vaccination.

111
 To determine the presence of antibodies in the blood against a
specific infectious agent either the patient’s serum or plasma is tested.
Serum is the fluid portion of blood that remains after blood clots; plasma
is the fluid portion of blood treated with an anticoagulant to prevent
clotting. Because serum is often used as a source of antibodies, the study
of in vitro antibody-antigen interactions is referred to as serology.
Direct serologic testing utilizes a known antiserum in order to
detect an unknown antigen, either foreign or self. Direct tests are
qualitative and provide results relatively quickly. They are used mostly
for screening purposes.
Indirect serologic testing utilizes antibodies from the patient that
may be specific for either self or foreign antigen. This test is based on
the concept that antibodies are produced in response to a specific disease
state. Indirect tests may be qualitative and used for screening purposes
or quantitative, which provides the amount of antibody in the patient’s
serum.
Most immunologic tests can be performed using direct or
indirect measures. Indirect tests are generally more specific, resulting in
fewer false-positives. The Coombs, ELISA, and fluorescent antibody
tests are all examples of tests that can be utilized in either a direct or
indirect manner.
To obtain known antibodies for revealing of certain infectious
agent laboratory animals are used which are immunized with either the
whole agent or part of it, and the resulting antibodies are then collected
by harvesting the animal’s serum. The antibody preparation will be
polyclonal, meaning that multiple naïve B cells responded to the
immunization, producing a mix of different antibodies that recognize a
variety of epitopes on the antigen. One problem with polyclonal
antibodies is that some may bind to closely related organisms, resulting
in a false positive reaction.
Monoclonal antibodies recognize only a single epitope. They are
obtained through a laborious process that involves isolating individual B
cells from an immunized animal, and then fusing those short-lived B
cells with other cells that will divide repeatedly in culture (figure 7.1).
112
Figure 7.1 Production of monoclonal antibodies

Monoclonal antibodies are difficult and expensive to develop

and thus are generally available only if they have commercial value due
to their widespread use. In addition to the diagnostic value of
monoclonal antibodies, some have been developed that can be used to
treat disease. Monoclonal antibodies derived from an animal such as a
mouse can be “humanized”, meaning that recombinant DNA techniques
were used to replace at least part of the mouse derived molecule with
the human equivalents. This gives the molecule a longer half-life in
humans because the human immune system is less apt to recognize it as
foreign. Humanized monoclonal antibodies are referred to as rhu-Mabs
(recombinant human monoclonal antibodies). Some rhu-Mabs can be
used to treat certain types of cancers.
Certain serological tests use antibodies that bind to human IgG
molecules. This are referred to as anti-human IgG antibodies. They are

113
produced by animals that have been immunized with IgG from human
serum.
2.Quantifying antigen-antibody reactions.
The concentration of antibody molecules in a specimen is
usually determined by making two fold or ten-fold serial dilutions, and
then antigen is added to each dilution (figure 7.2).
The titer (concentration) is expressed as the reciprocal of the last
dilution that gives a detectable antigen-antibody reaction, or the
antibody titer of a serum is the highest dilution of the serum that shows
an observable reaction with the antigen in the particular test. The titer
of a serum is influenced by the nature and quantity of the antigen and
the type and conditions of the test. Thus, if a positive reaction is
observed in the dilution 1:10, but not in 1:100, then the antibody titer is
1:10.

Figure 7.2 Serial dilutions

Serology tests can be done in test tubes, but this requires many
tubes and large quantities of reagents. Therefore, the tests are usually
done using plastic microtiter plates (figure 7.3), which have 96, 384 or
114
768 wells. Either antigen or antibody can be permanently affixed to the
tiny plastic wells and tests can be done on minute samples.

Figure 7.3 Microtiter plate

Two important parameters of serological tests are sensitivity and
specificity:
• Sensitivity refers to the ability of the tests to detect even very
minute quantities of antigen or antibody. When a test is highly
sensitive, false negative results will be absent or minimal.
• Specificity refers to the ability of the test to detect reactions
between homologous antigens and antibodies only, and with no
other. In a highly specific test false positive reactions are absent
or minimal.
In general, the sensitivity and specificity of a test are in inverse
proportion.
3.Zone of equivalence.
As mentioned above, interaction of antigen and antibody occurs
in vivo, and in clinical settings it provides the basis for all serologically
based assays. The formation of immune complexes produces a visible
reaction that is the basis of precipitation and agglutination assays.
Agglutination and precipitation are visible when multiple antibody

115
molecules share the binding of multiple antigenic determinants, a
condition known as equivalence.
The amount of immune complexes formed is greatly influenced
by the relative proportions of antigen and antibodies. If increasing
quantities of antigens are added to the same amount of antiserum in
different tubes, precipitation will be found to occur most rapidly and
abundantly in one of the middle tubes in which the antigen and
antibody are present in optimal or equivalent proportions. In the
preceding tubes in which the antibody is in excess and in the later tubes
in which the antigen is in excess, the precipitation will be weak or even
absent. For a given antigen-antibody system, the optimal or equivalent
ratio will be constant, irrespective of the quantity of the reactants. If the
amounts of precipitate in the different tubes are plotted on a graph, the
resulting curve will have three phases (figure 7.4):
1. Prozone phenomenon. This is caused by excess antibody in
the test system. Failure of a visible reaction is due to
inhibition of lattice formation by the excess antibody.
2. Zone of equivalence. The antigen and antibody are in
optimum proportions. Lattice formation and visible
reactions are enhanced.
3. Post-zone phenomenon. No visible reaction will occur
because of excess antigen in the test system.
The prozone and post-zone phenomena may be corrected by
making serial dilutions of serum, thereby, reducing the concentration of
antigen or antibody in the test serum, and optimizing the concentrations
of antigen and antibody.
Zoning occurs in agglutination and some other serological
reactions. The prozone is of importance in clinical serology, as sera rich
in antibody may sometimes give a false negative precipitation or
agglutination result, unless several dilutions are tested (prozone
phenomenon in secondary syphilis).

116
Figure 7.4 Zone of equivalence

The antibody response during many infectious diseases shows

the following progression:
• At the start of the infection the patient is in a state of antigen
excess; the pathogen is proliferating in the host and the synthesis
of specific antibodies has not yet begun.
• As the patient begins to make an adequate antibody response, he
enters the equivalence zone, all available antigen is complexed
with antibody, and neither free antigen nor free antibody can be
detected in the serum (window period in the hepatitis B)
• As the infection is resolved, the patient enters the antibody
excess zone, when more antibody is being produced than is
necessary to bind the available antigen.

117
1.Precipitation reactions. Ring precipitation test.
2.Immunodiffusion tests.
3.Immunoelectrophoresis.
THE AIM OF THE LESSON:
1.to know the applications of precipitation reactions.
2.to know the principle of performing of precipitation reactions.
METHODICAL INSTRUCTIONS:
1.Precipitation reactions. Ring precipitation test.
Precipitation reactions involve the reaction of soluble antigens
with IgG or IgM antibodies to form large, insoluble interlocking
aggregates called lattices (insoluble precipitate). Precipitation reactions
occur in two distinct stages. First, there is the rapid interaction between
antigen and antibody to form small antigen- antibody complexes. This
interaction occurs within seconds and is followed by a slower reaction,
which may take minutes to hours, in which the antigen-antibody
complexes form lattices that precipitate from solution. Precipitation
reactions normally occur only at certain relative concentrations of
antigen and antibody. The optimal ratio is produced when separate
solutions of antigen and antibody are placed adjacent to each other and
allowed to diffuse toward each other. A precipitate will form in a
distinct region called the zone of optimal proportions.
The precipitation test may be carried out as a qualitative or
quantitative test. It is sensitive in the detection of antigens and as little
as 1μg of protein can be detected. It is relatively less sensitive for the
detection of antibodies.
Precipitation tests have several applications, including
identification of blood and seminal stains in forensics, testing for food
adulterants, grouping of streptococci by the Lancefield technique, the
VDRL test for syphilis, standardization of toxin and toxoids, testing of
toxigenicity of diphtheria bacilli.

118
 Ring precipitation test is the simplest type of precipitation. This
test is done by layering the antigen solution over a column of antiserum
in a narrow tube and a cloudy line of precipitation (ring) appears in the
area of liquid, where the optimal ratio of antigen and antibody has been
reached (figure7.5). Typing of streptococci and pneumococci, C-reactive
protein test and Ascoli’s thermo-precipitation test for the diagnosis of
anthrax, are some of the uses of ring test. This technique is also used in
detection of adulteration of food stuffs.

Figure 7.5 The ring precipitation test

2.Immunodiffusion tests (precipitation in gel).

Immunodiffusion tests are precipitation reactions carried out in
a gel such as agarose. Precipitation in gel has several advantages over the
precipitation in liquid medium. The reaction appears as a distinct band
of precipitation which can be stained for better visibility and
preservation. Since each antigen-antibody reaction gives rise to one line
of precipitation, therefore, different number of antigens in a mixture can
be detected. This technique also indicates identity, cross-reaction and
non-identity between different antigens.

119
 There are many modifications of the immunodiffusion test. One
method used is the Ouchterlony technique – double diffusion in gel,
which can be done in a Petri dish or on a slide.
This is qualitative test, which is most widely employed and helps
to compare different antigens and antisera directly. Antiserum is placed
in the central well, and different antigens in separate surrounding wells
cut in the gel and are allowed to diffuse toward each other. If the
antibody molecules recognize the antigen, they will form antigen-
antibody complexes, resulting in a line of precipitate at the zone of
optimal proportions. If two adjacent antigens are identical, the lines of
precipitate formed by them will fuse. If they are unrelated, the lines will
cross each other. Cross-reaction or partial identity is indicated by spur
formation (figure 7.6).

Figure 7.6 Double diffusion according to Ouchterlony Double

immunodiffusion in gel can detect antigens that are the same, or
partially the same.

A special variety of double diffusion in two dimensions is the

Elek test for toxigenicity in diphtheria bacilli. When diphtheria bacilli
are streaked at right angles to a filter paper strip carrying the antitoxin
implanted on a plate of suitable medium, arrowhead-shaped lines of
precipitation appear on incubation, if the bacillus is toxigenic (figure
7.7).
Unlike the Ouchterlony technique, the radial immunodiffusion
test is quantitative. Here the antibody is added to the melted, cooled
agarose before it hardens, generating a uniform concentration of
antibody molecules throughout the gel.

120
 The antigen is added to the wells cut on the surface of the gel. It
diffuses radially from the well and forms ring-shaped bands of
precipitation concentrically around the well.

Figure 7.7 Elek’s immunodiffusion test

The higher the concentration of the antigen in the sample, the

further the ring will form from the well. In order to determine the
actual antigen concentration, the radial immunodiffusion test includes a
separate set of wells into which a series of standards of known
concentrations of antigen have been added. The diameter of the ring of
precipitation around each of the standards can be used to construct a
standard curve, which can then be used to determine the concentration
of the unknown (figure 7.8). The radial immunodiffusion test is
commonly used to measure the level of the various complement system
proteins in order to diagnose inherited deficiencies. It can be used for
the estimation of the immunoglobulin classes.
3.Immunoelectrophoresis.

121
 Immunoelectrophoresis is a variation of the precipitation in gel
technique that combines electrophoresis and immunodiffusion. This
method can be used to analyze complex antigens in biological fluids
using electrophoresis for separation of mixture of protein antigens on
the basis of their movement through an electrical field. Different
antigens will migrate at different rates or even in different directions,
depending upon their size and charge and the conditions of
electrophoresis. A glass slide is covered with molten agar or agarose. A
well for antigen and a trough for antibodies are cut on it.

Figure 7.8 Radial immunodiffusion Is used to measure the concentration

of antigen in a sample

122
 Antigen well is filled with antigen mixture. The slide is then
placed in an electrical field for about an hour to allow the
electrophoretic migration of various antigens. The antibodies are placed
in a trough and allowed to diffuse toward the separated protein antigens,
resulting in the formation of precipitin bands in zones of optimal
proportions in 18-24hours. By this method, over 30 different antigens
can be identified in human serum (figure 7.9).
One of the many applications of immunoelectrophoresis is to
determine different classes of immunoglobulins. The person’s serum
being tested for immunoglobulins, the antigen in this case, is placed in
the well and after electrophoresis antiimmunoglobulin antibodies are
placed in the trough and allowed to diffuse toward the antigen. In
normal serum, precipitation lines will form for IgA, IgG and IgM. IgD
and IgE are normally present in such small quantities that they do not
yield visible precipitates. Some patients lack immunoglobulins, and their
sera will give no lines of precipitation with anti-human antibodies.
Other patients have myeloma tumors, in which a single plasma cell has
given rise to a tumor. All the cells of the tumor produce the same
immunoglobulin that the original cell produced. In this case the serum
from the myeloma patient will give a heavy, thick line for the class of
immunoglobulin being produced. Thus, abnormally high or low
production of immunoglobulins is readily detected by
immunoelectrophoresis.

123
Figure 7.9 Immunoelectrophoresis Electrophoresis is used to separate the
antigens; then, immunodiffusion is used to identify them.

124
1.Agglutination reactions. Direct agglutination tests.
2.Passive agglutination tests.
3.Coomb (antiglobulin) test.
THE AIM OF THE LESSON:
1.to know how to perform slide agglutination reaction.
2.to know how to perform tube agglutination reaction and define the
titer.
3.to know the meaning of Coombs reaction to reveal incomplete
antibodies.
METHODICAL INSTRUCTIONS:
1.Agglutination reactions. Direct agglutination test.
Agglutination reactions like precipitation reactions depend on
cross linking and lattice formation, however, in agglutination reactions
the antigen consists of relatively large particles (cell wall, flagella,
capsule) instead of soluble molecules, so larger aggregates of antigen and
antibody are formed, which are much easier to see. Reaction of
agglutination is the sticking of microbes (agglutinogens) or other cells
with specific antibodies (agglutinins) which is expressed by the
formation of visible conglomerates (agglutinate) in the presence of
electrolyte (figure 7.10).

Figure 7.10 Agglutination of erythrocytes in abo blood typing

125
 In direct agglutination test a suspension of specific antibody is
mixed with insoluble antigen, such as red blood cells, bacteria or fungi.
Readily visible clumping is a positive test. The agglutination of red blood
cells by antibody or other means is referred to as hemagglutination, and
is used in ABO blood typing (figure 7.11).

Figure 7.11 Agglutination test for blood typing

Some viruses, such as those causing infectious mononucleosis,

influenza, measles, and mumps, agglutinate red blood cells by an
interaction between the surface components of the virus and the red
cell. This is not a serological reaction because of the absence of
agglutinating antibodies and positive reaction can speak about the
presence of virus. Specific antiviral antibodies neutralize the viruses and
inhibit the agglutination - hemagglutination inhibition. Serum is serially
diluted and mixed with the virus and red blood cells. Dilutions of serum
containing specific antibodies inhibit the usual agglutination of virus
and red cells. The titer of antibodies is determined by the highest
dilution of serum in which agglutination does not occur (figure 7.12).

126
Figure 7.12 Agglutinated cells form a rough pattern over the bottom of
the well, while unagglutinated cells fall into a small button at the bottom.

• Slide agglutination: (orientating reaction of agglutination) is done

on the slide to serotype the unknown culture of microbes.
Procedure: For this reaction diagnostic agglutinating serum,
experimental culture on MPA, physiological solution are needed.
A drop of diagnostic serum is put on the slide and the physiological
solution near it (control). Using inoculating loop experimental culture
from MPA is placed in the first and the second drop, and then mixed
carefully. Agglutination reaction is indicated by the clumping of
microbes and the clearing of the drop. Depending on the titer of the
serum, agglutination may occur instantly or within seconds. In the
control drop the uniform turbidity remains. Agglutination is usually
visible to the unaided eye but may sometimes require confirmation
under the microscope.
• Tube agglutination reaction: This is a standard quantitative method
for the measurement of antibodies. When a fixed volume of a
particulate antigen suspension is added to an equal volume of serial
dilutions of an antiserum in test tubes, the agglutination titer of the
serum can be estimated. Tube agglutination is routinely used for the
serological diagnosis of typhoid, brucellosis and typhus fever.
127
 In the Widal test used in typhoid, two types of antigens are used. The
“H” or flagellar antigen on combining with its antibody forms large,
loose, fluffy clumps resembling wisps of cotton wool (diagnostic titer is
>320). The “O” or somatic antigen forms tight, compact deposits
resembling chalk powder (diagnostic titer is >160). Agglutinated bacilli
spread out in a disk-like pattern at the bottom of the tubes (figure 7.13).
Nowadays microtiter plates are also used.
Procedure:
1.Label 10 wells in the microtiter plate 1-10.
2.Add 80μl saline on the first well and 50μl to each of the remaining
wells.
3.Add 20μl patient’s serum to the first well. Mix up and down three
times and, with a new pipette tip, transfer 50μl to the second well.
Change pipette tips, mix, and transfer 50μl to the third well, and so on
until the ninth well. Discard the 50μl from ninth well. The patient’s
serum is now diluted.
4.Carefully add 50μl of the antigen to each well.
5.Shake the plate carefully in a horizontal direction to mix the
contents in each well. Place the plate in a 35°C incubator for 60 minutes.
6.Refrigerate until the next laboratory period.
7.Observe the bottom of each well. There shouldn't be agglutination
in the 10-th control well. Weighted flakes in the experimental wells
show positive reaction. The complete agglutination is marked by 4
pluses - the liquid absolutely transparent, there is precipitation from
glued microbe flakes on the bottom. The smaller number of microbes is
agglutinated, the more turbid the liquid is and the less the precipitation
is on the bottom.

128
Figure 7.13 Widal test

2.Passive agglutination test.

Passive agglutination amplifies the outcome of antibody-antigen
aggregate formation by attaching either the antibody or the soluble
antigen to the red blood cells, latex beads or other particles (bentonite).
Agglutination of these insoluble particles is much easier to see than the
precipitate of soluble molecules.
• Passive hemagglutination: Human or sheep erythrocytes adsorb
a variety of antigens. Polysaccharide antigens may be adsorbed
by simple mixing with the cells. For adsorption of protein
antigens, tanned red cells are used.
• Latex agglutination: Latex beads, to which specific antibodies
have been attached are produced commercially and used to test
for various bacteria, fungi, viruses and parasites, as well as drugs,
hormones and other substances (latex is manufactured as
uniform spherical particles, 0,8-1,0 μm in diameter). The beads
are mixed with a drop of a body fluid (serum, urine, spinal fluid,
any other specimen) or suspended microbial culture. If the
specific antigen is present, easily visible clumps will form (figure
7.14). Latex agglutination tests are available for the diagnosis of

129
 cerebrospinal infections caused by Streptococcus species,
N.meningitidis, H.influenzae type b, Cryptococcus.

Figure 7.14. Latex beads coated with antibodies that bind specifically
antigen of bacteria are mixed with a suspension of a suspected culture.

Visible clumping confirms the presence of bacteria.

• Co-agglutination: This is the example of reversed passive
agglutination, when instead of the antigens, the antibody is adsorbed
to carrier particles in tests for the estimation of antigens. This method
is used to diagnose bacterial antigens like Legionell, N.gonorrhoea,
S.pyogenesin clinical samples. It is based on agglutination of the
antibody sensitized protein A-bearing Staphylococcus aureus in the
presence of bacterial antigens in clinical specimen.
3.Coombs (antiglobulin) test.
The antiglobulin test was devised by Coombs for the detection of
anti-Rh antibodies that do not agglutinate Rh-positive erythrocytes in
saline. When sera containing incomplete anti-Rh antibodies are mixed
with Rh-positive red cells, the antibody globulin coats the surface of the
erythrocytes, though they are not agglutinated. When such erythrocytes
coated with the antibody globulin are washed free of all unattached
protein and treated with a rabbit antiserum against human
gammaglobulin (antiglobulin or Coombs serum), the cells are
agglutinated.
Two variations of the Coombs test exist. The direct Coombs is
designed to identify maternal anti-Rh antibodies that are already bound
to infant RBCs or antibodies bound to RBCs in patients with
130
autoimmune hemolytic anemia. In this case the sensitization of the
erythrocytes with incomplete antibodies takes place in vivo (figure 7.15).

Figure 7.15 Direct Coombs test

The indirect Coombs test is designed to identify Rh-negative

mothers who are producing anti-Rh antibodies of the IgG isotype,
which may be transferred across the placenta harming Rh-positive
fetuses. The indirect Coombs is also used in the diagnosis of transfusion
reactions (figure 7.16).
Originally employed for detection of anti-Rh antibodies the
Coombs test is useful for demonstrating and type of incomplete or non-
agglutinating antibody, as, for example, in brucellosis.

Figure 7.16 Indirect Coombs test

131
1.Complement fixation test (CFT).
2.Neutralization tests.
THE AIM OF THE LESSON:
1.to know how to perform complement fixation test and interpret the
results.
2.to know the practical application of complement fixation (Bordet-
Gangou test) and neutralization tests.
METHODICAL INSTRUCTIONS:
1.Complement fixation test (CFT).
Bacteria, red blood cells, or other cells may sometimes lyse as
the result of complement activity. Recall that complement is a complex
system of proteins, that interact with antibody bound to antigenic
components of cells and cause the cells to lyse. This phenomenon is the
basis for the complement fixation test (CFT), which is used to test for
specific antibodies in a patient’s serum. CFT is a complex procedure
consisting of two steps and five reagents – antigen, antibody,
complement, sheep erythrocytes and rabbit antibody to sheep red cells.
Each of these reagents has to be separately standardized.
The patient’s serum sample is heated at 56°C for half an hour to
inactivate any complement present.
The source of the complement is guinea pig serum, which
should be titrated for complement activity. One unit or minimum
hemolytic dose (MHD) of complement is defined as the highest dilution
of the guinea pig serum that lyses one unit volume of washed sheep
erythrocytes in the presence of excess hemolysin (amboceptor) within a
fixed time (usually 30 or 60 minutes) at a fixed (37°C) temperature
(reaction of immune hemolysis).
The hemolytic serum is obtained by 3 or 4 times intravenous
immunization of a rabbit by the suspension of sheep red blood cells.The

132
amboceptor should be titrated for hemolytic activity. MHD of
amboceptor is defined as the least amount (or highest dilution) of the
inactivated amboceptor that lyses one unit volume of washed sheep
erythrocytes in the presence of excess complement within a fixed time
(usually 30-60 minutes) at a fixed temperature (37°C). The diluent for
CFT is physiological saline with added Ca and Mg ions.
The suspension of sheep red blood cells is obtained from
defibrinated sheep blood. The sheep red blood cells are washed up and a
3% suspension is made of it.
The reaction takes place in 2 phases: in the 1-st phase the
patient’s serum, the antigen and the complement are incubated together
at 37°C for one hour. The process of complement fixation is not visible
reaction. To find out whether the complement is fixed or remained
independent in the first step, the second- indicatory hemolytic system is
used - the system, which consists of the sheep red blood cells suspension
and rabbit hemolytic serum received by immunization of sheep's red
blood cells.
If the serum contains specific antibody, the complement will be
utilized during the 1-st phase. If the serum does not contain the
antibody, no antigen-antibody reaction occurs and the complement will
therefore be left intact (figure 7.17).
Testing for complement in the post-incubation mixture will
indicate whether the serum had antiserum or not. This consists of
adding sensitized cells (sheep erythrocytes coated with4MHD
hemolysin), and incubating at 37°C for 30 minutes.
Interpretation of results:
• Lysis of the erythrocytes indicates that the complement was not
fixed in the first step and, therefore, the serum did not have the
antibody (negative CFT).
• Absence of erythrocyte lysis indicates that the complement was
used up in the first step and, therefore, the serum contained the
antibody (positive CFT).

133
 Figure 7.17 Complement fixation test

Appropriate controls should be used, including the following:

• antigen and serum controls to ensure that they are not anti-
complementary
• complement control to ensure that the desired amount of
complement is added
• cell control to observe that sensitized erythrocytes do not
undergo lysis in the absence of complement
The immobilization test is another complement-dependent
reaction. In the Treponema pallidum immobilization test, a highly
specific test formerly considered the “gold standard” for the
serodiagnosis syphilis, the test serum is mixed with a live motile
suspension of T.pallidum in the presence of the complement. On
incubation, the specific antibody inhibits the motility of treponemes.
Cytolytic or cytocidal tests are also complement dependent. When a
134
suitable live bacterium, such as the cholera vibrio, is mixed with its
antibody in the presence of the complement, the bacterium is killed and
lysed. This forms the basis of the vibriocidal antibody test for the
measurement of anti-cholera antibodies.
2.Neutralization tests.
Neutralization tests are another example of useful tests that are
often superseded by other, easier methods.
In the virus neutralization test serum is mixed with a known
viral suspension. If antibodies to that particular virus are present, they
will bind to it and neutralize the virus, preventing infection of cells in
culture.
Toxin neutralization test can be used in vivo and in vitro.
Specific antibodies against the toxin (antitoxin) will neutralize the
effects of the toxin. For example, in a suspected case of botulism, a lethal
form of food poisoning, leftover food thought to be the source of the
toxin can be tested by injecting some of the food into mice. The
antigenic type of toxin is determined by mixing part of the food sample
with specific antitoxin before injection. Untreated food, containing
botulinum toxin kills the mice, but they are protected if the food is first
mixed with specific antitoxin that neutralizes the toxin.
The Schick test is based on the ability of circulating antitoxin to
neutralize the diphtheria toxin given intradermally, and indicates
immunity or susceptibility to the disease.
Negler’s reaction is a test for the identification of alpha toxin of
Clostridium perfringens in clinical specimens. This toxin, on addition of
antitoxin to cultures grown on agar medium containing egg yolk (as a
source of lecithin), prevents visible opacity due to lecithinase action
which is normally observed around colonies.

135
1.Labeled antibody systems. Fluorescent antibody tests.
2.Enzyme-linked immunosorbent assay (ELISA).
3.Fluorescence activated cell sorting (FACS).
THE AIM OF THE LESSON:
1.to know how to perform fluorescent antibody tests, ELISA and FACS.
2.to know the practical application of fluorescent antibody tests, ELISA
and FACS.
METHODICAL INSTRUCTIONS:
1.Labeled antibody systems. Fluorescent antibody tests.
Labeled antibody systems are utilized for the detection of
antigens, which may be either self or foreign. These antigens can be
visualized using a combination of specific antibody that is labeled or
tagged with a compound used for its detection. Common tags include
fluorescent compounds and enzymes.
Each of the following discussed is an example of a labeled
antibody system. Additionally, fluorescent antibody tests and ELISA can
be done using either direct or indirect tests as described previously.
In 1942 Coons and his colleagues showed that fluorescent dyes
can be conjugated to antibodies and that such “labelled” antibodies can
be used to locate and identify antigens in tissues. Nowadays fluorescent-
antibody tests are used to identify microorganisms or to detect the
presence of antibodies in a patient’s serum and has several diagnostic
and research applications.
The direct fluorescent antibody test (DFA) is used for
identification of bacteria, viruses or other antigens. The tissue sample to
be tested is treated with antibodies against that particular antigen that
have been labeled with a fluorescent dye (fluorescein isothiocyanate –
FITC, or rhodamine). In this procedure, the specimen containing the
antigen is fixed onto a slide. Known antibodies labeled with fluorescein
are added, the mixture is incubated, and the slide is washed. Antibody

136
that binds the microorganism will not wash away. The slide is then
examined under a fluorescence microscope with a special light source to
permit light of the desired wavelength (UVL) to excite the dye. The dye
then emits light of a different wavelength, which is viewed by using
special filters in the microscope. If microorganism has bound the
antibody, they will glow a yellow-green color. If rhodamine is used
instead of fluorescein, the microorganisms will glow a red color. By
using various fluorescent dyes, it is possible to locate different antigens
in the same cell or preparation (figure 7.18).

Figure 7.18 Direct fluorescent antibody testing

Variations of this test are used to diagnose respiratory syncytial

virus, herpes simplex 1 and 2, rabies in animal tissues, and Pneumocystis
infections.
The indirect fluorescent antibody test (IFA) is used to detect the
presence of specific antibodies in the patient’s serum. In this case, a
known species of microorganism is mixed to a slide, a sample of the test
serum is added, and the mixture on the slide is incubated. If specific
antibodies are present in the serum, they will bind to the antigen of the
microorganism. This complex cannot be seen. Unfixed material is
washed off and anti-human-gamma-globulin (anti-HGG) antibodies that
have been labeled with fluorescein are added to the slide which is
incubated. Anti HGG is prepared by immunizing animals with the
gamma globulin portion of human serum. The anti-HGG attaches to any
antibody remaining on the slide from the unknown serum. Then the
slide is examined in the fluorescent microscope and the organisms

137
appear as yellow-green (figure 7.19). This technique can be used to
detect autoantibodies in various autoimmune diseases.

a) b)
Figure 7.19 Fluorescent antibody testing (a) Indirect test (b)Treponema
pallidum stained with fluorescein-tagged antibodies in an indirect test

2.Enzyme-linked immunosorbent assay (ELISA).

Enzyme labelled conjugates were first introduced in 1966 for
localization of antigens in tissues, as an alternative to fluorescent
conjugates. ELISA is so named because the technique involves the use of
an immunosorbent, an absorbing material specific for one of the
components of the reaction: the antigen or antibody. ELISA is usually
done using 96-well microtiter plates suitable for automation.
The ELISA is an extremely sensitive test, as little as 10–9g of
material can be detected. It employs antibodies that have been labeled
with a detectable enzyme such as peroxidase and can be used to detect
the presence of hormones, drugs, antibiotics, serum proteins, infectious
disease antigens, and tumor markers. When the labeled antibodies bind
directly or indirectly to an antigen fixed to a surface, their location can
be determined using colorimetric assay. It does so by utilizing a
chromogenic substrate that undergoes an enzyme-mediated color
change.

138
 The direct ELISA is used to detect an unknown antigen. A
specimen suspected of containing that antigen is affixed to a surface such
as a well in a microtiter plate. Oftentimes, the antigen is not affixed
directly, but is instead “captured” by antibodies, that have been attached
to the surface. This modification is known as sandwich ELISA. After
attachment of the antigen, enzyme labeled antibodies known to bind the
antigen are added. The unbound antibodies are then washed away. To
detect remaining labeled antibodies, a colorless substrate is added, that
develops a color if acted upon by the enzyme (figure 7.21 a)). Examples
of commercial modifications of ELISA test include rapid Group A strep
tests done in doctors’ clinics, and home pregnancy kits (figure 7.20).

Figure 7.20 ELISA test for pregnancy

The indirect ELISA is typically used to detect the presence of given

antibody specificity. The known antigen is affixed to a surface, such as a
well in microtiter plate. The serum to be tested is added, and then a
washing step removes unbound antibodies. To allow the antigen-
antibody complex to be detected, enzyme-labeled anti-human IgG
antibodies are added, which bind any IgG antibodies remaining from the
serum. Unbound labeled antibodies are then washed away. As with
direct ELISA, a colorless substrate of the enzyme is then added.
Development of color indicates a positive reaction.

139
 The indirect ELISA is routinely used to test donated blood for
antibodies against HIV p24 capsid antigen before the blood is used for
transfusion. A small percentage of the results may be false positive,
because of which a complicated but more reliable test, Western blotting
is used to confirm the positive ELISA results (figure 7.21 b)).

a) b)
Figure 7.21 ELISA test (a) Direct ELISA (b) Indirect ELISA

In the Western blot technique a mixture of antigens is separated

by electrophoresis in a polyacrylamide gel. Smaller proteins migrate
faster than larger ones. The resulting bands separated antigens can react

140
with specific antibodies, but antibodies do not diffuse well into these
gels. Therefore, it is necessary to transfer the antigens present in the
bands from the gel by blotting them onto a filter. The steps after that are
very similar to those of an ELISA. To test for antibodies specific for the
separated proteins in a patient, a serum sample is added to the blot and
unbound antibodies are washed off. Enzyme or radioactively-labeled
anti-human IgG antibodies are then added, which attach to any
antibody remaining from the serum. Unbound labeled antibodies are
then washed off. Finally, the label is detected, generating a bar code-like
pattern that indicates which proteins were recognized by the patient’s
antibodies.
3.Fluorescence activated cell sorting (FACS).
Fluorescence activated cell sorting (FACS) is a procedure used to
rapidly analyze cell types in a complex mixture. This is done by sorting
the cells into different populations based on their binding to specific
fluorescently labelled antibodies.
By using antibodies against cell-surface markers conjugated to
different fluorescent dyes, it is possible to analyze the relative numbers
of cells present in a specific tissue location.
Cells in a mixture are reacted with specific labeled antibody and
then forced into a stream so fine that single cells move in the stream and
through a laser beam. The laser causes the fluorescent label to emit light,
which is measured with sensitive electronic detection apparatus and a
computer-generated graph is produced, plotting the intensity and color
of fluorescence of each cell along the axes. Each dot on the graph
reflects the passage of a cell with a certain level and color of
fluorescence, so the darkly dotted areas of the graph reflect the presence
of many cells of similar attributes (figure 7.22).

141
Figure 7.22 Fluorescence activated cell sorting/©2018 by Kaplan, Inc.

142
1.Which are the revealing methods of capsule:
1.staining by Gram
2. staining by Hinss-Burry
3.hanging drop method
4.staining by Ionne
a)1.4. b)2.3. c)1.3. d)2.4.

2.Capsule can be revealed by the following method:

a)gram staining
b)dark field microscopy
c)hanging drop
d)Ione staining

3.Capsule:
1.protects microorganism from external unfavorable factors
2.is an antiphagocytic factor
3.contains lipopolysaccharides
4.is revealed by Hinss-Burry staining method
a)1.3.4. b)2.4. c)1.2.4. d)1.3.

4.Which of the following are typical of capsule, Except:

a)protects microorganisms from external unfavorable factors
b)is an antiphagocytic factor
c)contains lipopolysaccharide
d)is revealed by Hinss-Burry staining method

5.Gram staining method includes treatment of smear by:

1.0.5% hydrochloric acid
2.gencian violet
3.Neisser’s blue
4.lugole solution
143
a)1.2.4. b)2.4. c)2.3. d)1.3.4.

6.Which of the following can restore synthesis of the cell wall:

1.protoplast
2. L-forms
3.spheroplast
4.mycoplasma
a)2.4. b)1.3.4. c)1.2.3. d)3.4.

7.Functions of the cell wall are the following, Except:

a)provides Neisser’s staining
b)provides the shape of bacteria
c)provides Gram staining
d)it is receptor for bacteriophages and colicines

8.Which of the following are used in Gram staining:

a)water fuchsine
b)HCl
c)0.5% sulfuric acid
d)Ziehl's fuchsine

9.Which of the following are typical of cytoplasmic membrane, Except:

a)is a three layer structure which consists of lipids and proteins
b)regulates metabolism of bacterial cell
c)participates in spore formation
d)has tinctorial property

10.Volutin granules are:

1.revealed by Ionne method
2.the same as metachromatic granules
3.protein synthesizing system
4.poly and metaphosphates
a)2.4. b)3.4. c)1.4. d)2.3

144
11.Which of the following is Not typical of volutin granules:
a)have differential diagnostic meaning
b)are also called Babesh Ernst granules
c)are stained by Ziehl-Neelsen
d)possess metachromazia property

12.Neisser’s staining method:

1.is used to find out spores
2.includes 5% H2SO4 usage
3.is used to find out volutin granules
4.includes the usage of vinegar sour blue and Lugol’s solution
a)1.3.4. b)3.4. c)1.2.3. d)2.4.

13.Motility of bacteria is revealed by:

a)Leoffler’s staining method
b)Hinss-Burry method
c)Ionne method
d)hanging drop method

14.Staining method by Ojeshki:

1.is used for detection of volutine granules
2.includes elaborating by Ziehl-carbolic fuchsine
3.is used for spore detection
4.includes cultivation by 5% H2SO4
a)1.2.4. b)2.3.4. c)1.4. d)3.4.

15.Resistancy of spores is determined by the following:

1.presence of lipoteichoic acid
2.presence of Ca-salts of dipicolinic acid
3.absence of free water
4.presence of cortex
a)1.3.4. b)2.4. c)2.3.4. d)3.4.

145
16.Which of the following has adhesive function:
1.capsule
2.flagella
3.teichoic acid
4.fimbria
a)1.2.4. b)2.4 c)1.3.4. d)1.2.

17.Motility of bacteria is detected by:

a)Loffler’s staining method
b)Neisser’s staining method
c)hanging drop method
d)acid- fast staining

18.Which of the following are the revealing methods for Spirochetes:

1.Osheshki
2.Romanovsky-Giemza
3.Neisser
4.Morozov
a)1.2. b)3.4. c)2.4. d)2.3.

19.Which of the following are typical of Rickettsia:

1.staining by Zdradowski
2.presence of cell wall
3.intracellular parasitism
4.growth on artificial nutrient media
a)1.4. b)1.2.3. c)1.3.4. d)2.4.

20.Chlamydia are:
1.obligate intracellular parasites which lack cell wall
2.obligate parasites which replicate by binary fission
3.obligate parasites which cannot grow on artificial nutrient media
4.obligate parasites which affect both human and animals
a)1.3. b)2.3.4. c)3.4. d)1.2.4.

146
21.Mycoplasma:
a)belong to kingdom “Vira”
b)affect respiratory and genital-urinary tract
c)cannot grow on artificial nutrient media
d)contain one type of nucleic acid

22.Which of the following are typical of Actinomycetes, Except:

a)are gram positive
b)form mycelium on artificial nutrient media
c)belong to kingdom Prokarya
d)are microaerophilic

1. d, 2. d, 3. c, 4. c, 5. b, 6. c, 7. a, 8. a, 9. d, 10. a, 11. c, 12. b, 13. d,

14. b, 15. c, 16. c, 17. c, 18. c, 19. b, 20. b, 21. b, 22. d

1.Note the important substances in the nutrient media for the growth of
microorganism:
1.Andrade’s indicator
2. amino-acids
3.microelements
4.vitamins
a)1.2.4. b)2.3.4. c)2.3. d)3.4.

2.Spores of bacilli are destroyed in:

a)pasteurization
b)prolonged dryness
c)autoclaving
d)heating

3.Which substance is used to obtain the solid consistence of nutrient

media:
a)carbohydrates
147
b)proteins
c)agar-agar
d)aminoacides

4.Differential-diagnostic media are:

a)universal and are used for cultivation of majority of microbes
b)favorable for some types of microorganisms and don’t inhibit the
growth of others
c)favorable for some types of microorganisms and inhibit the growth of
others
d)used for orientated differentiation of microorganisms

5.Solid nutrient media are represented by, Except:

a)smooth agar
b)curved agar
c)plate agar
d)column agar

6.Viral inclusions can be revealed by the following method:

a)Zdradowski
b)Ionne
c)Romanowsky-Giemsa
d)Ziehl-Neelsen

7.The growth of viruses takes place in:

1.experimental animal organism
2.bile broth
3.cell culture
4.embryonated eggs
a)2.3.4. b)1.3.4. c)1.2. d)2.4.

8.Titration of bacteriophage is carried out in:

a)liquid media
b)embryonated egg
148
c)animal’s organism
d)tissue(cell) cultures

1. b, 2. c, 3. c, 4. d, 5. a, 6. c, 7. b, 8. a

1.Which microbes are included in mouth normal microflora:

1.Streptococci
2.Treponema orale
3.Candida albicans
4.E. coli
a)1.3. b)2.4. c)1.2.3. d)1.3.4.

2.What does mutualism mean:

a)is an association in which one partner benefits but the other remains
unharmed
b)is an association in which both partners benefit
c)is an association in which one organism derives benefit at the expense
of the other organism
d)is an association in which one organism increases physiological
functions of the other organism

3.What does commensalism mean:

a)is an association in which one partner benefits but the other remains
unharmed
b)is an association in which both partners benefit
c)is an association in which one organism derives benefit at the expense
of the other organism
d)is an association in which one organism increases physiological
functions of the other organism

149
4.What does parasitism mean:
a)is an association in which one partner benefits but the other remains
unharmed
b)is an association in which both partners benefit
c)is an association in which one organism derives benefit at the expense
of the other organism

1. c, 2. b, 3. a, 4. c

1.Modifications of microorganisms:
1.do not depend on environmental factors
2.are characterized by changes of genotype
3.are provided by changes of phenotype within the same genotype
4.can revert to previous properties
a)1.3. b)1.4. c)3.4. d)2.4.

2.The copying of a DNA sequence into mRNA is called:

a)conjugation
b)transcription
c)transformation
d)translation

3.The conversion of the mRNA information into protein at the ribosome

is called:
a)translation
b)transduction
c)transformation
d)transcription

4.Which of the following depends on hereditary variability, Except:

a)L forms production
b)transformation

150
c)transduction
d)conjugation
1. c, 2. b, 3. a, 4. a

1.Bacteremia is a:
a)fresh infection of the host by the same bacteria in which the main
disease has not ended
b)repeated infection of the host by the same bacteria in which primary
disease has been treated
c)circulation and multiplication of bacteria in the blood
d)circulation of bacteria in the blood

2.Due to the origin infections are divided into:

1.exogenous
2.endogenous
3.superinfection
4.mixed infection
a)2.3.4. b)1.2.3. c)1.2. d)2.4.
3.Note the consequent stages of infectious disease development:
1.re-convalescent
2.incubation period
3.specific illness period
4.prodrome period
a)2.3.4.1. b)2.4.1.3. c)3.1.4.2. d)2.4.3.1.

4.The stages of infectious disease are, Except:

a)incubation
b)relapse
c)specific illness
d)prodrome period

1. d, 2. c, 3. d, 4. b,
151
1.ß-lactamates interfere with:
a)cell wall synthesis
b)plasma membrane function
c)protein synthesis
d)DNA function

2.Antibiotic sensitivity tests are the following:

1.disc diffusion method
2.method of sequential dilutions
3.tuberculin test
4.fluorescent antibody test
a)1.2.3. b)2.4. c)1.2. d)2.3.

3.Which of the following may form resistance to antibiotics:

a)Col. plasmid
b)R-plasmid
c)constitutive enzymes
d)F- plasmid

4.The following causes the resistance to antibiotics:

1.Col-plasmid
2.R-plasmid
3.adaptive enzymes
4.F-plasmid
a)2.3. b)1.3. c)1.2.3. d)2.3.4.

5.The antibacterial chemotherapy preparations are:

1.phythoncides
2.preparations of arsenic
3.sulfanilamides
4.formaldehydes
a)1.3. b)2.3.4. c)2.4. d)1.2.3.
152
6.The following are the complication of antibacterial therapy:
1.disbacteriosis
2.rash
3.formation of L-form
4.hormonal changes
a)1.2. b)2.3.4. c)1.2.3. d)3.4.

1. a, 2. c, 3. b, 4. a, 5. d, 6. c

1.For classical complement fixation reaction (Bordet-Gangou reaction) is

necessary:
1.patient’s serum
2. non specific antigen
3.specific antigen
4.sheep erythrocytes
a)1.2.3. b)1.2. c)1.3.4. d)2.4.

2.Which of the following is typical of precipitation reaction:

a)is expressed by formation of white ring in liquid media
b)reveals incomplete anti-Rh antibodies
c)is usually not visible to the naked eye
d)is expressed by formation of sediment

3.Serological reaction is used, Except:

a)to reveal unknown antigen
b)to diagnose infectious diseases
c)to reveal cultural properties of bacteria
d)to find unknown antibodies

4.Agglutination reaction is described by the following, Except:

a)is used to reveal unknown antigen
b)is used to reveal unknown antibody

153
c)reveals soluble antigen
d)may be performed on slide
5.Which of the following statements are correct:
1.anatoxin is obtained from exotoxin
2.killed vaccines are also named corpuscular
3.alive vaccines are also named subunite
4.anatoxins have high antigenic and immunogenic properties and are
obtained from endotoxins
a)2.4. b)1.2.4. c)1.2. d)3.4.

6.Which of the following statements are correct:

1.anatoxins are obtained from endotoxins treated with 0.3-0.4% of
formalin, during 1 month
2.subunite vaccines are less reactogenic compared with alive and killed
3.alive vaccines don’t multiply in the organism
4.adjuvant increases the size of molecule and form depot in the organism
a)1.2. b)1.3. c)1.2.4. d)2.4.

7.Vaccines and immune sera:

a)must be less reactogenic and should not cause complications
b)must change their structure after penetration into the cell
c)should be kept at room temperature
d)should be provided by low immunogenic properties

8.Which of the following statements is Wrong:

a)there are monovaccines, divaccines, trivaccines etc.depending on the
quantity of prevented infections
b)alive vaccines are obtained by decreasing the virulence of bacteria,
using biological factors
c)alive vaccines multiply in the organism and form prolonged, stable
immunity
d)alive vaccines are used only in solved condition, and parenterally

1. c, 2. a, 3. c, 4. c, 5. c, 6. d, 7. a, 8. d
154
1. Anantanarayan and Paniker’s “Textbook of Microbiology”, India,
710 pages, 2014
2. Waren Levinson, Ernest Jawetz, “Medical Microbiology and
Immunology”, New-York, 644 pages, 2004
3. Nester, Anderson, Roberts, Peasrsall, Nester “Microbiology”, 817
pages, 2011
4. Ted R.Jonson, Christine L. Case, “Laboratory Experiments in
Microbiology”, 418 pages, 1995
5. Ivan Roit, Jonathan Brostoff, David Male, “Immunology”,
London, 480 pages, 2001
6. Jeffrey K. Aktor, Immunology and Microbiology, Texas, 172
pages, 2007
7. Kaplan, USMLE Step 1 Lecture Notes, Immunology and
Microbiology, 433 pages, 2017
8. Patrick R., Murray Ken S. Rosenthal, Michael A. Pfaller,
“Medical Microbiology”, 932 pages, 2016
9. Paul G. Engelkirk, Gwendolyn R. W. Burton, Burton’s
Microbiology For The Health Sciences, 398 pages, 2007
10. Շեկոյան Բ.Ա., Մանուկյան Կ.Ղ. «Բժշկական
Մանրէաբանություն Վիրուսաբանություն և
Իմունաբանություն», ԵՊԲՀ, 478 էջ, 2015

155
 .................................. 7

.................................................................. 11
.......................................... 13
..................... 15

.................................................................................... 18

................................................................................ 22

..................................... 26
............................................................... 29

....................................... 32

...................................................................... 38

............................................................................ 42

................................. 52

.................................................................... 57

156
 ...................... 61

....................................................................... 65

..................................... 72

................................................................................. 77
........................... 81

............................................... 89

.................................................................... 98

G.M. Poghosyan

................................................................................................... 102

.. 111
................................................. 118
............................................. 125

....................................................................................... 132
.......................................... 136

157
 143

158
Հրատարակությունը ԼեգալՊրինտ տպարանի
Տպաքանակը՝ 100 օրինակ

159