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Sechenov Moscow Medical Academy

Medical Faculty
Department of Microbiology, Virology and Immunology

By Ang Hui Min


Medical Faculty, English Medium, Grou
Scientific Supervisor: Prof. E.P. Pashkov
Purpose:

1. To assist the physician to do a correct


diagnosis of infection and give correct
treatments to the patient against the
invading pathogen.
2. To know the cause of the disease,
epidemiological significant.
Classification of Microbes

MICROBIAL WORLD
Non Cellular Microbes
cellular
microbes
Prions Domain Domian MICROBIAL WORLD
Bacteria Archaea Domain Eukarya
Viriods
PROKARYOTES EUKARYOTES
Viruses
Bacteria Archaebacte Protozoa Fungi
ria
Microbes are too small to be seen with our naked
eye.

Microscopes are used to help us to differentiate


the morphological differences between the
microbes.

Different types of microscope is used


oStandard bright field microscope
oDark field microscope (Phase-contrast
microscope)
oFluorescence microscope
o
Microscop
e
Differential
Stains
**Microorganisms E.g. Gram stain, Acid-fast
are
invisibile Structural
without Stains
staining, they E.g. Negative stain,
are Anjeskys Stain
colorless.
What we can see by using microscope?


Bacilli
Cocci
Spiral
Filamentous

BAC
K
Bacilli
Cocci
Spiral
Filamentous

BAC
K
Bacilli
Cocci
Spiral
Filamentous

BAC
K
Bacilli
Cocci
Spiral
Filamentous

BAC
K
(Bacteriological Examinatio

Why Cultural Examination?

Microbes are morphological identical


To grow..
Microbes
Energy Physical Condition
Chemical Condition
Nutrients
Free from Competitors
1 2
3 4

1- obligate anaerobe
2- facultative anaerobe Aerobic
Anaerobic
3- microaerophile Culture
culture
4- strict aerobe
Microaerophili
c culture

1 2
3 4

Aerobic Culture
1 2
3 4

Anaerobic Culture
1 2
3 4

Microaerophilic culture
** Essential Media
It should contain essential food elements
It should have satisfactory moisture content
It should have correct pH (most bacteria are
neutrophiles. They grow best at pH between 6-8.
The consistency of the medium should be favourable
for cultivation.
It should be sterile.
It should be isotonic.
It should be transparent.
It should be oxygen-free if the cultivation of strict
anaerobes is desired.
Solid
Liquid
What we need for Inoculation??

Inoculating loops

START!!!
Petri-dishes with solid medium
Sterilize the inoculating loop Aseptic of instruments:
or needle by heating it in the
flame. Hold the instrument
like a pencil and at an angle
so that the wire is pointing
downward. Pass the entire
wire portion through the
flame starting at the base,
where it is attached to the
holder, and continue all the
way through the loop or end
of the needle. Heat the wire
until it is glowing red then
allow it to cool before
picking up any
microorganisms. The
instrument is now
considered to be sterile. Do
Streaking Technique:

A small amount of sample is


transferred onto the surface of a
suitable, solid agar medium either
by loop or transfer needle. This is
then streaked in such a Way as to
provide successive dilutions and
ultimately to have well isolated
colonies. Streaking may be done in
any of the ways. In each case the
sample becomes progressively
diluted and at the end of streak
one would expect the well isolated
colonies. Quadrant Streak Method
Quadrant Streak Method

2 2
1st quadrant
can be 3
1
inoculated with
a loop or swab.
Each of the
remaining three
quadrants must
be streaked 4
with a sterile
instrument
such as flamed
Pour Plate technique :
1.Dilute specimen to yield
approximately 30 to 300 CFU
per aliquot to be plated
2.Diluted sample is
added directly to the
empty plate
(Inoculation)

The dishes are incubated


3.Melted plate at suitable temperature.
count agar, 45 After few days, different
C kinds of microbe: grow as
is added to separate colonies. Cells
sample and from individual colony may
mixed be picked up for a
subculture. Purpose:
Purpose
Spreading Plates : : for counting microbes

Small volume Sterile bent


of the diluted glass rod is
sample is used to
added to the spread the
surface of the small volume
medium of sample on
the surface
or solid
medium.

**Colonies grow on the agar surface after incubation.


Measuring microbial concentrations Plate
count By
spreading

After incubation, colonies


can be seen.
Calculate the colonies
and multiply with the
Colonie
s
Colonies:

size
type of
margin
colony
elevation
colony
texture
Light
transmission
colony
pigmentation
2. Isolation
To obtain pure culture
Pure culture
colonies will be
If different types of obtained after
colonies can be seen on incubation.
the plate

Choose one colony from the plate,


Inoculate in the slant agar.
Evaluate water quality
1.Heterotrophic Plate count ( Pour Plate Method is
used)
2.Multiple-Tube fermentation Technique
3.Membrane Filter Technique
Typical
growth is
observed
and these
tubes are
used to
determine
the Water
quality.

Lactose Bile Broth EC broth


Examining Air Contamination
1.Sedimentation Method (Settling Plate)
- Solid medium directly expose to Air then
incubated
2. Aspiration Method

Aspiration Method (slit Air-borne Microbes


colonies
Media Guide
Urea Broth EMB agar
Sheeps blood Agar Plate
Motility Test
Media
MacConkey
Agar Triple
Sugar Iron
MacConkey Agar
A culture medium
designed to grow Gram-
negative Bacteria and
stain them for lactose
fermentation
The organism growing at
the top is a Gram
negative organism that
does not ferment
lactose. The organism
growing at the bottom is
a Gram negative
organism that does
ferment lactose.
Urea Broth

The tube on the left is a


urease positive organism
and the tube on the right
is a urease negative
organism.


EMB
Agar
Each of these plates
contain Gram negative
organisms that can
ferment lactose. The
left plate is
contaminated. Notice
the white colonies
interspersed with the
pink colonies. When
organisms ferment the
lactose in EMB they
produce acid which
turns the colonies pink,

purple or green,
depending on the pH.
Sheeps Blood Agar Plate
This media is used to test if an
organism has hemolysins
(enzymes that lyse red blood
cells). If an organism lyses red
blood cells in the media then
clearing zones are produced
around the culture. An organism
can be classified as alpha, beta or
gamma hemolytic. The culture
on the top portion of this plate is
showing alpha hemolysis (partial
clearing of red blood cells). The
culture on the bottom of the
plate is showing gamma
Triple Sugar Iron Slant (TSI
Slant)
TSI slants are used to test if an
organism ferments glucose and
sucrose and/or lactose and if the
can reduce sulfate. The tube on
the far left is uninoculated. The
second tube shows a yellow butt
and a yellow slant. This means
the organism fermented glucose
and lactose and/or sucrose. The
third tube has a yellow slant and
a yellow butt. This means that
the organism fermented glucose
and lactose and/or sucrose and
reduced sulfate. The fourth tube
from the left shows a yellow butt
and a red slant. This means that
the organism only fermented
Motility Test Media

Motility Test Media is a


indirect method to
determine the
presence of flagella of
bacteria.

From left to right: motile, non-


motile, motile


3.
Identificatio
Identification of the pure culture of
n microbe on the basis of its cultural,
morphological and tinctorial
properties, biochemical and antigenic
characteristics
Cultural Examination (Bacteriological
method) allows microbiologist to
perform epidemiological typing of
isolated microbe and to choose the
drugs for antimicrobial therapy
Techniques for Epidemiology typing

1.Biotyping (biovars)
2.Serotyping (serovars)
3.Bacteriophage (phagovars)
4.Toxigenicity
5.Bacteriocin
6.Nucleic acid restriction analysis and other
genetic tests
7.Antibiograms
- Disk Diffusion Method
- Minimum Inhibitory Concentration (MIC)
Phagotyping Antibiogram
When pathogen cannot be isolated on the
culture media in laboratory, animated models
of culture media is used.
Incubation of
chicken
embryo

Duodenum tissue culture Experimental


animals
Specimen: Patients Blood serum
Object : To determine specific antibodies
against the
pathogen responsible for an
infection
Serial dilution agglutination test
Indirect hemagglutination test
Complement-fixation tests
Immunologic reactions with labeled
components (e.g. IF-test, RIA, ELISA)

** Determine antibody titer in the


patients serum
1. Serial Dilution agglutination

Add a fixed amount of


particles (e.g., red blood
cells) carrying antigen
on their surface to a
series of tubes
containing increasing
(usually doubling)
dilutions of the
antiserum. The titer of
the antiserum is the
reciprocal of the highest
dilution in which
agglutination occurs.
Hemagglutina
tion
can be used to measure the
level of antibodies to
particulate antigens. In this
test, serial dilutions are made
of a sample to be tested for
antibody and then a fixed
number of red blood cells or
bacteria or other such
particulate antigen is added.
Then the maximum dilution
that gives agglutination is
determined. The maximum
dilution that gives visible
agglutination is called the titer.
The results are reported as the
Complement-fixation test
Antigen/antibody complexes can
also be measured by their ability
to fix complement.
An antigen/antibody complex will
"consume" complement if it is
present
Antigen is mixed with the test
serum to be assayed for antibody After allowing complement
and antigen/antibody complexes fixation by any
are allowed to form. A control tube antigen/antibody complexes,
in which no antigen is added is also a standard amount of red
prepared. If no antigen/antibody blood cells, which have been
complexes are present in the tube, pre-coated with anti-
none of the complement will be erythrocyte antibodies is
fixed. If antigen/antibody added.If all the complement
complexes are present, they will fix was still, all the red cells will
complement and thereby reduce be lysed.
Positive
Localized delayed
hypersensitivity
(redness and
swelling) occurs.
The person has the
disease.

Negative
No reactions.
No contact with the
pathogens

** second contact hypersensitivity


Polymerase chain reaction (PCR)

IIF-test (Indirect Immunofluoresence)

Enzyme-linked Immnosorbent Assay


(ELISA)
Polymerase Chain Reaction
(PCR)
PCR allows scientists to extract
and analyze bits of microbial DNA
from samples, meaning they
dont need to find and grow
whole cells.
PCR is an essential element in
DNA fingerprinting and in the
sequencing of genes and entire
The technique makes use of
genomes.
the DNA repair enzyme
polymerase.
Polymerase uses the intact
half of the DNA molecule as a
template and attaches the
right nucleotides, which
circulate constantly in the cell,
to the complementary
Indirect
Immunofluorescence test
Samples are added on a
substrate slide for primary
reaction of antibodies and
antigens. After washing,
Fluorescein isothiocyanate
(FITC) conjugated antibodies
are added to make complexes
of antigens - antibodies - FITC
conjugated antibodies. After
washing, the fluorescence
from FITC is observed by
fluorescent microscope.
ELISA
Samples are added to microwell coated with antigen (or antibody),
allowing antibody (or antigen) in samples to react with the
immobilized antigen (or antibody) in the microwell (Sample
incubation). After wash to remove any unbound serum proteins,
enzyme conjugated antibodies are added and incubated
(Conjugate incubation). Following another washing step, the
enzyme substrate is added and allowed to incubate for an
additional period of time (Substrate incubation). Acid solution is
then added to each well to terminate the enzyme reaction and to
stabilize the color development. The assay can be quantified by
measuring the reaction photometrically.
By reading the tests
result, the laboratory
staff and physician
maybe able to cure the
patient and minimize
the danger of epidemic
spread.

These findings also


alert the attending
physician to
immunologic
deficiencies or other
---THE END---
1. Medical Microbiology and Immunology LANGE Warren
Levinson, Ernest Jawertz
2. Sechenov Moscow Medical Academy- Department of
Microbiology General Microbiology, Part I : Morphology of
Microorganisms
3. Sechenov Moscow Medical Academy- Department of
Microbiology General Microbiology, Part II: Physiology and
Genetics of Microorganisms
4. Sechenov Moscow Medical Academy- Department of
Microbiology General Microbiology, Part III : Applied
Infectology and Immunology
5. Sechenov Moscow Medical Academy- Department of
Microbiology Special Microbiology, Part I and Part II
6. www.answers.com , www.wikipedia.com
7. Color Atlas and Textbook of Diagnostic Microbiology by Elmer
W. Koneman (Author), Stephen D. Allen (Author), William M.,
Ph.D. Janda (Author), Paul C. Schreckenberger (Author),
Washington C., Jr., M.D. Winn (Author)
8. www.googles.com