Division
Division
Division
Culture Media:
Liquid media such as nutrient broth can be used for the propagation
of large members of microorganisms, fermentations tests and other
biochemical tests.
Semisolid media contain less than 1% agar and it can be used for motility
and fermentation tests.
Solid media contain less than 1.5% agar and it can be used for culture isolation,
colonial appearance and tests for extracellular enzymes.
Selective media: permit the growth of kinds of bacterial and inhibit others.
Enrichment media: are liquid media which encourage the growth of certain
kinds of bacteria in mixed population.
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Transport media
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Background:
Since bacteria are so small, they are difficult to see with the light
microscope. Bacteria are usually colorless and therefore cannot be seen
because of the lack of contrast with the surrounding medium. Simple
stain is the use of basic dye to increase the contrast of cells for
microscopy; the scope of simple staining technique to determine cell
morphology (shape) relative size and arrangement.
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Gram stain
Background:
It is one of the most important and widely used procedures in microbiology
for characterizing most bacteria into two groups based on the structure and
chemical difference of the cell wall.
Those organisms which retain the crystal violet (appear dark blue or
violet) are designated gram positive; those which lose the crystal violet
and stained by the safranine (appear red) are designated gram negative.
Gram Stain
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Background:
• A few genera, particularly the members of the genus
mycobacterium, are resistant and can only be visualized by the
acid-fast method.
• Since M. tuberculosis and M. leprae represent bacteria that are
pathogenic to humans, the stain is of diagnostic value in
identifying these organisms.
• The characteristic difference between mycobacteria and other
microorganisms is the presence of a thick waxy (lipoidal) wall
that makes penetration by stains extremely difficult. Once the
stain has penetrated, however, it can not be readily removed even
with the vigorous use of acid alcohol as a decolorizing agent.
• Because of this property, these organisms are called acid-fact,
while all other microorganisms, which are easily decolorized by
acid-alcohol, are non-acid- fast.
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Negative stain
Background:
The negative stain technique does not stain the bacteria but stains the
background. The bacteria will appear clear against a stained
background. The negative stain does not stain the bacteria due to the
ionic repulsion of the negative charge on the bacterial surface and the
acidic stain (negative charge). No heat fixing or strong chemical are
used, negative stain can be used to determine cell morphology, size and
capsule.
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Endospore stain
Background:
• Endospores are formed by members of two genera Bacillus and
Clostridium which are of great medical importance.
• Endospores are metabolically inactive and are resistant to
heating, various chemicals and many harsh environmental
conditions.
• Sporogensis is not for reproduction, but it is resistant to
unfavorable environment, Endospore can remain dormant for
long time. However, Endospore may return to its vegetative or
growing state.
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Background:
• Antibiotics are substance isolated from a biological source that is
antagonistic to microorganisms.
• Today there are many antibiotics commercially available for use
against a variety of microorganisms.
• Antimicrobial chemicals absorbed or used internally whether
natural (antibiotics or synthetic) are called chemotherapeutic
agents.
• Using disc diffusion methods, disks impregnated with different
chemotherapeutic agents are placed on culture-plated pre-
inoculated with the organism to be tested, after overnight
incubation the degree of sensitivity is measured from the
diameter of zone of inhibition which it self is depend on variables
such as :
• The ability and rate of diffusion of the antibiotic into the medium, and its
interaction with the test organism.
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Background:
• The genus staphylococcus is composed of both pathogenic and
nonpathogenic organisms.
• They are gram-positive cocci and occur most commonly as irregular
clusters or spherical cells.
• They are mesophilic non-spore –formers; however, they are
generally highly resistant to drying, especially when sequestered in
organic matter such as blood, pus, and tissue fluids.
• The three major species include S. aureus, S. saprophyticus, and S.
Epidermidis. Strains of the last two species are generally avirulent;
however, under special circumstances in which a suitable portal of
entry is provided, S. epidermidis may be the etiological agent for
skin lesions and endocarditis and S. saprophyticus has been
implicated in some urinary tract infections.
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Catalase test
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Background:
• Members of the genus Streptococcus are perhaps responsible for
a greater number of infectious diseases than any other group of
microorganisms.
• Morphologically, they are cocci that divide in a single plane
forming chains. They form circular, translucent to opaque,
pinpoint colonies on solid media.
• All members of this group are gram-positive, and many are
nutritionally fastidious, requiring enriched media such as blood
for growth.
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Classification
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Optochin test
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Bacitracin test
a filter paper disc impregnated with 0.04 unit of bacitracin is applied to the
surface of a blood agar plate previously streaked with the organism to be
identified. Following incubation, the appearance of a zone of growth
inhibition surrounding the disc is indicative of group A streptococci. The
absence of this zone suggests a non-group A organism.
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CAMP test
group B streptococci produce a peptide, the CAMP substance that acts in
concert with the beta-hemolysins produced by some strains of
staphylococcus aureus, causing an increased hemolytic effect. Following
inoculation and incubation, the resultant effect appears as an arrow-shaped
zone of hemolysis adjacent to the central streak of S. aureus growth. The
non group B streptococci do not produce this reaction.
Camp test
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Background:
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IMViC tests:
IMVIC Tests
IMVIC REACTIONS
IMVIC reactions are a set of four useful reactions that are commonly employed in the
identification of members of family enterobacteriaceae. The four reactions are:
Indole test, Methyl Red test, Voges Proskauer test, and Citrate
utilization test.
INDOLE TEST:
Principle: Some bacteria can produce indole from the amino acid tryptophan
using the enzyme typtophanase.
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Principle: While MR test is useful in detecting mixed acid producers, VP test detects
butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the
production of butylene glycol. In this test two reagents, 40% KOH and alpha-naphthol
are added to test broth after incubation and exposed to atmospheric oxygen. If acetoin
is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts
with guanidine components of peptone, in the presence of alphanaphthol to produce red
color. Role of alpha-naphthol is that of a catalyst and a color intensifier.
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Principle: This test detects the ability of an organism to utilize citrate as the sole
source of carbon and energy. Bacteria are inoculated on a medium containing sodium
citrate and a pH indicator bromothymol blue. The medium also contains inorganic
ammonium salts, which is utilized as sole source of nitrogen. Utilization of citrate
involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate.
Oxaloacetate is further broken down to pyruvate and CO2.
Production of Na2CO3 as well as NH3 from utilization of sodium citrate and ammonium
salt respectively results in alkaline pH. This results in change of medium’s color from
green to blue.
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Agglutination Reactions
Background:
The classical reaction in immunology is antibody-antigen reaction, that is
resulted or known as antibody-antigen complex formation. The reactions of
antibody (Ab) with a multivalent antigen (Ag) that is particulate (i.e. insoluble
particle) results in the cross-linking of the various Ag particles by antibodies.
This cross-linking eventually results in the clumping (agglutination) of the
antigen particles by the antibody.
Precipitation Reactions
Background:
A. Precipitin reaction in liquid
The precipitation reaction takes place when antibodies and soluble antigens are
mixed in the correct proportions. The lattice theory states that precipitation is a
result of the increase in size of antigen-antibody aggregates such that each
molecule of antigen is linked to more than one antibody molecule and vice
versa. When the aggregates exceed some critical volume, they settle out of the
solution (i.e. precipitate) spontaneously.
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Ouchterlony method
Single radial immunodiffusion (SRID) test is a variation of the immuno-
doublediffusion test. The wells contain antigen at different concentrations,
while the antibodies are distributed uniformly in the agar gel. Thus, the
precipitin line is replaced by a precipitin ring around the well. The diameter of
the precipitin ring is directly proportional to the concentration of the antigen in
the well. From the results obtained with known concentrations, a calibration
curve is constructed; permitting quantitation of the Ag concentration, figure 6.
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Blood Types
ABO is the best-known system for grouping blood types, though
there are other methods. There are four major categories within the
ABO group: A, B, O, and AB.
When a person needs a transfusion, doctors must give them the right
type. The wrong blood type can trigger an adverse reaction that
could be life-threatening.
What makes a blood type?
The blood group will depend on which antigens are on the
surface of the red blood cells. Antigens are molecules. They can be
either proteins or sugars. The types and features of antigens can vary
between individuals, due to small genetic differences.
The ABO blood group system classifies blood types according to the
different types of antigens in the red blood cells and antibodies in the
plasma.
Group A: The surface of the red blood cells contains A antigen, and
the plasma has anti-B antibody. Anti-B antibody would attack blood
cells that contain B antigen.
Group B: The surface of the red blood cells contains B antigen, and
the plasma has anti-A antibody. Anti-A antibody would attack blood
cells that contain A antigen.
Group AB: The red blood cells have both A and B antigens, but the
plasma does not contain anti-A or anti-B antibodies. Individuals
with type AB can receive any ABO blood type.
antigens. Since these antigens are not present, a person with any
ABO blood type can receive this type of blood.
Rhesus factor
Some red blood cells have Rh factor, also known as the RhD antigen. Rhesus grouping
adds another dimension.
If the red blood cells contain the RhD antigen, they are RhD positive. If they do not, they
are RhD negative.
If two parents have different blood types, the mother will not necessarily have the same
blood type or Rh factor as the child.
If the mother has Rh-negative blood, and the child has Rh-positive, this can pose a risk
during pregnancy and delivery.
A small number of red blood cells from the fetus’ circulation can
cross the placenta and enter the mother’s bloodstream. Anti-RhD
antibody can then develop in the mother’s plasma, in a process
known as sensitization.
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Having a higher or lower number of WBCs than normal may indicate an underlying
condition.
A WBC count can detect hidden infections within your body and alert doctors to
undiagnosed medical conditions,
Infants are often born with much higher numbers of WBCs, which
gradually even out as they age.
Having a higher or lower percentage of a certain type of WBC can
also be a sign of an underlying condition.
Leukocytosis and Leukopenia
Definition
• Leukocytosis
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Leukopenia
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