Practical Microbiology

Culture Media & Isolation of Pure culture

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.

Agar, as solidifying agents, agar is a complex polysaccharide extracted

from marine algae agar, is not digested by most microorganisms, melt
at 100c and solidify at 45c.

Media used for routine laboratory may be:

Enriched media:
Are prepared to meet nutritional requirements of more exacting
bacteria, by the addition of substance such as blood, serum and egg to
basal medium. These media can be formulated to have special
functions.

Differential media: manifest reaction which helps to differentiate between

bacterial species.

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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Nutrient Broth Nutrient Agar MacConkey Agar

Blood Agar Chocolate Agar CLED Agar

Simmons Citrate Agar Cooked Meat media Lowenstein Jensen

media

Loeffler serum medium Semisolid medium

Transport media

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Smear Preparation & Simple stain

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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Acid fast stain

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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Antibiotic Susceptibility Test

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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Antibiotic Susceptibility Test

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Identification of Human Staphylococcal Pathogens

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

During aerobic respiration, microorganisms produce hydrogen peroxide,

accumulation of this substance will result in death of the organism unless
it can be enzymatically degraded. Organisms capable of producing catalase
rapidly degrade hydrogen peroxide ad illustrated

2H2O2 Catalase 2H2O +O2

Laboratory tests for Identification of Staphylococcal sp.

Gram +ve cocci

Staphylococcus sp. +ve Catalase test -ve Streptococcus sp.

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Identification of Human Streptococcal Pathogens

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.

• The streptococci are classified by means of two major methods:

(1) Their hemolytic activity, and (2) the serologic
classification of lancefield. The observed hemolytic reactions on
blood agar are of the following three types:

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Classification

I- Hemolytic activity on Blood Agar

β-hemolytic α- hemolytic γ- hemolytic

Complete lyses to RBCs Partial lyses Non-hemolytic
due to production of Pneumococcus e.g. Enterococcus
- Streptolysin O Viridans group Esculin+ve
- Streptolysin S

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Optochin test

This is a growth inhibition test in which 6 mm filter paper discs

impregnated with 5 mg of ethylhydrocupreine hydrochloride (optochin)
and called P-discs are applied to the surface of a blood agar plate streaked
with the test organisms. The S. pneumoniae, being sensitive to this surface-
active agent, are lysed with the resultant formation of a zone of inhibition
greater than 15 mm surrounding the P-disc. Nonpneumococcal alpha-
hemolytic streptococci are resistant to optochin and fail to show a zone of
inhibition or produce a zone less than 15 mm.

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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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Identification of Enteric Pathogens (Enterobacteriaceae)

Background:

• The Enterobacteriaceae are a significant group of bacteria that

are endogenous to the intestinal tract or that may gain access to
this diet via a host's ingestion of contaminated food and water.
• The family consists of a number of genera whose members
vary in their capacity to produce disease.
• Salmonella and Shigella are considered to be pathogenic.
Members of other genera, particularly Escherichia and
Enterobacter, and to a lesser extent Klebsiella and Proteus
constitute the natural flora of the intestines and are generally
considered to be avirulent. However; that all can produce
disease under appropriate conditions.
• The Enterobacteriaceae are gram-negative; short rods. They
are mesophilic; non fastidious organisms that multiply in many
foods and water sources. They are all non-spore formers and
susceptible to destruction by common physical and chemical
agents. They are resistant to destruction by low temperatures
and can therefore frequently survive in soil, sewage, water, and
many foods for extended periods of time.
• From a medical point of view, members of the
Enterobacteriaceae account for about 50% of all clinically
significant bacterial isolates. They account for about 50% of
septicemia cases, 60 – 70 % of bacterial enteritis, and >70% of
urinary tract infections.
• A provisional identification based on the pattern of reactions in
a restricted range of tests (key tests) is usually considered
sufficient for a general hospital bacteriology laboratory. Further
identification and typing requires the use of extended
biochemical tests, serological tests, and other typing
techniques.

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Laboratory tests for the identification of enteric bacteria:

Gram –ve bacilli

Enterobacteriaceae -ve Oxidase test +ve non-

Enterobacteriaceae

Identification based on their biochemical properties:

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.

Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent.

Indole reacts with the aldehyde in the reagent to give a red color. An alcoholic
layer concentrates the red color as a ring at the top.

Example: Escherichia coli: Positive; Klebsiella pneumoniae: Negative.

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METHYL RED (MR) TEST:

Principle: This is to detect the ability of an organism to produce and maintain

stable acid end products from glucose fermentation. Some bacteria produce large
amounts of acids from glucose fermentation that overcome the buffering action of
the system. Methyl Red is a pH indicator, which remains red in color at a pH of
4.4 or less.

Example: Eschericihia coli: Positive; Klebsiella pneumoniae: Negative

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VOGES PROSKAUER (VP) TEST:

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.

Examples: Escherichia coli: Negative; Klebsiella pneumoniae: Positive

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CITRATE UTILIZATION TEST

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.

Examples: Escherichia coli: Negative; Klebsiella pneumoniae: Positive.

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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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Precipitation reactions can be performed in either liquids or gels. If performed

in liquid, the assay is called the precipitin test. The precipitin test is performed
by adding increasing amounts of antigen to a series of tubes containing a
constant volume of antiserum. The amount of protein precipitated increases
with the amount of antigen added, to a maximum beyond which larger amounts
of antigen lead to progressively less precipitation. In the equivalence zone, a
maximal amount of antibody is precipitated by an optimal amount of antigen.
Like agglutination reactions, precipitation reactions are subject to the prozone
and postzone effects (figure 4).

B. Precipitation reactions in gels

When soluble antigen and antibodies are placed in wells cut in an agar gel, the
reactants diffuse in the gel and form gradients of concentration, with the highest
concentrations closest to the well. Somewhere between the two wells, the
reacting antigen and antibodies will be present at proportions that are optimal
for formation of a precipitate (zone of equivalence), figure 5. This assay is
called the Ouchterlony or double radial immunodiffusion (DRID) technique.

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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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Single Radial immunodiffusion (SRID)

Immunoelectrophoresis is used to separate antigens in complex mixtures on the
basis of their charges in an electrical field. Antigens are separated in an agar
gel by applying an electric charge. The pH is chosen so that positively charged
proteins move toward the negative electrode and negatively charged proteins
move toward the positive electrode. A trough is then cut between the wells and
filled with
antibody, which is allowed to diffuse. The antigens and antibodies form
precipitin arcs, figure 7. This method is often used to characterize human serum
proteins.

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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 antigens in blood have various functions, including:

• transporting other molecules into and out of the cell

• maintaining the structure of red blood cells
• detecting unwanted cells that could cause illness

White blood cells produce antibodies. These antibodies will target an

antigen if they consider it a foreign object. This is why it is essential to
match blood types when a person needs a transfusion.

ABO and the most common blood types

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.

There are four ABO groups:

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.

Group O: The plasma contains both anti-A and anti-B antibodies,

but the surface of the red blood cells does not contain any A or B
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antigens. Since these antigens are not present, a person with any
ABO blood type can receive this type of blood.

Universal donor and universal recipient

O negative blood contains no A, B, or RhD antigens. Almost anyone

with any blood type can receive these red blood cells. A person with
group O negative blood is a universal donor.

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.

Blood types in pregnancy

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.

A problem can arise if this antibody then detects a “foreign” antigen

in the fetus’ blood cells. The antibodies may start to attack the fetus’
red blood cells as a defense mechanism.

In some cases, severe jaundice can result, and possibly brain

damage.

An injection of anti-RhD immune globulin G can help prevent the

mother from producing this antibody and reduce the impact of a
sensitizing event on the fetus.

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WBC (White Blood Cell) Count

A white blood cell (WBC) count is a test that measures the number
of white blood cells in your body.
There are several types of white blood cells, and your blood usually
contains a percentage of each type. Sometimes, however, your white
blood cell count can fall or rise out of the healthy range.
Purpose of a WBC count

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 is an elevation in the absolute WBC count (>10,000 cells/μL).

• Leukopenia is a reduction in the WBC count (<3500 cells/μL).

• Leukocytosis

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o An elevated WBC can be the normal BM response to an infectious or

inflammatory process, corticosteroids, β-agonist or lithium therapy, or
splenectomy, and is usually associated with an absolute neutrophilia.

Leukopenia

• Leukopenia can occur in response to infection (i.e., HIV), inflammation, primary

BM disorders (i.e., malignancy), autoimmune disorders, medications,
environmental exposure (i.e., heavy metals or radiation), and vitamin deficiencies.

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