Transcript Slide 1

In the name of God
Yasuj University of Medical Sciences
Department of Microbiology
By: Dr. S. S. Khoramrooz
Department of Microbiology, Faculty of Medicine,
Yasuj University of Medical Sciences, Yasuj, Iran
Intended Use

Blood Agar Base (Infusion Agar), with the addition of
sterile blood, is used for the isolation, cultivation and
detection of hemolytic activity of streptococci and other
fastidious microorganisms.
Summary and Explanation
 Infusion Agar is an all-purpose medium which has been
used for many years as a base for the preparation of
blood agars.

In a study of viability of streptococci, Snavely and Brahier
performed comparative studies of horse, rabbit and sheep
blood
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
with Blood Agar Base, and found that sheep blood gave the
clearest and most reliable colony and hemolysis
characteristics at both 24 and 48 hours.

In the course of the investigation, about 1,300 isolations of
streptococci were made with Blood Agar Base containing
5% sheep blood.

Blood Agar Base media are specified in standard methods
for food testing.

Infusion Agar has been largely replaced as a blood agar
base by the Tryptic/Trypticase™ Soy Agar formulations,
which contain milk and plant peptones in place of the
variable infusion component.
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Principles of the Procedure

Infusion from heart muscle, casein peptone and yeast
extract provide nitrogen, carbon, amino acids and vitamins
in Blood Agar Base.

Supplementation with blood (5-10%) provides additional
growth factors for fastidious microorganisms, and is the
basis for determining hemolytic reactions.

Hemolytic patterns may vary with the source of animal
blood or type of base medium used.
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1. Suspend 40 g of the powder in 1 L of purified
water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute
to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. For preparation of blood agar, cool the base to 4550°C and aseptically add 5% sterile, defibrinated
blood. Mix well.
5. Test samples of the finished product for
performance using stable, typical control cultures.
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Expected Results

Colonial morphology on blood agar containing 5% sheep blood is
as follows:
1. Hemolytic streptococci may appear as translucent or opaque,
grayish, small (1 mm), or large matte or mucoid (2-4 mm)
colonies, encircled by a zone of hemolysis.
Gram stains should be made and examined to check the macroscopic
findings. (Other organisms which may cause hemolysis include
Listeria, various corynebacteria, hemolytic staphylococci,
Escherichia coli and Pseudomonas.)
Approximate quantitation of the number of colonies of hemolytic
streptococci may be helpful to the clinician.
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2. Pneumococci usually appear as very flat, smooth,
translucent,grayish and sometimes mucoid colonies
surrounded by a narrow zone of “green” (alpha)
hemolysis.
3. Staphylococci appear as opaque, white to gold-yellow
colonies with or without zones of beta hemolysis.
4. Listeria may be distinguished by their rod shape in
stains, and by motility at room temperature.
Small zones of beta hemolysis are produced.
5. Other organisms representing minimal flora and
clinically significant isolates can also be expected to
grow on this nonselective formulation.
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Intended Use
 Brain Heart Infusion (BHI) is a general-purpose liquid
medium used in the cultivation of fastidious and
nonfastidious microorganisms, including aerobic and
anaerobic bacteria, from a variety of clinical and
nonclinical materials.

It serves as a base for supplemented media containing
0.1% agar, Fildes enrichment or 6.5% sodium chloride.

A supplemented pre-reduced formulation in tubes is
especially recommended for the cultivation of anaerobes.
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
In the formulation containing 6.5% sodium chloride, the
salt acts as a differential and/or selective agent by
interfering with membrane permeability and osmotic and
electrokinetic equilibria in salt-intolerant organisms.

Fildes enrichment (peptic digest of sheep blood) is
incorporated into one tubed formulation for the cultivation
of fastidious microorganisms, such as Haemophilus
influenzae.

The addition of 0.1% agar aids in the cultivation of
anaerobic microorganisms because its consistency yields
conditions of reduced oxygen tension.
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 The
pre-reduced medium in Hungate tubes is
based on Hungate methods of culturing anaerobic
microorganisms outside of an anaerobic chamber.

The tubes provide a reduced medium in a selfcontained, anaerobic tube sealed using a Hungate
screw cap.
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
Growth in the tubes is indicated by the presence of turbidity
compared to an uninoculated control.

If growth appears, cultures should be examined by Gram stain
and subcultured onto appropriate media; e.g., a Trypticase™
Soy Agar with 5% Sheep Blood and/or Chocolate II Agar plate,
EMB Agar or MacConkey II Agar plate.

If anaerobes are suspected, subcultures should be incubated
anaerobically, as in a GasPak EZ anaerobic system.

Enterococci will grow in the 6.5% NaCl broth within 24-48
hours.

Nonenterococcal group D streptococci fail to grow in the
medium after 48 hours of incubation.
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Intended Use
 Brain Heart Infusion (BHI) Agar is a general-purpose
medium suitable for the cultivation of a wide variety
of organism types, including bacteria, yeasts and
molds.
 With
the addition of 5% or 10% sheep blood, it is
used for the isolation and cultivation of a wide variety
of fungal species, including systemic fungi, from
clinical and nonclinical sources.
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Intended Use

LB Agar, Lennox and LB Broth, Lennox are used for maintaining and
cultivating recombinant strains of Escherichia coli.
Summary and Explanation

LB Agar, Lennox and LB Broth, Lennox are nutritionally rich
media developed by Lennox for the growth and maintenance of
pure cultures of recombinant strains of E. coli.

These strains are generally derived from E. coli K12, which are
deficient in B vitamin production.
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 This
strain of E. coli has been further modified
through specific mutation to create an
auxotrophic strain that is not capable of growth
on nutritionally deficient media.
 LB Agar,
Lennox provides all the nutritional
requirements of these organisms.
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Expected Results
 After
sufficient incubation, the agar medium should
show growth as evidenced by formation of colonies
and/or a confluent lawn of growth.
 In
the broth medium, growth is evident by the
appearance of turbidity.
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Intended Use
 LB Agar, Miller and LB Broth, Miller (Luria-Bertani)
are used for maintaining and propagating Escherichia
coli in molecular microbiology procedures.
Expected Results

Growth should be evident on the agar medium by the
appearance of colonies and/or a confluent lawn on the
surface of the medium.

In the broth medium, growth is evident by the
appearance of turbidity.
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Intended Use
 Luria Agar Base, Miller and Luria Broth Base, Miller are
used for maintaining and propagating Escherichia coli
in molecular microbiology procedures with or without
added glucose.
Principles of the Procedure
 Peptone and yeast extract provide nitrogen, carbon,
vitamins (including B vitamins) and certain trace
elements.

Sodium chloride provides essential ions. Agar is the
solidifying agent.
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Intended Use
 Each lot of Mueller Hinton Agar and Mueller Hinton
II Agar has been tested according to, and meets the
acceptance limits of, the current M6 protocol
published by the CLSI.
 Mueller
Hinton Agar is recommended for
antimicrobial disc diffusion susceptibility testing
of common, rapidly growing bacteria by the
Bauer-Kirby method, as standardized by the
Clinical and Laboratory Standards Institute
(CLSI).
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 Mueller
Hinton Agar with 5% Sheep Blood is
recommended for antimicrobial disc diffusion
susceptibility testing of Streptococcus pneumoniae
with selected agents; i.e., chloramphenicol,
erythromycin, ofloxacin, tetracycline and
vancomycin,
 In
addition to oxacillin screening for susceptibility
to penicillin, as standardized by the Clinical and
Laboratory Standards Institute (CLSI).
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
NOTE: The recommended medium for disc diffusion
susceptibility testing of Streptococcus pneumoniae is
Mueller Hinton agar with 5% sheep blood.

The recommended medium for Haemophilus influenzae is
Haemophilus Test Medium (HTM) gar.

The recommended medium for Neisseria gonorrhoeae is
GC Agar with 1% defined growth supplement (GC II
Agar with BBL™ IsoVitaleX™ Enrichment or
equivalent).

Interpretive criteria are provided in the CLSI Document
M100 (M2),5 which is included with CLSI Document
M2,
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 The
Bauer-Kirby procedure is based on the diffusion
through an agar gel of antimicrobial substances
which are impregnated on paper discs.
 In
contrast to earlier methods which used discs of high
and low antimicrobial concentrations and which used
the presence or absence of inhibition zones for their
interpretation, this method employs discs with a single
concentration of antimicrobial agent and zone
diameters are correlated with minimal inhibitory
concentrations (MIC)
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
In the test procedure, a standardized suspension of the
organism is swabbed over the entire surface of the
medium.

Paper discs impregnated with specified amounts of
antibiotic or other antimicrobial agents are then placed
on the surface of the medium, the plate is incubated and
zones of inhibition around each disc are measured.

The determination as to whether the organism is
susceptible, intermediate or resistant to an agent is
made by comparing zone sizes obtained to those in the
CLSI Document M100(M2)
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 Various
factors have been identified as influencing
disc diffusion susceptibility tests.

These include the medium, excess surface moisture on the
medium, agar depth, disc potency, inoculum
concentration, pH and β-lactamase production by test
organisms.
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Intended Use

Nutrient Agar is used for the cultivation of bacteria and
for the enumeration of organisms in water, sewage,
feces and other materials.
Principles of the Procedure

This relatively simple formulation provides the
nutrients necessary for the replication of a large
number of microorganisms that are not excessively
fastidious.
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Procedure
 Liquefy the agar if prepared tubes are used, cool to
45-50°C and pour into Petri dishes. Allow to
solidify for at least 30 minutes.
 Use
standard procedures to obtain isolated
colonies from specimens. Incubate plates at 35 ±
2°C for 18-24 hours and 42-48 hours, if necessary.
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 Tubed
slants are used primarily for the
cultivation and maintenance of pure cultures.
 They
should be inoculated with an inoculating
loop and incubated under the same conditions as
the plated medium.
Expected Results
 Examine plates for growth.
 Growth from tubes inoculated with pure cultures
may be used for biochemical and/or serological
testing.
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Intended Use
 Nutrient Broth is used for the cultivation of many
species of nonfastidious microorganisms.
 It is one of several nonselective media useful in
routine cultivation of microorganisms.
Principles of the Procedure
 This relatively simple formulation supports the
growth of nonfastidious microorganisms due to
its content of peptone and beef extract.
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Procedure
 Inoculate tubes of the broth medium with the test
samples.

Incubate tubes for 18-24 hours at 35 ± 2°C in an aerobic
atmosphere.
Expected Results
 After incubation, growth is evidenced by the appearance
of turbidity in the broth.


Aliquots of the broth can be used for subculturing to
solid media for purification and identification purposes.
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Intended Use
 Fluid Thioglycollate Medium (FTM) is used for
the sterility testing of biologics and for the
cultivation of anaerobes, aerobes and
microaerophiles.
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 Thioglycollate
Medium, Brewer Modified is used for
the cultivation of obligate anaerobes,
microaerophiles and facultative organisms.
 Fluid
Thioglycollate Medium with Beef Extract is
used in cultivating microorganisms from normally
sterile biological products.
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Principles of the Procedure

Dextrose, peptone, L-cystine and yeast extract provide
the growth factors necessary for bacterial replication.

Sodium thioglycollate is a reducing agent that prevents
the accumulation of peroxides which are lethal to some
microorganisms.

The L-cystine is also a reducing agent, since it contains
sulfhydryl groups which inactivate heavy metal
compounds and maintain a low redox potential, thereby
supporting anaerobiosis.
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 Methylene
blue is an indicator of the level of
oxidation/reduction in the medium; increased
oxidation raises the Eh, causing the methylene blue
indicator to become green.
 Resazurin
is an oxidation-reduction indicator, being
pink when oxidized and colorless when reduced.
 The
small amount of agar assists in the maintenance
of a low redox potential by stabilizing the medium
against convection currents, thereby maintaining
anaerobiosis in the lower depths of the medium.
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Precautions

Do not reheat the media more than once; continued
reheating gives rise to toxicity.
Expected Results

After incubation, growth is evidenced by the presence of
turbidity compared to an uninoculated control.

Strict aerobes tend to grow in a thin layer at the surface of
the broth; obligate anaerobes will grow only in that
portion of the broth below the upper oxidized layer.
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THE END
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