Transcript Lab 13,14

Media Preparation and Sterilization
A medium is sterilized (living organisms removed) before usage
in the lab.
Sterilization methods include; autoclaving, dry-heat, filtration, UV
exposure and ethylene oxide.
Culture: Is part of specimen grown in culture media.
Culture Media: is a medium (liquid or solid) that contains
nutrients to grow bacteria in vitro. Because sometimes we
cannot identify with microscopical examination directly, and
sometimes we do culture for antibiotic sensitivity testing.
Medium is a nutrient blend used to support microbial
growth.
There are three physical forms of media, broth, solid,
and semisolid.
Solid media are more versatile in their usage.
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Promote surface growth
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Used to isolate pure cultures
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Ideal for culture storage
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Helpful in the observation of biochemical
reactions
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Used to make slants, deeps, and plates (named
by medium)
Properties of Media:
Support the growth of the bacteria.
 Should be nutritive (contains the required amount of
nutrients).
 Suitable pH (neutral to slightly alkaline 7.3-7.4).
 Suitable temperature, and suitable atmosphere.
(Bacteria grow at 370C)
 Note: media are sterilized by autoclaving at 1210C
and 1.02 atmosphere (15 p.s.i.) for 15-20 minutes.
With the autoclave, all bacteria, fungi, viruses, and
spores are destroyed. Some media can’t be
sterilized by autoclaving because they contain eggs
or carbohydrates .
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Forms of culture
Solid (agar): Is Broth plus agar (seaweed).
Are prepared by adding a solidifying agent (agar 1.5 3%).Prepared mainly in Petri dishes, but also in tubes and
slopes. After growth the bacterial colonies are visible. e.g.
blood agar, chocolate agar, MacConkey agar.
 Semisolid agar: (soft agar): Contains small amounts of agar
(0.5-0.7%). Used to check for motility and also used as a
transport media for fragile organisms. Can have semisolid
agar in Petri dishes or in tubes. In tubes it is usually slanted to
increase surface area.
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Liquid (Broth): Mostly used for biochemical tests
(blood culture, Broth culture). Growth of bacteria is
shown by turbidity in medium. e.g. Nutrient broth,
Selenite F broth, alkaline peptone water.
Properties of agar:
Some what like gelatin.
It melts at 850C and solidifies at 40-320C.
Comes as sold powder and then you add water to it.
Types of Culture Media
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Simple (basal, ordinary): Culture Media: are
media that contain the basic nutrients (growth
factors) that support the growth of bacteria without
special nutrients, and they are used as basis of
enriched media. E.g. Nutrient broth, nutrient agar,
peptone water. They are for the growth of nonfastidious organisms like E. coli.
Enriched Culture Media: are media that are
enriched with: Whole blood e.g. blood agar. Lysed
blood (heated to 85C) e.g. Chocolate agar.
Selective Media: it is a media, which contains
substances that prevent or slow the growth of
microorganisms other than the bacteria for which the
media is prepared for. For example
EMB (Eosin Methylene blue): enteric isolation media
Differential Media (indicators): Contains indicators,
dyes, etc, to differentiate microorganisms. E.g.
MacConkey agar, which contains neutral red (pH
indicator) and is used to differentiate lactose
fermenter and non-lactose fermenter. (E.g. E. coli and
Salmonella).
Common media used in Microbiology
Laboratory:
Chocolate Agar: blood agar prepared by heating
blood to 85C until medium becomes brown or
chocolate in color heating the blood releases broth
X and V growth factors and also destroys the
inhibitors of V factor. These factors are required for
the growth of most species of Haemophilus and
also Neisseria gonorrhoear.
 MacConkey Agar: an inhibitory and differential
medium used to distinguish lactose- fermenting
Gram- negative organism from non fermentation.
Crystal violet, bile salts and neutral red are inhibitor
agent. neutral red is the PH indicator.
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Mannitol Salt Agar ( MSA ): for selective isolation for
coagulase positive, mannitol-fermenting staphylococcus.
Mannitol fermentation by pathogenic staphylococci is
indicated by a yellow halo surrounding the
colonies.Sodium chloride is the inhibitor agent.Phenol red
is the PH indicator.
Mueller Hinton Agar: rich medium that support the
growth of most microorganism. It is commonly used for
antibiotic susceptibility testing: disk diffusion antibiotic
susceptibility; antibiotic serum level measurements; MBC
determination.
Salmonella Shigella ( SS ) Agar : isolation and
differential medium for pathogenic Gram-negative bacilli in
particular, Salmonella and Shigella. Inhibitor for Coliforms.
Triple Sugar Iron Agar (TSI): this a key medium for use
in beginning the identification of a Gram- negative bacilli of
the enteric group. It contains glucose (0.1% ), Lactose
(1%), sucrose(1%). And peptone (2%) as nutritional
sources. Sodium Thiosulfate serves as the electron
receptor for reduction of sulfur and production of H2S.
Detects fermentation of sucrose, lactose, glucose, as well
as production of hydrogen sulfide and /or gas . Phenol red
is the PH indicator; ferric ammonium citrate is H2S
indicator.
SINGLE MEDIA / MULTIPLE
TESTS
SINGLE MEDIA / MULTIPLE TESTS
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Several media are designed to yield more than one
biochemical reaction. Among the more commonly used media
in this category are:
SIM medium.
Kliger's Iron agar (KIA).
Triple Sugar Iron agar (TSIA).
Lysine Iron agar (LIA).
Motility Indole Ornithine (MIO) medium.
SIM MEDIUM
INGREDIENTS
 0.4% agar ( semisolid).
 Peptone ( which rich in treptophan amino acid ).
 Sodium thiosulfate
 Ferrous ammonium sulfate. H2S INDICATOR
The ingredients in SIM Medium enable the determination of three
activities by which enteric bacteria can be differentiated.
Sodium thiosulfate and ferrous ammonium sulfate for indication of
hydrogen sulfide production. The ferrous ammonium sulfate reacts with
H2S gas to produce ferrous sulfide, a black precipitate.
The peptone is rich in tryptophan, which is attacked by certain
microorganisms resulting in the production of indole. The indole is
detected by the addition of Kovac’s reagent.
Motility detection is possible due to the semisolid nature of the medium
growth radiating out from the central stab line indicates that the test
organism is motile.
SIM MEDIUM
INDOLE NEGATIVE (KOVAC'S REAGENT DID NOT TURN RED) AND
HYDROGEN SULFIDE NEGATIVE (AGAR DID NOT TURN BLACK).
SIM MEDIUM
IF INDOLE IS PRODUCED FROM THE BREAKDOWN OF THE
AMINO ACID TRYPTOPHAN, THE KOVAC'S REAGENT, WHEN
ADDED, WILL TURN RED.
SIM MEDIUM
INDOLE NEGATIVE (KOVAC'S REAGENT DID NOT TURN RED)
AND HYDROGEN SULFIDE POSITIVE (AGAR TURNED BLACK)
KLIGLER'S IRON AGAR (KIA)&
TRIPLE SUGAR IRON AGAR (TSI)
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INGREDIENTS
Enzymatic Digest of Casein.
Enzymatic Digest of Animal Tissue.
Yeast Enriched Peptone.
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Dextrose...................0.1 %.
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Lactose......................1.0 %.
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Sucrose......................1.0 %. NOT PRESENT IN KLIGlER’s IRON AGAR
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Ferric Ammonium Citrate. AS H2S PRODUCTION INDICATOR
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Sodium Chloride.
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Sodium Thiosulfate.
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Phenol Red. AS PH INDICATOR
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Agar............................1.5 %.
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Final pH: 7.4 ± 0.2 at 25°C.
PRINCIPLE
Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue,
and Yeast Enriched Peptone provide the nitrogen, carbon, and
vitamins required for organism growth.
Triple Sugar Iron Agar contains three carbohydrates, Dextrose,
Lactose and Sucrose. When the carbohydrates are fermented, acid
production is detected by the Phenol Red pH indicator.
Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen
sulfide reacts with an iron salt yielding the typical black iron
sulfide.
Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator.
Sodium Chloride maintains the osmotic balance of the medium.
Agar is the solidifying agent.
RESULTS
An alkaline slant-acid butt (red/yellow) indicates
fermentation of dextrose only.
An acid slant-acid butt (yellow/yellow) indicates
fermentation of dextrose, lactose and/or sucrose.
An alkaline slant-alkaline butt (red/red) indicates
dextrose ,lactose or/& sucrose were not fermented
(non-fermenter).
Cracks, splits, or bubbles in medium indicate gas
production.
A black precipitate in butt indicates hydrogen sulfide
production.
FIG. B The small amount of acid produced in the slant of the tube
during dextrose fermentation oxidizes rapidly, causing the medium to
remain red or revert to an alkaline pH. In contrast, the acid reaction
(yellow) is maintained in the butt of the tube because it is under lower
oxygen tension.
Results (slant/butt)
Symbol
Interpretation
Red/yellow
K/A
Glucose fermentation only;
Peptone catabolized
Yellow/yellow
A/A
Glucose and lactose and/or
sucrose fermentation
Red/red
K/K
No fermentation; Peptone
catabolized
Red/no color change
K/NC
No fermentation; Peptone
used aerobically
Yellow/yellow with bubbles
A/A,G
Glucose and lactose and/or
sucrose fermentation; Gas
produced
Red/yellow with bubbles
K/A,G
Glucose fermentation only;
Gas produced
K/A,G, H2S
Glucose fermentation only;
Gas produced; H2S
produced
Red/yellow with black
precipitate
K/A, H2S
Glucose fermentation only;
H2S produced
Yellow/yellow with black
precipitate
A/A, H2S
Glucose and lactose and/or
sucrose fermentation; H2S
produced
Red/yellow with bubbles and
black precipitate
LYSINE IRON AGAR (LIA)
INGREDIENTS
 Enzymatic Digest of Gelatin.
 Yeast Extract.
 Dextrose...............0.1 %.
 L-Lysine...............1.0 %.
 Ferric Ammonium Citrate. AS H2S INDICATOR
 Sodium Thiosulfate.
 Bromocresol Purple. AS PH INDICATOR
 Agar…………......1.5 %.
 Final pH: 6.7 ± 0.2 at 25°C.
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BROMOCRESOL PURPLE
PH INDICATOR
LYSINE IRON AGAR (LIA)
MOTILITY INDOLE ORNITHINE (MIO)
MEDIUM
o INGREDIENTS
Yeast Extract.
 Peptone.
 L-Ornithine…….. 0.5%.
 Dextrose ..............0.1%.
 Agar ……………. 0.4%.
 Bromcresol Purple. PH INDICATOR
 Final pH 6.5 ± 0.2 at 25°C.

PRINCIPLE
MIO Medium is prepared to provides three differentiating tests in one
culture tube: motility, indole production, and ornithine decarboxylation.
The principles of each are outlined below:
o MOTILITY: Organisms are stabbed into the semisolid medium using a
straight wire. If the organisms are motile, they will migrate from the stab
line by means of their flagella, producing turbidity or cloudiness
throughout the medium. Non-motile organisms grow only along the stab
line, leaving the surrounding medium clear.
o INDOLE: Tryptophan is an ingredient contained in the medium by the
inclusion of peptones. If the organism possesses the enzyme
tryptophanase, with the addition of Kovacs reagent, a red color will form
at the top of the medium if indole is present. This reaction occurs as a
result of the indole reacting with the aldehyde to yield a quinone or
quinone-type structure, resulting in a red color in the alcohol layer. A
negative test results in no color change.
PRINCIPLE CONTINUE….
o ORNITHINE:
The medium also tests for the
presence of the enzyme ornithine decarboxylase by
including L-ornithine in the agar. If the organism
possesses the enzyme, it will be activated in an acid
environment created by the initial fermentation of
glucose. Once the amino acid is decarboxylated, the
by-product diamine putrescine is produced. The
result is a shift in pH to the alkaline range, turning
the medium a dark purple. Organisms, which do not
possess the enzyme, will stay in the acid range due
to the fermentation, resulting in a yellow color in the
medium.
BROMOCRESOL PURPLE
PH INDICATOR
MOTILITY INDOLE ORNITHINE (MIO)
MEDIUM