Transcript Single Media – Multiple tests

SINGLE MEDIA / MULTIPLE
TESTS
Medical Microbiology Laboratory
MEDI 3101
Mr.Shadi Alashi
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.
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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