Transcript Single Media & Multiple Tests{ S }

Single Media & Multiple Tests
Several media are designed to yield more than one
biochemical reaction. Among the more commonly used
media in this category are:
1) SIM medium.
2) Kliger's Iron agar (KIA).
3) Triple Sugar Iron agar (TSIA).
4) Lysine Iron agar (LIA).
5) Motility Indole Ornithine (MIO) medium.
SIM Medium
SIM Test Medium is a semisolid medium used as differential
test medium .
 { S } “sulfide”, discriminates organisms that can take up
sodium thiosulfate, they can reduce it to sulfite using the
enzyme thiosulfate reductase, with the release of hydrogen
sulfide gas.
 { I } “indole”, discriminates organisms that can produce
tryptophanase to hydrolyze the amino acid tryptophan into
indole, ammonia and pyruvic acid.
 { M } “motility” discriminates motility (presence of flagella),
ability to “swim” through media production.
INGREDIENTS
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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.
1) 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.
2) 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.
3) 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.
 The ability to reduce sulfur is specific to two members of the
enteric family: Salmonella and Proteus and aids in
distinguishing them from other enterics.
 Salmonella (H2S positive and motile),
 Proteus (H2S positive and motile),
 Klebsiella (nonmotile and does not produce H2S).
Procedure :
1) Label each of the SIM agar deep tubes with the
name of the bacterium to be inoculated, your name,
and date.
2) Using aseptic technique inoculate each tube with the
appropriate bacterium by stabbing the medium of the way to
the bottom of the tube. Do the same for the three motility test
medium deeps.
3) Incubate the cultures for 24 to 48 hours at 35°C.
Note :
If Bacteria can produce hydrogen sulfide , we can not detect the
motility of organism . Unfortunately, the motility aspect of this
test typically gives false negative results.
Indole negative (kovac's reagent
did not turn red) and hydrogen
sulfide negative (agar did not turn
black).
Indole negative (kovac's reagent
did not turn red) and hydrogen
sulfide negative (agar did not turn
black).
If indole is produced from the
breakdown of the amino acid
tryptophan, the Kovac's reagent,
when added, will turn red.
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)
KIA and TSIA are widely used in the identification of gram
negative bacteria particularly the Enterobacteriaceae. The media
are identical except that TSI contains sucrose in addition to the
dextrose and lactose found in KIA. The media are poured as
slants and are inoculated with a stab to the butt followed by a
streak of the slant surface. The bacteria therefore are exposed to
both an anaerobic environment (butt) and an aerobic one (slant).
Phenol red is present as an indicator. Do not tighten the cap on
the tube.
If the bacteria are nonfermenters, such as Pseudomonas, they
can grow on the slant by the aerobic degradation of protein
components in the medium.
INGREDIENTS
 Enzymatic Digest of Casein.
 Enzymatic Digest of Animal Tissue.
 Yeast Enriched Peptone.
 Dextrose..........0.1 %.
 Lactose............1.0 %.
 Sucrose...........1.0 %. Not present in Kligler's Iron Agar.
 Ferric Ammonium Citrate. As H2S Indicator H2S.
 Sodium Chloride.
 Sodium Thiosulfate.
 Phenol Red. As PH Indicator.PH
 Agar............................1.5 %.
 Final pH: 7.4 ± 0.2 at 25°C.
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.
 SIGNIFICANCE:
This test is of a great value in the initial identification
of the family Enterobacteriaceae.
 TSIA detects three primary characteristics of a bacterium:
1) The ability to produce gas from the fermentation of sugars,
2) The ability ferment lactose and sucrose
3) The production of large amounts of hydrogen sulfide.
PRINCIPLE:
For fermentative organisms, glucose is the first sugar
catabolized. If glucose is used up, a bacterium reaches
a metabolic fork in the road.
If an organism produces the enzymes to hydrolyze the
disaccharide lactose, the organism will continue fermenting to
assist in ATP generation. However, if the glucose is limiting and
the organism does not produce the necessary enzymes to
catabolize lactose, the organism can utilize the protein in the
medium via the process of deamination.
Procedure :
1) Inoculate the test organism on TSIA slants by
stabbing the butt and streaking the slant completely.
2) Incubate for 24 hours at 37 oC.
Note :
Timing is critical in reading KIA/TSIA results. An early reading
could reveal yellow throughout the medium, leading one to
conclude that the organism is a lactose fermenter when it simply
may not yet have exhausted the glucose. A reading after the
lactose has been depleted could reveal a yellow butt and red slant
leading one to falsely conclude that the organism is a glucose only
fermenter.
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 (nonfermenter).
Cracks, splits, or bubbles in medium indicate gas
production.
A black precipitate in butt indicates hydrogen sulfide
production.
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
RESULTS From left to right: Morganella morganii (K/A, atypically
not producing gas), Pseudomonas aeruginosa(K/NC),uninoculated
control, Proteus mirabilis(K/A,H2S), and Escherichia coli (A/A,G).
Lysine Iron Agar (LIA)
INGREDIENTS
 Yeast Extract.
 Dextrose..........0.1 %.
 L-Lysine..........1.0 %.
 Enzymatic Digest of Gelatin.
 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.
LIA agar is used to determine whether a Gramnegative rod decarboxylates or deaminates Lysine
and forms Hydrogen sulfide (H2S).
 Lysine deamination is an aerobic process which occurs on the
slant of the media. Lysine decarboxylation is an anaerobic
process which occurs in the butt of the media.
Salmonella: Alkaline/alkaline/H2S
Citrobacter: Alkaline/yellow/H2S
Procedure :
1) LIA is slanted when made and is inoculated by
streaking the slant and stabbing the butt and slant.
2) Incubate at 35◦C ± 2◦C for 18 to 24 h.
Results:
Motility Indole Ornithine (MIO) Medium
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:
 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.
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 byproduct 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.
 INDOLE: Tryptophan is an ingredient contained in
the medium by the inclusion of peptones.
A Positive test results: red color will form at the top
of the medium, If the organism possesses the
enzyme tryptophanase, with the addition of Kovacs reagent.
A negative test results: no color change.
END OF LECTURE