SINGLE MEDIA / MULTIPLE TESTSMedical Microbiology LaboratoryMEDI 3101Mr.Shadi Alashi

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:SIM medium.Kliger's Iron agar (KIA).Triple Sugar Iron agar (TSIA).Lysine Iron agar (LIA).Motility Indole Ornithine (MIO) medium.

SIM MEDIUM

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 testorganism is motile.

INGREDIENTS

0.4% agar ( semisolid). Peptone ( which rich in treptophan amino acid ). Sodium thiosulfate Ferrous ammonium sulfate. H2S INDICATOR

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)

SIM MEDIUM

KLIGLER'S IRON AGAR (KIA)&TRIPLE SUGAR IRON AGAR (TSI) 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 PRODUCTION INDICATOR Sodium Chloride. Sodium Thiosulfate. Phenol Red. AS PH INDICATOR Agar............................1.5 %. Final pH: 7.4 ± 0.2 at 25°C.

PRINCIPLEEnzymatic 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 loweroxygen tension.

Interpretation Symbol Results (slant/butt)Glucose fermentation

only; Peptone catabolizedK/A Red/yellow

Glucose and lactose and/or sucrose fermentation

A/A Yellow/yellow

No fermentation; Peptone catabolized

K/K Red/red

No fermentation; Peptone used aerobically

K/NC Red/no color change

Glucose and lactose and/or sucrose

fermentation; Gas produced

A/A,G Yellow/yellow with bubbles

Glucose fermentation only; Gas produced

K/A,G Red/yellow with bubbles

Glucose fermentation only; Gas produced; H2S

produced

K/A,G, H2S Red/yellow with bubbles and black precipitate

Glucose fermentation only; H2S produced

K/A, H2S Red/yellow with black precipitate

Glucose and lactose and/or sucrose

fermentation; H2S produced

A/A, H2S Yellow/yellow with 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.

BROMOCRESOL PURPLE PH INDICATOR

LYSINE IRON AGAR (LIA)

MOTILITY INDOLE ORNITHINE (MIO) MEDIUMoINGREDIENTS 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.

PRINCIPLEMIO 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