MIO Medium

 

MIO Medium

3. After incubation, incubation, examine examine tubes tubes for evidence of lysine deaminase, motility, lysine decarboxylase reactions and, after addition of Indole Reagent Kovacs, indole production.

Expected Results Lysine deaminase deaminase is indicated by a red or red-brown color in the top centimeter of the medium. Motility is indicated by a clouding of the medium or by growth extending from the inoculation line. Lysine decarboxylase is indicated by a purple color throughout the medium. This This color may vary in intensity intensity and may may be bleached out to a pale light color due to reduction of the indicator. Lysine-negative cultures produce a yellow medium that may be purple or red on the top. Tubes that show a purple reaction with a red color on top should be incubated for a longer period of time. After examining the medium for lysine deaminase, motility and lysine decarboxylase reactions, add 3 or 4 drops of Indole Reagent Kovacs (Cat. No. 261185) to the top of each tube. The appearance of a pink to red color in the reagent is interpreted as a positive indole test.

M  

Positive and negative reactions are based on 90% or more occurrences. When an aberrant reaction occurs, subcultures should be plated on differential media to ensure the purity of the culture.

Limitations of the Procedure 1. Do not add Indole Reagent Kovacs until the final lysine deaminase, lysine decarboxylase and motility results have been interpreted. 2. Occasionally Occasionally,, the indole test produces produces false-negativ false-negativee or falsely weak reactions.4

References  2:247. 1. Reller and Mirrett Mirrett.. 1975. J. Clin. Clin. Microbiol. Microbiol. 2 :247. 2. Murray, Baron, Pfaller, Tenover and Yolken. Yolken. (ed.). 1999. Manual of clinical clinical microbiology, 7th 7th ed. American Society for Microbiology, Washington, Washington, D.C. 3. Forbes, Sahm and Weissfeld. Weissfeld. 1998. Bailey and and Scott’s diagnostic diagnostic microbiology, 10th ed. Mosby Inc., Inc., St. Louis, Mo. 4. MacFaddin. 1985. 1985. Media for isolation-cultivation-identif isolation-cultivation-identification-maintenance ication-maintenance of medical bacteria, vol.1. Williams & Wilkins, Baltimore, Md.

Availability Difco™ MIL Medium Cat.. No. Cat

218041 218 041

Dehydr Deh ydrate ated d – 500 g

Difco /BBL  Indole Reagent ™

Cat. No.

™

261185 26118 5

Droppers, Drop pers, 0.5 mL – Ctn. of 50

MIO Medium • Motility Indole Ornithine Medium Intended Use

Summary and Explanation

Motility Indole Ornithine (MIO) Medium is used to demonstrate motility, motility, indole production and ornithine decarboxylase activity for the differentiation of Enterobacteriaceae.

MIO Medium was formulated by Ederer and Clark 1 and Oberhofer and Hajkowski2 for detection of motility, indole and ornithine decarboxylase production in one tube as an aid in the identification of members of the Enterobacteriaceae Enterobacteriaceae family.  family.

User Quality Control  Identity Specifications Difco™ MIO Medium Dehy De hydr drat ated ed App Appea eara ranc nce: e:

Beig Be ige, e, fre freee-fl flow owin ing, g, hom homog ogen eneo eous us..

Solution:

3.1% solution, soluble in purified water upon boiling. boiling. Solution is purple, clear to slightly opalescent.

Prep Pr epar ared ed App Appea eara ranc nce: e:

Purp Pu rple le,, slig slight htly ly opa opale lesc scen ent, t, sem semii-so soli lid. d.

Reaction of 3.1% Solution at 25°C:

pH 6.5 ±  0.2

Cultural Response Difco™ MIO Medium Prepare the medium medium per label directions. Inoculate with fresh cultures using an inoculating needle and incubate with caps loosened at 35 ± 2°C for 24-48 hours. Detect the presence of indole by the addition of 3-4 drops of Kovacs’ Reagent. OR G A N I S M

ATCC™

M OT OT IL IL IT IT Y

I ND ND OL OL E

O RN RN IT IT HI HI NE NE

Enterobacter aerogenes Escherichia coli  Klebsiella pneumoniae subsp. pneumoniae subsp.  pneumoniae Proteus mirabilis

13048 25922

+ +

– +

+ +

13883 25933

– +

– –

– +

Uninoculated Tube

Enterobacter  aerogenes

Escherichia coli  ATCC™ 25922

™

ATCC  13048

329

 

Section III M MIO Medium, cont.

Principles of the Procedure Peptones, yeast extract and dextrose provide amino acids and other nitrogenous and carbonaceous substances, vitamins and minerals essential for bacterial metabolism. Motility can be read because of the semi-solid consistency of the medium. Organisms that possess the enzyme “tryptophanase” degrade the amino acid tryptophan to indolepyruvic acid, from which indole can be formed through deamination.3 When ornithine decarboxylase is present, the ornithine is decarboxylated to putrescine which causes a rise in the pH and corresponding color change of the bromcresol purple from yellow to purple.

Formula Difco™ MIO Medium Approximate Formula* Per Liter Yeast Extract ..............................................................3.0 Peptone ................................................................ ................................................................... ... 10.0 Tryptone ..... ........... ............ ............ ............ ............ ............ ............ ........... ........... ............ .........10.0 ...10.0 L-Ornithine HCl ....... ............. ........... ........... ............ ............ ............ ............ ........... ..........5.0 .....5.0 Dextrose............... Dextrose......... ........... ........... ........... ........... ........... ........... ........... ........... ........... ..........1.0 .....1.0 Agar ..... ........... ........... ........... ........... ........... ........... ........... ........... ........... ............ ............ ............ ........ .. 2.0 Bromcresol Purple ......................................................0.02

g g g g g g g

*Adjusted and/or supplemented as required to meet performance criteria.

from a primary isolation plate or other pure culture. Incubate all tubes for 18-24 hours at 35 ± 2°C in an aerobic atmosphere.

Expected Results Read motility and decarboxylase activity prior to the addition of the reagent for the detection of indole production. 1. Motility is indicated by growth extending extending from the line of  inoculation. Nonmotile organisms grow only along the line of inoculation. 2. Decarboxylation of ornithine is indicated indicated by the developdevelopment of a turbid purple to a faded faded yellow-purple color. color. A negative reaction is indicated by a yellow color. 3. Indole production is indicated by the formation of a pink to red color after the addition of three or four drops of  Kovacs’ reagent to the surface of the medium and gentle shaking. A negative reaction is indicated by the development of a yellow color. Refer to appropriate texts for typical reactions produced by various members of the Enterobacteriaceae Enterobacteriaceae..4-6

References

Directions for Preparation from

1. Edere Edererr and Clark. Clark. 1970. 1970. Appl. Micro Microbiol. biol. 2:849. 2. Oberhofer and Hajkowski. Hajkowski. 1970. Am. J. Clin. Pathol. 54 54:720. :720. 3. MacFaddin. 2000. Biochemical tests tests for identification of medical medical bacteria, 3rd ed. Lippincott

Dehydrated Product 1. Suspend 31 g of the powder in 1 L of purified water water.. Mix

Williams & Wilkins, Baltimore, Md. 4. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae Enterobacteriaceae,, 4th ed. Elsevier Science Publishing Co., New York, N.Y. 5. Holt, Krieg, Sneath, Sneath, Staley and Williams Williams (ed.). 1994. Bergey’ Bergey’ss Manual of determinative determinative bacteriology, bacteriology, 9th ed. Williams & Wilkins, Baltimore, Md. In Murray, 6. Farm Farmer er.. 199 1999. 9. In  Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 7th ed. American S ociety for Microbiology, Washington, D.C.

thoroughly. 2. Heat with with frequent agitation and boil for for 1 minute to completely dissolve the powder. 3. Aut Autocl oclav avee at 121°C for 15 minutes. 4. Test samples of the finished finished product for for performance using stable, typical control cultures.

Procedure To prepare the stored medium for use in motility mo tility studies, loosen caps, heat the medium to boiling and cool to room temperature prior to inoculation. inoculatio n. Inoculate tubes of medium by a single stab to 1/4 inch from the bottom of the tube using growth

Availability Difco™ MIO Medium BAM

Cat.. No. Cat

273520 273 520

Dehydr Deh ydrate ated d – 500 g

BBL™ Motility Indole Ornithine Medium BAM

Cat. No.

221517 221517 221518

Prepared Deeps (K Tubes) Prepared ubes),, 5 mL mL – Pkg. Pkg. of 10* Prepared Deeps (K Tubes), 5 mL – Ctn. of 100*

*Store at 2-8 C. °  

MR-VP Medium • MR-VP Broth Intended Use MR-VP Medium and MR-VP Broth (Methyl Red-Voges Proskauer Medium/Broth, also known as Buffered PeptoneGlucose Broth) are used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer Voges-Proskauer reactions.

Summary and Explanation Voges and Proskauer, in the latter part of the 19th century, reported the initial observations regarding the production of a red color after the addition of potassium hydroxide to specific culture media in which various organisms had grown.1 Clark and Lubs,2 in 1915, found that the addition of methyl red to cultures of Escherichia coli resulted coli resulted in a red color due to the high acidity produced during the fermentation of dextrose. 330

The smaller amount of acid produced by Klebsiella pneumoniae and Enterobacter and  Enterobacter aerogenes is aerogenes is converted to acetoin resulting in an alkaline reaction (negative methyl red test). In the Voges-Proskauer test, Reagent A (5% [w/v] alpha-naphthol in absolute alcohol) contains a catalyst enhancing the formation of specific metabolic products that form a red complex upon the addition of Reagent B (40% [w/v] potassium hydroxidee in purified water). hydroxid MR-VP Medium/Broth was developed to enable both the MR and the VP tests to be performed in the same medium, although in different tubes or on aliquots from the same tube.