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Section III

H-K

3. Inoculate tubes of Herrolds Egg Yolk ANV Agar with andwithout Mycobactin J with no more than 0.25 mL (5-7

drops from a sterile transfer pipette) of the final processedand decontaminated material.

4. Incubate tubes at 35-37C in a slanted position with thecaps loose.

5. Tighten caps when medium surface is dry (1-2 weeks) and

place in an upright position in the incubator.6. Read and evaluate tubes for growth and contamination

every week for up to 16 weeks.

Expected ResultsColonies appearing should be evaluated for typical acid-fast-

ness and morphological appearance ofM. paratuberculosis.M. paratuberculosis-like colonies should not appear on the

medium lacking Mycobactin J.

To confirm Mycobactin J dependency, suspend suspected colonies

in 0.5 mL autoclaved purified water so that the turbidityis equivalent to a No. 1 McFarland nephelometer standard.

Dilute the specimen 100-fold in autoclaved purified water.Inoculate 0.1 mL of the suspension onto a single tube of

Herrolds Egg Yolk ANV Agar with Mycobactin J and 0.1 mLonto a single tube of the medium without Mycobactin J. Incu-

bate tubes for 1 week with loosened caps at 35-37C. Tighten

caps and re-incubate. Examine tubes weekly for the presenceof growth. Acid-fast cultures that grow only in the presence of

Mycobactin J are identified as M. paratuberculosis.

Limitations of the ProcedureSubcultures ofM. paratuberculosis may occasionally lose theirdependency on Mycobactin. Reduced growth of the organism

may be observed after multiple transfers.

References1. Chiodini, van Kruinigen and Merkal. 1984. Cornell Vet. 74: 218.2. Twort. 1910. Proc. R. Soc. Lond. Ser. B. 83: 156.3. Twort and Ingram. 1912. Proc. R. Soc. Lond. Ser. B. 84: 517.4. Whipple, Callihan and Jarnagin. 1991. J. Vet. Diagn. Invest. 3: 368.5. Whitlock, Rosenberger and Spencer. 1989. Proc. Annu. Meet. U.S. Animal Health Assn.93: 382.6. Whitlock and Sweeney. 1990. Proc. Annu. Meet. Livestock Conservation Inst., p. 24.7. Stabel. 1997. J. Vet. Diagn. Invest. 9: 375.8. Merkal and McCullough. 1982. Curr. Microbiol. 7: 333.9. Thoen and Baum. 1988. J. Am. Vet. Med. Assoc. 192: 1609.

AvailabilityBBL Herrolds Egg Yolk Agar with Mycobactin Jand ANV

Cat. No. 222232 Prepared Slants Pkg. of 10*

222233 Prepared Slants Ctn. of 100*BBL Herrolds Egg Yolk Agar without Mycobactin Jwith ANV

Cat. No. 222240 Prepared Slants Pkg. of 10*222241 Prepared Slants Ctn. of 100*

*Store at 2-8C.

ISP Medium 1 ISP Medium 2 ISP Medium 4

Intended UseISP Medium 1, ISP Medium 2 and ISP Medium 4 are usedfor characterizing Streptomyces species according to the Inter-

national Streptomyces Project (ISP).1

Summary and ExplanationISP media were developed by Difco Laboratories for the Inter-national Streptomyces Project (ISP) in order to select stableproperties and reproducible procedures for characterizationofStreptomyces species.1

ISP Medium 1 is also referred to as Tryptone-Yeast Extract Broth.

ISP Medium 2 is also referred to as Yeast Extract-Malt Extract

Agar.

ISP Medium 4 is also referred to as Inorganic Salts-Starch Agar.

Principles of the ProcedurePeptone and yeast extract provide nitrogen, vitamins, carbonand amino acids in ISP Medium 1.

Yeast extract and malt extract provide nitrogen, amino acidsand vitamins in ISP Medium 2. Dextrose is the carbon source.Agar is the solidifying agent.

ISP Medium 4 is composed of many inorganic salts and solublestarch to provide essential nutrients for organism growth. Agaris the solidifying agent.

FormulaeDifco ISP Medium 1

Approximate Formula* Per Liter

Pancreatic Digest of Casein ........................................ 5.0 gYeast Extract ..............................................................3.0 g

Difco ISP Medium 2

Approximate Formula* Per LiterYeast Extract ..............................................................4.0 gMalt Extract..............................................................10.0 gDextrose .....................................................................4.0 gAgar .........................................................................20.0 g

Difco ISP Medium 4

Approximate Formula* Per LiterSoluble Starch ..........................................................10.0 gDipotassium Phosphate ..............................................1.0 gMagnesium Sulfate USP .............................................1.0 gSodium Chloride ........................................................1.0 gAmmonium Sulfate .................................................... 2.0 gCalcium Carbonate .................................................... 2.0 g

Ferrous Sulfate ...........................................................1.0 mgManganous Chloride ..................................................1.0 mgZinc Sulfate ................................................................1.0 mgAgar .........................................................................20.0 g*Adjusted and/or supplemented as required to meet performance criteria.

Herrolds Egg Yolk Agar, cont.

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Directions for Preparation fromDehydrated Product1. Suspend the powder in 1 L of purified water:

Difco ISP Medium 1 8 g;

Difco ISP Medium 2 38 g;

Difco ISP Medium 4 37 g.Mix thoroughly.

2. Heat with frequent agitation and boil for 1 minute to com-pletely dissolve the powder.

3. Autoclave at 121C for 15 minutes.4. Mix thoroughly while dispensing ISP Medium 4.

5. Test samples of the finished product for performance usingstable, typical control cultures.

ProcedureFor details on the use of these media for characterization ofStreptomyces species, consult the reference.1 For a completediscussion on the isolation and maintenance of Strepto-myces species refer to appropriate references.2,3

Expected ResultsRefer to appropriate references and procedures for results.

References1. Shirling and Gottlieb. 1966. Int. J. Syst. Bacteriol. 16:313.2. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for

Microbiology, Washington, D.C.3. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed.

American Society for Microbiology, Washington, D.C.

AvailabilityDifco ISP Medium 1

Cat. No. 276910 Dehydrated 500 g

Difco ISP Medium 2

Cat. No. 277010 Dehydrated 500 g

Difco ISP Medium 4

Cat. No. 277210 Dehydrated 500 g

User Quality Control

Identity SpecificationsDifco ISP Medium 1

Dehydrated Appearance: Beige, free-flowing, homogeneous.

Solution: 0.8% solution, soluble in purifiedwater upon boiling. Solution is lightamber, clear to very slightly opalescent.

Prepared Appearance: Light amber, clear to very slightly opal-escent.

Reaction of 0.8%Solution at 25C: pH 7.0 0.2

Difco ISP Medium 2

Dehydrated Appearance: Beige, free-flowing, homogeneous.

Solution: 3.8% solution, soluble in purifiedwater upon boiling. Solution is lightto medium amber, very slightly toslightly opalescent.

Prepared Appearance: Light to medium amber, sl ightly opal-escent.

Reaction of 3.8%Solution at 25C: pH 7.2 0.2

Difco ISP Medium 4Dehydrated Appearance: White to light beige, free-flowing,

homogeneous.

Solution: 3.7% solution, soluble in purifiedwater upon boiling. Solution is whiteto off-white, opaque with precipitate.

Prepared Appearance: White to off-white, opaque, may havea precipitate.

Reaction of 3.7%Solution at 25C: pH 7.2 0.2

Cultural ResponseDifco ISP Medium 1, ISP Medium 2 or ISP Medium 4

Prepare the medium per label directions. Inoculate tubes of preparedISP Medium 1 with the test organisms and incubate at 30 2C for upto 96 hours. Inoculate prepared ISP Medium 2 and ISP Medium 4 withthe test organisms by placing approximately 0.1 mL of inoculum nearthe edge of the plate. Five parallel streaks across the plate are madefrom this 0.1 mL of inoculum, followed by four perpendicular streaks.Incubate inoculated plates at 30 2C for 48-96 hours.

ORGANISM ATCC INOCULUM CFU RECOVERY

Streptomyces albus 3004 102-103 Good

Streptomyces lavendulae 8664 102-103 Good

Indole Nitrite Medium (Trypticase Nitrate Broth)

Intended UseIndole Nitrite Medium is used for the identification of micro-organisms by means of the nitrate reduction and indole tests.

Summary and ExplanationIndole Nitrite Medium was developed to serve the dual role ofdetecting indole production and nitrate reduction of a wide

range of microorganisms. Due to its nutritive content, themedium will support the growth of aerobes, microaerophiles

and facultative and obligate anaerobes.

Indole Nitrite Medium can be used for nitrite tests withmembers of the Enterobacteriaceae but is not recommendedfor the indole test with these organisms since they reduce

nitrate to nitrite, which prevents the detection of indole.1

Tryptophan (Trypticase) 1% Solution is the medium of choice

for indole test with enteric bacilli.

Principles of the ProcedureThe casein peptone contains tryptophan, which is attackedby certain microorganisms, resulting in the production of

indole, detectable by the addition of chemical reagents to 18-

Indole Nitrite Medium