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Section III

L Litmus Milk, cont.

2. Fermentation of lactose and/or dextrose in the milkwith production of acid (pink color), including stormyfermentation (strong evolution of gas) by certain strainsof Clostridium.

3. Action of proteolytic enzymes on lactalbumin withproduction of ammonia or basic amines resulting in analkaline reaction (blue color).

4. Coagulation of casein as evidenced by the formation ofa curd or clot. If the casein is converted to paracasein bythe enzyme rennin, a clear, watery liquid called “whey” isproduced at the top of a thoroughly coagulated tube.1

5. Peptonization due to digestion of the milk protein asevidenced by a clearing of the medium and dissolutionof the clot.

6. Reduction of the litmus in the depths of the tube due to theaction of reductase enzymes with the resultant removal ofoxygen to form the decolorized leucolitmus compound.

FormulaBBL™ Litmus Milk

Approximate Formula* Per LiterSkim Milk ...............................................................100.0 gAzolitmin ................................................................... 0.5 gSodium Sulfite ............................................................ 0.5 g*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation fromDehydrated Product1. Dissolve 100 g of the powder in 1 L of purified water,

preferably preheated to 50°C. Mix thoroughly.

2. Autoclave at 115°C for 20 minutes or by fractional steamsterilization for 30 minutes on three successive days. Avoidoverheating and the consequent caramelization whichoccurs.

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

ProcedureInoculate tubes of Litmus Milk with 18- to 24-hour purecultures. For the study of anaerobic organisms, sterile mineraloil can be layered over the medium following inoculation. In-cubate tubes at 35 ± 2°C for up to 14 days and recordreactions at various intervals during the incubation process.

Expected ResultsConsult an appropriate reference for the expected reactionsfor specific microbial species.2

References1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,

vol. 1. Williams & Wilkins, Baltimore, Md.2. Sneath and Holt (ed.). 1986. Bergey’s Manual™ of determinative bacteriology, vol. 2. Williams &

Wilkins, Baltimore, Md.

AvailabilityBBL™ Litmus MilkCat. No. 211343 Dehydrated – 500 g

221657 Prepared Tubes – Pkg. of 10**Store at 2-8°C.

Littman Oxgall AgarIntended UseLittman Oxgall Agar is used for isolating and cultivating fungi,especially dermatophytes.

Summary and ExplanationIn 1947, Littman1 described Littman Oxgall Agar, a selectivemedium for the primary isolation of fungi, including patho-genic fungi.

Littman demonstrated that Littman Oxgall Agar is valuablefor culturing the dermatophytes. Molds and yeasts formnonspreading, discrete colonies, that are easy to isolate inpure culture. He also suggested that the medium be used forestimating the normal fungal flora of feces, sputum and otherhuman discharges. The medium can also be used for single cellisolation of fungi and plate counts of viable saprophytic fungiin foodstuffs and air.

In a comparative study, Littman2 compared this medium withSabouraud Dextrose Agar using a large variety of pathogenicand saprophytic fungi. He reported the isolation of three timesas many fungi from feces, sputum, skin scrapings and hairon Littman Oxgall Agar and four times as many pathogenicdermatophytes on the selective medium compared withSabouraud Dextrose Agar.

Principles of the ProcedurePeptone provides nitrogen, amino acids and carbon. Dextroseis an additional carbon source. Oxgall restricts the spreadingof fungus colonies. Crystal violet and streptomycin are selectivebacteriostatic agents. Agar is the solidifying agent.

FormulaDifco™ Littman Oxgall Agar

Approximate Formula* Per LiterPeptone ................................................................... 10.0 gDextrose ................................................................... 10.0 gOxgall ....................................................................... 15.0 gAgar ......................................................................... 20.0 gCrystal Violet .............................................................. 0.01 g*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation fromDehydrated Product1. Suspend 55 g of the powder in 1 L of purified water. Mix

thoroughly.2. Heat with frequent agitation and boil for 1 minute to

completely dissolve the powder.3. Autoclave at 121°C for 15 minutes. Cool to 46°C.

311

LL

Liver Infusion Agar

4. Add 30 µg streptomycin per mL of medium.5. Test samples of the finished product for performance using

stable, typical control cultures.

ProcedureSee appropriate references for specific procedures.

Expected ResultsRefer to appropriate references and procedures for results.

Limitations of the Procedure1. Although culture techniques are primary in the identification

of etiological agents of mycotic infections, they are notabsolute. Often identification must be accomplished by

using one or more of the following techniques to corrobo-rate cultural findings: direct microscopic examination ofthe specimen; animal inoculation; biochemical determina-tion; or serological procedures.

2. Do not use to culture Nocardia asteroides, Streptomycesor any other microorganism sensitive to streptomycin.3

References1. Littman. 1947. Science 106:109.2. Littman. 1948. Am. J. Clin. Pathol. 18:409.3. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,

vol. 1. Williams & Wilkins, Baltimore, Md.

AvailabilityDifco™ Littman Oxgall AgarCat. No. 229410 Dehydrated – 500 g

User Quality Control

Identity SpecificationsDifco™ Littman Oxgall AgarDehydrated Appearance: Grayish-blue, free-flowing, homoge-

neous.

Solution: 5.5% solution, soluble in purified waterupon boiling. Solution is blue, veryslightly to slightly opalescent.

Prepared Appearance: Blue, very slightly to slightly opalescent.

Reaction of 5.5%Solution at 25°C: pH 7.0 ± 0.2

Cultural ResponseDifco™ Littman Oxgall AgarPrepare the medium per label directions without (plain) and with theaddition of streptomycin. Inoculate and incubate at 25-30°C for up to 72hours.

UninoculatedPlate

INOCULUM RECOVERY RECOVERYORGANISM ATCC™ CFU PLAIN w/STREPTOMYCIN

Candida albicans 10231 102-103 Good GoodEscherichia coli 25922 102-103 Fair to good InhibitionSaccharomyces cerevisiae 9763 102-103 Good GoodSaccharomyces cerevisiae 9080 102-103 Good GoodTrichophyton mentagrophytes 9533 102-103 Fair to good Fair to good

Saccharomyces cerevisiaeATCC™ 9763

Trichophytonmentagrophytes

ATCC™ 28185

Liver Infusion Agar • Liver Infusion BrothIntended UseLiver Infusion Agar is used for cultivating Brucella and otherpathogenic organisms.Liver Infusion Broth is used for cultivating a variety of organ-isms, particularly Brucella and anaerobes.

Summary and ExplanationBrucellosis is a zoonotic disease with a domestic animal reservoir.Transmission by milk, milk products, meat and direct contactwith infected animals is the usual route of exposure.1

Most strains of Brucella will grow on chocolate or blood agar.However, special media such as liver infusion, tryptose, tryptoneor brucella agar are preferred.2 The nutritive factors of LiverInfusion media permit luxuriant growth of Brucella and otherfastidious pathogens.

For isolating Brucella strains from contaminated milk, crystalviolet (gentian violet) can be added to Liver Infusion Agar tosuppress gram-positive organisms.3 Five percent (5%) heatedhorse or rabbit serum enhances growth of Brucella.4

Liver Infusion Agar at approximately half strength may be usedto prepare Endamoeba medium for cultivating Endamoebahistolytica.5

Liver Infusion Broth maintains a degree of anaerobiosiswell suited to support growth of anaerobic microorganisms,especially Clostridium species.