Lysine decarboxylase test

Formula in grams per liter

Gelatin Peptone.................. 5,00 L-Lysine.arginine,ornithine.5,00 Yeast Extract .................... 3,00 Dextrose ...........1,00 Bromcresol Purple ............ 0,02 Final pH 6,8 0,2 at 25C Preparation Dissolve 14 grams of the medium in one liter of distilled water. Dispense in quantities of 5 ml in screw-capped tubes. Sterilize in autoclave at 121C (15 lbs sp) for 15 minutes. Let the cap a bit loose to allow a good gas exchange. Close it well after sterilization.
Uses
The tubes are inoculated with the microorganism samples and incubated for 24 hours at 32 to 35C, or if preferred, at 37C. The enteric bacilli produce acid in the initial fermentation of dextrose with a change to a yellow color. The cultures that decarboxylate lysine form cadaverine and the color returns to the alkaline purple. A yellow color after 24 hours indicates a negative result. The following chart indicates the typical reactions of the important groups of the Enterobacteriaceae: With the substitution of arginine or ornithine for lysine, this medium (Falkow Broth Base) can be used to study the decarboxylation of these amino acids.

OF BASAL MEDIUM
(HUGH AND LEIFSON)
Formula in grams per liter Casein Peptone ................................................... 2,00 Sodium Chloride...............5,00 Dipotassium Phosphate ...................................... 0,30 Bacteriological Agar ..........2,50 Bromthymol Blue ................................................. 0,03 Final pH 7,1 0,2 at 25C Preparation Suspend 9,8 grams of medium in one litre of distilled water. Heat with frequent agitation until dissolved. Sterilize in an autoclave at 121 C (15 lbs. sp.) for 15 minutes. Add 10 ml. of 10% glucose (or any suitable sugar) solution sterilized by filtration to 100 ml. of liquid medium. Mix and dispense aseptically 5 ml. per tube. If preferred, add 1,0 grams of carbohydrate directly to 100 ml. of medium and sterilize in an autoclave at 118 C (12 lbs.) for 10 minutes to avoid the degradation of the sugar. The color of the prepared medium is green. Uses Inoculate 2 fresh tubes by stabbing with a fresh culture of the organism in study. If the medium has been prepared and stored, remelt in a water bath to expel the dissolved gases. After inoculation add to one of the tubes a layer of 4 to 5 mm. of paraffin oil. It is not recommended to use mineral oil. Incubate both tubes at 35 C for 48 hours or more, up to 7 days with the caps loose. To

facilitate the identification of Gram-negative non-fermenting bacilli, usealso Indol Nitrate Medium

PHENYLALANINE AGAR
Used for the differentiation of enteric bacilli which deaminate phenylalanine to phenyl pyruvic acid

Formula in grams per liter D-L Phenylalanine ............................................... 2,00 Yeast Extract..3,00 Sodium Chloride.................................................. 5,00 Sodium Phosphate...............................................1,00 Bacteriological Agar........................................... 12,00 Final pH 7,3 0,2 at 25C Preparation Suspend 23 grams of the medium in one liter of distilled water. Mix well. Heat with frequent agitation and boil for one minute. Dispense and sterilize in autoclave at 121C (15 lbs. sp.) for 10 minutes. Allow the tubes to solidify in a slanted position. Uses Proteus and Providencia are the only enterobacteria which have a positive reaction, the others are negative. To differentiate Proteus and Providencia seed heavily the suspicious organisms in Urea Agar Base (Christensen), or Urea Broth. Proteus hydrolyzes the urea. The Providencia is negative for urease production. Inoculate heavily with the sample organism. Incubate for 18 to 24 hours at 35C. Add 4 to 5 drops of 10% ferric chloride. The immediate appearance of an intense green color (1-5 minutes) indicates the presence of phenylpyruvic aci

DNASE TEST AGAR (DEOXYRIBONUCLEASE)

Preparation
Suspend 42 grams of the medium in one litre of distilled water. Mix well to obtain a homogeneous suspension. Heat with frequent agitation and boil for one minute. Sterilize in an autoclave at 118-121C (15 lbs. sp.) for 15minutes. Cool to 45-50C and pour into sterile Petri dishes. If desired, add 5% blood to the medium without mannitol to prepare a blood agar medium.

Uses
Make a heavy band streak (2 cm. in length) of the test organism (e.g.Staphylococci, Pseudomonas, Serratia,Bacillus, etc.) on the surface of the plate. You can simultaneously place in the same plate 4 to 5 different samples. Incubate for 18 to 24 hours at 35C. After good growth add a drop of 1N hydrochloric acid or a few drops of 0,1% toluidine blue solution. With some strains it is necessary to increase the concentration ofappearance of a well defined bright clear halo around the bacterial streak. In presence of diluted hydrochloric acid, the reaction with DNA in the culture medium forms a hazy precipitate. Colonies producing desoxiribonuclease appear surrounded by a zone or a clear halo which contains fractions of soluble nucleotides from the degradation of DNA, which are not precipitated by the hydrochloric acid.

ASPARAGINE BROTH
For the presumptive identification and enumeration (MPN) of Pseudomonas aeruginosa Formula in grams per liter Monopotassium Phosphate .............................. 10,00 Asparagine .................2,00 Dipotassium Phosphate ...................................... 1,00 Magnesium Sulfate ..................0,50 Final pH 7,0 0,2 at 25C

Preparation
Suspend 13,5 grams of the medium in one litre of distilled water with 8 ml. of glycerol. Heat agitating until completely dissolved. Dispense and sterilize at 121C (15 lbs. sp.) for15 minutes. To obtain a double strength broth, dissolve 27 grams of the medium and add 16 ml. of glycerol.

Uses
This medium is an excellent enrichment broth for P.aeruginosa because the formula contains a strictly mineral base with asparagine as the sole source of carbon. Asparagine Broth is recommended for enumeration by the MPN method with 5 tubes/series inoculating 10 ml., 1 ml. and 0,1 ml. All tubes are incubated at 37C for 48 hours.The appearance of growth with or without pigmentation is considered a presumptive test for the presence of P.aeruginosa and counts are determined using the MPN tubes. Confirmation is made by subculturing a loopful from each turbid tube into Acetamide Broth

ACETAMIDE BROTH
For the confirmation of Pseudomonas aeruginosa in bottled water Formula in grams per liter Acetamide.......................................................... 10,00 Sodium Chloride...................................................5,00 Dipotassium Phosphate ...................................... 1,39 Monopotassium Phosphate....................................0,73 Phenol Red .......................................................... 0,012 Final pH 7,0 0,2 at 25C

Preparation
Dissolve 17,2 grams of the medium in one litre of distilled water. If needed, heat gently to dissolve completely. Sterilize by filtration. DO NOT AUTOCLAVE. Aseptically dispense into sterile test tubes.

Uses
In this medium the acetamide is the sole source of carbon, whose utilization by many bacteria indicates deamination which is shown by a color change from orange-red to purple-red. It is adopted by the CeNAN, for confirmation of Pseudomonas aeruginosa (presence). Inoculate with one or two loopfuls from a tube of presumptive medium (Asparagine Broth) and incubate at 37C for 48 hours. A positive reaction turns the medium to an intense purple-red. P. aeruginosa is confirmed by a positive asparagine test and a positive acetamide test.