King’s Medium B Base w/ 1.5% Agar

Kings Medium B Base w/ 1.5% Agar is recommended for the non-selective isolation, cultivation and pigment production of Pseudomonas species in accordance with FDA BAM, 1998.

Composition**

Final pH ( at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 38.00 grams of dehydrated medium in 1000 ml distilled water containing 10 ml of glycerol. Heat to boiling to dissolve the medium completely. Mix well. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Aseptically pour into sterile Petri plates.

Principle And Interpretation

Pseudomonas aeruginosa is known to produce two types of pigments, pyocyanin and fluorescein which is a characteristic property and aids in its isolation from clinical and food samples. An additional pigment entitled pyorubin was reported by King(1). Pyocyanin is green, fluorescein is fluorescent yellow and pyorubin is reddish brown in colour. Some strains produce all the three pigments while the others produce one or two. Kings Medium B Base w/ 1.5% agar, recommended by FDA BAM is particularly suited for fluorescein production(2). This medium can be used as a general medium for the non-selective isolation and pigment production of Pseudomonas species from foods, cosmetics etc (3). This media contain proteose peptone, which provides carbonaceous and nitrogenous compounds for the growth of bacteria. Glycerol serves as a source of energy and also as an enhancer in pigment production. Magnesium sulphate also enhances pigment production. Pigments and/or their derivatives produced by Pseudomonas species play a role as siderophores in the iron uptake systems of bacteria, and hence, their production is markedly enhanced under conditions of iron deficiency. The production of pigments especially non-fluorescent blue pigment, pyocyanin is readily demonstrated by culturing on Kings Medium B Base w/ 1.5% Agar, which contains no added iron (4). The addition of dipotassium phosphate increases the phosphorus content of the medium thereby enhancing production of fluorescent pigment.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Light yellow coloured, clear to slightly opalescent gel forms in Petri plates

Reaction: Reaction of 3.8% w/v aqueous solution (containing 1.0 %v/v glycerol) at 25°C. pH : 7.2±0.2

pH: 7.00-7.40

Cultural Response: Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

Cultural Response

Storage and Shelf Life

Store below 30°C in tightly closed container and prepared medium at 2-8°C. Use before expiry period on the label.

Reference

1. King, E. O, M. K Ward, and D. E Raney. 1954. J. Lab and Clin. Med 44: 301-307.
2. FDA, U.S. 1998. Bacteriological Analytical Manual. 8 ed. Gaithersburg, MD: AOAC International.
3. Ann, G, and Matthysse. 1998. The Genus Agraobacterium. The Prokaryotes 3 ed.
4. Todar K., Todars Online Textbook of Bacteriology, University of Wisconsin -Madison, Department of Bacteriology.