PLET AGAR

Cat. no. G153 PLET Agar, 15x100mm Plate, 20ml 10 plates/bag
Cat. no. P15 PLET Agar, 15x60mm Contact Plate, 15ml 10 plates/bag

INTENDED USE

Hardy Diagnostics PLET Agar (Polymyxin-Lysozyme-EDTA-Thallous acetate) is used for the detection and isolation of Bacillus anthracis from environmental samples, animal products, carcasses, and clinical samples from non-sterile sites. PLET Agar can be useful as a component of a screening program for anthrax.

SUMMARY

Bacillus anthracis is the causative agent of anthrax. Humans become infected when they are inoculated with the spores either by inhalation during exposure to contaminated animal products, or by traumatic introduction. (3) Other Bacillus species, such as B. cereus , can cause serious infections, but the relative virulence of this and other Bacillus spp. is trivial compared with that of B. anthracis .

PLET (polymyxin-lysozyme-EDTA-thallous acetate) is a selective media used for the isolation of Bacillus anthracis from contaminated specimens. PLET Agar inhibits most contaminating organisms and spore-formers closely related to B. anthracis , such as B. cereus . The specimens are first heat- or alcohol-shocked, then dilutions of these preparations are spread across the PLET plates. (2) Circular creamy-white to gray-white colonies with a ground-glass texture are subcultured onto appropriate media to test for gamma phage, penicillin susceptibility, lack of motility, and lack of beta-hemolysis for complete identification of B. anthracis . (2)

Organic nitrogen, carbon, sulfur, vitamins and trace substances are provided by pancreatic digest of casein, peptic digest of animal tissue, yeast extract, and beef heart infusion. Sodium chloride is added to maintain the osmotic equilibrium. Polymyxin inhibits gram-negative organisms. The combination of EDTA and thallous acetate results in the unique action whereby B. anthracis strains are easily recovered, while B. cereus strains are generally inhibited. The specific interactions that may be involved are not known at this time. (4)

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 9.0gm
Peptic Digest of Animal Tissue 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 4.0gm
Beef Heart Infusion 2.0gm
Sodium Carbonate 0.325gm
EDTA 300.0mg
Thallous Acetate 40.0mg
Lysozyme 300,000U
Polymyxin 30,000U
Agar 15.0gm

Final pH 7.4 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

The expiration date applies to the product in its intact packaging when stored as directed.

PRECAUTIONS

PROCEDURE

I. For environmental samples or samples submitted from non-sterile sites: (2)

1. Solid samples should first be emulsified in sterile deionized water or saline (1:1 wt/vol).

2. Heat-shock the spores and effectively destroy vegetative cells of contaminating organisms by heating at 65ºC. for 15-30 minutes in a waterbath or heatblock (*see note below). Alternatively, alcohol-shock by adding filter-sterilized 95-100% ethanol to the specimen to a final concentration of 1:1 (wt/vol) and hold for 30-60 minutes at room temperature.

3. Using a sterile, plastic spreader (Cat. no. SPRLS01), spread 0.25ml aliquots of undiluted and 1:10, 1:100, and 1:1000 dilutions of a heat- or alcohol-treated suspension of the specimen across PLET, Blood Agar 5% (Cat. no. A10), and Nutrient Agar plates (Cat. no. W10).

4. Incubate at 35-37ºC.

5. Inspect plates for growth after 8 hours and up to 48 hours of incubation.

* Note: Treat at 67ºC. for 45-50 minutes if the solution is contained in a polypropylene tube and heat-shocked in a heatblock.

II. For clinical specimens from normally sterile sites:

Clinical samples from normally sterile sites should be cultured directly on to Blood Agar 5% (Cat. no. A10) or Nutrient Agar (Cat. no. W51). Do not use the heat- or alcohol-shock procedures on samples from normally sterile sites (e.g. blood, CSF) since B. anthracis does not usually form spores while inside the body. B. anthracis is easily recoverable on these media after overnight incubation. (2)

III. For contact plate method of use:

Allow plates to warm to room temperature, and agar surface to dry. Select a surface to test. Sample the surface by firmly pressing the agar against the test area, using the thumb and second finger to hold the plate, and the first finger to press firmly and evenly on the base. The same amount of pressure should be used for each sample. Do not move the plate laterally, as this spreads contaminants across the agar surface. A rolling motion may be used when slightly curved surfaces are sampled. Areas to be assayed may be divided into grids or sections, and days, as required. Incubate and observe for growth per above instructions.

INTERPRETATION OF RESULTS

Bacillus anthracis will appear as circular creamy-white to gray-white colonies, 1 to 3mm in diameter, with a ground-glass texture. Colonies with this appearance are then subcultured on Blood Agar plates to test for gamma phage, penicillin susceptibility, motility and hemolysis.

Colonies of B. anthracis may grow more slowly and will appear smaller and smoother on PLET Agar than on non-selective media, such as Blood Agar 5% and Nutrient Agar.

Consult the listed references for further procedures for identification of isolates. (1-4)

LIMITATIONS

Some strains of spore-forming Bacillus , other than B. anthracis, may be resistant to the inhibitory agents in PLET Agar. Most gram-negative bacilli will be inhibited on PLET Agar, although some exceptions may occur, such as Proteus vulgaris . Staphylococci and enterococci are not inhibited on PLET Agar. A thorough characterization and identification of each isolate is required.

A level C Public Health Laboratory must be informed of any isolates presumptively identified as Bacillus anthracis .

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, spreaders, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacillus thuringiensis
ATCC ® 33679
A 12-48hr 35°C Aerobic Growth
Bacillus cereus
ATCC ® 14579
A 24-48hr 35°C Aerobic Growth
Bacillus megaterium
ATCC ® 14581
B 24-48hr 35°C Aerobic Partial to complete inhibition
Escherichia coli
ATCC ® 25922
B 12-48hr 35°C Aerobic Partial to complete inhibition
Pseudomonas aeruginosa
ATCC ® 27853
B 12-48hr 35°C Aerobic Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

PLET Agar should appear clear, and light to medium amber in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Kinsely, R.F. Selective Medium for Bacillus anthracis. J. of Bacteriology. Sept., 1966.

5. Turnbull, P.C.B., Boehm R., Cosivi O., Doganay M., Hughs-Jones M. E., Laitha M.K. and DeVos, V. 1998. Guidelines for the Surveillance and Control of Anthrax in Humans and Animals. WHO/EMC/ZDI/98.6. World Health Organization, Geneva, Switzerland.

ATCC is a registered trademark of the American Type Culture Collection.

040416gr