PLET Agar: Introduction, Principle, Preparation, Test Procedure, Colony Morphology and Keynotes

Introduction of PLET Agar

PLET agar is the selective medium for the isolation and cultivation of Bacillus anthracis.

Principle of PLET Agar

PLET Agar Base is the Knisley formulation. It is the best selective medium for the cultivation of Bacillus anthracis. Components of the medium like Beef heart infusion from solids and tryptose provide the carbonaceous and nitrogenous compounds necessary for growth whereas sodium chloride (NaCl) provides the osmotic equilibrium. Thallous acetate, polymyxin, lysozyme (FD185) are inhibitory agents allowing growth of B.anthracis while inhibiting contaminants. Lysozyme specifically suppresses the growth of gram-negative contaminants.  Colonies of B.anthracis appear in 36-40 hours after incubation at 37°C. Roughly circular, creamy-white colonies with a ground-glass texture.

Composition of PLET Agar

Ingredients  Gms / Litre

• Beef heart, infusion from: 500.0
• Tryptose: 10.0
• Sodium chloride: 5.0
• EDTA: 0.3
• Thallous acetate: 0.04
• Agar: 15.0
• Final pH (at 25°C): 7.3±0.2

Preparation of PLET Agar

1. Suspend 4.034 grams, PLET agar, in 99 mL purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 50°C and aseptically add rehydrated contents of 1 vial of Anthracis Selective Supplement (FD185).
5. Mix well before dispensing.
6. Pour PLET agar into each plate and leave plates on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of PLET Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( Clinical samples or bacterial growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains ( Bacillus anthracis ATCC 14578 and Bacillus cereus ATCC 10876 )

Test Procedure of PLET Agar (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Take a plate for one specimen.
3. Inoculate and streak the specimen as soon as possible after collection.
4. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
5. Streak for isolation with a sterile loop.
6. Incubate the plate aerobically at 35-37°C for 36-40 hours.
7. Examine colony characteristics.

Colony Morphology of PLET Agar

Bacillus anthracis: Luxuriant growth

Other bacteria or Bacillus species other than Bacillus anthracis: No growth

Control stains

Bacillus anthracis ATCC 14578: Luxuriant growth

Bacillus cereus ATCC 10876:  inhibited

Keynotes on PLET Agar

• Man is relatively resistant to anthrax and laboratory workers are rarely infected.
• Anthrax cannot spread directly from human to human but anthrax spores can be transported by human clothing, shoes, etc.
• In humans, anthrax is caused by exposure to dead infected animals, consumption of infected animal tissue, or exposure to light density anthrax spores from animal wool, fur, hide, etc.
• Some strains of B. cereus from the soil on PLET agar form colonies but they are smaller than those of B. anthracis, minute after 24 hours and moderately sized after 48 hours.
•  Great care should be taken to avoid the escape of the long-surviving spores into laboratory environment because of high-risk group concern organism and therefore all the procedures should be carried out in biosafety cabinet.
•  In human anthrax, the bacillus is usually demonstrable in material from a malignant pustule, sometimes in sputum from pulmonary anthrax, and also in the blood in the septicemic stage of all forms of the infections.

Further Readings

• Knisely R. F. 1966, J. Bacteriol, 92:784-786.
• Norris J. R., Berkley C. W., Logan N. A., and ODonnell A. G., 1981, In M. P. Starr et al (Ed) The Prokaryotes: a Handbook
• on Habitats, Isolation, and Identification of Bacteria, Vol. 2, Springer-Verlag, Berlin.
• Parry J. M., Turnbull P. C. B. and Gibson J. R., 1983, A Colour Atlas of Bacillus species. Wolfe Medical Publications, London, United Kingdom.
• Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology,
• 8th Ed., American Society for Microbiology, Washington, D.C.