Bird Seed Agar: Introduction, Principle, Composition, Test Procedure, Colony Characteristics and Uses

Bird Seed Agar: Introduction, Principle, Composition, Test Procedure, Colony Characteristics and Uses

Introduction of Bird Seed Agar

Bird Seed Agar sounds seed of a bird but actually, it is a niger seed, botanical name Guizotia abyssinica. Niger seed contains caffeic acid that serves as a substrate for the detection of phenoloxidase, an enzyme produced by Cryptococcus neoformans. The action of phenoloxidase on caffeic acid results in the production of melanin which is occupied by the yeast cell wall forming a tan to reddish-brown pigmentation. Bird seed agar in brief is also called BSA.  Stab’s formulation BSA  is a selective and differential medium for both C. neoformans and C. gattii while modification of Staib’s formulation ignores C. gattii.

Principle of Bird Seed Agar

Niger seeds, creatinine, and glucose provide nutrients for the growth of Cryptococcus neoformans. Chloramphenicol inhibits the bacteria as well as rapidly growing molds that often overgrow the slow-growing dimorphic fungi. The addition of diphenyl also assists in preventing the growth of molds and fungus and is therefore used as a selective agent in the medium.

Composition of Bird Seed Agar

Ingredients         Gms / Litre

  • Guizotia abyssinica (niger) seeds: 70.000
  • Creatinine:  0.78
  • Dextrose (Glucose): 10.0
  • Chloramphenicol: 0.05
  • Agar: 20.0
  • Distilled water: 1000 mL
  • Final pH ( at 25°C):  6.7±0.2

Preparation of Bird Seed Agar

  1. Suspend 10.8 grams Bird Seed Agar ( BSA) 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 45-50°C and add 100 µg diphenyl per ml of medium (1 ml of sterile 1% w/v aqueous solution of diphenyl).
  5. Mix well before dispensing.
  6. Pour BSA 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 Bird Seed 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-Cerebrospinal Fluid (CSF), tissue, exudates, sputum, blood, and urine or fungal growth)
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains ( Cryptococcus neoformans ATCC 32045
    and Candida albicans ATCC 10231)

Test Procedure of Bird Seed 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 30°C for 7 days.
  7. Examine colony characteristics.

Colony Characteristics of Bird Seed Agar

Development of brown to black pigmented smooth colonies-Cryptococcus neoformans

Non-pigmented colonies-Cryptococcus laurentii, Candida, Saccharomyces cerevisiae

Uses of Bird Seed Agar

BSA is the recommended medium for selective isolation and differentiation of Cryptococcus neoformans from other Cryptococcus and other yeasts.

Keynotes on Bird Seed Agar

  • Staib’s Medium (Bird Seed Agar) is formulated for selective isolation and differentiation of C.neoformans and  C. gattii.  from other Cryptococcus species and other yeasts.
  • The above medium is formulated by Staib and Shields and Ajello which is selective isolation and differentiation of only C. neoformans from other Cryptococcus species and other yeasts.
  • Compositiuon of Staib Formunation

Ingredients             Gms/Litre

  • Guizotia abyssinica (niger seed): 50.0
  • Dextrose (glucose): 1.0
  • KH2PO4 (potassium dihydrogen orthophosphate): 1.0
  • Creatinin: 1.0
  • Agar: 15.0
  • Final pH:  6.5 +/- 0.3 at 25ºC.

Additives: to each 500 ml bottle

  • Penicillin G (20 units/ml) 1 mL
  • Gentamicin (40 mg/ml) 1 mL
  • Other species of Cryptococcus are-
  1. C. albidus ( nitrate positive)
  2. C. laurentii ( melibiose fermentation)
  3. C. gattii ( Trehalose positive)
  4. C. neoformans: All the above tests are negative.
  5. C. neoformans var. grubii ( serptype A)- worldwide distribution
  6. C. neoformans var. neoformans  (serotype D)-Restricted distribution and prevalent in France, Italy, and Denmark.
  • Diphenyl prevents the growth of molds and fungus and is therefore used as a selective agent in the medium.
  • Bird seed agar is also known as Caffeic acid agar or niger seed agar or Staib agar.

Further Readings

  1. https://www.himedialabs.com/TD/M675.pdf
  2. http://www.essentialpaservices.com/listing/birdseed-agar-thermo-fisher-scientific-425b-ce549f
  3. https://catalog.hardydiagnostics.com/cp_prod/product/g213-caffeic-acid-agar-bird-seed-agar-for-cryptococcus-15x100mm-plate-order-by-the-package-of-10-by-hardy-diagnosticsbr-bminimum-order-10-packsb-media-prepared
  4. https://en.wikipedia.org/wiki/Biphenyl
  5. Casadevall A, Perfect J. R., 1998, Cryptococcus neoformans, ASM Press, Washington, D.C.
  6. A Text-Book of Medical Mycology. Editor: Jagdish Chander. Publication Mehata, India.
  7. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC
  8. Description of Medical Fungi-David Ellis, Stephen Davis, Helen Alexiou, Rosemary Handke, Robyn Bartley
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