Bird Seed Agar Bird Seed Agar is an effective solid medium to isolate selectively and differentially of Cryptococcus Neoformans from clinical specimens. It is utilized for the specific isolation from Cryptococcus neoformans as well as C. Gattii. C. Neoformans typically grows as yeast (unicellular) and reproduces via budding. Cryptococcus Neoformans is an enclosed yeast, which makes the enzyme phenoloxidase an enzyme that is essential to melanin production.
The brown-colored colonies of Cryptococcus Neoformans were discovered by Staib during 1962 when he was able to grow the yeast in media that contained Guizotia Abyssinica seeds. The seeds were later found to contain the seeds contained caffeic acid, which acts in the production of melanin as a substrate. Birdseed agar was a common designation for the Staib’s Agar medium. The year was 1966. Shields as well as Ajello altered the Staib’s Birdseed Agar by adding the antibiotic to enhance its selectiveness. It’s also referred to by the name of Caffeic Acid Agar or Niger Seed Agar or Staib Agar.
Principle of Bird Seed Agar
Birdseed agar consists of Guizotia abyssinica seedsas well as dextrose and creatinine that supply nutrients to the development of C. Neoformans. The Guizotia abyssinica seeds has caffeic acid that acts as a substrate to aid in the analysis of phenoloxidase which is an enzyme made by C. Neoformans. Its action caffeic acid causes melanin being produced which is absorption in the cells of the yeast and results in the appearance of a reddish-brown to tan coloration.
The energy source is glucose within the medium. Creatinine increases the melanization rate of certain varieties from C. neoformans. The addition of antibiotics such as chloramphenicol hinders the growth of the bacteria as well with rapidly growing molds which frequently overtake slower-growing dimorphic fungi. Agar is the agent that helps to solidify.
Intended Use
Used for selective isolation and differentiation of Cryptococcus neoformans from other Cryptococcus and other yeasts.
Composition of Bird Seed Agar
| Ingredients | Gm / Litre |
| Guizotia abyssinica seeds | 70g |
| Creatinine | 0.780g |
| Dextrose | 10g |
| Chloramphenicol | 0.050g |
| Agar | 20g |
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
Preparation of Bird Seed Agar
- Suspend the prescribed amount (as as per the manufacturer’s instructions) to 1 liter distillate water.
- Bring the temperature to a boil to completely dissolve the medium.
- Sterilize with autoclaving with 15 lbs tension (121degC) over 15 mins.
- Cool to 45°C and then add 100 mg diphenyl to each ml medium (1 one ml of unsterile 1% w/v diphenyl aqueous solution).
- Mix well before pouring into sterilized Petri plates.
Note: The composition and the method to prepare media differ depending on the manufacturer. Some media are pre-added with the phosphate as well as other antibiotics such as penicillin G or gentamicin. Other media require supplements.
Result Interpretation of Bird Seed Agar
| Result | Colony characteristics | Organisms |
| Positive | Development of brown to black pigmented smooth colonies | Cryptococcus neoformans |
| Negative | Non-pigmented colonies | Cryptococcus laurentii, Candida, Saccharomyces cerevisiae |
Test Procedure of Bird Seed Agar (specimen/organism inoculation)
- Let the plates warm at 37degC or until temperatures of room temperature. Then allow the agar surface to air dry prior to inoculating.
- Get a plate for a example.
- Streak and inoculate your specimen the earliest is possible following collection.
- If the sample to cultivated will be on a swab place the swab on only a small portion of the surface of the agar.
- Streak to isolate with the use of a unsterile loop.
- Incubate the plate aerobically 30degC for seven days.
- Review the colony’s characteristics.
Uses of Bird Seed Agar
- It’s used to specific isolation and differentiation of Cryptococcus Neoformans in comparison to other yeasts which includes other Cryptococcus species that are isolated from different samples from the environment and clinical.
Storage and Shelf life of Bird Seed Agar
- Store at 2-8oC far from direct light.
- Media is not recommended in the event of any indications of degradation (shrinking cracks, cracks or discoloration) or if there is any evidence of contamination.
- The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.
Limitations of Bird Seed Agar
- Organs which produce coloration on agar made from bird seeds must be compared to the similar growth on a different medium, such as Sabouraud Dextrose agar in order to establish and cross-check whether the organism naturally has pigmentation.
- Particularly contaminated specimens can block growth and/or the coloration of C. Neoformans.
- Rare varieties of C. Neoformans might not have pigmented colonies.
- Aureobasidium, Sporothrix, Wangiella and Phialophora could make dark brown colonies but the coloration does not result of an enzyme that is evident through the pigmentation that develops in colonies across every medium.
Quality control
- Cryptococcus neoformans ATCC 32045 can be used as a positive control that shows brown to black pigmented colonies.
- Escherichia coli ATCC 25922 can be used as a negative control in which growth is inhibited partially or completely.
References
- https://himedialabs.com/TD/M675.pdf
- https://microbeonline.com/bird-seed-agar-principle-composition-uses/
