I am using the single spore isolation,terrestrial twig fungi using the 2% water agar and 2 or4% malt extract agar,
Some spores unable to grow in the media,
suggest any modification or alternate?
You can try with other culture media such as potato dextrose agar or Sabouraud dextrose agar.
If you are using antibiotics to inhibit the growth of bacteria replace it for NaCl (7.5%). This also will enable the growth of osmophilic fungi and not affect the growth of mesophilic fungi.
You must take into account that there are non-culturable fungi or is not easily develop in culture media.
Good luck.
If you isolate the spores with use of micromanipulator and trying to isolate single spore per Petri dish, you should take into account that single spores alone cannot germinate in some cases. They need a "company" to initiate its germination tube/bubble development .
So the ancient method of successional spore suspension dilution may be also acceptable. Such a way, you isolate the single spores after the germination (young colonies). But the accurate microscopic control of germinating spores is needed in this case to make sure you isolate really single spore culture.
For the spore suspension with hydrophobic surface you may use TWIN80.
Certain antibiotics may also inhibit the spore growth. So sometimes it is not so easy to find the appropriate chemical to inhibit contaminants.
And as Alian suggested, the growth media is also critical for germination. In some cases a very gentle media is needed. For example, the media contain amino-acids and simple sugars instead of long-chained substances. Such parameters as media pH, temperuture, agar / water ratio are also very important.
Regards,
Maria
Consider the possibility that some species may have evolved responses to environmental clues for specific seasons. So maybe a heat "shock" or drying (of the twigs) followed by simulated rains? Would depend, of course, on the seasonal changes in the area where you collected and, perhaps, the season in which the materials were collected. (I'm guessing they wouldn't need a period of freezing temperatures such as some fungi might need around here, especially in the mountains.) I don't know of any specific examples to guide you, however.
We used the following protocol for excystment of Tetrahymenas. I am not sure that it fits for fungi but some tips might be useful.
1. Transfer cysts in the diluted Brain Heart Infusion (BHI) medium (0,37%) supplemented with 0,25 % fetal serum.
2. Incubate at 25 C and 500 L illuminance for 5-7 days.
3. Dilute 1:4 in the fresh medium with increased serum percentage (0,5 %). We don’t mix the culture to take the sample from the volume but always take the sediment to dilute
4. Incubate as at the step 2.
5. Repeat steps 3 and 4 increasing the serum percentage up to 1% with 0,25% increment. Spores germinate after 3-5 passages.
Simplest of all is use Glucose yeast extract medium supplemented with 0.5% of any surfactant (Tween 80, polysorbate etc.). Composition of medium is
Glucose-10g, Yeast extract powder-10g, Peptone-5g, distilled water-1000mL and set pH to 5- 5.5.
Give preincubation of 50 to 60 degtree centigrade to the sporulating culture and then suspend the culture in above listed broth medium. Incubate at optimum temperature in a shaker incubator at 100-150RPM. The spore will germinate for sure.
Regards
Mukesh Chander
increased glucose concentration may enhance the sporulation in fungi.
For Alian Molina Veloso:
Do we put NaCl before or after autoclaving the media ?
For Niranjan M :
1. Photoperiod may enhace or induce the sporulation of your spores
2. Try to change a media such as PDA( Potato dextrose agar) and you should put few media
Regards
Narimene
And you should little media in each petri dish ( instead of putting 20ml of media, put 15ml or 10ml)
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