Methods of molecular identifications and lab. protocols
In my study of morphology of Trichoderma I have tried CMD (cornmeal agar + 2% dextrose), Malt agar (2% malt extract), Walter Gams' oatmeal agar, potato dextrose agar and SNA. In my opinion only SNA and CMD provide morphological characters that more or less approximate what is found in nature. Maybe malt agar but it is not as useful to me as CMD or SNA. Trichoderma species are essentially the same when grown on CMD and SNA but conidial production is less rich on SNA than on CMD. A drawback to use of these two media is that colonies must be studied soon after conidia are formed, otherwise the phialides will disappear.
I typically incubate cultures used for morphology at 20C, 12 h darkness/12 h cool white fluorescent light. The only difference between 20 and 25 C is that conidia form faster at 25 C, so that is not critical. Many species also grow well and sporulate at 30 C but some do not, so this is not such a good temperature for routine use. It is essential to incubate at 35 C because relatively few species grow well at that temperature: growth at 35 C is an important taxonomic criterion. Growth at low temperature (I only use 15 C) is not so informative. The optimum for most species is between 25 and 30 C.
Microscope preparations are made from pustules where there are still white conidia, otherwise the pustule will probably be overmature. For most species, this is within a week of incubation at 20 or 25 C. I place a small amount of material in a drop of 3% KOH on a slide. the KOH wets the conidia and allows conidiophores to spread; it is replaced with water after the prep has been made. It is critical to use a small amount of material in order to get a flat preparation and thus to observe branching. Before placing a cover slip on the prep I usually separate hyphae and conidiophores using insect pins. Once the cover slip is in place, I often flood the prep with water and then, under low power of the compound microscope, tap the conidiophores etc with the tip of a #11 scalpel in order to further separate the elements.
Then I draw the water out by applying a small piece of tissue to the edge of the cover slip. This gives me a really flat preparation. I never use any mountant other than KOH and water.
posted by Gary Samuels on 25 Oct, 2004