Quantitative determination of perithecium formation

Quantitative determination of perithecium formation Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol19/iss1/9

and with qn &orbonce of 0.05, is pipetted onto the surface of the ogar in the inoculated plate.This time the conidial suspension is spreed over the wrfoce of the agar with on wtoclavcd 5 x 7.5 cm gloss slide.The slide is used for the spreading procedures, instead of a rod, since the inoculum can be applied faster and more evenly, according to our experience in the laboratory, by this method.

Dark perithecia farm in seven days on Cc&b's cornmeal agw. The rpreadicg technique allows
for even distribution on the surface of the agar, so that the perithecia cw be more easily counted with the aid of a bacterial colony counter.
Before counting, however, the plater are washed with 2% Clorox to inactivate the conidiosparer which hove formed.
A control plate, one in which the mote,nol strain has not been treated, is found to contain approximately 250 p&the&.
The 1:loO dilutiar of Em52970 conidia used in this technique might be varied by the investigator depending on the perithecia producing capacity of the stroir.used.This method has been found in our laboratory to be applicable to seveml strains that were tested.It may be concluded that tbir method is excellent for measuring changer in the ability of the chemically or physicallytreated strain to produce perithecia when mated to an untreated paternal strain, as compared to on untreated control cross. This of sterile cotton in D disposable 10 ml syringt'in order to wnove hyphol fragments fra the c&dial ulspension and to obtain microconidia.Sterile water is used to dilute this suspension until an absorbance reoding of 0.05 is reached cm D Bausch and Lomb Spctrophotometer 20, set at 500 nm.One ml of the suspension is diluted 1:1&l with sterile water.Sterile 35mm Brewer pletes with metal tops holding abbsotbent discs are filled with 15 ml of sterile &Lab's Cxoid cornmeal agar, which is mode according to the specifications on the label with I .5% agar.The Brewer plater are used since the discs absorb the water that condenser in the plater.Fewer perithecia ore formed when other brands of commwl agar, such 01 Difco'r or BBL'r arc employed.Wertergaard's medium (Westergo& and Mitchell 1947 Am.J.Botany 34:573) can not be used since conidiol growth is enhanced, thereby making it more difficult to ccunt the prithecia.After the plota are cool, 0.05 ml oliquoh of the Em 52970 surpmion, diluted 1:lW and containing less than 150 but more than 100 microconidia, are delivered to the centers of the plater, using a Schwartz BioRewarch Autopipettor.The disposable tips for the pip&or are sterilized in a petri plate for 24 hr before use with seveml drops of ethylene oxide, since the tips connot be wtoclaved.The automatic pip&or is used since it allows fast, accurate dispensicm of the conidial suspension onto the plates.Inowlclted cornmeal plater me left at 25°C until protoperithecia develop, after which 0.05 ml of N. crassa St. Lawrence 74A inoculum (with the -same micmconidial concentration and obtained in the same manner as described for Em52970 work was supported in part by Research Grant No. MlW from the Robert A. Welch Foundation and from an Institutional Grant from the Texor Woman's University.---Department of Biology, Texas Woman's University, Denton, Texas 76204.