Ward's World Working with Guides

Page 5 Special Culture Techniques General Hints • Sterilize all transfer pipets before beginning culture transfers. • Wash all glassware thoroughly, rinse several times, and soak in hot water for a final rinse. • Examine each culture under a microscope before transferring. Check the culture's condition and look for possible contamination. Use only the best cultures for subculturing. Sterilize all algal media before using. Algal media can be sterilized by autoclaving at 15 psi for 15 – 20 minutes. Some marine media cannot be autoclaved due to high salt concentrations. Pasteurize these media before using by heating the medium to 73˚C for 15 minutes. Repeat this procedure for three consecutive days. Subculturing Flasks, tubes, bottles, or Petri dishes can be used as culture vessels. If using a 250 mL Erlenmeyer flask, fill the flask to approximately 150 mL with freshly prepared media and sterilize the media. Add a small amount (5 – 10 mL) of inoculum from a stock culture, handling the inoculum according to the instructions for the particular species that is being subcultured. Always prepare more than one subculture in case one of the new cultures becomes contaminated. To culture on agar, sterilize the media and, using sterile technique, transfer the algae from stock culture to a tube containing fresh media with a sterile cotton swab. Be sure to cover the entire surface of the agar and replace the cap loosely to allow for gas exchange. Some algae can be grown on agar and kept viable for up to a year. However, cultures maintained on agar may begin to exhibit abnormal morphology over the course of time and may need to be trans- ferred to liquid before normal morphology can once again be observed. To do this , remove some of the cells from the surface of the agar with a sterile cotton swab and place them in fresh liquid medium. If pressed for time, add sterile distilled water to the culture tube to allow some of the cells to regain their normal morphology. Demonstrations of Sexual Reproduction in Algae Various forms of algae can be used to demonstrate sexual reproduction. These are only guidelines, so you should expect some variation in reaction time due to differences in light intensity, tempera- ture, and media. Because the sexual process in most algae is associated with light, it is not possible to supply cultures which will be immediately sexually active. Therefore, be sure to obtain cultures well enough in advance so that they may be cultured in or acclimated to a new environment. Chlamydomonas, Plus strain and Minus strain 1. Grow the heterothallic strains separately, on soil extract agar or Bristol's agar, for about one week under 350 foot candles of illumination. 2. The day before the demonstration, wash the cells of each strain from the agar using approximately 5 mL of distilled water. Place the suspensions of the strains in separate flasks. 3. Illuminate the flasks for several hours and then place in a dark area. 4. Two hours before the demonstration, illuminate the flasks again. After this period, mix a drop of each culture together on a clean glass slide. Sexual reproduction is evidenced by the immediate clumping of the flagellated gametes. Pairs can be observed within a few minutes after mixing. The pairs swim about, joined at the anterior end. Fusion (plasmogamy) occurs approximately six to eight hours later. Use the usual 16-hour light period alter- nating with an 8-hour dark period.

• Cover