Methods 31 Race Tubes

3.1.1. What Is a Race Tube?: Purchasing/Manufacturing

Race tubes are long hollow glass tubes with a thin layer of agar medium, in which growth of Neurospora can be assayed accurately for several days (see Fig. 2). Race tubes are generally approx 400 mm in length, of which 45 to 50 mm on both ends are bent upward at an angle of 30 to 45° (see Note 5). Race tubes have an internal diameter of approx 14 mm and a glass thickness of about 1 mm. Uniformity of race tubes is an important parameter in obtaining reproducible results. Race tubes can be purchased "custom made" (see Note 6) or brought in as long glass tubes and cut and shaped by an in-house workshop (or even by hand on the bench; see Note 7).

3.1.2. Preparation of Race Tube "Six-Packs"

1. Using autoclave tape, tape together clean and dry race tubes, creating "six-packs" (see also Note 8; for cleaning of race tubes, refer to Subheading 3.1.3.).

Fig. 2. Graphic representation of a race tube. Race tubes are long, hollow glass tubes with a thin layer of agar growth medium. Neurospora is inoculated at one end of the tube, causing its growth to be restricted to one direction only (here from left to right). When grown in constant darkness (black bar), defined areas of the mycelium differentiate and aerial hyphae with bright orange conidia (white "bands" in top view picture) appear once every circadian day (free-running period of 22 h). These conidial bands are separated by areas with undifferentiated mycelia (gray "interbands" in top view picture). Race tubes are marked every 24 h (black marks).

Fig. 2. Graphic representation of a race tube. Race tubes are long, hollow glass tubes with a thin layer of agar growth medium. Neurospora is inoculated at one end of the tube, causing its growth to be restricted to one direction only (here from left to right). When grown in constant darkness (black bar), defined areas of the mycelium differentiate and aerial hyphae with bright orange conidia (white "bands" in top view picture) appear once every circadian day (free-running period of 22 h). These conidial bands are separated by areas with undifferentiated mycelia (gray "interbands" in top view picture). Race tubes are marked every 24 h (black marks).

2. Prepare race tube medium in a large glass beaker or flask and bring to a boil (on a heater under constant stirring) to allow the agar to dissolve (see Note 9).

3. Fill each race tube with 13 to 18 mL (see Note 10) of hot medium using a 25-mL pipet or a Fill-O-Matic dispenser (see Fig. 3). When large numbers of race tubes are to be filled, it is recommended to keep the agar medium on a hot plate at low setting to prevent the agar from cooling down too quickly.

4. Prior to autoclaving, plug each race tube on both ends with nonabsorbent cotton or foam stoppers (50 x 25mm, cut in halves; see Note 11).

5. Stack race tube six-packs, cover the plugged ends with foil, and autoclave, making sure all tubes stay horizontal at all times (see also Note 12).

6. During the autoclaving process, free enough level bench space to lay out all six-packs next to one another.

7. After autoclaving carefully remove six-packs from the autoclave, when still very hot (~80°C; see Note 13).

8. Taking a small number of six-packs at a time, make two or three circular swirling movements while keeping the race tubes horizontal (see Fig. 4 and Note 14), before laying the six-packs out flat on a level lab bench. When dealing with large

Fig. 3. Filling race tube six-packs with Fill-O-Matic dispenser. (Pictures by S. K. Crosthwaite, University of Manchester, UK.)
Fig. 4. "Swirling" of race tubes. Two or three circular movements are performed as indicated while holding the race tubes horizontally. The swirling of hot race tubes is an essential step that prevents condensation within the race tubes (see also Note 14).

numbers of six-packs, work quickly. Make sure each six-pack, as well as each individual race tube, is level. 9. While agar medium is still liquid, remove all air bubbles (see Note 15). Again, work quickly when dealing with large numbers of six-packs. 10. Replace wet or soiled cotton wool plugs or foam stoppers with fresh, clean ones (there is no need for replacement plugs or stoppers to be sterile). Once the agar medium has solidified, six-packs can be stacked.

11. Leave race tubes to dry at room temperature at least overnight before inoculation (see Subheading 3.2.1.). However, drying for 2 to 3 d is normally better. Quick drying in an incubator or drying cabinet is not very effective and therefore is not recommended.

3.1.3. Cleaning (See Also Note 16)

Once the race tubes have been scanned and analyzed, the six-packs should cleaned for reuse. It is recommended to clean the race tubes sooner rather than later after the end of the experiment, as drying out of the agar makes the cleaning process more difficult.

1. Reautoclave the six-packs in stacks, placed in a large tray to prevent spillage.

2. Remove six-packs from autoclave when still hot (~80°C) (see also Note 13).

3. Take out foam stoppers or cotton wool plugs with forceps (see also Note 17). When dealing with large numbers of six-packs, work quickly.

4. Pour out hot molten agar into a container (to set and waste) and immediately run hot tap water under great pressure through each race tube. Run tap water through from both ends of the race tubes. Again, work quickly. Water pressure can be easily increased by squeezing the end of the tube attached to the tap. If possible, leave six-packs intact.

5. Afterward, rinse the race tubes thoroughly with distilled H2O and dry in a drying cabinet.

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