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ExoLab-3 – Biological Preparation
ExoLab-8 Astrobotany Research
Introduction
The following protocols are for use with the “student kit” sans ExoLab.
Materials
- 38 x 200 mm test tubes and caps (2x) › growth chambers
- Test tube rack – assembly required
- Agar agar powder › 1.5 g packet
- Seed Packet – Trifolium pratense included (red clover)
- Rhizobium inoculant › powder in 1.5 ml tube
- pipette
- forceps
- 250 ml plastic water bottle (full, user-supplied)
this is used for measured volume of water as a substitute for a graduated cylinder › if using graduated cylinder, distilled water will also be needed - 500 ml beaker or microwave-safe jar or measuring cup (user supplied)
- 100-200 ml beaker, measuring cup or bottle
used for preparation of inoculant
using the empty water bottle from above may also be used - alcohol (user supplied)
used for surface sterilization of seeds
either isopropyl (rubbing) alcohol or ethanol may be used - distilled water (user supplied)
- paper towels, several sheets (user supplied)
- plastic wrap or foil (user supplied)
- small container about 50 ml (user supplied)
small refrigerator containers, shot glasses, small ramekins, or similar may be used in lieu of 50 ml beaker
- goggles (user-supplied – recommended)
- hot pads (user supplied)
- gloves – clean, unused (user-supplied – recommended) › disposable examination gloves or kitchen gloves
- pot or microwavable container (user supplied)
sufficient size to accommodate test tubes for sterilization - tongs (user supplied)
used for removing test tubes and caps from sterilization bath
Workflow
1. Hygiene: prepare a clean, heat resistant work surface, wash hands (5 min) 2. Prepare media for test tube growth chambers (30-60 min)
3. Prepare Seeds for sowing (30 min)
4. Sterilize test tubes and caps (15 min)
5. Prepare Rhizobium inoculant (10 min)
6. Prepare growth chambers for sowing (30-60 min)
7. Sow seeds (10 min)
8. Initiate experiment (20-30 days) log observations daily (30 min)
9. SAFETY ADVISORY: gloves and goggles recommended
10. clean work surface with detergent and water, alcohol, disinfecting wipes or mild bleach solution 11. wash hands frequently to prevent cross-contamination of materials
12. SAFETY ADVISORY: goggles recommended, hot pads required
13. Empty agar powder into a 250 ml bottle of water
14. Replace cap and agitate solution until powder is completely dissolved
15. Pour the solution into a beaker or other microwave-safe container
16. Microwave to boiling point
use extreme care as media will be extremely hot
heat media in 1 – 2 intervals to prevent boiling over
once the solution starts to boil, remove from microwave and set aside to cool cover with plastic wrap or foil to prevent contamination during the cooling period
17. place six (6) seeds in 50 ml beaker (or substitute suggested above)
18. add a few ml of isopropyl or ethanol ensure seeds are completely covered with alcohol
19. swirl for 30-60 seconds
20. set aside to soak for 5 minutes
21. pour seeds and alcohol onto a paper towel and allow alcohol to completely evaporate
22. transfer 0.5 ml of powder to beaker or suitable substitute with capacity of at least 100 ml
23. measure and add 100 ml of distilled water to beaker
24. swirl gently or stir with pipette to get powder into suspension
25. SAFETY ADVISORY: goggles recommended, hot pads and tongs required
26. Sterilize test tubes and caps by submerging in boiling in distilled water
do not use tap water as it contains dissolved minerals and salts that will leave spots on the test tubes
boil in a pot of sufficient size on the stovetop for 15 min or in a microwavable container of sufficient size for 5 minutes
27. carefully remove test tubes and caps with tongs, set aside in test tube rack to cool and air dry 28. Once test tubes are cool to the touch, pipette 1 ml of Rhizobia solution to each tube
29. Once media is cool to the touch, pour half of the media into each test tube and cap
30. Allow media to completely gel
31. sterilize tips of forceps with alcohol, shake dry (or flame off with a lighter under adult supervision)
32. carefully transfer 3 seeds to each test tube with forceps try to place seeds evenly around the media surface near the edge of the test tube wall try not to dig forceps into the media surface
33. Place growth chamber with a rack under bright light
100 watt equivalent white LED bulb suspended 20 cm above growth chamber or windowsill be careful to ensure the growth chambers do not overheat