2. Lab Procedure – Session 1

2.1 Set-up the Investigation

You will inoculate your petri dishes with C. richardii (hereafter referred to as fern) spores that have been pre-sterilized and suspended in sterile distilled water. The plates will be incubated under constant lights, 27°C, and high humidity. The spores will germinate and over the next few weeks, will grow into gametophytes. Use aseptic technique during set-up.

1. Label the outer surface of the bottom plate with the following information: group initials, lab section, and plate number (A-F). Having the outer part labelled will make it easier to observe the gametophytes.
2. Serial dilution and plating. Working as a pair and maintaining proper aseptic technique is essential.

a) Label pipette bulbs as “spore” or “water” – do not remove from packaging until ready to use. Never put the pipette down! It is important to use the spore pipette only when transferring and resuspending spores, and the water pipette only when adding water to the spore vial.

b) Use the sterile spore pipette to carefully and thoroughly mix the suspended spore solution (i.e., draw up and expel liquid repeatedly, being careful to not draw the liquid into the bulb).

c) Immediately sow 6 drops of this suspension onto plate “A”. Return the remaining suspension in the pipette back into the vial of fern spores. Set plate “A” to the side, keeping the plate with the agar side down to allow spores and liquid to absorb into the agar.

d) Dilute the spores by adding 6 drops of water to the spore vial, resuspending the spores, and sowing 6 drops onto “B”. Return the remaining suspension in the pipette back into the vial of fern spores and set the plate aside.

e) Repeat step d for each of the remaining plates (C-F; i.e., complete steps for “C” before diluting etc. for “D”, and so on):

• • dilute the spore vial with 6 drops of water
  • resuspend,
  • sow 6 drops
  • return excess back to the spore vial
  • set plate aside

Tip: You should always have approximately the same amount of liquid in the spore vial.

You should now have 6 plates, each plate having a different number of spores (A = highest, F = lowest)!

3. Spread the spores. Starting with “Plate F”, spread the spores across the whole surface of the plate using the sterile spreader provided. Try to get an even distribution of spores across the plate (why do you think that is?). Do the same with the rest of plates in order of increasing density (i.e., F→A)
4. Stack the plates and place tape around the stack to keep everything together – put your initials and lab section on the tape. Place the stack of plates in the incubator as instructed – you will check on the plates in the next 1-3 weeks!