Blanes Plankton Isolation (BPI) - IBEMicrobeHub/culturing GitHub Wiki

By using the RS medium, cultivate the most abundant protists (not still in culture) present in the ocean.

Before starting:

Send a mail to Clara ( [email protected] ) a few days before the sampling to get the sample. Specify if you want the unfiltered or 200 mesh filtered.

Decide which dilution of RS medium you want to do.

Book the biological hood!

Clean the biological hood and the lab materials we are gonna use with EtOH 70%.

Leave on the UV for 10-15 minutes.

Clean the previous inoculum flasks and the RS medium (diluted or not, it’s up to the culture) by using EtOH 70% before putting them under the hood.

A) ISOLATION BY DILUTION

Materials:

  • Plankton sample from Blanes collected the same day of the experiment and unfiltered by size.
  • Prepare cut tips: cut the blue tips (P1000) and yellow ones (P200) to also promote the picking up of microorganisms with range-size 180-2000 μm. Autoclave before the use.
  • Three or more 6-well-plates (it’s up to how much dilution of the RS medium you want to do).
  • Pipettes 5 or 10 mL.
  • RS medium (NNM+NM), falcons and/or autoclavable bottles to prepare the RS medium diluted. Use the NNM to dilute, not H2O.

ISOLATION BY DILUTION INTO 6-WELL PLATE

Under the biological hood.

  1. By using a marker, mark the three or more 6-well-plates as shown here:

Screenshot 2023-09-21 at 12 11 35

NOTE: here an example of dilutions - 1:10, 1:100 and 1:1000 -.

  1. Add in each well the relative volume of RS medium. The total volume in each well is 5mL.
  2. Mix the marine sample.
  3. By using the cut tips, add X μL of the sample in each well.
  4. Parafilm the plates.
  5. Place the plates to the light to promote also the growth of photosynthetic microorganisms.
  6. Leave them at 18℃ - culture room.

Steps 6 & 7: place the plates on your own conditions (light/dark - TºC).

  1. By microscopy, check along the days (for two weeks) if the planktonic cells are able to grow or how the marine community changes in laboratory conditions.
  2. After about two weeks, move the cells - which are growing - from a well into the 50mL flask. In other words we do the SPLIT.

B) SPLIT

INOCULUM PROTOCOL

Under the biological hood.

  1. By using the “pipetador”, add 17mL of the relative medium - RS medium diluted or not, according to the growth condition of the relative culture.
  2. Then, by using the P1000 inoculate 3mL of the specific well in 17mL previously added.
  3. Store the flasks at 18°C.
  4. Check the flasks very frequent to see if the culture is going to stabilize.

Only after its “stabilization” proceed with DNA, PCR, Cloning etc.

At the end:

Tidy everything up and clean the biological hood.