Plating and Cell Colony Counts

Assessment of control (100%) viability level

1. Perform a haemocytometer count to obtain the cell density of the culture to be cryopreserved.

2. Using 1-mL aliquots and logarithmic dilutions in sterile medium dilute to a defined culture cell density that would result in 50-500 cells in 1-mL. (e.g. for a culture with a cell density of 5 x 10⁷ dilute the culture by 10⁵ i.e. through 5 logarithmic dilutions.)

3. Transfer 1-mL aliquots of the dilution obtained in step 2, and place in replicate (3) identical Petri dishes (50-mm diameter).

4. Pour approximately 2.5-mL of agar (BBM+V) containing agar (1% w/v) into the Petri dish and agitate gently to insure uniform mixing. (Note you need to hold the agar at 40^oC prior to dispensing. It is optimal to restrict to small flasks/bottles containing ~ 50-mL agar).

5. The number of algal units in the agar should be such that between 50 and 200 colonies will be produced.

6. After gelation, seal the plates with Parafilm or Clingfilm to prevent excessive dehydration.

7.For sensitive strains plates should be incubated in the dark/ very low light for 8-24h prior to transfer to standard culture conditions. Note, this step should be omitted for Chlorella vulgaris 211/11b.

8. Incubate the plates, inverted, under standard algal culture conditions (see above).

9. Depending on CFU density count the number of colonies present on each plate after 7-14 days (Counting on one occasion). Note; periodically check plates for growth, if there is a risk that colonies will merge, then count as soon colonies are distinguishable.

10. Calculate the mean 100% viability level

Assessment of frozen/thawed material

1. Stored vials should be transferred from the Cryostore to a small Dewar containing liquid nitrogen and transferred to the laboratory for thawing.
2. Vials are thawed by placing in a pre-heated water bath (40^oC) and agitate until the last ice crystal has melted
3. On thawing rapidly transfer to a laminar flow cabinet and wipe the outside of the vial with 70% (v/v) ethanol.
4. Using a disposable plastic pipette transfer the 1-mL of each of 3 thawed samples to separate logarithmic dilutions that would result in 50 –500 cells in 1-mL (see above).
5. Transfer 1-mL aliquots of the dilution obtained in step 4, and place in replicate (3) identical agar plates.
6. Pour approximately 2.5-mL of agar (BBM+V) containing agar (1% w/v) into the Petri dish and agitate gently to insure uniform mixing. (Note you need to hold the agar at 40^oC prior to dispensing. It is optimal to restrict to small flasks/bottles containing ~ 50-mL agar).
7. The number of algal units in the agar should be such that between 50 and 200 colonies will be produced.
8. After gelation, seal the plates with Parafilm or Clingfilm to prevent excessive dehydration.
9. Incubate the plates, inverted, under standard algal culture conditions (see above).
10. Depending on CFU density count the number of colonies present on each plate after 7-14 days (Counting on one occasion). Note; periodically check plates for growth, if there is a risk that colonies will merge, then count as soon colonies are distinguishable.
11. Percent viability of the cryopreserved culture is then determined as:

% viability = (post-treatment colony count)* / (control colony count) X 100.

*Note, take into account that frozen/thawed material has been diluted 1:1 with cryoprotectant solution prior to cryopreservation, thawing and logarithmic dilution.

Date: 2015-12-17; view: 928