The effect of repeated freeze-thaw cycles on cryopreserved Neurospora
crassa samples.
Kevin McCluskey, Aric Wiest and Sheera Walker. Fungal Genetics Stock Center,
University of Missouri- Kansas City
Fungal Genetics Newsletter 53:37
To better characterize handling parameters for the arrayed mutants prepared for the Neurospora functional genomics program, we have put 7 day old conidia from strain FGSC 2489 through a series of cycles of freezing at -80 C in 25% glycerol and 3.5% reconstituted non-fat dry milk. This is prepared by mixing autoclaved 50% glycerol with 7% milk that had been sterilized in a pressure cooker for 20 minutes. Samples were frozen in 2 ml screw-cap vials without control of freezing rate. Thawing was carried out on the bench. We diluted spores in sterile water and plated them on agar-solidified medium containing 1X Vogels salts and 1X FGS. Plates were incubated at 30 C until countable colonies were visible. Two replicates beginning with independent conidiating cultures were carried out and the results are presented in Table 1.
Table 1
|
Viability of conidia1 |
|||||
|
Freeze- thaw cycles |
1 |
2 |
3 |
4 |
5 |
|
First Replicate |
ND2 |
2.7 X 10-1 |
2.0 X 10-1 |
1.1 X 10-1 |
7.0 X 10-2 |
|
Second Replicate |
8.3 X 10-1 |
2.3 X 10-1 |
3.8 X 10-1 |
1.8 X 10-1 |
5.0 X 10-2 |
1
Viability is calculated as the ratio of the viable conidia to the plated conidia.Freeze thaw cycles do decrease the recovery of Neurospora crassa conidia and the decrease is linked to the number of freeze-thaw cycles. For practical uses, the ability to recover a strain following cryopreservation is robust when one begins with a significant number of conidia. In the first replicate, we started with 5 X 106 conidia per ml and in the second we started with 6.5 X 106 conidia per ml.
Because Neurospora conidia are hydrophobic they have a tendency to form clumps. This may account for some of the variability in numbers (for example the apparent increase in viability between cycles 2 and 3 in the second replicate). This is not a practical problem when one begins with great numbers of conidia and the goal is to maintain viability.
The ability to recover mutant strains may be reduced depending on the specific characteristics of the strain. Nevertheless, the ability to thaw and re-freeze strains arrayed as glycerol stocks demonstrates the usefulness of this resource.
The FGSC is supported by grant 0235887 from the US National Science Foundation and by National Institutes of Health Grant P01 GM068087.