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PROTOCOL 1

How to determine the number of bacterial cells needed to produce 1 detectable ice nucleus at a given T°C.

At a training course on characterization of bacterial ice nuleators held in Avignon, France (end of 2008 and early 2009), this is the protocol we taught for quantifying ice nucleation activity of bacterial strains. This technique is the basis of the technique used for detection of biological ice nucleators in environmental samples (Christner et al 2008 Science 319:1214 and Proc. Nat. Acad. Sci. 105: 18854–18859 ).

1. Verify that the sterile distilled water to be used in the test has no ice nuclei (at -10°C, i.e. colder than the coldest test temperature)

2. Grow bacteria under standard conditions (for fluorescent pseudomonads: King’s medium B, 3 days, 25°C)

3. Prepare bacterial suspensions in sterile, non ice-nucleation-active water (from step 1)

4. Adjust the initial concentration of the suspensions (10e8 cfu/ml) and prepare the necessary dilutions.
Be sure to prepare enough total volume of suspension for the number of droplets needed.
If you use a reliable calibration curve (optical density vs. plate counts) you do not need to systematically verify bacterial density via dilution plating. Nevertheless, verify the concentration of at least some of the suspensions via dilution plating.

5. Put the suspensions at 4°C for at least 1 hour (take into account the time needed for the entire suspension to reach 4°C). This induces maximum ice nucleation activity.

6. Turn on the cold bath ahead of time so that it will be at the initial test temperature when you begin step 7.

7. Distribute the aliquots on a metal plate sprayed with silicon (lubricant spray)

► for example, 20-30 droplets each of 20-30 µl per suspension
For this step the spacing between the drops should be large enough to prevent initiation of neighbor drops by the crystals that form around the drops when they freeze.
Also pay attention that the drops do not dry out during placement on the plate or during the test.

►or for example, 20-40 aliquots of a larger volume in plastic epindorph tubes.
The tubes should be transparent enough to be able to see the ice formation

8. Expose the aliquots to successive temperatures (1° intervals) from -2°C to -9°C
For the small volume drops on a metal plate, the drops reach the initial cold temperature about 30 after putting the plate on the cold bath, and then ca. 5 min after the bath has reached each successive temperature (for a covered, well insulated bath)

For aliquots in epindorphs, you will need floating racks that allow the liquid in the tube to be fully submerged.
The time to reach the initial temperature will likely be longer than for small drops on plates.

9. Calculate the number of cells per ice nucleus (or the reciprocal) using the formula of Vali (1971)

N° = the number of drops or aliquots tested
Nx = the cumulative number of drops or aliquots frozen at a given temperature

For a given temperature:

number of nuclei / drop or aliquot (K) = ln(N°) – ln(N° – Nx)
number of nuclei / bacterial cell (X) = K/ number of bacteria per drop or aliquot
number of bacteria needed for 1 nucleus = 1/X

To make the calculation you need to use a dilution where at least one droplet or aliquot does not freeze. When data are available for several dilutions, there has been no discussion in the literature about  which dilution is best for the calculation. From our own experience we have noticed that the data are most robust if the log(number of cells/nucleus) is calculated for each dilution for which the calculation is possible and then the mean value is calculated over the whole range of dilutions used.  Any comments on this would be welcome.

Vali G (1971) Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J Atmos Sci 28: 402-409.

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