1. Cells are grown aerobically at 37° in Mcllvaine's citrate-phosphate buffer, pH 7 (containing per liter: 8.58 g Na2HPO4, 1.34 g citric acid,

4 Note that we have rigorously established that the surviving cells are not mutants that have been adapted to the loss of the channels, but represent physiological variants. There is no doubt that survival of hypoosmotic shock could be used to select for resistant mutants and that such an approach would be very informative. However, it would require a secondary screen to detect those few cells that are actually mutants among the many that survive.

0.87 g K2HPO4), supplemented with (per liter) 1 g NH4SO4, 0.1 g MgSO4, and 0.002 g (NH4)2SO4-FeSO4-6H2O (added from 100x concentrated stock dissolved in 0.1 M HCl), 0.001 g thiamine, and 2 g glucose (Levina et al., 1999). This medium has a final osmolarity of 220 mOsm.

2. A single colony is used to inoculate a 5-ml overnight culture in a 25-ml MacCartney in which the glucose concentration is lowered to 0.4 g/liter to limit growth to OD650 «0.4 after overnight growth. The next morning the glucose is increased to 0.2 g/liter, and the culture is returned to the shaking incubator. After a 1-h incubation to restore exponential growth the culture is diluted 10-fold into fresh growth medium in a 100-ml Erlenmeyer flask and grown to mid-exponential phase (OD650 «0.35 to 0.4). The culture is then diluted into fresh prewarmed growth medium supplemented with 0.5 M NaCl (to obtain faster growth one can lower the NaCl to 0.3 M) and incubated in the shaking incubator until OD650 «0.2 to 0.3.

3. Hypoosmotic shock is achieved by diluting the culture 20-fold into fresh prewarmed McIlvaines buffer containing supplements in the presence (control) or absence of 0.5 MNaCl (shock). Note that one can vary the NaCl concentration at this point to achieve a range of osmotic shocks.

4. To determine viability, 50-ml samples are taken at timed intervals and diluted serially in the same growth medium, and four 5-ml samples are spotted onto Luria broth plates in either the presence (control) or the absence of 0.5 MNaCl (shock). To allow for the greatest accuracy in the final count of colonies, the 5-ml spot is gently spread using the Gilson tip from which the sample is dispensed.

5. Plates are incubated at 37° overnight (shock) or for up to 36 h (control) and the colonies counted. On high salt medium growth is somewhat slower and to achieve accurate colony counts one must wait longer than for the test samples.

6. A useful modification of this assay protocol is the combined acid and osmotic shock (Levina et al., 1999; McLaggan et al., 2002). E. coli K-12 cells survive quite well when incubated at pH 3.6, but not at lower pH values. If a MS channel opens, however, the cytoplasm is flooded with low pH buffer, causing cell death through protein unfolding and so on. By combining the hypoosmotic shock with a shift to pH 3.6, one can obtain an indication of channel gating. Note that if the channel has shown no protective effect in the aforementioned protocol, one must limit the change in osmolarity to one that does not compromise cell integrity. McIlvaine's buffer can be adjusted to acid pH by varying the ratio of citric acid and phosphate. The technique is otherwise the same but the range of serial dilutions that must be assayed is greater because acid hypoosmotic shock kills > 99.99% of cells if the channel has fired but less than 1% if the channel is closed.

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