Solution I 0.01M Na2HPO4, 0.15M NaCl, pH 7.0 Solution II O.5% (W/V) lysozyme in Solution I
1. Centrifuge the culture in a autoclaved 50ml Corning Orange Cap tube in the International centrifuge at 2,500 rpm for 15 min.
2. Transfer the pellet to a 1.5ml microtube and microfuge for 10 min.
3. Pour off the supernatant, and dry the pellet with the twisted corner of a Kimwipe. Freeze the pellet at -20deg C.
4. Thaw and resuspend in 1.25ml of Solution I.
5. Add 150ul of 0.5% lysozyme (Solution II). Vortex.
6. Incubate at 37deg C for 2h with reciprocal shaking in a water bath.
7. Stand the tubes O/N at 4deg C.
8. Mix contents of tube by briefly vortexing.
9. Spin in the microfuge for 30 min.
10. Remove 1ml from the supernatant with a P1000 Pipetman. Avoid contamination with the pellet. Transfer this 1ml to a tube of at least 2ml capacity.
11. Bring to 2ml by adding 1ml of Solution I.
12. Scan solution from 750-500 nm using the Perkin Elmer 330 Spectrophotometer. Dilute if necessary using Solution I.
5 (alt). Use only 30 ul of lyzozyme solution.
10 (alt). Remove only 200 ul of the supernatant, and scan in the black-walled microcells with 0.5 cm path length.
BENNETT, A., and BOGORAD, L. (1973). Complementary Chromatic Adaptaption in a Filamentous Blue-Green Alga. J. Cell Biol. 58, 419-435