TABLE 1.

Summary of steps for FISH and CARD of marine bacteria

StageStep no.Descriptiona
Embedding1Prepare subsamples on membrane filters (37).
2Dip filters in 0.2% low-gelling-point agarose; place filters face up onto glass slides and air dry at 35°C.
3Dehydrate filters in 96% ethanol (1 min, RT).
4Air dry filters.b
Permeabilization and inactivation of peroxidases5 6 7Incubate in lysozyme (37°C, >30 min). Sediment samples: incubate them in 0.1% active diethyl pyrocarbonate in PBS (37°C, overnight). Wash filters twice in MQ (1 min, RT).
8Wash filters in 96% ethanol (1 min, RT).
9Air dry filters.b
Hybridization10Cut filters into sections.
11Place sections in reaction vial (0.5 ml, 10 to 20 sections per vial).
12Mix 400 μl of hybridization buffer and 4 μl of probe working solution, and add to filter sections.
13Incubate filters at 35°C for at least 2 h.
14Wash filters in prewarmed washing buffer (10 min, 37°C); do not air dry filter sections after washing them.
Tyramide signal amplification15 16Remove excess liquid with blotting paper but do not let the filters run dry. Incubate filters in 1× PBS amended with 0.05% of Triton X-100 (50 ml, RT, 15 min, mild agitation).
17Dab filters on blotting paper but do not let them run dry.
18Incubate filters in substrate mix (1 part Cy3-tyramide, 10 parts of amplification diluent) (RT, 10 min, in the dark).
19Dab filter on blotting paper.
20Wash filter for 15 min as described in step 16.
21Wash filter in 10 ml of MQ (RT, 1 min).
22Wash filter in 10 ml of 96% ethanol (RT, 1 min).
23Air dry preparations.b
24Counterstain filters with DAPI.b
  • a RT, room temperature.

  • b Preparations may be stored at −20°C for several days to weeks without an apparent loss in signal.