Fig. 2

HCR-FISH signal intensity of a Gramella forsetii pure culture, fixed with formaldehyde (1%) or ethanol, as measured by microscopy. FISH with probes containing 4 fluorochromes was compared to two different HCR-FISH protocols (prior to and after optimization) and CARD-FISH. The optimized HCR-FISH protocol includes 30 min denaturation at 65°C prior to 2 h of hybridization with hybridization buffer B and 120 min of amplification. The original protocol did not include a denaturation step and 2 h of hybridization with hybridization buffer A were followed by 45 min of amplification