Comparative techniques to quantify cyanobacteria dominated epilithic biofilms on tropical rocky shores

Abstract

The epilithic biofilm of tropical shores is dominated by cyanobacteria with only a sparse cover of diatoms. Geographical comparison of biofilms is difficult, however, due to variation in enumeration techniques. This paper describes techniques to quantify the biofilm in an attempt to standardize comparative studies. Species richness and relative abundance were best enumerated using scanning electron microscope (SEM) images of air dried rock chips, as compared to light, epifluorescence and confocal laser microscopy. SBM preparations provided clear images and allowed identification of most species. Air dried and Cryo-stage preparations provided the least damaged images of species whereas the harsh dehydration process in critical-point dried specimens damaged morphology and removed loosely attached species resulting in underestimation of species richness and abundance. A combination of SEM techniques (e.g. Cryo-stage, air dried and critical-point dried samples) is recommended for initial species identification. Chlorophyll a extraction from rock chips, as an indirect estimate of biomass, was more efficient using methanol or ethanol as compared to acetone. Methanol heated for 2 min and cooled for 12 h yielded 100% chlorophyll a extraction while cold methanol, methanol heated for 2 min and cooled for 3 and 6 h, and ethanol heated for 5 min, extracted greater than 95% chlorophyll a. No significant loss of chlorophyll a was recorded in rock samples stored in moist or dry conditions at 4°C or room temperature for 24 h or at -20°C for a month, but in samples stored at 4°C or room temperature for a week, 60 to 75% loss occurred. Chlorophyll a, once extracted, can be stored for a week in the dark prior to measurement without significant loss. To standardize techniques for tropical, cyanobacteria-rich epilithic biofilms, chlorophyll a extraction of rock chips using cold methanol is recommended.