A quick epifluorescence microscopy method for sediment bacteria enumeration

Bacterial abundance is an important parameter in assessing the roles of bacteria in sediment. Direct enumeration by epifluorescence microscopy is widely accepted as a standard method for counting planktonic bacteria. Due to particle masking effects and variable extraction efficiencies; however, this method is far from satisfactory for counting sediment bacteria. In this study, a modified epifluorescence enumeration method was developed to count sediment bacteria; design goals were to improve the recovery efficiency of bacteria and decrease masking effects of sediment particles. Briefly, sediment bacteria were detached, diluted with dispersing buffer, stained with SYBR and enumerated using a hemocytometer under an epifluorescence microscope. To test the recovery efficiency of the hemocytometer method, SYBR I-stained E. coli DH5α cultures were counted using the standard epifluorescence microscope method with an Anodisc filter or mixed with a sediment suspension and counted with the hemocytometer under epifluorescence microscope. The DH5α culture counts acquired using the two methods did not differ significantly, which indicates that the bacterial recovery efficiency was satisfactory in the hemocytometer method. Particle masking effects and counting accuracy were also tested using serially diluted sediment samples. Less than 200 times dilution resulted in masking effect and rendered the enumeration difficult. Masking effects were negligible at dilutions over 200 times, but the counting result was highly variable as dilution reaching 1000 times. Thus, more than 24 bacteria per counting square and over 200 times dilution were suitable to avoid masking effects and obtain relatively precise counting results in the hemocytometer method. In conclusion, the hemocytometer method is a useful supplement to epifluorescence microscopy methods and will facilitate enumeration of sediment bacteria.