Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

Abstract

Abstract:The common practice of counting bacteria using epifluorescence microscopy involves selecting 5-30 random fields of view on a glass slide to calculate the arithmetic mean which is then used to estimate the total bacterial abundance. However, not much is known about the accuracy of the arithmetic mean when it is calculated by selecting random fields of view and its effect on the overall abundance. The aim of this study is to evaluate the accuracy and reliability of the arithmetic mean by estimating total bacterial abundance and to calculate its variance using a bootstrapping technique. Three fixed suspensions obtained from a three-week-old marine biofilm were stained and dispersed on glass slides. Bacterial cells were counted from a total of 13,924 fields of view on each slide. Total bacterial count data obtained were used for calculating the arithmetic mean and associated variance and bias for sample field sizes of 5, 10, 15, 20, 25, 30, 35 and 40. The study revealed a non-uniform distribution of bacterial cells on the glass slide. A minimum of 20 random fields of view or a minimum of 350 bacterial cells need to be counted to obtain a reliable value of the arithmetic mean to estimate the total bacterial abundance for a marine biofilm sample dispersed on a glass slide.

<em>In Situ</em> Filtration Rates of Blue Mussels (<em>Mytilus edulis</em>) Measured by an Open-Top Chamber Method

“…Samples were filtered through a 0.2 µm black polycarbonate sheet filter (Whatman Nuclepore Track Etch Membrane) and subse-quently stained for at least 4 min with 100 µl 4'-6'-diamidino-2-phenylidole (DAPI). When used in combination with epifluorescence microscopy (Leica, type 020-505.030) and UV excitation (Leica Hg-lamp Osram HBO 50 W L2; filter cube A: excitation filter: BP 340 -380 nm, emission filter: LP 425 nm) individual cells were identified (1000-times magnification), and > 250 bacteria were counted each time according to Muthukrishnan et al [20].…”

Use of Ratiometric Probes with a Spectrofluorometer for Bacterial Viability Measurement

“…DAPI is widely used to stain DNA for quantification and fluorescent microscopy observation [26,27]. Firstly, the limit of detection of the microvolume spectrofluorometer was determined by bacterial enumeration with DAPI.…”

Glacier ice archives fifteen-thousand-year-old viruses

“…The filter was mounted on a glass slide with freshly-made anti-fade solution (1 mg ascorbic acid : 100 μl PBS : 100 μl glycerol) and a 25 mm 2 cover slip. Cells on the filter were counted using epifluorescence microscopy (Zeiss Axio Imager.D2) with >350 cells or >20 fields counted, which was a reliable threshold to estimate the total bacterial abundance (76).…”