Evaluating the reliability of counting bacteria using epifluorescence microscopy

Thirumahal Muthukrishnan, Anesh Govender, Sergey Dobretsov, Raeid M.M. Abed

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7 Citations (Scopus)

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.

Original languageEnglish
Article number4
JournalJournal of Marine Science and Engineering
Volume5
Issue number1
DOIs
Publication statusPublished - Mar 1 2017

Fingerprint

field of view
microscopy
Bacteria
Microscopic examination
glass
Glass
bacterium
Biofilms
biofilm
bootstrapping

Keywords

  • Arithmetic mean
  • Bacteria
  • Bootstrapping
  • Count
  • Epifluorescence
  • Normal distribution
  • Random

ASJC Scopus subject areas

  • Civil and Structural Engineering
  • Ocean Engineering
  • Water Science and Technology

Cite this

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