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Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms
Author(s) -
Thomas P.,
Mujawar M.M.,
Sekhar A.C.,
Upreti R.
Publication year - 2014
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12412
Subject(s) - microbiology and biotechnology , microbacterium , pantoea , biology , chocolate agar , bacillus pumilus , agar plate , paenibacillus , enterobacter , bacteria , staphylococcus epidermidis , pseudomonas , staphylococcus aureus , escherichia coli , biochemistry , genetics , 16s ribosomal rna , gene
Aims To understand the factors that contribute to the variations in colony‐forming units ( CFU ) in different bacteria during spread plating. Methods and Results Employing a mix culture of vegetative cells of ten organisms varying in cell characteristics (Gram reaction, cell shape and cell size), spread plating to the extent of just drying the agar surface (50–60 s) was tested in comparison with the alternate spotting‐and‐tilt‐spreading ( SATS ) approach where 100 μ l inoculum was distributed by mere tilting of plate after spotting as 20–25 microdrops. The former imparted a significant reduction in CFU by 20% over the spreader‐independent SATS approach. Extending the testing to single organisms, Gram‐negative proteobacteria with relatively larger cells ( E scherichia, E nterobacter, A grobacterium, R alstonia, P antoea, P seudomonas and S phingomonas spp.) showed significant CFU reduction with spread plating except for slow‐growing M ethylobacterium sp., while those with small rods ( X enophilus sp.) and cocci ( A cinetobacter sp.) were less affected. Among Gram‐positive nonspore formers, S taphylococcus epidermidis showed significant CFU reduction while S taphylococcus haemolyticus and actinobacteria ( M icrobacterium, C ellulosimicrobium and B rachybacterium spp.) with small rods/cocci were unaffected. Vegetative cells of B acillus pumilus and B . subtilis were generally unaffected while others with larger rods ( B . thuringiensis, B revibacillus, L ysinibacillus and P aenibacillus spp.) were significantly affected. A simulated plating study coupled with live–dead bacterial staining endorsed the chances of cell disruption with spreader impaction in afflicted organisms. Conclusions Significant reduction in CFU could occur during spread plating due to physical impaction injury to bacterial cells depending on the spreader usage and the variable effects on different organisms are determined by Gram reaction, cell size and cell shape. The inoculum spreader could impart physical disruption of vegetative cells against a hard surface. Significance and Impact of Study Possibility of CFU reduction in sensitive organisms and the skewed selection of hardier organisms during spread plating, and the recommendation of SATS as an easier and safer alternative for CFU enumerations.