Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU

Abstract

Aims: To elucidate the cause of high variations and inconsistencies in bacterial CFU observed within and between different experiments while assessing viable bacterial counts through spread plating (SP). Methods and Results: Following the inconsistent results, CFU estimations were undertaken through conventional SP using the spreader, or a modified approach that did not use spreader employing four organisms. The latter approach involving spotting‐and‐tilt‐spreading of inoculum on agar surface [spotting spreading (SS)] yielded higher CFU by 11–120% over the weighted average depending on the organism and diluent. The adverse effect owing to the spreader was the most obvious in Escherichia coli followed by Staphylococcus epidermidis, Enterobacter cloacae and Bacillus pumilus. Plate attributes that determined the surface moisture levels of agar medium and the spreading practice adopted by the personnel formed two other major influencing factors. Plating for shorter periods (<60 s) using fresh 15/20 ml plates caused loss of 3–12% CFU owing to inoculum adhesion to spreader irrespective of glass or polypropylene make. On the other hand, prolonging the plating brought down the CFU significantly. Spreader movement on agar surface subsequent to the exhaustion of free moisture, which was marked by the experiencing of some friction to smooth spreader movement, was detrimental to vegetative cells, while Bacillus spores were less affected. Conclusions: The study brings out that the way SP is carried out exerts significant effects on CFU influenced by plate conditions. Prolonged use of spreader on dry agar surface could be highly detrimental to bacterial cells. A mild use of spreader accounting for spreader‐adhering inoculum or the practice of SS not involving the spreader is recommended. Significance and Impact of the Study: This study unravels the effects owing to the spreader on bacterial cells and the CFU and recommends an alternate approach of SS to minimize CFU inconsistencies and to maximize the viable bacterial counts.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation

“…; Thomas et al . ) therefore, we speculate that the hydrophobic nature of Peptide MSal020417‐coated bead contributed to the MS + plate counts variation observed in this study.…”

Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms

“…Our recent investigations have brought out that the spreader could significantly alter the CFU during spread plating depending on the extent of its usage on agar surface in comparison with the alternate approach that did not involve the use of spreader (Thomas et al . ). While a brief plating operation (≤10 s) caused some inoculum adhesion to the spreader, prolonged plating imparted a significant reduction in CFU leading to variable results.…”

Effect of chlorine dioxide on decontamination of fresh‐cut coriander and identification of bacterial species in fresh‐cutting process

“…Coriander (25 g) was added in a conical flask containing 675 ml of sterile saline in a sterile environment. After being homogenized for 20 min, the solution was serially diluted (1:10) with sterile double‐distilled water, spotted on beef extract‐peptone medium (Thomas, Sekhar, & Mujawar, ) and incubated at (36 ± 1) °C for (48 ± 2) hr. Colony growth was observed till the fifth generation and the bacteria were purified by employing single colony isolation technique.…”