Evaluation of Methods for Sampling, Recovery, and Enumeration of Bacteria Applied to the Phylloplane

Abstract

Determining the fate and survival of genetically engineered microorganisms released into the environment requires the development and application of accurate and practical methods of detection and enumeration. Several experiments were performed to examine quantitative recovery methods that are commonly used or that have potential applications. In these experiments, Erwinia herbicola and Enterobacter cloacae were applied in greenhouses to Blue Lake bush beans (Phaseolus vulgaris) and Cayuse oats (Avena sativa). Sampling indicated that the variance in bacterial counts among leaves increased over time and that this increase caused an overestimation of the mean population size by bulk leaf samples relative to single leaf samples. An increase in the number of leaves in a bulk sample, above a minimum number, did not significantly reduce the variance between samples. Experiments evaluating recovery methods demonstrated that recovery of bacteria from leaves was significantly better with stomacher blending, than with blending, sonication, or washing and that the recovery efficiency was constant over a range of sample inoculum densities. Delayed processing of leaf samples, by storage in a freezer, did not significantly lower survival and recovery of microorganisms when storage was short term and leaves were not stored in buffer. The drop plate technique for enumeration of bacteria did not significantly differ from the spread plate method. Results of these sampling, recovery, and enumeration experiments indicate a need for increased development and standardization of methods used by researchers as there are significant differences among, and also important limitations to, some of the methods used.

Short-term community dynamics in the phyllosphere microbiology of field-grown sugar beet

“…This reflects the highly selective nature of the phyllosphere: often only a few species dominate at any one time and distinctive populations occur at specific times of the year over several seasons . The community analysis and the quantitative data, support reports that bulking individual leaves reduces the degree of variance between samples [5,21]. Because replicate samples were not taken within quadrats, only limited conclusions can be drawn about the spatial variation in phyllosphere microbiology of plants taken from different locations in the field.…”

Toxin YafQ increases persister cell formation by reducing indole signalling

“…To determine the number of persister cells from producing YafQ, overnight cultures were diluted to a turbidity of 0.05 and grown in LB medium with chloramphenicol (30 μg ml −1 ) to a turbidity of 1.0, then 1 mM IPTG was used to induce yafQ. After 2 h, cells were washed, adjusted to a turbidity of 1.0 in LB and were exposed to 100 μg ml −1 ampicillin or 5 μg ml −1 ciprofloxacin with 1 mM IPTG for 3 h. Cells were washed and diluted by 10 2 to 10 7 via 10-fold serial dilution steps in 0.85% NaCl solution and applied as 10 μl drops on LB agar to determine cell viability (Donegan et al, 1991). For BW25113 ΔtnaA, cells were grown to a turbidity of ∼ 0.6 or ∼ 2.0 at 600 nm, and indole was added at 0, 0.5, 1 and 2 mM (from 500 mM stock; dimethylformamide was used as negative control).…”

Autecology of a genetically modified fluorescent pseudomonad on sugar beet

“…Pseudomonads are believed to be less susceptible to reverting to a non-culturable state than other genera, particularly in a habitat such as the phyllosphere, which is susceptible to continual environmental fluctuations [34,35]. Such fluctuations may also account for the large variation in propagules numbers detected in replicate samples, a common feature encountered in phyllosphere studies [36]. In this study the variation between samples was particularly large, certainly when compared to wheat [23].…”