Evaluation of whole cell fixation methods for the analysis of nanoscale surface features of Yersinia pestis KIM

Abstract

Manipulation of viable Yersinia pestis (etiologic agent of plague) in the laboratory usually necessitates elevated biosafety and biocontainment procedures, even with avirulent or vaccine strains. To facilitate downstream biochemical or physical analyses in a Biosafety Level 1 laboratory environment, effective inactivation without affecting its intrinsic properties is critical. Here, we report on the morphological and biochemical changes to Y. pestis surfaces following four different fixation methods that render the cells nonviable. The results, obtained at the single cell level, demonstrate that methanol inactivation is best able to preserve bacterial morphology and bioactivity, enabling subsequent analysis. This nanoscale evaluation of the effects of inactivation on cell morphology and surface bioactivity may provide a crucial preparatory approach to study virulent pathogens in the lab setting using high-resolution microscopic techniques such as atomic force microscopy.

Nanoscale Phenotypic Textures of Yersinia pestis Across Environmentally-Relevant Matrices

“…In each case, after inoculation, cultures were grown at 30 • C for 48 h. in a stationary incubator. Following 48 h. growth, cells were harvested from plates using sterile cell scrapers and inoculation loops and inactivated with 100% MeOH, washed twice, then re-suspended in deionized water until further analysis [23]. Growth curves for Y. pestis KIM were generated in Tryptic Soy Broth (TSB) as well as four different SESOM formulations (i.e., liquid broths containing only soil-derived organic matter) which vary in organic carbon content, ranging between~3% and~10%.…”

“…All cells were inactivated using previously reported protocols using methanol, that was earlier shown by our group to not affect the cell surfaces [23]. This ensured that the cells were completely inactivated prior to imaging which was conducted outside of a biological safety cabinet.…”

“…However, to date, Y. pestis has not been well studied at the nanoscale. Our group earlier presented some of the first reports using nanoscale imaging and biochemical mapping at the single cell level, for Y. pestis cultured across standard laboratory media and across different temperatures [23,33].…”

A Stable and Sensitive Engineering Bacterial Sensor via Physical Biocontainment and Two-Stage Signal Amplification

“…Traditional physical biocontainment materials, such as agar and alginate, have improved the protection of bacteria. However, the low free diffusion efficiency of target pollutants will lead to a lower detection sensitivity and a longer response time, which seriously limits their performance in environmental analysis. − Therefore, to effectively prevent engineered bacteria from escaping, maintain cell growth activity in harsh water environments, and maximize the sensitivity of environmental analysis, it is necessary to design robust and signal amplification capabilities and physical biocontainment strategies.…”

Effects of fixation on bacterial cellular dimensions and integrity

“…Importantly, while cells are thought to remain stable after fixation for at least one week (Levin, 2002), the impacts of fixation on cell morphology and on fluorescence levels remain mostly unknown on both short and long timescales. Multiple studies reported decreased cell numbers and altered fluorescence histograms after storage of fixed marine bacteria (Kamiya et al, 2007;Troussellier et al, 1995), and another study found fixativedependent effects on Yersinia pestis cell morphology (Wang et al, 2016). Thus, it is critical to evaluate whether and how fixed cells change over time, especially given that certain labeling protocols (e.g.…”