Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining

Abstract

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.

Keywords: Fluorescent microscopy; Phosphotungstic acid; Plate count; Scanning electron microscopy; Viability.

Graphical abstract

Fig. 1

Proof-of-concept workflow for bacterial viability assay using SEM and PTA.

Fig. 2

A. Polyethylene terephthalate glycol (PETG) 3D printed slide developed in-house for Energy Dispersive X-Ray (EDX) analysis - eliminating the overlapping Kα peak of Silicon Si with the M peak for Tungsten W. B. Stitched images of blank slide imaged using SEM.

Fig. 3

Proof-of-concept on E. coli. Pure fresh cultures were mixed with dead bacteria (heat shocked at 90 °C) and imaged with and without PTA staining. PTA staining revealed a clear dark and bright contrast among bacteria, which was clearly absent in the unstained bacteria. Dark contrast referring to PTA localization around the live bacterial cell. (Green Circles) Bright contrast referring to PTA penetration into the dead bacterial cell. (Red Circles).

Fig. 4

Contrast analysis and different morphological profiles detected based on the internal contrast as observed on the micrographs and validated by the line pixel profile of the bacterium generated using image-J.

Fig. 5

Proof-of-concept by Viability Count. A. The viability count performed using scanning electron microscopy. B. The viability count performed using fluorescence microscopy. C. The viability count performed using the plate count method. D. Statistical comparison (Mann-Whitney test (two-tailed t-test)) of the bacterial viability using SEM, FM and CFU methods; demonstrating non-significant difference between the three methods. All experiments were realized in triplicate. Refer to Supplementary Figure 5 for SEM micrographs.

Fig. 6

E. coli live/dead artificial mixtures, assessed by fluorescence microscopy. Right panel: Green fluorescence for SYTO9 staining all bacteria. Middle panel: Red fluorescence for PI staining dead bacteria only. Left panel: Merged channels. Scale bars: 20 µm. Refer to Supplementary Figure 6.

Fig. 7

A. Micrographs of PTA-stained Akkermansia muciniphila after one-hour non-exposure or exposure to oxygen in Mueller Hinton Broth (MHB) and in protectant medium (PM). Scale bars: 10 µm. Black arrows: live dark bacteria. White arrows: dead bright bacteria. B. The viability count performed by scanning electron microscopy. C. The viability count performed by the plate count method. D. Statistical comparison (Mann-Whitney test (two-tailed t-test)) of the bacterial viability using SEM (blue) and plate count (pink) methods. All experiments were realized in triplicate.

Fig. 8

A-B. Histograms comparing (Mann-Whitney test (two-tailed t-test)) SEM bacterial viability count to the plate count method in Protectant medium and MHB after freezing and freeze-drying. Experiments were realized in triplicate. Refer to Supplementary Figure 7 for SEM micrographs.

Fig. 9

SEM-PTA viability assay applied to fresh E. coli (upper panel), E. coli subjected to killing by ethanol (middle panel), and a mixture of both (lower panel). Micrographs show not stained and PTA-stained bacteria. Acquisition settings are visible on each micrograph in the following format: Instrument, Accelerating Voltage, Working Distance, Magnification, and Detector.

Fig. 10

SEM-PTA viability assay applied to fresh E. coli (upper panel), E. coli subjected to killing by imipenem at 0.5 mg/L (middle panel), and E. coli subjected to killing by imipenem at 5 mg/L (lower panel). Micrographs show PTA-stained bacteria at different time points (0 and 120 min) with clear distinction of live (green circles), dead (red circles) and total lysed/disintegrated bacteria (yellow circles). Acquisition settings are visible on each micrograph in the following format: Instrument, Accelerating Voltage, Working Distance, Magnification, and Detector. Refer to Supplementary Figure 8 for detailed SEM micrographs at all time-points.

Fig. 11

Proof of PTA Stain Localization using Energy-dispersive X-ray spectroscopy analysis. EDX visual representation of PTA localization according to viability status of PTA-stained bacterial cells. Hypo-dense (dark) bacterium, considered live, shows a clear external contour signal of Tungsten, as compared to the hyper-dense (bright) bacterium, considered dead, showing a complete overlap of Tungsten and Carbon indicating the presence of tungsten within the bacterium.