Table 1.
Preservation conditions with corresponding abbreviations (left) and MOB type strains with corresponding species name, type of MOB and standard cultivation conditions (right).
Figure 1.
Effect of long-term preservation on MOB viability and culturability after 3, 6 and 12 months.
The average viable (black triangle) and culturable (black square) log transformed cells per mL of all tested strains in all preservation conditions was plotted over time: before preservation & 3, 6 and 12 months post-preservation. For both the viable as well as the culturable counts, counts were significantly lower after preservation (p-value<0.05), while the 3 tested time points post-preservation were similar compared to one another (p-value>0.05).
Table 2.
The percentage of strains that were culturable or viable per preservation condition (left) and the percentage of preservation conditions for which culturability or viability was achieved per strain (right) after 12 months of preservation.
Table 3.
Ranking and separation into homogenous subsets using Scheffé’s test of tested preservation conditions based on average log transformed culturability data.
Figure 2.
Average viable (dark grey bars) and culturable (light grey bars) log drops of all tested strains plotted for each preservation condition.
Log drops were obtained by subtraction of 12 month post-preservation counts by pre-preservation counts using log transformed data. Following preservation, the drop in culturability is mostly more extensive (larger bars) than the drop in viability indicating that a vast number of cells became viable-but-non-culturable (VBNC) after the preservation process (arrow indicating VBNC as difference between viable and culturable log drop for condition LPA-GB). Bars that are not visible indicate that no drop was observed after preservation (e.g. DMSO/TT for both viable and culturable log drop counts). The use of TT medium before, during and after preservation reduced the amount of VBNC cells significantly (p-value<0.05; e.g. T/HS/TT compared with T/HS) in a comparison between the four conditions using TT with the four corresponding conditions in standard medium. When the viability drops, the culturability also drops but to a higher extent, it may indicate that FCM can be used to partially predict the success of subsequent cultivation attempts following long-term preservation.
Figure 3.
Live-dead staining of Methylocystis parvus NCIMB 11129T prior to preservation (A) and preserved for 12 months using LPA-GB (B), glycerol (C) and DMSO/TT (D) measured by flow cytometry.
Green fluorescence (FL1 detector) was plotted against red fluorescence (FL3 detector). Cells emitting more green than red were considered viable (V), cells emitting more red than green were considered non-viable (NV). Cytocount beads (Be) were added to calculate the amount of viable cells per mL. These types of counts were encircled for indicative purposes only. Background counts found at the bottom left are not shown for clarity. Since obtained non-viable counts were expected to be underestimated (detritus of cells causing increase in background), these were not used in actual calculations. Before preservation (A) the majority of cells were viable. However, lyophilization of NCIMB 11129T with LPA-GB (B) killed the majority of cells (shift from viable to non-viable), which corresponded with an MPN count below detection limit (Table S1). In contrast, a drop in viability could not be observed between pre-preservation of NCIMB 11129T and post-preservation using glycerol (C) and DMSO/TT (D). Interestingly, despite their similar flow cytometric counts and profiles, the culturable fraction was a lot higher after preserving with DMSO/TT (MPN log drop 0.7; Table S1) than with glycerol (MPN log drop 2.6; Table S1) indicating that the VBNC fraction was limited in the former and extensive in the latter condition.