Clonal heterogeneity test

Heterogeneity in colony morphologies (colony phenotype switch) and frequency of petite mitochondrial mutants in packed products were assessed by plating samples directly after the first pre-culturing (as described above) onto YPD agar plates (for colony morphologies) and onto GlyYP (glycerol yeast extract peptone) + 0.1% glucose agar plates with cell densities of approx. 200/plate (after cell counting in a haemocytometer). Plates were incubated for 10 days at 30°C (with agar surface facing down) and were visually scored for various phenotypes on YPD (rough, wrinkled, sectored, stalk-like, and very small colonies) and for potential petite mutants on GlyYP. Presumed petites were transferred to YPD and after overnight culturing, were inoculated onto GlyYP plates without glucose. Subclones unable to grow on glucose-free GlyYP were scored as petites. Finally, YPD colonies were washed under tap water to determine the frequency of invasivitiy into agar. At least 1,000 subclone colonies were counted for each strain and for each assay.<br><br>Clonal heterogeneity ins tress tolerance was assessed by using the same stress conditions as in the spot plate assays, supplemented with assays on SD and YPD media. Freshly grown cells (as described for spot plates) were counted in a haemocytometer and spread to land about 200 cells/plate. Plates were incubated at 30°C for 2 days (YPD and SD media), or for 2, 3, 4, and 6 days (stress media) as colonies reached sizes that were visible but not yet close to each other. For each condition, three replicate plates were used for each sample. Photographs were taken 4 and 6 days after inoculation with a DSLR camera. Data on colony area was gathered by using the Fiji software package CountPHICS (Brzozowska et al., 2019) with circularity set to 0.8. Pixel to mm ratios were measured and area calculations were randomly verified by manual measurment in ImageJ for altogether ten colonies (Rueden et al., 2017). Plate photographs and all colony area values were uploaded to FigShare.

通过在首次预培养后直接将样品涂布于YPD琼脂平板（YPD agar plates，用于菌落形态分析）以及GlyYP（glycerol yeast extract peptone）+0.1%葡萄糖琼脂平板（细胞密度约为200个/平板，经血细胞计数板（haemocytometer）计数细胞后调整），对批量产品中的菌落形态异质性（菌落表型转换）以及小菌落线粒体突变体（petite mitochondrial mutants）频率进行评估。将平板倒置（琼脂面朝下）于30℃培养10天，随后对YPD平板上的多种表型（粗糙、褶皱、嵌合、柄状及极小菌落）以及GlyYP平板上的潜在小菌落线粒体突变体进行目视评分。将推定的小菌落突变体转接至YPD培养基，过夜培养后接种于无葡萄糖的GlyYP平板上，无法在无葡萄糖GlyYP平板上生长的亚克隆即判定为小菌落突变体。最后，将YPD平板上的菌落用自来水冲洗，以测定其琼脂侵袭频率。每个菌株的每项测定均需计数至少1000个亚克隆菌落。

克隆性胁迫耐受异质性的评估采用与点板实验相同的胁迫条件，并补充SD培养基与YPD培养基上的测定。将新鲜培养的细胞（参照点板实验的制备方法）经血细胞计数板计数后涂布，使每平板约接种200个细胞。将平板置于30℃培养：YPD与SD培养基培养2天，胁迫培养基则分别培养2、3、4及6天，直至菌落可见且尚未相互融合。每种条件下每个样品设置3个重复平板。分别于接种后4天和6天使用数码单反相机（DSLR）拍摄平板照片。菌落面积数据通过Fiji软件包中的CountPHICS工具（Brzozowska等，2019）获取，圆形度阈值设为0.8。同时测量像素与毫米的转换比例，并通过ImageJ手动测量共计10个菌落以随机验证面积计算结果（Rueden等，2017）。平板照片及所有菌落面积值均上传至FigShare平台。