Chang C W, Hwang Y H, Grinshpun S A, Macher J M, Willeke K
Department of Environmental Health, University of Cincinnati, Ohio 45267-0056.
Appl Environ Microbiol. 1994 Oct;60(10):3732-8. doi: 10.1128/aem.60.10.3732-3738.1994.
Colony counting error due to indistinguishable colony overlap (i.e., masking) was evaluated theoretically and experimentally. A theoretical model to predict colony masking was used to determine colony counting efficiency by Monte Carlo computer simulation of microorganism collection and development into CFU. The computer simulation was verified experimentally by collecting aerosolized Bacillus subtilis spores and examining micro- and macroscopic colonies. Colony counting efficiency decreased (i) with increasing density of collected culturable microorganisms, (ii) with increasing colony size, and (iii) with decreasing ability of an observation system to distinguish adjacent colonies as separate units. Counting efficiency for 2-mm colonies, at optimal resolution, decreased from 98 to 85% when colony density increased from 1 to 10 microorganisms cm-2, in contrast to an efficiency decrease from 90 to 45% for 5-mm colonies. No statistically significant difference (alpha = 0.05) between experimental and theoretical results was found when colony shape was used to estimate the number of individual colonies in a CFU. Experimental colony counts were 1.2 times simulation estimates when colony shape was not considered, because of nonuniformity of actual colony size and the better discrimination ability of the human eye relative to the model. Colony surface densities associated with high counting accuracy were compared with recommended upper plate count limits and found to depend on colony size and an observation system's ability to identify overlapped colonies. Correction factors were developed to estimate the actual number of collected microorganisms from observed colony counts.(ABSTRACT TRUNCATED AT 250 WORDS)
对因难以区分的菌落重叠（即掩盖）导致的菌落计数误差进行了理论和实验评估。使用一个预测菌落掩盖的理论模型，通过对微生物收集和发育为菌落形成单位（CFU）的蒙特卡罗计算机模拟来确定菌落计数效率。通过收集雾化的枯草芽孢杆菌孢子并检查微观和宏观菌落，对计算机模拟进行了实验验证。菌落计数效率降低的情况如下：（i）随着可培养微生物收集密度的增加；（ii）随着菌落尺寸的增加；（iii）随着观测系统将相邻菌落区分为独立单位的能力下降。在最佳分辨率下，当菌落密度从1个微生物/平方厘米增加到10个微生物/平方厘米时，2毫米菌落的计数效率从98%降至85%，相比之下，5毫米菌落的计数效率从90%降至45%。当使用菌落形状估计CFU中单个菌落的数量时，实验结果与理论结果之间未发现统计学显著差异（α = 0.05）。由于实际菌落大小不均匀以及人眼相对于模型具有更好的辨别能力，在不考虑菌落形状时，实验菌落计数是模拟估计值的1.2倍。将与高计数准确性相关的菌落表面密度与推荐的平板计数上限进行比较，发现其取决于菌落大小和观测系统识别重叠菌落的能力。开发了校正因子，以根据观察到的菌落计数估计实际收集的微生物数量。（摘要截断于250字）
