Omar A A, Newman H N
J Clin Periodontol. 1986 Oct;13(9):814-24. doi: 10.1111/j.1600-051x.1986.tb02236.x.
This study considers false results which may arise due to problems in the preparation or examination of specimens for darkground microscopy of subgingival plaque. Subgingival plaque samples obtained with a sterile curette were placed in 0.1-0.3 ml sterile full or 1/4 strength Ringer's solution: 0.85% saline, 1% gelatin in 0.85% saline, formal saline or pyrogen-free water for injection. Test slides were prepared from the original dispersion, and control slides from the corresponding sterile solution. Optimal dispersion solution, syringe dispersion frequency and the effect on motility of delay in processing samples were tested. Slides were also prepared from dispersions of 11 representative subgingival "periodontopathic" organisms. Problems in sampling included variability in counts between sites with comparable pocket depths, contamination of the sample and reduction of the sample volume after scaling. Problems in dispersion included contamination, uneven distribution of the different morphotypes and destruction of delicate organisms. Problems in slide preparation included slide contamination, limitation in the number of samples that can be assessed by one examiner at a given time without loss of activity of motile cells, and preparation of a cell monolayer. Problems in identification and counting included confusion of Brownian movements with motility, coccoid particles with cocci, spirochetes with campylobacter, flagella with flagella-like structures, size of cocci, counting of fragmented spirochetes and non-motile flagellated organisms and motile cells, and also bias in counting. Problems in morphotype grouping included the observation that many (10 of the 11 representative) periodontitis-related organisms were in the non-motile groups and not all cells of the motile species (Campylobacter, Capnocytophaga) showed motility. The results indicate that each stage of subgingival plaque darkground microscopy, sampling, dispersion, slide preparation, counting, morphotype grouping and interpretation may lead to false results if not representative or reproducible. Procedures are suggested for the minimisation of problems in the preparation and examination of subgingival plaque specimens for darkground microscopy.
本研究考虑了在龈下菌斑暗视野显微镜检查标本的制备或检查过程中可能出现的错误结果。用无菌刮匙获取的龈下菌斑样本置于0.1 - 0.3 ml无菌全强度或1/4强度的林格氏液中：0.85%生理盐水、0.85%生理盐水中含1%明胶、甲醛生理盐水或无热原注射用水。从原始悬液制备测试玻片，从相应的无菌溶液制备对照玻片。测试了最佳分散液、注射器分散频率以及样本处理延迟对活力的影响。还从11种代表性龈下“牙周病”微生物的悬液中制备了玻片。采样问题包括具有相似牙周袋深度的部位之间计数的变异性、样本污染以及洁治后样本体积的减少。分散问题包括污染、不同形态类型的分布不均以及脆弱微生物的破坏。玻片制备问题包括玻片污染、在不损失活动细胞活力的情况下，一名检查者在给定时间内可评估的样本数量有限以及细胞单层的制备。鉴定和计数问题包括将布朗运动与活力混淆、球菌样颗粒与球菌混淆、螺旋体与弯曲杆菌混淆、鞭毛与鞭毛样结构混淆、球菌大小、破碎螺旋体和无运动能力的鞭毛生物及活动细胞的计数，以及计数偏差。形态类型分组问题包括观察到许多（11种代表性微生物中的10种）与牙周炎相关的微生物属于无运动能力组，并且并非所有运动型物种（弯曲杆菌、二氧化碳嗜纤维菌）的细胞都显示出运动能力。结果表明，如果龈下菌斑暗视野显微镜检查的每个阶段，即采样、分散、玻片制备、计数、形态类型分组和解释不具有代表性或不可重复，都可能导致错误结果。建议采取相应程序以尽量减少龈下菌斑标本制备和检查过程中用于暗视野显微镜检查的问题。
