肺炎链球菌荚膜教学标本的制作方法研究(1)
[摘要]目的 探索并優化肺炎链球菌荚膜教学标本片的制作条件和染色过程，以期找到适合我校荚膜教学标本片需求的制作和保存方法，提升微生物实验课堂的教学质量。方法 将肺炎链球菌传代活化后制成悬液，以腹腔注射法(0.2 ml悬液)感染10只昆明小鼠并传代培养，提取发病待濒死小鼠的腹腔液涂片，分别使用石炭酸复红染色法和改良Hiss荚膜染色法对其进行染色，从菌体形态、数量、荚膜大小、染色效果等方面综合比较两种染色方法的差异。最后将镜检合格的标本用中性树胶封固，贴上标签后保存备用。于2018年12月，抽查我校2017年12月使用改良Hiss荚膜染色法所染制的35片标本片，评估标本片的质量。结果 随着传代的进行，受染小鼠体内的肺炎链球菌增多，荚膜增大。传代两次即可见宽大的荚膜，且第二次传代感染实验小鼠的发病时间明显缩短，两次感染发病时间分别为10～12 h和8～10 h。同传统的石炭酸复红染色法比较，改良Hiss荚膜染色法所染制的标本，背景均匀，染料颗粒少，菌体呈现深紫色，同淡紫色背景对比明显。同时，抽查已保存1年的改良Hiss荚膜染色法所染制的标本共35片，抽查结果表明，改良Hiss荚膜染色法制作、保存的标本荚膜仍清晰可见，无褪色和霉变现象，标本片完好率达到100%。结论 在实际教学中，小鼠体内传代两次即可观察到明显的荚膜，改良Hiss荚膜染色法的染色效果优于传统的石炭酸复红染色法，且封片保存的标本可反复多次使用，能满足我校教学需要。
[关键词]肺炎链球菌;荚膜;石炭酸复红染色;改良Hiss荚膜染色法;实验教学
[中图分类号] R378.12 [文献标识码] A [文章编号] 1674-4721(2019)12(c)-0025-04
[Abstract] Objective To explore and optimize the preparation conditions and dyeing process of making streptococcus pneumoniae capsule teaching specimens， in order to find the most suitable preparation and preservation methods for the requirements of making and preserving capsule teaching specimens of our school， so as to eventually improve the quality of experimental teaching class in Microbiology. Methods To make the suspension of streptococcus pneumoniae followed the process of subculture and activation， ten Kunming mice were infected by intraperitoneal injection (0.2 ml suspension) and subcultured. Peritoneal fluid of mice were extracted before the mice dying for smear， and carbonic acid complex red staining and modified Hiss capsule staining were used to stain the capsules， respectively. The differences between the two staining methods were compared between the carbonic acid complex red staining and modified Hiss capsule staining in terms of morphology， quantity， capsule size and dyeing effect. Finally， the qualified specimens were sealed with neutral gum， labeled and stored for future use. In December 2018， 35 specimens dyed by the modified Hiss capsule staining method in December 2017， were sampled to evaluate the quality of the specimens. Results With the passage， the number of streptococcus pneumoniae increasing， the size of capsule become enlarged. And the wide capsule could be seen in only two passages， meanwhile， the infection onset time in the second was obviously shorter than that in the first one. The infection onset time of the two infections passages was 10～12 h and 8～10 h， respectively. Compared with the carbonic acid complex red staining method， the modified Hiss capsule staining method showed that the specimen had a uniform background， fewer dye particles， dark purple cells， and obvious contrast of capsule with light purple background. At the same time， a total of 35 samples were dyed by the modified Hiss capsular staining method， which had been preserved for 1 year. The results showed that the capsule of the specimens preserved by the modified Hiss capsular staining method was still clearly visible， without fading or mildeutriation， and the percentage of specimens in good condition was highly up to 100%. Conclusion During the practical teaching， the obvious capsule can be observed in mice with only two passages and the dyeing effect of the modified Hiss capsule staining method has obviously advantages than the carbonic acid complex red staining method. And the sealed specimen can be reused many times. All of these merits can completely meet the need of experimental teaching in our college., 百拇医药(王燕 曾燏 张富斌)
[关键词]肺炎链球菌;荚膜;石炭酸复红染色;改良Hiss荚膜染色法;实验教学
[中图分类号] R378.12 [文献标识码] A [文章编号] 1674-4721(2019)12(c)-0025-04
[Abstract] Objective To explore and optimize the preparation conditions and dyeing process of making streptococcus pneumoniae capsule teaching specimens， in order to find the most suitable preparation and preservation methods for the requirements of making and preserving capsule teaching specimens of our school， so as to eventually improve the quality of experimental teaching class in Microbiology. Methods To make the suspension of streptococcus pneumoniae followed the process of subculture and activation， ten Kunming mice were infected by intraperitoneal injection (0.2 ml suspension) and subcultured. Peritoneal fluid of mice were extracted before the mice dying for smear， and carbonic acid complex red staining and modified Hiss capsule staining were used to stain the capsules， respectively. The differences between the two staining methods were compared between the carbonic acid complex red staining and modified Hiss capsule staining in terms of morphology， quantity， capsule size and dyeing effect. Finally， the qualified specimens were sealed with neutral gum， labeled and stored for future use. In December 2018， 35 specimens dyed by the modified Hiss capsule staining method in December 2017， were sampled to evaluate the quality of the specimens. Results With the passage， the number of streptococcus pneumoniae increasing， the size of capsule become enlarged. And the wide capsule could be seen in only two passages， meanwhile， the infection onset time in the second was obviously shorter than that in the first one. The infection onset time of the two infections passages was 10～12 h and 8～10 h， respectively. Compared with the carbonic acid complex red staining method， the modified Hiss capsule staining method showed that the specimen had a uniform background， fewer dye particles， dark purple cells， and obvious contrast of capsule with light purple background. At the same time， a total of 35 samples were dyed by the modified Hiss capsular staining method， which had been preserved for 1 year. The results showed that the capsule of the specimens preserved by the modified Hiss capsular staining method was still clearly visible， without fading or mildeutriation， and the percentage of specimens in good condition was highly up to 100%. Conclusion During the practical teaching， the obvious capsule can be observed in mice with only two passages and the dyeing effect of the modified Hiss capsule staining method has obviously advantages than the carbonic acid complex red staining method. And the sealed specimen can be reused many times. All of these merits can completely meet the need of experimental teaching in our college., 百拇医药(王燕 曾燏 张富斌)