松材线虫脂滴4种染色方法的比较

  目的  从4种脂滴染色方法中筛选最适合松材线虫Bursaphelenchus xylophilus的脂滴染色方法。  方法  采用苏丹黑B染色、尼罗红染色、油红O染色和后置油红O染色等4种染色方法来固定和表征松材线虫体内脂滴的分布,对染色后的线虫进行显微观察和拍摄,并用ImageJ软件对皮下和肠道内染色的脂滴像素进行统计。  结果  4种脂滴染色方法对松材线虫的脂滴都有一定的染色效果。显微拍照后观察脂滴与ImageJ软件图像处理后像素强度比较结果显示:苏丹黑B染色脂滴像素为200.00×1017 m2,尼罗红染色脂滴像素41.64×1012 m2,油红O染色脂滴像素52.12×1017 m2,后置油红O染色脂滴像素83.85×1017 m2。苏丹黑B染色脂滴平均像素强度最高,尼罗红染色脂滴平均像素强度最低,而后置油红O法染色脂滴经计算转换产生了与原始图像一致的表征,实现单个脂滴的良好分离。  结论  综合染色方法的简易、染色时间的长短和染色后的效果,改良后的后置油红O法是最佳的松材线虫脂滴染色方法,能清晰显示脂滴大小及分布。图4表1参17

Comparison of four lipid droplets staining methods in Bursaphelenchus xylophilus

  Objective  With a comparison of the four commonly used methods for staining Caenorhabditis elegans lipid droplets, this study is aimed to figure out the most suitable dyeing method for Bursaphelenchus xylophilus lipid droplets.  Method  Four were used to fix and characterize After the distribution of lipid droplets in B. xylophilus was defined with its features revealed employ four dyeing methods including Sudan black B staining, Nile red staining, oil red O staining, and post-fix oil red O staining, the stained nematodes were observed and photographed microscopically, and then ImageJ software was used to count the subcutaneous and intestinal lipid droplet pixels.  Result  1) The four dyeing methods have a certain dyeing effect on the lipid droplets of B. xylophilus. 2) Upon the observation of the lipid droplets after microphotographing and the comparison of the pixel intensity after image processing by ImageJ software, the stained lipid droplets pixels with Sudan black B, Nile red, oil red O and post-fix oil red O are 200×1017 m2, 41.64×1012 m2, 52.12×1017 m2, and 83.85×1017 m2 respectively. 3) Lipid droplets stained by Sudan black have the highest average pixel intensity, and lipid droplets stained by Nile red have the lowest average pixel intensity, whereas the lipid droplets stained by post-fix oil red O, after calculation and conversion, produced a consistent characterization with the original image, with a favorable separation of individual lipid droplet.  Conclusion  In conclusion, in light of the simplicity of the dyeing method, the length of dyeing time and the effect of dyeing, the improved post-fix oil red O method is the optimal method for dyeing lipid droplets in B. xylophilus, with the size and distribution of lipid droplets clearly shown. [Ch, 4 fig. 1 tab. 17 ref.]