Öncel Koray
Mehmet Akif İnan Training and Research Hospital, Microbiology Laboratory, Şanlıurfa, Turkey.
Mikrobiyol Bul. 2019 Oct;53(4):419-433. doi: 10.5578/mb.68633.
Aim of the present study was to identify protozoones which are difficult to define through wet slide in fresh fecal samples by using different fixatives with modified Trichrome stain within five minutes. Two different fixatives prepared for the alternative approach. The slides were fixed by two different fixatives, one of them (fixative-1) was based ethylalcohol, formalin, acetic acid, distilled water and the other one (fixative-2) based ethylalcohol, formalin, citric acid, distilled water included a mordant [divalent or polyvalent metals which make coordination complex with some dyes] consisted copper sulphate pentahydrate (CuSO4 .5H2 O). Slides prepared by the two different fixatives were stained by a different modification of Gomori's trichrome stain that we made. Samples fixed by Schaudinn fixative including mercury chloride were stained by Wheatley modification of Gomori's trichrome stain as a gold standard for control and comparison. We worked with 50 fecal samples which we thought included human intestinal protozoones after the wet slide examination. Comparing the methods, slides prepared with the method including citric acid gave almost similar results with the classical method excluded Entamoeba coli cystes. Slides prepared with the methode including acetic acid gave low performance compared with the classical method especially E.coli cystes and Blastocystis spp., Endolimax nana, Iodamoeba bütschlii, E.hartmanni. Both new fixatives gave superior performance at the slides included Dientamoeba fragilis and approximately shorten the procedure process ten times than the classical method. When the both alternative methods compared in each other, the slides prepared with fixative-2 exposed better performance for the protozoones Blastocystis spp., E.nana, I.bütschlii and E.hartmanni while the fixative-1 displayed minimal superiority for D.fragilis including criterias that we based. The fixative-2 and modified stain methode that we used in our study, makes available the diagnostic phase ten times faster than the classical method in human stool parasitological tests excluding the E.coli cystes at parasitology and microbiolgy laboratories. It seems to be a good option to the classical method for routine usage.
本研究的目的是通过使用不同的固定剂和改良的三色染色法,在五分钟内识别新鲜粪便样本中难以通过湿片法定义的原生动物。为替代方法制备了两种不同的固定剂。玻片用两种不同的固定剂固定,其中一种(固定剂-1)以乙醇、福尔马林、乙酸、蒸馏水为基础,另一种(固定剂-2)以乙醇、福尔马林、柠檬酸、蒸馏水为基础,包含一种媒染剂[与某些染料形成配位络合物的二价或多价金属],即五水硫酸铜(CuSO4·5H2O)。用这两种不同固定剂制备的玻片用我们改良的Gomori三色染色法进行染色。用含氯化汞的Schaudinn固定剂固定的样本用Gomori三色染色法的Wheatley改良法进行染色,作为对照和比较的金标准。我们对50份粪便样本进行了研究,这些样本在湿片检查后被认为含有人体肠道原生动物。比较这些方法,用含柠檬酸的方法制备的玻片与经典方法得到的结果几乎相似,但不包括结肠内阿米巴包囊。用含乙酸的方法制备的玻片与经典方法相比性能较差,尤其是对于大肠杆菌包囊、芽囊原虫属、微小内蜒阿米巴、布氏嗜碘阿米巴、哈氏内阿米巴。两种新的固定剂在含有脆弱双核阿米巴的玻片上表现出优异的性能,并且比经典方法大约缩短了十倍的操作过程。当比较这两种替代方法时,用固定剂-2制备的玻片对芽囊原虫属、微小内蜒阿米巴、布氏嗜碘阿米巴和哈氏内阿米巴表现出更好的性能,而固定剂-1在我们所依据的标准下对脆弱双核阿米巴显示出最小的优势。我们在研究中使用的固定剂-2和改良染色方法,在人体粪便寄生虫学检测中,比经典方法快十倍完成诊断阶段,在寄生虫学和微生物学实验室中不包括大肠杆菌包囊。对于常规使用来说,它似乎是经典方法的一个不错选择。