dos Santos Samuel Ricardo, Branco Nilson, Franco Regina Maura Bueno, Paterniani José Euclides Stipp, Katsumata Masakazu, Barlow Peter W, Gallep Cristiano de Mello
School of Technology, University of Campinas/Limeira, SP, Brazil.
School of Agricultural Engineering, University of Campinas/Campinas, SP, Brazil.
Luminescence. 2015 Nov;30(7):1139-47. doi: 10.1002/bio.2872. Epub 2015 Feb 18.
Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA-SE (comprising living, non-stressed but aged cysts, heat-killed samples and UV-C-stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double-exponential decay kinetics, with the decay constant k2 being five times higher than k1. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples.
一系列测试表明，新鲜、完整的十二指肠贾第鞭毛虫和微小隐孢子虫（卵）囊样本不会被羧基荧光素 - 琥珀酰亚胺二乙酸酯（CFDA - SE）、C12 - 刃天青和SYTOX® Green等荧光探针标记，这可能是由于它们坚固的细胞壁。这些染料无法指示此类原生动物的活力，从而导致对活的且具有传染性的样本记录为阴性反应。微小隐孢子虫对化学物质的隔离性更强，提取后活的卵囊在长达90天的时间内都不会被探针染色。然而，在使用CFDA - SE对十二指肠贾第鞭毛虫样本进行染色的进一步荧光衰减（FD）实验中（包括活的、未受胁迫但老化的囊肿、热灭活样本和紫外线C胁迫样本），每个样本都呈现出不同的FD衰减曲线，在此对每个样本进行了7组测试，每组5次重复。FD曲线采用双指数衰减动力学拟合，衰减常数k2比k1高5倍。这种FD方法速度快，可通过10个步骤轻松重现，对于已经纯化的样本，大约需要1小时的实验室操作时间。
