Sedinová V, Urbanová E, Skarda J, Jukl A
Vet Med (Praha). 1985 Aug;30(8):467-75.
The rapid reaction of the diphenylamine agent with DNA was used for the determination of the counts of somatic cells in cow's milk, using the DNA filter method. The method is based on the filtration of a warmed (65-70 degrees C) mixture of milk with Triton X-100 through the Synpor nitrocellulose membrane filter, pore size 2 to 5 microns, and subsequent DNA determination of the collected somatic cells by the colour reaction of diphenylamine. A 2ml quantity of distilled water and 4 ml of diphenylamine reagent were added to the membrane filters with somatic cells. The mixture is warmed in water bath at 90 to 100 degrees C for 20 min., then it is cooled, centrifuged (3500 X g, 15 min.), and the optical density is measured at 595 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content to the counts of cells. The average variation coefficient of the determination was 5.9% and the coefficient of correlation between the diphenylamine DNA filter method and the direct microscopy of the somatic cells on membrane filters was r = 0.997. Using the diphenylamine DNA filter method, the counts of somatic cells can also be determined from milk samples stored in frozen condition or from the filters with collected cells kept at the temperature of 4 degrees C (10 days) or 25 degrees C (3 days). Milk stabilized with formaldehyde can also be used for the determination if stored at 4 degrees C.
利用二苯胺试剂与DNA的快速反应，采用DNA过滤法测定牛奶中的体细胞数。该方法基于将加热至65 - 70摄氏度的牛奶与吐温X - 100的混合物通过孔径为2至5微米的Synpor硝酸纤维素膜过滤器过滤，随后通过二苯胺的显色反应对收集到的体细胞进行DNA测定。向含有体细胞的膜过滤器中加入2毫升蒸馏水和4毫升二苯胺试剂。将混合物在90至100摄氏度的水浴中加热20分钟，然后冷却、离心（3500×g，15分钟），并在595纳米处测量光密度。使用8微克 = 100万个细胞的关系将DNA含量转换为细胞数。测定的平均变异系数为5.9%，二苯胺DNA过滤法与膜过滤器上体细胞直接显微镜检查之间的相关系数r = 0.997。使用二苯胺DNA过滤法，也可以从冷冻保存的牛奶样品或保存在4摄氏度（10天）或25摄氏度（3天）的含有收集细胞的过滤器中测定体细胞数。用甲醛稳定的牛奶如果保存在4摄氏度也可用于测定。
