Goihman-Yahr M, Pine L, Albornoz M C, Yarzabal L, de Gomez M H, San Martin B, Ocanto A, Molina T, Convit J
Mycopathologia. 1980 Jul 1;71(2):73-83. doi: 10.1007/BF00440612.
Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimated viability by means of vital dyes. Methylene blue Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans. Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium. In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25%. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.
采用温和超声处理获得巴西副球孢子菌的单细胞悬液。根据显微镜标准,这些细胞是完整的。通过对给定接种物进行直接细胞计数以及在各种培养基上形成菌落来确定平板接种效率。使用超声处理和未超声处理的细胞悬液来研究平板接种效率,并通过活体染料评估活力。亚甲蓝、赤藓红B和詹纳斯绿与巴西副球孢子菌一起使用时不可靠,但与白色念珠菌测试时,活体染料是准确的。吖啶橙给出了更有意义的活力结果。然而,由于悬浮培养基组成的相对微小变化,活力估计值发生了显著变化。在最初的实验中,巴西副球孢子菌的平板接种效率极低。随着接种物稀释度的增加,其效率急剧下降。如果向脑心浸液平板中添加马血清,或者将葡萄糖甘氨酸酵母提取物(GGY)平板在室温下孵育并计数菌丝菌落,则效率会大大提高。采用我们报道的技术,超声处理悬液目前的平板接种效率约为25%。我们的结果和方法对于那些涉及体外杀灭悬浮液中的巴西副球孢子菌或从临床或环境标本中分离该真菌的研究具有重要意义。