Dallavecchia Daniele Lourinho, da Silva Filho Renato Geraldo, Aguiar Valéria Magalhães
Laboratory of Diptera Studies of the Microbiology Laboratory, Department of Microbiology and Parasitology, Biomedical Institute, Federal University of the State of Rio de Janeiro, Post-graduate Program in Biological Sciences: Neotropical Biodiversity, Rio de Janeiro, Brazil
Laboratory of Diptera Studies of the Microbiology Laboratory, Department of Microbiology and Parasitology, Biomedical Institute, Federal University of the State of Rio de Janeiro, Post-graduate Program in Biological Sciences: Neotropical Biodiversity, Rio de Janeiro, Brazil.
J Insect Sci. 2014 Jan 1;14:160. doi: 10.1093/jisesa/ieu022. Print 2014.
Large-scale, quality-controlled laboratory production of fly larvae is needed for biotherapy. The objective of this study was to assess the action of glutaraldehyde on the sterilization of Chrysomya putoria eggs by applying pharmaceutical sterility tests. Egg masses with 0.600 g were divided into three parts of 0.200 g, the eggs were separated using sterile distilled water, and the suspensions obtained were mixed with activated 2% glutaraldehyde solution. After 15-min contact, the suspensions were filtered through Whatman filter paper, and the glutaraldehyde residue obtained in the filtrate was neutralized by rinsing with Tryptone Soy Broth. The treated eggs were placed aseptically on Petri dishes containing gauze moistened with sterile saline solution. About 10% of the sterilized mass was transferred to test tubes containing Tryptone Soy Broth and Fluid Thioglycollate Broth. The tubes were incubated, respectively, at 22.5 and 35.0°C for 14 d to verify egg mass sterility. The plates containing the rest of the eggs (90%) were sealed with plastic film and kept in a climatized chamber at 30°C/d, 28°C per night, 60 ± 10% relative humidity, and under a 12-h light period to assess insect viability and survival. Each experiment was carried out in triplicate using a biological class II safety cabinet. No change in color or turgidity was observed with the agent tested, proving the sterility of the product and that there was no trace of contamination. Forty larvae (in three replications) in the periods of 12, 24, and 48 h after sterilization, when transferred to diet, produced larvae, pupae, and total viability similar to the control (larvae without sterilization). However, for the 72-h treatment, larvae and total viability were significantly lower than for the other treatments. There was no significant difference for the pupal stage. The product tested was shown to be efficacious for use as a sterilizer of C. putoria eggs for all the parameters assessed.
生物疗法需要大规模、质量可控的实验室生产蝇幼虫。本研究的目的是通过应用药品无菌试验来评估戊二醛对致腐金蝇卵的灭菌作用。将0.600克卵块分成三份，每份0.200克，用无菌蒸馏水分离卵，所得悬浮液与活性2%戊二醛溶液混合。接触15分钟后，将悬浮液通过沃特曼滤纸过滤，滤液中得到的戊二醛残留物用胰蛋白胨大豆肉汤冲洗中和。将处理过的卵无菌地放置在含有用无菌盐溶液湿润的纱布的培养皿中。将约10%的灭菌卵块转移到含有胰蛋白胨大豆肉汤和硫乙醇酸盐流体培养基的试管中。将试管分别在22.5℃和35.0℃下孵育14天，以验证卵块的无菌性。装有其余卵（90%）的培养皿用塑料薄膜密封，保存在气候控制室中（白天30℃，夜间28℃，相对湿度60±10%，光照12小时），以评估昆虫的活力和存活率。每个实验使用二级生物安全柜进行三次重复。所测试的试剂未观察到颜色或浊度变化，证明产品无菌且无任何污染痕迹。灭菌后12、24和48小时的四十只幼虫（三次重复）转移到饲料中后，产生的幼虫、蛹和总活力与对照（未灭菌的幼虫）相似。然而，对于72小时处理，幼虫和总活力显著低于其他处理。蛹期没有显著差异。对于所有评估参数，所测试的产品被证明可有效用作致腐金蝇卵的消毒剂。
