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果蝇螨虫(Drosophila mite)

(2012-05-26 02:32:13)
分类: 科研总结
2012.6.22:简单有效的方法:
把果蝇转到新的vial里面,四五天后去除果蝇,加入几毫升70%酒精,搅动food,把酒精(含food和larvae)转入新的vial。十分钟后,倒去酒精,用水清洗两遍。加入kimwipe吸水。盖好塞子。六七天后,幼虫孵化出来就没有mite了。

试过在有larvae的vial里面直接加酒精,十分钟后倒掉(没有清洗),结果larvae全死了。



螨虫是对果蝇威胁最大的生物。杂食动物,一般以酵母为食,也会吃果蝇的胚胎和蛹。发育时间比果蝇稍长,及时flip果蝇可以抑制其增长。如任其发展,可能导致果蝇全军覆没。

观察mite,必须用解剖显微镜放大很多倍。因为mite很小,其egg更小。  

实验室用的mite-proof foam plug非常有效,用了这个plug的vial完全没有mite。预防和治疗mite感染的好方法是:stock用mite proof的plug。bottle感染后,从vial里面重新expand。  

对于感染的果蝇,我们实验室的常规做法是:丢掉大部分感染的果蝇,尤其是很老的vial和bottle。然后,感染的果蝇每三天转移到新的瓶子中,瓶子里面加tedion paper(可以导致螨虫不育)。连续转三四次(转的时候,大部分mite会留在原来的vial内从而减少mite的量)。酒精喷洒桌面消毒。

从其他实验室得到的果蝇先要经过六周时间的检疫。急于用果蝇的话,要把果蝇在解剖显微镜下仔细检查,确认没有螨虫及卵。

这样的方法太耗时且tedion paper有毒。我正在尝试两种简单而无毒的方法:

1. 把幼虫挑出来,放到dissecting plate里面,用酒精浸泡幼虫十分钟。幼虫具有非常强的抵抗力,在4%甲醛,70%酒精中浸泡半个小时都不会死。而螨虫对于酒精高度敏感,且螨虫一般不会在液化的食物里面产卵和活动,所以,正常情况下,幼虫身上是不带螨虫的。保险起见,70%酒精浸泡十分钟。然后转到新的vial里面。

优点:比常规方法快,简单,无毒。缺点:仍然比较麻烦。

2. 把果蝇转到新的vial里面,三四天后,去除果蝇,vial里面存留幼虫。酒精喷洒到vial壁面,一共三四毫升左右,然后浸泡十分钟。去掉酒精。三四天后重复一次,因为egg可能对酒精的抵抗力很强,等三四天后egg孵化为mite出来,再用酒精杀灭。

优点:比法1更简单,适合大量处理。缺点:不如1可靠。可靠性有待验证。

我现在验证法2,不过是用20天的老的vial,里面有许多pupae。处理前,可见大量mite,包括大的。处理三天后观察,不见大的mite了,但是仍有小的mite,看起来是刚孵化出来的样子,因为大小和egg差不多。同时可见一些egg,说明酒精难以杀死egg。又等了三天再看,还有egg。看来,egg的孵化潜伏期很长。如果换用新的vial,没有pupa case,估计会好很多。

补充:还见egg,不代表是活的。

2012.6.11补充:酒精浸泡含有adult和pupae case的vial 10分钟后,发现mite还是活的,提示酒精不能杀死mite。有论文研究表明可以杀死吗?此外,部分pupae case酒精不能浸入。总之,这个方法不太可行。



Mites
The single biggest threat to Drosophila stocks is a mite infestation. While some mites eat only the flies' food, others eat embryos and pupae, and can completely wipe out a fly lab. Mites are very small, and therefore difficult to detect. The adults are smaller than Drosophila embryos, and can most easily be seen (under the dissecting microscope) walking on empty Drosophila pupal cases inside old vials. Mite embryos are even tinier, and often are found in pearly strands of 10-20 eggs on the Drosophila pupal cases.

Mites love the detritus that accumulates in fly trays and incubators, so keeping things clean is essential. Since mites and their eggs can be killed by ethanol, wiping down your dissecting scope, bench, CO2 pad, and brushes with 70% ethanol before or after you handle your flies is always a good idea. The mite life cycle is slightly longer than that of the fly, so if flies are turned over as soon as they eclose, mites will be unable to overcome a stock. The best insurance against mites is: a) to quarantine incoming stocks; and b) to turn over fly stocks regularly.

If you find mites in a stock, you should immediately discard the vial somewhere outside of the lab, wipe down everything with which the vial has come in contact with 70% ethanol, and carefully check all the stocks housed near it for mites. If you need to rescue a mite infested stock, you can select 2-3 females and 1-2 males from it, making sure their bodies aren't carrying any mites or mite eggs, and place them in a clean vial in quarantine.

Quarantine
To guard against mites, flies coming into the lab from other labs or stock centers should be quarantined for 6 weeks in an office or room where no other flies are being maintained. Quarantine procedure:
a) write the date on the original vial (and keep it till the 6 weeks are up);
b) turn the flies over every 14-17 days;
c) periodically inspect the pupal cases in the original vial under a dissecting microscope, looking for adult mites and/or mite eggs; 
d) wipe down the microscope and the bench around it with 70% ethanol; 
e) at the end of 6 weeks, carefully inspect the original vial and some of the more recent ones for adult mites and/or mite eggs; if none are seen, the stock can be brought into the lab.
f) if mites are detected, you can rescue the stock as described above, but will need to keep the flies in quarantine for 6 weeks following the attempted rescue, and to check them carefully at the end of the six weeks.

If you are anxious to work with a stock that is in quarantine, you can set up your crosses in the quarantine room. If at the end of 6 wks, the original stock vial is mite free, you can move your entire experiment into the lab/incubators.




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