Y代表“美味”（Yummy）
蛋白质是包括细菌、真菌等微生物的美味大餐。这些微生物可以分泌蛋白酶，将你的样品降解成小肽段，你的结晶实验也将因此而毁于一旦。除了你的样品，其实你的试剂，包括多聚的聚乙二醇等，甚至还有缓冲液，都有可能是这些微生物的美餐。就算这些可恶的家伙们没有将你的样品“嚼碎”，它们也可能影响你的试剂，改变溶液的pH，产生可能干扰结晶的化学物质，使得你的实验很难重复出来。为了避免你的蛋白样品或者试剂变成这些微生物的阳光早午餐，你需要考虑以下建议：
1. 使用0.2 μm孔径的滤膜除菌；
2. 样品在室温放置不要太久，暂时不用的样品应在低温保存(4, -20, or -80°C）；
3. 对于以前从事机械、物理、计算机等学科的人而言，他们经常面对的夸克、胶子、硬盘等这些东西都跟细菌扯不上关系，他们来做结晶实验时，应让他们保持对微生物污染或化学物质污染的警惕；（问:为什么以前好好的能长出晶体的溶菌酶储液这次长不出来了？答：当你不用这些储液的时候放置在什么地方了？“跟其它试剂在一起啊”“在哪儿？”“就在实验台上”“放了多久？”“没多久，几天吧，最多两周” 他们问这些问题没有什么可奇怪的，我们曾经也学过。耐心去给他们讲解，就像机械师给你讲解新式ATM机如何使用或给你演示高通量机械手一样，给他们讲解蛋白可是微生物的美餐。谁知道呢，或许下次你的电脑要更新系统时他们会帮助你噢）
4. 试验中用到的水，缓冲液、盐等等都要过滤除菌。
5. 放置了超过半个月的溶液，使用前应该先摇一摇，看有没有长菌。有时候可能是溶液中出现沉淀，为了区分是不是杂菌，可以用手将溶液捂热，看它会不会消失；
6. 使用灭菌的枪头；
7. 结晶的实验台用布盖着，只有点晶体操作的时候才掀开它；
8. 当你的实验室里也在培养细菌的时候，或者实验楼里有培养细菌的，就要更加注意防止细菌或真菌污染你的实验。
原文：Protein samples are yummy treats for microbes such as bacteria, yeast and fungus which secrete proteases that like to chop your sample into tasty bites, ruining your crystallization setup. These same microbial menaces also like to dine on polymer based crystallization reagents such a polyethylene glycols and even buffers. So even if the bugs and their secreted proteases do not chop your sample into bits, they can degrade reagents, alter the pH of the solution, or generate chemical species which can influence crystallization and even make reproducing conditions a pain in the tush. To prevent or at least minimize your sample and reagents from becoming Sunday Crystallization Brunch for microbes, consider the following suggestions:
Sterile filter the sample into a sterile tube using a 0.2 micron filter before setup to remove microbes. Do not leave the sample on the bench at room temperature for extended periods. Store unused sample appropriately (4, -20, or -80°C; only you know best). Those of you working with engineers, physicists, computer scientists, or folks from outside the life science field, take a moment to realize that quarks, gluons, hard drives and other physical things these folks are used to handling do not support microbial growth. Take a little time to help these crystallization rookies understand what happens to samples left on the bench for a day or three and the significance of keeping things clean to prevent microbial (as well as chemical) contamination. Case in point – Caller: “My lysozyme stock that was growing crystals no longer grows crystals.” Tech Support: “Where are you storing the lysozyme when it is not being used?” Caller: “Same place as the reagents.” Tech: “Which is?” Caller: “On the bench (i.e. room temperature).” Tech Support: “For how long?” Caller: “Oh, not long, maybe a few days, a couple weeks at the most”. Hey, nothing wrong with these types of questions. We all gotta learn some time. Just be sure and help that engineer, computer scientist and new post doc from a physics lab who is helping you with your new AFM machine or testing the high throughput robot, to understand proteins are food for microbes. Who knows, maybe they will help you with the next Windows update installation.
Sterile filter water into a sterile container before formulating crystallization reagents that cannot or should not be filtered (detergents, gels, and some high molecular weight polymeric agents too big for filtering).
Sterile filter (0.2 micron pore size) buffers, salts, polymers, and diluted organics into sterile containers. Sterile containers such as polypropylene or PETG plastic are readily available today and are cost-effective, time-saving alternatives to glass and autoclaving.
When using a stock that has been sitting about for more than a couple of weeks, give the bottle a swirl and look for signs of microbial growth such as a settle, faint white, off white or yellow to brown precipitate. Sometimes slightly precipitated salts will resemble microbial growth to the untrained eye. To help differentiate precipitate from microbial growth, try warming the solution in your hands for 5 to 10 minutes. If the material disappears it might well be precipitate. Microbial growth will not dissolve. Although we are not endorsing the sniffing of reagents, especially since some crystallization reagents can be hazardous, precipitates will not smell, while microbial growth will often stink.
Use sterile pipet tips when pipetting reagents, sample, and water into plates. Keep your filthy paws away from your pipet tips and do not touch pipet tips to the counter, your lab partner or other non-sterile items. If you set your pipet down it had better not have a tip on it or the tip will likely touch something non-sterile.
Keep crystallization plates in their sealed wrappers until just before use. This will prevent airborne microbes from setting up home in your crystallization plates. Most crystallization plates are not offered with a claim of sterility but most makers of these plates operate the injection molds in clean areas and package the plates straight out of the mold so there is little risk of contamination. Use them as is and put the worry into keeping them clean once the plate is out of the package.
If you do crystallization in a lab that also grows microbe bugs, especially fungal microbes, be especially careful as these airborne spore forming nasties can set up camp in crystallization plates quite easily.
