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Search our Tips from A to Z for more information. Still having trouble finding what you want? Contact Hampton Research technical support.
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| Y is for Yummy |
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| 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 |
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TECHNICAL SUPPORT
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Tel: 949-425-1321
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Fax: 949-425-1611
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Email: tech@hrmail.com
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LOCATION
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Hampton Research
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34 Journey
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Aliso Viejo, CA 92656-3317
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Tel: 949-425-1321
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Fax: 949-425-1611
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