'crusty food' solutions

'crusty food' solutions

Ryder, Elizabeth F

From: Ryder, Elizabeth F Subject: 'crusty food' solutions Date: 11 Jan 2005 21:29:47 -0000 Hi again fellow wormers -- I posted my 'crusty food' problem yesterday. As always, a whole bunch of you wrote back with many suggestions, which I've summarized in case this info may help someone else in the future. I've also included some of the longer replies with more details. I'm sure some of these suggestions will solve our problem. Thank you all so much!! Liz Ryder Most of the suggestions had to do with saltiness in the plates, rather than problems with the bacteria. Here are some common suggestions: Possible solutions:to crusy food problem: 1) Concentrations, pH of solutions for plates; in particular, too much calcium was mentioned by several people 2) Try using 'NGM lite' (from Eric Lambie's WBG article. In particular, no CaCl2 or MgSO4 is added). This solution worked for Andrew Spencer, who had emailed the newsgroup concerning this problem awhile back. 3) Water source could cause problem, especially if too acidic 4) Quality of media components may vary, especially agar (may be too salty) 5) Plates may be getting too dry (e.g. from wintertime low humidity) 6) Agar being poured at the wrong temp; may get precipitation during cooling of media (one person reported less crustiness when precipitation does occur) 7) Broth in which bacteria are grown may matter (recommendation for TY medium) 8) Bacterial strain may have accumulated mutations over time; get new OP50 from CGC Here are more details: Erik Jorgensen ...here are also notes from Eric Lambie's recipe for NGM lite: Calcium: Calcium or magnesium causes the bacterial lawn to be thick and granular. Addition of 1M CaCl2 to an NGM-lite plate caused bacteria to become crusty. For this reason calcium was eliminated in the NGM-Lite recipe. Because autoclaving of CaCl2 and MgSO4 causes the salts to precipitate these solutions had to be added after autoclaving in the NGM recipe. Because they have been eliminated from the NGM-lite recipe, no solutions need to be added after autoclaving. The absence of calcium and magnesium does not alter the viability of sick strains (E. Lambie, personal communication). Creg Darby -- Witchcraft it ain't, I've seen this too, though not recently. Sometimes OP50 seems to change phenotypes with prolonged passage, and I know from work with a different bacterium that certain mutants make a crusty lawn. Since you said you went back to frozen stocks that shouldn't be the problem, but you might try ordering a different OP50 stock to be sure. I've seen rather large differences between OP50 from different labs. I've found that the texture of the lawn varies with the broth the OP50 is grown in before it's spotted on plates. LB seems to make it crustier, so my lab uses TY broth, which is 5 g tryptone, 2.5 g yeast extract per L and nothing else (i.e. no NaCl). Laura Berkowitz-- ...the solution was to switch to Eric Lambie's NGM Lite (to be found in WBG) or reduce the amount of Ca+2 in NGM to 1/10. The person that had this problem with their OP-50 was Andrew Spencer at U of Wisconsin Robert Reis -- I think many of us have had similar problems at one time or another (and the rest will, if they keep at it long enough!). I know you can intentionally simulate the problem by growing worms on rich medium (high-tryptone) plates, so maybe even when there is no cause evident, you could get rid of the crustiness by making the agar "poorer" with respect to nutrients. Josh Mcelwee -- ... it usually has something to do with the salt in the plates. I'd check to make sure that the calcium and magnesium stocks you're using are the correct molarity. Also, we used to find that if we didn't find salt precipitates in the plates, that this problem was more common (dunno exactly what makes the salt crystals form in the plates, however). Morris Maduro -- 1. Dryish lawns. The lawns chip like you described. Solution seemed to be make new seeding stock from frozen stock; cause may be related to dryness of plates or age of seeding stock (i.e. multiple causes?) 2. The impenetrable lawn. The lawns don't chip; rather, worms crawl 'around' the bacteria. A worm pick won't even chip the bacteria. We concluded the E. coli were somehow lysing; the problem could be resolved by using water from a different source. Our building distilled water in Santa Barbara flowed through 'scrubbers' that seemed to have different effects on E. coli growth depending on when they were last changed. Making enriched plates, with extra uracil, also seemed to alleviate the problem. (We never resolved if it was the media or the uracil that solved the problem here though.) Since starting my lab at UC Riverside we have used our own water filtration system (Barnstead, but Millipore is probably fine -- the resistance of the water is about 18 megohm) with good results. It also probably wouldn't hurt to get OP50 from the CGC. Peter Barrett: 1) Overdry plates (this is the major one), especially if the lab humidity is very low (as is often the case with New England winters)-- if the plates are too dry, the worms will also mostly sit on the edges of the lawn and will be too thin 2) Plates which have been allowed to sit too long (ditto) 3) Problems with the media; specifically, something wrong with or a change in supplier of any of the agar, Bacto-tryptone/peptone, etc. You didn't mention in your post if this was NGM, or MYOB (I switched our lab over to MYOB when I arrived there-- in general, I like this media better, it's much easier to make, and grows far fewer contaminants than NGM) ... Since you've been doing stuff for years without problems, then in your position I would begin to suspect things like the media reagents. If I remember right, Eric Lambie, in his original description of the MYOB media, suggests switching from peptone to tryptone for making the MYOB, as this makes the lawns "smoother" and less "crusty" or "bumpy". In my lab, we've also had the experience of the media reagents being different from different sources (e.g., agar more or less granular, peptone and tryptone more or less finely milled and/or of a different color... suggesting that the middleman has switched suppliers and/or quality control isn't what it used to be...) At least, that's a good place of where to start looking for ideas. Another possibility (that just occurred to me) is your autoclave. We've had the experience that it is not possible to re-autoclave the media (if you wait too long before pouring, such that it cools down)-- theoretically there should not be any problem with re-autoclaving, but we've recently had the experience with our current agar that if you do that, you can't then use the plates because the worms burrow too much, implying that the agar is changing chemically as a result of the autoclaving. (This is a new problem, so I think it's because the agar source has changed over time.) So the autoclaving can affect the media... which could in turn affect the bacteria... Hope this helps! Ryder, Elizabeth F wrote: > Hi -- > > We are having trouble with our OP50 lawns being 'crusty' -- that is, > they > form a hard surface that the worms have a hard time moving around on. > The > worms often get under the crust, which is a pain, and it 'chips' off > like > paint chips, which makes worms difficult to manipulate. We have been > pouring > and spotting plates for years with no problem, and have gone back to > frozen > strains of OP50 with no big improvement seen. We have grown the > bacteria in > LB and Hershey broth with similar results. We did move to new lab > space > recently, but the problem started a couple months later. > > Has anyone ever encountered this problem? If you found a solution to > it, we > would be eternally grateful if you would share it with us. We are > starting > to suspect witchcraft... Thanks! > > Liz Ryder > --- > > > > ---