From owner-celegans@net.bio.net Mon Nov 09 22:00:00 1998 Path: biosci!biosci!not-for-mail From: Erich Schwarz Newsgroups: bionet.celegans Subject: Cleaning worms with antibiotics besides streptomycin Date: 10 Nov 1998 10:55:42 -0800 Organization: Columbia University Lines: 105 Sender: daemon@net.bio.net Approved: wmorgan@acs.wooster.edu Distribution: world Message-ID: <72a27e$sb0@net.bio.net> NNTP-Posting-Host: net.bio.net Worm strains aren't always effectively decontaminated by streptomycin (Str), and can be inefficiently decontaminated by bleach -- either because the strains are poorly fertile, or because one has many dozens to decontaminate, or both. This motivated me to ask bionet.celegans if other antibiotics besides Str work. First, my own brief take on all the responses: Ampicillin works sometimes, and doesn't hurt the worms. You can feed them on XL-1 carrying pBluescript, and XL-1:pBlue will grow (a bit slowly) on NGM with Amp. If you are in a hurry to get rich XL-1 lawns, try the "enriched peptone plate medium", plus ampicillin. En. pept. medium is described by Lewis and Fleming in Ch. 1 of the _C. elegans_ methods book (Academic Press). Ampicillin doesn't always work. Another option is to grow the worms on MYOB (described by Eric Lambie in WBG 13(2): 12a). It is very restrictive and seems to only grow _E. coli_. It is not totally reliable -- some worm strains die on it -- but worth trying if Amp fails. A final option is tetracycline, which is very powerful at cleaning up slimes but also somewhat toxic to worms. Again, you can grow XL-1 upon NGM + Tet or en. pept. + Tet, and then grow the worms on that. One might want to try using a LOW concentration of Tet (1 ug/ml instead of 10 ug/ml) to select against slime while trying not to kill the worms too. Next, a lengthier and detailed summary of the responses: 1. If you want to be maximally rigorous, use bleach. John Thaden emphasized this point, noting that protocols exist that allow bleach to be used on a small number of gravid hermaphrodites. For example, one is available at: http://www.dartmouth.edu/artsci/bio/ambros/protocols/worm_protocols.html under the header, "Cleaning Worm Stocks: Michael Koelle". 2. Ampicillin (Amp): Raffi Aroian reports that this works well, given _E. coli_ carrying pBluescript. I've also found it apparently effective in stopping at least one slime contaminant. However, Thaden found Amp to be ineffectual against at least one contaminant in his lab. 3. Tetracycline (Tet): Peter Barrett reports that this is the *one* antibiotic effective against the most persistent contaminants. One caveat reported by Barrett and Thaden is that Tet visibly reduces brood size and can prevent worm growth completely. Another possible caveat is that Tet is antagonized by magnesium. Mg^2+ is a major component of NGM, but not of MYOB or NGM Lite (more below). 4. Gentamycin (Gen): Thaden finds that 60 and 200 ug/mL concentrations inhibited growth of an Amp-resistant contaminant, while lower concentrations failed. Gen did not visibly harm worms at 200 ug/mL. 5. Pen Strep aminoperin (PSa): Thaden finds that PSa is harmless to worms, and inhibited growth of at least one contaminant. 6. Chloramphenicol (Chl): Several XL-1-Blue derivatives from Stratagene are resistant to Chl at <40 ug/mL, so Chl might be worth trying, especially since its mechanism of action (inhibition of the bacterial 50S ribosomal subunit) is like that of Tet (30S inhibition). However, like Tet, Chl may well be toxic to worms (it hasn't been tested by anybody as far as I know). 7. Changing the pH: Barrett reports that this can eliminate unwanted microbes picked up at pH 8.0 in NGM. An easy way to change pH from 8 to 6 is to switch the worms to Eric Lambie's MYOB medium, described in WBG 13(2): 12a. MYOB is harmless to most, though not all, mutant _C. elegans_ strains; Hodgkin has observed one mutant strain totally unable to grow on MYOB. Where MYOB is unusable one can instead try Lambie's "NGM Lite", described in WBG 13(5): 11. 8. Antibiotics versus E. coli: For exotic antibiotics for which a plasmid conferring resistance to E. coli isn't available, one might plaster OP50 or HB101 onto the antibiotic plates in the form of a thick paste. Thaden: "A typical lawn on a normal 10 cm NGM plate is about 18 A600 optical density units, so that is about how much I would spread. We use a stock slurry of OP50 in S buffer that is usually 80-100 A600 units per mL, as determined by reading a serial dilution at about 0.3 A600 units." This would sidestep the need for resistant _E. coli_; HB101 might be preferable, being less sticky and easier for worms to eat. Alternatively, one might plate OP50 on several antibiotic plates, select rare resistant colonies, and grow them as an OP50-derivative strain. 9. XL-1 and XL-2 are consumable by worms, and are commercially available as competent cells. Also, XL-1 and XL-2 have intrinsic Tet and Chl resistance. 10. Not all contaminants are created equal. Thaden found a contaminant that was resistant to Amp. In general, no one antibiotic is guaranteed to remove all contaminants or any given contaminant. --Erich Schwarz schwarz@cubsps.bio.columbia.edu .