Difference between revisions of "Dionne:Protocols"
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Brewer's Yeast <br>
8 litres water<br>
8 litres water<br>
Revision as of 09:40, 13 May 2009
- 1 Setting up a small-scale Drosophila kitchen
- 2 Working with Mycobacterium marinum
- 3 In future, this page will also include:
Setting up a small-scale Drosophila kitchen
When I started my own lab in January 2007, I found myself needing to make my own fly food for the first time. This is how I set up my own fly kitchen, on the relatively-cheap. Much of this was done based on the advice of Ralf Stanewsky, to whom I am very much indebted.
Cooking and dispensing the food
Very small batches of fly food (<1 litre at a time) can be made in a microwave.
In order to make larger quantities, I bought an 1800-watt portable induction cooker from John Lewis for £60. This is basically just a hot-plate, but it is more efficient (and hence puts out more heat at a given power consumption) than a normal electric hot-plate. The induction cooker is also a bit less of a fire-hazard than other kinds of cooker. I got a 12-litre pot from a catering-supply shop (£30). Note that many types of pot will not work with an induction top: the pot must be ferrous, and I'm told some steels still won't work. Ask at the shop if you're in doubt.
For mixing during cooking, I bought a Heidolph RZR-2041 (£500) with a PR-30 "Pitched-blade Impeller" (£25). There are probably cheaper mixers available, but this one was particularly recommended. It seems to work adequately (but see below for proper mixing).
To dispense the food, I got a Watson-Marlow 323S/D Peristaltic Pump (£755). This model is nice because it can be programmed to dispense a specific volume at the touch of a button. Others have recommended the Wheaton Unispense pump, which costs more. In any case, I strongly recommend a pump for pouring food: it makes this chore much easier.
Recipes and ingredients
We currently use the following recipe, which we designed ourselves. For 8 litres of food:
Brewer's Yeast 800g
8 litres water
Put all the dry ingredients in the pot, add 2 litres water, and mix everything together by hand. There should be no significant clumps remaining. Then add 5 litres water.
Bring to boil, stirring constantly, on highest heat. It is important that the food reach a full boil in order to dissolve the agar.
Simmer 10 minutes with heat on low. Watch to make sure it doesn't boil over!
Turn off the heat, then add the remaining litre of water.
Allow to cool to below 70°C, then add:
40ml 15% Nipagin in ethanol
60ml Propionic acid
(Nipagin = Nipagin M = tegosept M = p-hydroxybenzoic acid methyl ester)
Dispense about 8ml per fly vial.
We have previously used a complex molasses recipe, a very simple sucrose-yeast recipe, and a glucose-yeast recipe. We disliked the molasses recipe because it had so many ingredients; we disliked the sucrose-yeast recipe because we had serious problems with Leuconostoc infections (slimy vials). This recipe seems to work at least as well as the others at supporting life, doesn't get slimy, and is easy to pump.
Working with Mycobacterium marinum
This is a brief summary of how we culture Mycobacterium marinum. We aren't bacterial geneticists, generally speaking; if you want information on genetically manipulating this organism, you'll be better off asking someone else.
Basic M marinum culture
We culture M marinum in Middlebrook 7H9 media (including glycerol), supplemented with 10% OADC, 0.2% Tween-80, and (as necessary) antibiotics. Cultures are grown standing at 29°C; we find that disposable vented plasticware intended for tissue-culture (such as Nunc 169900) is convenient for this. (Others often grow these cultures shaking, but in my experience this is unnecessary.) For solid-phase culture, we use 7H9 agar, but we avoid solid culture whenever possible, because it's sllloooow. (7H11 agar is more selective but for our purposes we find no advantage in its use.)
Culture of M marinum from biological samples
Culture of mycobacteria from biological samples is difficult, because mycobacteria grow very slowly and are easily out-competed. This problem is painfully apparent in growing M marinum from Drosophila: at 29°C, the temperature at which we grow M marinum, we get vast numbers of competing bacterial and yeast species out of flies.
That said, we've managed to find a combination of antibiotics that will kill almost everything that comes out of our flies and allow M marinum to thrive. We smash flies thoroughly in PBS supplemented with 0.02% SDS and then plate them on 7H9 agar supplemented with the following (all in micrograms/ml): gentamicin 10; nalidixic acid 20; carbenicillin 50; cycloheximide 500; amphotericin B 10. You'll note that the cycloheximide makes this stuff both really toxic and fairly expensive, which is one of the reasons we avoid doing this experiment wherever possible.
The other reason we avoid this is because it works very poorly as a method of quantitation. M marinum stick together, and in flies at least are tightly tissue-associated. The 0.02% SDS in the smashing-buffer helps this, but nowhere near enough to get good bacterial quantitation (the freed bacteria are by and large single cells, but many many bacteria remain associated with the fly cuticle). We haven't been able to homogenize the fly well enough to free all, or even most, of the bacteria. Instead, we have developed a quantitative RT-PCR assay, detailed here, which seems to work at least as well.
Preparing M marinum for injection
As mentioned above, M marinum tends to clump. For infection experiments, we usually want a single-cell suspension, not least because this allows us to accurately quantify the dose by measuring optical density. People disrupt the clumps in various ways; we find the method that works best for us is to separate out single cells by centrifugation. We just take a nice, thick culture, spin at 3000x g for 5 minutes at room temperature to pellet all bacteria, resuspend in PBS + 0.2% Tween-80, then spin at 200x g for five minutes at room temperature to pellet clumps; single bacteria (and some doublets) will remain in suspension after this spin. These bacteria are now ready for infection. (This protocol originally came from Lian-Yong Gao.)
In future, this page will also include:
Measuring triglyceride, glycogen and glucose from Drosophila