I would like to study environmental bacteria under starvation. I would like to dilute a freshwater bacterial concentrate with a medium without any nutrients during several hours incubated on different temperature. What kind of water can I use in order to put the bacteria in starvation condition. Do you think that freshwater ultra-filtrate sterilized by autoclave would be suitable? Or physiological water?
The freshwater ultra-filtrate is a good idea but you may need to test the DOM or DOC, which is normally pretty low. Another option would be some artificial minimum medium without any carbon source in it. Hope it helps.
I think it is always better to use saline water instead of autoclaved water
Vital et al. descibe very nicely the preparation of low nutrient water, see DOI:10.1016/j.watres.2012.08.043
Under "starvation", you mean lack of carbon sources, not the other nutrients. Therefore, a setrile medium with a buffer and main mineral nutrients (N, P, K,) prepared with ultra-filtrated water will do. Make sure you wash out your bacterial inoculum with the same medium before you inoculate them, or they will have residual carbon sources adsorbed by cells and in their hydrate shells. To wash them, simply add the smedium, shake, and pellet them 2-3 times.
Water condensed (distilled) from steam, then bring Dissolved Oxygen (DO) content to minimum 6ppm (or greater) IF the microbiology you study is aerobic, Their nutrient starvation regimen excludes all carbon and nitrogen sources.
there are recipes for artificial freshwater. Start with ultra purified water (Milli-Q or similar), but that process removes essential ions that need to be replaced. Use salts that have been combusted at 500deg C to remove any organics.
I certainly would go for a prepared minimal medium (only inorganic sources of high purity). You may also check for low amounts of DOC and TOC in the water you are using for dilution and media preparation. However, if you are using a fresh water concentrate of bacteria, the existing heterotrophs will use virtually all organic matter in few hours. Be cautios when using highly pure water with no salts added since this will cause lysis of some cells and usable organic matter will be present in the medium. I really like Dr Barkovskii comment. In addition using a prepared medium instead of natural water of unknown composition enables you to repeat the experiment at the very same conditions.
If you want an aquatic environment with no kind of any nutrient at all, in order to study your bacterial population under complete starvation conditions, then you should use either distilled or deionized water which should be autoclaved. Otherwise you might use a basic buffer, however a buffer contains some nutrients (such as P, K) and so you do not create the complete starvation environment. When I used a phosphate buffer to test aerobic bacteria survival under starvation conditions they survived for months.
I suggest the distilled water which should be autoclaved. Otherwise low concentration phosphate buffer would be OK.
If you use distilled water, you need to use glass distilled (best doubled distilled) tap water. Do not use deionized water because it containes amines and sometimes phenolics from the ion exchanger. -depnding on the supplier). (except you se the high end Millipore system, wichhas the advantage that it tells you how clean your water is)
Those chemicals distill al to some extend also over withyour water --especially if you use the distille for a longer time. The doubled distilled water gives you the opportunity to tailor exactly what nutrients you want to add and which (C, N, P, S, Oxygen, trace minerals) you want to limit. T can become important in instances you observe some interesting results ---you want to have exact/defined conditions so you can make possible but meaningful explanations. Also it is important to wash the cell inocculum as Barkovski (see above) said. For some bacteria a few hours of starvation is not enough, you may need several days and for some conditions months especially when you get into the area of the viable-but-non-cuturable state.
Good luck
I think the most suitable would be natural freshwater from the same environment, sterilized by ultrafiltration or by autoclave supplemented with NaCl 0,5%-0.9% to reach the salt concentration of physiological water. Before to add NaCl it is important to know the salinity of fresh water, because it could happen that the ionic strength would be enough; that is the natural water has salt enoughsterilized
under "starvation", I mean without any nutrients (C,N,P), thank you very much for all your interesting answers.
Does NaCl allow to avoid the risk of osmotic shock of the bacteria?
Hi Stephanie,
The choice of water used to test starvation may also depend on what quantity, how many replicates and the time-periods of you repeats. It's a question of having equal conditions and limit ionic heterogeneity from one repeat to another.
One possibility is to supplement distilled water with oligoelements and buffer it withs TRIS for instance, but you have to make sure the same oligoelement solution is used for all repeats and that it doesn't change during storage between experiments/repeats.
Another possibility is to use mineral water from a natural source (one of the best I know is not so far from where you work ;) and you are not obliged to provide the commercial brand in a paper), that have well known and constant mineral contents. You may still have to buffer it, though !
For the quantity, I will make 9 replicates of 5L under 3 different temperatures so I will need a total volume of 135L.
Yes, NaCl will prevent the osmotic shock. Determine total salinity (not only NaCl) in your original sample, and add enough NaCl to ultra-filtrated and sterilized water in equimolar concentration to compensate for other salts. Of course, the dissociation constants for these salts vary, but I don't believe you want to go into calculating these variabilities, though you can if you want. As a rule of tomb, 2+ cations -based salts dissociate to alesser extent that 1+-based ones.
Use a complete salts recipe. If you use only NaCl, you will harm gram negative bacteria that use divalent cations to bridge outer polysaccharides to the cell membrane. See: Separation and localization of cell wall layers of a gram-negative bacterium. Forsberg CW, Costerton JW, Macleod RA. J Bacteriol. 1970 Dec;104(3):1338-53
I usualy use Artificial freshwater from Standard Methods (192 mg l-1 NaHCO3,
120 mg l-1 CaSO4 ¥ 2H2O, 120 mg l-1 MgSO4, 8 mg l-1 KCl;) This is DOC- free if you use Milli-Q water. see: APHA, AWWA, WPCF, 2005, See Marxsen et al FEMS 2009, Fazi et al 2008 Environ. Microbiol.
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