Bacterial Growth in Water Reservoirs

[tatep]

I have a question regarding water reservoirs in prep HPLC. Our prep instruments have 10L coated glass reservoirs and over time the water reservoir frits will get slimy from bacteria. We are currently adding 1% 2-propanol to the water reservoir but I don't think this is hindering the bacteria much, if at all.

The lowest amount of ACN in any gradient is 5%, would adding this 5%ACN to the water reservoir be enough to prohibit bacterial growth? Is there another volatile solvent that can be used without changing the retention much (I have heard a little Toluene will work), or possibly a UV light under the reservoir. Salts and such are not an option as we don't want to add further steps for drying down.

These instruments are in a "walk-up" environment where people add solvents as needed, so the simpler the solution, the better chance of complete compliance.

Thank you,

[Victor]

I am not sure whether you have bacterial growth, or whether these could be fungi or yeasts-maybe it is everything. But micro-organisms are happy in 5% alcohol or more, otherwise we could not have beer! On the other hand acetonitrile is something they do not like at all, and I am sure 5% ACN will do the trick. However, before you do anything I would clean out the water lines thoroughly first, perhaps even with pure ACN at some stage in the cleaning process. This should kill off everything and you can make a fresh start.

[Peter Apps]

Hi Peter

Bacteria, moulds and yeast need a source of organic carbon to grow, so adding solvents to the reservoir is counterproductive. If you add ACN you will sooner or later deveop a microbial culture that can thrive in its presence.

Filling the reservoir with pure (i.e. HPLC grade), filtered water deprives the bugs of their food source.

If you add a UV light it will have to be suspended in the tank, because the biocidal wavelengths are absorbed by glass.

Peter

[chhubert]

My colleague working in Biochemistry laboratory told me that they add a few drops of chloroform into water to inhibit growth of mould and bacteria. I never try it 'cos I always use water of Milli-Q quality but it sounds logical as chloroform is antiseptic.

[skunked_once]

Chhubert's suggestion does work. There is an article by Decedue and Unruh from BioTechniques, volume 81 (March/April) 1984, pp. 78-81 which describes the use of chloroform to remove suspended particles from water. A side benefit is the prevention of microbial growth. They used 5-10 mL of spectral grade chloroform per 500 mL of water and allowed the water to stand for a few days to allow the chloroform to settle out. This worked very well for me in the past but I did occasionally have a baseline anamoly from stabilizer in the chloroform.

[skunked_once]

About the use of chloroform. I forgot to mention the issues of chloroform vapors gradually exiting mobile phase reservoirs. Some safety officers may have comments on this.

[james little]

We have a LC-MS method that uses 2.5 mmolar ammonium acetate in water for the solvent and were having a lot of problems with growth of bugs in the solvent reservoir.

We washed our system well with pure methanol, used brown instead of clear bottle, added 3% acetonitrile to the reservoir since that was acceptable as a starting point for gradient. Usually change the solvent once every 1-2 weeks. Have no problems anymore..

[Uwe Neue]

Our experience agrees with that of James Little. A small concentration of MeCN, commonly 5%, prevents the growth of the little critters.

[Mark Tracy]

Has anyone tried Kathon CG? I once did a Google search on that and there were several papers where that was added to the mobile phase as an antibacterial. Supposedly it is effective at 15mg/mL.

[HW Mueller]

Victor, if you put enough alcohol into the soup from which the beer is made your buggies will desist from making any more, ever.
Peter Apps, probably there are already some bugs that love ACN, etc., at some concentrations, but does anybody really have unequivocal evidence that they produced mutants in their lab??? Could it be that they grew in some encapsulated areas, centering around some dust, dirt....? Or the organic had partially evaporated? If you have a good ACN, etc., eating bug you could get rich.
I have also noticed that cleanliness goes a long way to almost eliminate microbe problems, but dust is everywhere......., so once in a while one gets a big surprise. On the other hand, I have allowed some mobile phases to stand around for years, just now I picked up a bottle of 25% phosphate buffer, 5% THF, 70% MeOH from 1997: Looks like freshly made (wouldn´t use this anymore, though).
Furthermore, some materials have a tendency to transform and precipitate in flakes which resemble microbe colonies (one example I can think of right now is solutionds containing NaI).

[syx]

In the past, we had dedicated one line for water and buffer. The buffer is always changed everyday, and every 2 days for water (reservoir and its content). Though we flushed the line using acetonitrile or methanol every weekend, after several times we found white layer inside the line and on the inlet filter.
Now we never dedicate flow-lines for certain solvent, we always change the position at least once a week.

[Victor]

HWM- I am puzzled at your comments. I have never seen a bottle of brandy or whisky growing bugs (have about 40% alcohol). Nor have I seen a bottle of sherry or port (about 18% alcohol) growing bugs-but maybe this is borderline, maybe in some climates 18% alcohol is not enough. So why are you surprised if your 70% methanol solution does not grow bugs?Of course you cannot make beer above a certain alcohol level, I was only being cautious in that some of your German beers contain somewhat more than 5% ethanol.

I have never seen bugs growing in 5% ACN-and this appears also to be the experience of Mr Neue and Mr Little.

Algae can utilise atmospheric C02 for photosynthesis. They may die and provide a source of carbon for other microorganisms-this may be a cuse of growths.

Having said all this my I just use water alone in my water bottle -it never grows bugs. This may be because:

a) The lab temperature is never above 25 Celsius
b) The water is highly purified and filtered as part of the purification.
c) It is stored in a dark bottle.
d) I replace the water every day.
e) The storage bottle is Pyrex. I think some cheap glass bottles may leach sodium and potassium into water which can be co-factors in micro-organism growth.

However, if these conditions cannot be satisfied in a lab, then I would try 5% ACN

[tom jupille]

does anybody really have unequivocal evidence that they produced mutants in their lab???

Actually, yes. I alluded to it in passing in an earlier thread (http://www.sepsci.com/chromforum/viewtopic.php?t=3612 ).

This occurred in the early '80s (1983 or thereabouts). I worked for a now-gone company doing "single column" ion chromatography. The original work had been done a few years earler at Iowa State University using dilute (5 mM or so) phthalic acid buffers with anion exchange columns. I started in mid-1981. We prepared our buffers fresh every day, but after a couple of years, we were seeing evidence of growth by the end of the day, and we had to pull the inlet frits off the pumps for cleaning once a week.

Some of the newer work from Iowa State had been done using p-hydroxybenzoic acid buffers. We tried it, it worked, and we found as a collateral benefit that our mobile phases would last several days before beginning to show signs of bug growth.

A bit later, one of the Iowa State people stopped by for a visit; he commented that he was surprised we were using pHBA; they had gone back to phthalic acid because the pHBA buffers were growing bugs too quickly (within 24 hours). Comparing notes we concluded that we were both doing inadvertent selective breeding experiments; each generation was being enriched in the strain of bug which could most efficiently metabolize the buffer being used.

We ended up adding 5% MeOH to the mobile phases. it inhibited bug growth and had negligible effect on the separation (this was anion exchange of inorganic ions, remember).

I'll admit that this is not exactly a parallel situation, but it suggests that any treatment which is marginally effective (i.e., which kills most but not all of the contaminating organisms) will become less effective over time.

[Victor]

Tom-that is a very nice story.

However, I think the question was how to inhibit growth in a water bottle, not one containing buffer?

Also your point about selective breeding is interesting. However, I think we all know that alcohols in a low % (say 5% ) do not inhibit bug growth. I can find methanol in some ciders as well as ethanol, by GC- I understand this is produced by the "wrong" kind of microbes. So your selective breeding argument in such a case is relevant.

However, do you have real evidence that bugs can grow in 5% ACN? It seems that several posters here have no evidence that this can occur, despite the hypothesis that it might be a carbon source.

[Bruce Hamilton]

It's known that bugs ( particularly yeasts ) can degrade dilute ( <1% ? ) aqueous solutions of acetonitrile - as that's how such organic pollution is removed from water and soils. But in those cases, the bug colonies usually exist in the equivalent of a five star hotel with other nutrients available from potable water and substrates.

Googling " microbial degradation acetonitrile water " should find some details.

The issue is probably more about whether a specific HPLC environment
could allow such colonies to develop ( *static rather than *cidal ), and 3%+ CH3CN seems to be effective *static for many cases.

I've not encountered the problem, and prefer regular replacement of Milli-Q water, rather than add any solvent. Sod's law tells me the next method would use methanol...

My guess is that many of the problems arise in the water purification system, I know our old Milli-Q used to have quite high TOC and bug counts until we introduced more frequent replacement of the final 0.22um filter - doesn't take long for bugs to grow on the filter outlet, unles protected. Especially in labs where water usage is intermittent.

Almost every pharm and fabrication facility has comprehensive programmes to stop biofilms developing in their sterile, purified water systems, as once established such colonies are very hard to remove.
That shows bugs don't need many nutrients to start.

Do whatever works for you, and please keep having fun,

Bruce Hamilton