Bile test for Lactobacillus - (Aug/06/2012 )

hi
sorry for replying this late as i've been away

pito,
so does that means if there's pieces of bile precipitations at the bottom of the MRS solution it will give a not-so-good spectro result(if i understanded it correctly lol)?
btw, i can't get my hands on the sterile filter. but with the sediments in the MRS, i was wondering if i could just proceed, or replace the MRS with nutrient broth? it's because nutrient broth does not have any probs with the bile solution.

El Crazy Xabi
no, it's no problem. yea you're right. i made a mistake there. when i mentioned that it stay cloudy in water it's because i didn't fully waited for it to dissolve in water, and it does stays cloudy if it was kept stirred.
i've checked the pH for a 10% bile solution (1g of bile in 10ml of water) and it's about pH 8.
it will turn the MRS cloudy once the bile is mixed into the MRS solution as the MRS solution is at pH5 (the brand of MRS that i'm using is Merck's)

my Supervisor suggested that i should change the pH of the bile mixed with MRS solution to pH 7 so that the cloudiness disappear (it did actually) as it might be the difference between the bile solution pH level and the MRS pH level. but i was wondering if that could affect the solution/environment that is as same as it is before i alter the pH level.

plus, when i autoclaved the bile mixed with MRS (pH that had been changed to 7) it didn't turn out with precipitates like how i attached a picture of it previously, but it had a little 'sediments'-kind of precipitates at the bottom.


so here ate the pictures attached.

first pic : (left to right) MRS broth before adding with bile salt solution, a 0.2% bile in MRS, a 0.4% in MRS, and 10% bile solution.

second pic : 0.2% and 0.4% bile in MRS with pH 7 after autoclave. looks way better compared to the ones previously. but there's some 'sediment'-like at the bottom.

extra. third pic : since my lacto's not really stable in growing in MRS, i've been using nutrient broth as a substitute (since it is a standard requirement for bacteria growth broth. i think). the lacto grew well in it and i've thought of replacing the MRS with nutrient broth for the Bile test. is it wise?

Did you test the pH7 MRS without bile salts? Just in case the precipitate would be just because of the pH...
Also, measure the pH after autoclaving, it might change and be another reason for the precipitates.

Nutrient broth is quite different from MRS so, I would suggest to keep the MRS if possible. But check it with your supervisor

yeah i've checked. the pH for MRS before adding in bile for both autoclaved and non--autoclave was around ph5. (after adding in was about pH6).

once the bile is added in, all i saw was cloudiness. and when it was autoclaved with the MRS, the cloudiness became a white clump. (this was before i changed the MRS pH level to 7.)

and i've tried another time by changing it exactly to a 10% bile solution pH level which is at pH8.36. the sediments dissolved completely and have no problems even after autoclaving.

so i guess it might be the pH's influence like how you've said it..(my supervisor said that she did it before but have encountered no problems like this when she autoclaved her MRS with bile salts. i think it might be the different MRS broth brands?)

thanks for suggesting =)
i was just wondering if it could affect the result since its pH had been changed to 8.36. wouldn't it will be the same as without adding bile into the MRS, but with the condition that the MRS pH is changed to pH8.36?..as in with or with out the bile salts, the pH of the MRS will still be 8.36.

man, i'm a lil but confused. sorry =/

Different MRS broth or different bile salts, there are many different bile salts depending on the origin.

The media with lowest pH I've seen is 6.9, most others are from 7.0-7.5. I would recommend to keep the pH as low as possible for the growth of Lactobacillus.

The pH change of MRS-bile salts pH7 might be because of the MRS or the bile salts interaction with the medium. So, I would consider 2 options to get, maybe, the desired pH:
- Test the initial pH of the media+bile salts to achieve a final pH 7 after autoclaving. Just make some mixture, dispense in tubes and adjust the pH individually in intervals of 0.2 units of pH (should be precise enough). Check the pH of the tubes after autoclaving and cooling. One will definitelly have the desired pH.
- MRS (adjusted pH) and bile salts autoclaved separately. Hopefully the buffer capacity of MRS will avoid big pH changes. You may try adjust the pH of the bile salts before, but I don't know what will happen... You also can mix the solutions, adjust pH and then filter sterilize, but that's a pain

Hope you find a solution soon

Tan Mei Yin on Fri Sep 28 06:38:13 2012 said:

dear pito and El Crazy Xabi

I've decided to change the MRS broth's pH to 8.36 (the pH level where bile dissolved completely in.). And the control will be 0% bile (which is just only normal MRS broth).
I think the problem with bile being clumped together after autoclave depends on the brand (the ingredients and the overall pH level of the broth too) of MRS that you're using. My supervisor said there's no problem with the one with Oxoid (sadly we couldn't get Oxoid's).

I've done the experiment on it and it seemed okay, the bacteria that was assumed to be lacto did grow in the 0.2% bile concentration. (i did it according to this journal).

Unfortunately there's another problem..we think that the spectro is not working well so we're using microplates instead. To save the broth usage too.

So..could I ask another question? I'm not sure how much microlitre for 1 well in the microplate needs. If i assume that it needs 1microlitres, is it fine if i inoculate 1% of the bacteria into it? (1% of inoculant in 1 microlits. broth = 10 microlits.) or 10%?

Tan Mei Yin on Sat Oct 27 06:05:32 2012 said: