16 replies to this topic

[Tan Mei Yin]

Hi,

I'm a student currently doing my Final Year Project and I needed help for biochemical tests on detecting the presence of Lactobacillus from a sample.

My project involves isolating the bacterias from a sample (fermented beverage) and the aim is to find/ detect the presence of Lactobacillus in it by doing biochemical tests, and molecular methods.


One of the tests is by using Bile salts test to detect the probiotic properties of the colonies that was isolated ( colonies that are picked are rod shaped and gram positive).

My question : When I prepared 0.4% of bile salt solution in MRS broth and distributed to 19 bijou bottles for 19 samples and I autoclaved it after that. Somehow, after autoclave, the solution turns out to have white precipitate in each of the bottles (the bottles are clean and washed) instead of how it was originally in cloudy form. Is it normal for it to be precipitated like that after autoclave? The solutions have to be autoclaved since my work involves bacteria it is to avoid any possible contamination. I was thinking of autoclaving the MRS broth first then add the bile salts later. Is it wise?

After inoculating the bacteria into the bottle with bile solution, the cultures are let to grow and readings are taken after 6hours and 24hours using the UV-Vis spectrophotometer. Since the precipitates present after autoclaving, I fear that it might interfere with the readings.

Hope that you could help. Thank you =)

[pito]

Its just MRS with bile salts? or?

It is possible that you get precipitations, not everything can be autoclaved, so sometimes you need to filter sterilize it.

Not sure about this one though.
MRS itself should not be a problem, so I guess its the bile salts.
I know there are some media out there with bile salts that can not be autoclaved, but I doubt its because of the salts.


What you were thinking is good: autoclave the MRS first, check for precipitations (if there is none, then its the bile salts thats cuasing it). After autoclaving it, let it cool down and add the bile salts using a filter in the laminair flow.

Make sure to work sterile.

(its the same with adding antibiotics : you also add those after autoclaving the medium and letting it cool down)

[El Crazy Xabi]

Clasical culture media didn't usually pay attention to slight precipitates, I suppose your precipitates appear like a cloudy medium?

MRS broth recipe containing phosphate and MgSO4 such the one preformulated by Difco (Difco™ Lactobacilli MRS Broth) can have slightly opalescence aspect, ie becomes hazy/whitish after autoclaving. Tween80 can change the appearance of the medium specially right after leaving the autoclave, but usually become clearer when cooling.

Don't know if bile salts may affect. In all media I saw they are added prior autoclaving but they are old formulations

As said by pito, try first to see the aspect of normal broth. If it still has that aspect, try to add the MgSO4 (and maybe MnSO4) after autoclaving if you want a clear broth. MgSO4 is added after autoclaving in many minimal media (as well as CaCl2) because they use to precipitate probably by forming phosphates.

[chandra3316]

Hi!
friend I think the same,of Veteran's answer. Since bile salts contain steroid nucleus, they gets destroyed upon autoclaving. like plant hormones we use. (your doubt about interference is reasonable) So you better add them after the Autoclaving. with in the laminar air flow(use flame if possible to maintain strict sterile condition during addition) to avoid precipitation.

[Tan Mei Yin]

Attached here is the (left to right) MRS broth after autoclaving, bile salt solution in MRS broth after autoclaving, and the settled precipitates in the broth



pito,
Yeah, it is just MRS with bile salts that had been dissolved in distilled water prior addition to the MRS.

Thank you for your idea, I shall try it out by using the filter. =)

Um, there's one more thing, is it okay if i filter the bile salts solution (without autoclaving) after dissolving it with distilled water? Will it make any difference to the cloudiness of the solution? I noticed that the solution will turn quite cloudy and the reading for the spectro could not be obtained as it is too cloudy.


El Crazy Xabi,
Yeah, the precipitates appear like a cloudy medium, and there's some that precipitates so badly that white clumps presents too.
The type of MRS that I am currently using is Merck's and it does contain phosphate but it does not turn cloudy after autoclaving. So I guess it must be the bile salts that's causing it.

Thank you so much for the information, I shall take it as a note =)

chandra3316
Hi =)
Oh, I see.. so if the bile salts solution is autoclaved then the chemicals that it contains will be gone? So that means it will be the same just like not putting in any bile salts into the MRS? LOL
Thank you for your advice =)

[El Crazy Xabi]

Interesting...

Reading the Difco manual of culture media (you can download it from the web) many culture media with bile salts are not autoclaved

For example for the TCBS agar, it says:

Quote

1. Suspend 89 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
3. Cool to 45-50°C and use immediately. DO NOT AUTOCLAVE.
4. Test samples of the finished product for performance using
stable, typical control cultures.

However, few others like the EC medium (and variations), MacConkey agar (if it will be used after more than 12 h, also variations) are autoclaved but seems to be exceptions:

Quote

1. Dissolve 37 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Warm slightly to completely dissolve the powder.
3. Dispense into tubes containing inverted fermentation vials.
4. Autoclave at 121°C for 15 minutes.
5. Test samples of the finished product for performance using
stable, typical control cultures

So, my recomendations:
- If use will be inmediate (and you are lazy ) just heat up the medium to disolve if required.
- If you are willing to used the next day, willing to store some or like me, you are more confident sterilizing everything, autoclave the MRS broth and filter sterilize a stock of bile salts and add it after cooling the medium

[pito]

It does seem that the bile salts are causing it.. so yeah, its best to filtersterilise them.
And yes, you can dissolve or dilute the bile salts with water , just use (to be save) autoclaved water and then filtersterilise the bilesalt dilution.

About being too cloudy: I dont know, never had this kind of problem before. If it turns cloudy when  adding water.. then doesnt it turn cloudy when you add it to your broth too..?

[Tan Mei Yin]

EL Crazy Xabi
Thanks! I will tyr out the method that you suggested =) Thank you so much again

pito
Autoclave the water..sounds a great idea. Thank you!
Yup, the bile salts will turn cloudy too when it is put into the MRS broth. I was thinking of reducing the cloudiness to that the reading from the spctro could not be affected. But I wasn't so sure if there's such a way to reduce it. =/

[pito]

Tan Mei Yin, on 11 August 2012 - 08:32 AM, said:

EL Crazy Xabi
Thanks! I will tyr out the method that you suggested =) Thank you so much again

pito
Autoclave the water..sounds a great idea. Thank you!
Yup, the bile salts will turn cloudy too when it is put into the MRS broth. I was thinking of reducing the cloudiness to that the reading from the spctro could not be affected. But I wasn't so sure if there's such a way to reduce it. =/

The reading of the spectro could still be ok? You normally measure first without any micro-organisms...
If the entire solution is cloudy (or more dark) then its ok..
(there only problem is when you have pieces floting around.. if you see what I mean... )

[El Crazy Xabi]

Gets cloudy only in water O_o

Is the product new? I've been checking some bile salts product sheet in Sigma and... (example of bile salts mixture B3426):

Quote

The Bile salts mixture is a white, free flowing,
fine powder. It is freely soluble in water and
forms a clear, colorless solution that
produces foam if shaken. When incorporated
into culture media, the bile salts mixture does
not affect the color of indicator dyes or their
subsequent color change.
Bile Salts: ³45% (expressed as cholic acid)
Moisture: £6.0%
Final pH: 7.0 ± 0.5 (1% Solution)

Bile salts B3301

Quote

The
powder freely dissolves in water. The aqueous solution is clear and yellow,
and produces foam when shaken. The solution remains clear after
autoclaving.
Analysis
Moisture: Not more than 6.0%.
Effective Concentration: 0.25% - 0.5%
Final pH of a 1% Solution: 7-9

Also, in one of the products specifies max solubility of 10%.

So what you are explaining seems pretty weird. I'm not saying that you lie, don't missunderstand me, but it seems that the product doesn't match the expected characteristics. Check your product code and manufacturer specifications to be sure

[Tan Mei Yin]

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?

Attached Thumbnails

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Edited by Tan Mei Yin, 27 September 2012 - 10:44 PM.

[El Crazy Xabi]

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

[Tan Mei Yin]

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 =/

[El Crazy Xabi]

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

[pito]

Tan Mei Yin, on 27 September 2012 - 10:38 PM, said:

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.

Cant you get any flters? Normally labs have these.. I would find it weird you dont have acces to it...

And yes, the sediment will indeed influence the results.. you should not have this sediment. Or you can try to get rid of the sediment...  Just just the supernatans. But not sure the bacteria will grow good enough then, but you can try it.
If it is a sediment, changes are high that the bacteria cant even use it.. if it is complexed then the bacteria cant use it as food anyway, so try the supernatans.