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

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 =)

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)

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.

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.

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 =)

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:

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:

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

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..?

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. =/

Tan Mei Yin on Sat Aug 11 16:32:41 2012 said:

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... )

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):

Bile salts B3301

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