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Addition of 2% glycine in MRS broth

glcyine mrs colour

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#1 Meg P. Anula

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Posted 02 August 2015 - 01:29 AM

I have prepared a MRS media with addition of 2% (w/v) glycine. I added 5g of glycine into 13.75g of MRS, mixed with 250ml of water and then autoclaved.

 

To my horror I found the media turned black after autoclaving. I also autoclaved a bottle of MRS media and it is brown color, as normal.

 

Do anyone here have the experience of adding glycine into media and can tell me if this is normal?

 

 

Thanks.



#2 phage434

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Posted 02 August 2015 - 02:51 AM

You may have rather dramatically changed the pH of the medium. Changes in pH can make some components react or fall out of solution. Try neutralizing yor glycine (perhaps in a higher concentration solution) before adding it to the MRS medium. You could tell if this might be the issue by measuring the pH of the normal MRS and your augmented MRS.



#3 Chris22

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Posted 03 August 2015 - 07:01 AM

Hello Meg,

 

I had a similar issue with MRS broth before.

 

I later found out that MRS broth should not be autoclaved at 121 deg for this very reason.

 

Instead, modify the autoclave conditions to 110 or 115 degrees for 10 min.

 

The broth should be a little darker than golden brown. I believe I used to include the powdered glycine in the broth before autoclaving.

 

However, I never got Glycine to work well for my Lactobacillus species.

 

Instead I found that low concentrations of penicillin work far better as a cell wall weakening agent. However, it was  much more toxic to the cells if left too long.

 

As I am sure you are aware, most strains require their own optimised conditions so you may have to tweak (increase/decrease) the concentrations if it does not work straight away.

 

A protocol for this is below, (from doi: 10.1111/2049-632X.12040.):

 

Electrocompetent L. salivarius cells were prepared using a modified version of the approach previously described by Sheehan et al. (2006); essentially, a stationary phase (16–18 h) culture was inoculated (1% v/v) into 50 mL of MRS.

Following 2 h incubation at 37 °C with shaking at 100rpm (or until OD600 0.25), penicillin was added to a final concentration of 8 microgram/mL , and the culture was incubated for a further 1.5 h (max time!) until reaching an OD600 of 0.4–0.6.

After holding on ice for 10 min, cells were harvested by centrifugation (4 °C at 1000 g), washed twice with 1-mL ice-cold 10 mM MgCl2 solution, once with ice-cold electroporation buffer (EB; 0.5 M Sucrose/10% Glycerol) and resuspended in 46 microL of the same EB buffer.

Electrocompetent cells were kept on ice and used within 30 min. Electroporation was carried out using a Gene Pulser X electroporator (Bio-Rad). Forty-six microlitre of competent cells were mixed with 4 microL of plasmid DNA (Total concentration of 1 microg plasmid DNA/ reaction) in a chilled 0.2-cm electrode gap electroporation cuvette (Bio-Rad) and incubated on ice for a minimum of 5 min. The cells within the cuvette were then subjected to the following electrical parameters: 1.75 kV, 600 Ω parallel resistance, and 25 lF capacitance. For phenotypic expression, the cells were immediately diluted with 0.95 mL of prewarmed MRS-SM buffer (MRS broth, 300 mM Sucrose, 80 mM MgCl2), transferred to a sterile PP tube (Sarstedt) and incubated at 37 °C without agitation for a minimum of 3 h. After incubation, dilutions were plated onto MRS agar containing selective antibiotics. Plates were examined after 48 h incubation at 37 °C for the presence of antibiotic resistant colonies.

 

Hope this helps

chris



#4 Meg P. Anula

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Posted 03 August 2015 - 05:51 PM

Thanks.

 

Chris, I autoclaved both MRS alone and MRS+2% glycine at the same time. The MRS alone is normal dark brown/amber color. The one added with glycine becomes black in color. So my guess is there's some reaction probably pH change as mentioned by phage434. My strain is Lactobacillus casei and I used protocol by Posno 1991. I wanna give glycine a try first. May I know is it correct you added glycine powder and MRS powder together and autoclaved at lower temperature and still manage to get the normal color?



#5 El Crazy Xabi

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Posted 03 August 2015 - 06:20 PM

Is it black and cloudy? like a suspension? If so, if could be a product of Mn oxidation to MnO2. Although, I have my doubts that glycine can do that... otherwise is just that sugars and/or proteins were burnt.

 

I checked a couple of media handbooks and they don't say anything about a different autoclave cycle for MRS, i.e. they indicate the classic 15 min 121C 15psi. By the way did you add the correct ammount of powdered media? If you add too much, thw color is stronger.

Also, 2% glycine is correct? sure is not 2g/L?

 

Some of the questions are a bit silly but sometimes we don't see the most obvious



#6 Chris22

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Posted 04 August 2015 - 07:43 AM

I see, well that is a bit different so.

 

If it keeps occurring even after dropping to 115-deg, maybe consider making a high stock concentration of glycine, filter sterilizing and then ascetically adding to golden brown MRS after autoclaving.

 

Best

Chris







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