5 replies to this topic

[SuMi]

I want to grow Bacteroides fragilis in liquid broth and I can't find the specific growth conditions anywhere, i.e. broth, supplements and length of time that it needs to be grown for.

Is there any source where you can look up the conditions for different bacteria? Or does anyone know the condtions for Bacteroides?!

[klinmed]

SuMi, on Jun 26 2009, 04:29 PM, said:

I want to grow Bacteroides fragilis in liquid broth and I can't find the specific growth conditions anywhere, i.e. broth, supplements and length of time that it needs to be grown for.

Is there any source where you can look up the conditions for different bacteria? Or does anyone know the condtions for Bacteroides?!

B. fragilis is easy to grow in liquid culture. You can look in any basic bacteriological textbook (from the 1920´s on) for a plethora of media. Media as primitive as "nutrient broth" or LB are fine. The bug is anaerobic (modern evidence suggests that it is not too demanding in this regard).  Grow at 37 oC in culture flasks full of freshly autoclaved (degassed) medium without shaking. Growing this bug in liquid culture is as simple as growing E. coli. On agar, all you need is a basic anaerobic jar.

I refer you to:  Eley, A  and Greenwood, DF.  Comparative growth of Bacterioides species in various anaerobic culture media. J. Med Microbiol 19 (1995) 195-201

Hope this helps.

Edited by klinmed, 26 June 2009 - 11:29 AM.

[GeorgeWolff]

[Also consider oxyrase for plate culture.

[HomeBrew]

We use so-called "basal media", also known as PYG media (for peptone/yeast/glucose).  Here are the details:

Basal media (1 L)

20 g proteose peptone
5 g yeast extract
5 g NaCl

Combine ingredients in dH2O, aliquot to 100 ml bottles, autoclave, and allow to cool.

Before use, add (per 100 ml):

2 ml glucose stock
2 ml potassium phosphate stock
1 ml L-cysteine stock
100 µl hemin stock
50 µl vitamin K1 stock

Glucose stock (500 ml, 25%)

125 g glucose

Dissolve in a final volume of 500 ml dH2O.
Filter sterilize (0.22 µm filter).
Store at RT.

Potassium phosphate stock (500 ml, 25%)

125 g potassium phosphate (dibasic, K2HPO4)

Dissolve in a final volume of 500 ml in dH2O.
Filter sterilize (0.22 µm filter)
Store at RT

L-cysteine HCl stock (500 ml, 5%)

25 g cysteine (Sigma C1276)

Dissolve in a final volume of 500 ml dH2O.
Filter sterilize (0.22 µm filter)
Store at RT


Hemin stock (500 ml, 0.5%)

2.5 g hemin (Sigma H5533)

Dissolve in a final volume of 500 ml of 1 N NaOH
Filter sterilize (0.45 µm filter)
Store at 4°C.

Vitamin K1 stock (500 ml, ~5 µg/ml)**

2.5 ml vitamin K1 (Sigma V3501)

Dissolve in 500 ml of 100% ethanol.
Store at 4°C protected from light.

** The density of vitamin K1 = 0.984 g/ml at 25°C, thus this makes a 4.92 µg/ml solution.


For growing on solid media, we use BHI plates (37 g Brain Heart Infusion, 15 g agar per L).  After autoclaving, cool to 50 - 60°C, and add 1 ml per liter of the hemin stock (above) and 0.5 ml per liter of the Vitamin K1 stock (above).

I presume you realize that B. fragilis is an anerobe -- we grow it at 37°C in an atmosphere composed of 80% N2, 10% CO2, and 10% H2.  We have an anaerobic chamber, but we've also successfully used the BD BBL GasPak Plus system (see here) and a suitable container to grow B. fragilis and related species.

Hope this helps!

[SuMi]

Thanks everyone. That's a briliant answer Homebrew and much appreciated! Thanks again

[retrograde]

If I could ask an additional question on this topic;
Do you use an antibiotics to select for B.fragilis on the BHI plates on in liquid culture ?

Cheers