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        Mycoplasma Broth Recipes-PPLO Broth

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        2305
           200 ml  1000 ml  4000 ml
         Mycoplasma broth base (Gibco)  4.5  22.5  90.0
         DNA (Sigma, Degraded free acid, type IV)  0.05  0.20  0.80
         Glass Distilled Water  142 ml  710 ml  2840 ml
         Phenol Red (1% aqueous)  0.4 ml  2.0 ml  8.0 ml
         For Agar Media using Noble Agar (Difco)  2.0  10.0  40.0
        Using Seaplaque Agarose (FMC corp. Marine Colloids Div.)  1.6  8.0  32.0

        Adjust pH to 7.8 - 8.0 with 1N NaOH

        Autoclave at 121°C for 15 min. at 15 lbs pressure, slow exhaust.

        Allow to cool for liquid medium, and bring to 50°C in a water bath for agar

        Add aseptically:

           200 ml  1000ml  4000 ml
         Agamma horse serum  40 ml  200 ml  800 ml
         25% Yeast Extract  10ml  50 ml  200 ml
         Penicillin G (100,000 units/ml)  1 ml  5 ml  20 ml
         Cefobid 2.5 mg/ml (50:50 EtOH:50 water)  2 ml  10 ml  40 ml
         50 % Dextrose (filter sterilized)  2 ml  10 ml  40 ml

        pH should be around 7.8-8.0

        Store at 4°C for one month or freeze for 3 months

        Aseptically dispense agar into petri dishes with a minimum depth of 4mm, store in sealed plastic bags at 4°C for 3 weeks.

        <center><strong><font>Mycoplasma Broth</font></strong></center>

         

           4 L 1 L 0.5 L
        Dist. Water 2640 ml 660 ml 330 ml
        PPLO Broth Base 84 g 21 g 10.5 g
        Glucose 20 g 5 g 2.5 g
        1% Phenol Red 8 ml 2 ml 1 ml

        Adjust pH to 7.8-8.2 & Autoclave

           4 L 1 L 0.5 L
        Yeast Extract 400 ml 100 ml 50 ml
        Penicillin (120,000 u/ml) 20 ml 5 ml 2.5 ml
        Thallium Acetate (1:50) 100 ml 25 ml 12.5 ml
        Horse Serum 800 ml 200 ml 100 ml

        readjust pH after addition of above! 

         Mycoplasma Agar

         Substitute Agar-PPLO for PPLO broth base

         4 L 1 L 0.5 L
        60 g 15 g 7.5 g

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