Mycoplasma Agar (ATCC Medium 555)

[所属分类:培养基配方] [发布时间:2021-6-15] [发布人:网站管理员2] [阅读次数:] [返回]

Mycoplasma Agar
(ATCC Medium 555)

山东拓普生物工程有限公司 培养基配方 http://www.topbiol.com

Composition per 103.0mL:

Noble agar.....................................................................................0.7g
Hartley’s digest broth...............................................................30.0mL
Pig serum .................................................................................20.0mL
Enzymatic hydrolysate of lactalbumin ....................................10.0mL
Hanks’ balanced salt solution, 10X ...........................................4.0mL
Fresh yeast extract solution........................................................2.0mL
Phenol Red (0.25% solution).....................................................1.0mL
pH 7.4 ± 0.2 at 25°C
Hartley’s Digest Broth:
Composition per 10.0L:
Ox heart..................................................................................3,000.0g
Pancreatin....................................................................................50.0g
Na 2 CO 3  , anhydrous (0.8% solution)............................................5.0L
HCl, concentrated ....................................................................80.0mL
pH 7.5 ± 0.2 at 25°C
Preparation of Hartley’s Digest Broth: Finely mince the ox
heart. Add the meat to 5.0L of distilled/deionized water. Gently heat
and bring to 80°C. Add the 5.0L of Na 2 CO 3  solution. Cool to 45°C.
Add pancreatin and maintain at 45°C for 4 hr while stirring. Add the
HCl and steam at 100°C for 30 min. Cool to room temperature. Adjust
pH to 8.0 with 1N NaOH. Gently heat and bring to boiling. Continue
boiling for 25 min. Filter while hot. Cool to room temperature. Adjust
pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C.
Pig Serum:
Composition per 100.0mL:
Pig serum...............................................................................100.0mL
Preparation of Pig Serum: Adjust pH of pig serum to 4.4 with
sterile 1N HCl. Do not let pH go below 4.2. Let serum stand at 4°C for
18-20 hr. Adjust pH to 7.0 with sterile 1N NaOH. Centrifuge at 9000
rpm for 20 min. Discard pellet. Filter supernatant solution through a
0.2μm membrane. Store at −70°C.
Fresh Yeast Extract Solution:
Composition per 100.0mL:
Baker’s yeast, live, pressed, starch-free......................................25.0g
Preparation of Fresh Yeast Extract Solution : Add the live Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Enzymatic Hydrolysate of Lactalbumin:
Composition per 100.0mL:
Enzymatic hydrolysate of lactalbumin .........................................5.0g
Preparation of Enzymatic Hydrolysate of Lactalbumin: Add
enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff-
ered saline, 1X, pH 7.0.
Phosphate Buffered Saline Solution, 1X:
Composition per liter:
NaCl..............................................................................................8.0g
Na 2 HPO 4 ·7H 2 O...........................................................................2.16g
KCl................................................................................................0.2g
KH 2 PO 4 .........................................................................................0.2g
MgCl 2 ·6H 2 O .................................................................................0.1g
CaCl 2 .............................................................................................0.1g
Phosphate Buffered Saline Solution, 1X: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Hanks’ Balanced Salt Solution, 10X:
Composition per liter:
NaCl............................................................................................80.0g
Glucose.......................................................................................10.0g
KCl................................................................................................4.0g
CaCl 2 .............................................................................................1.4g
MgCl 2 ·6H 2 O .................................................................................1.0g
MgSO 4 ·7H 2 O................................................................................1.0g
Na 2 HPO 4 ·7H 2 O.............................................................................0.9g
KH 2 PO 4 .........................................................................................0.6g
Preparation of Hanks’ Balanced Salt Solution, 10X: Add
components to distilled/deionized water and bring volume to 1.0L. Mix
thoroughly.
Preparation of Medium: Combine components, except agar, in the
following order: Hanks’ balanced salt solution, 10X, Phenol Red, Hart-
ley’s digest broth, pig serum, enzymatic hydrolysate of lactalbumin,
and fresh yeast extract solution. Mix thoroughly. Adjust pH to 7.4 with
1N NaOH. Filter sterilize through a 0.2μm membrane. Add 0.7g of No-
ble agar to 36.0mL of distilled/deionized water. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to 56°C. Warm basal medium to 56°C.
Aseptically combine the two solutions. Pour into sterile Petri dishes or
distribute into sterile tubes.
Use: For the cultivation of Mycoplasma species.