[所屬分類:培養(yǎng)基配方] [發(fā)布時間:2021-4-12] [發(fā)布人: ] [閱讀次數(shù):] [返回]

Histoplasma capsulatum Agar

培養(yǎng)基配方

Composition per liter:
Agar ............................................................................................15.0g
Glucose .......................................................................................10.0g
Potato starch..................................................................................2.0g
α-Ketoglutaric acid.......................................................................1.0g
L- Cystine·HCl·H 2 O .......................................................................1.0g
Glutathione, reduced.....................................................................0.5g
L- Asparagine .................................................................................0.1g
L- Tryptophan...............................................................................0.02g
Solution 1...............................................................................250.0mL
Solution 3.................................................................................40.0mL
Solution 2.................................................................................10.0mL
Solution 4.................................................................................10.0mL
Solution 8.................................................................................10.0mL
Solution 5...................................................................................1.0mL
Solution 6...................................................................................0.1mL
Solution 7...................................................................................0.1mL
pH 6.5 ± 0.2 at 25°C
Solution 1:
Composition per liter:
KH 2 PO 4 .........................................................................................8.0g
(NH 4 ) 2 SO 4 .....................................................................................8.0g
MgSO 4 ·7H 2 O..............................................................................0.86g
CaCl 2 , anhydrous........................................................................0.08g
Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Store at 5°C.
Solution 2:
Composition per liter:
FeSO 4 ·7H 2 O..................................................................................5.7g
MnCl 2 ·6H 2 O..................................................................................0.8g
NaMoO 4 ·2H 2 O............................................................................0.15g
HCl, concentrated ......................................................................1.0mL
Preparation of Solution 2 : Add the 1.0mL of concentrated HCl to 100.0mL of distilled water in a 1.0L volumetric flask. Dissolve each component completely in the sequence given. Bring volume to 1.0L with distilled/deionized water. Store at 5°C. Discard if red color or red precipitate appears.
Solution 3:
Composition per 100.0mL:
Casein, acid-hydrolyzed, vitamin-free........................................10.0g
Preparation of Solution 3:  Add casein to distilled/deionized water and bring volume to 100.0mL. Do not use enzymatically digested casein.
Solution 4:
Composition per liter:
Calcium pantothenate ...................................................................0.2g
Inositol..........................................................................................0.2g
Riboflavin.....................................................................................0.2g
Thiamine·HCl...............................................................................0.2g
Nicotinamide.................................................................................0.1g
Biotin..........................................................................................0.01g
Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Store at −20°C.
Solution 5:
Composition per 100.0mL:
Hemin ...........................................................................................0.2g
NH 4 OH, concentrated................................................................0.3mL
Preparation of Solution 5:  Add hemin to approximately 30.0mL of distilled/deionized water. Add NH 4 OH. Mix thoroughly until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5°C.
Solution 6:
Composition per 10.0mL:
DL- Thioctic acid..........................................................................0.01g
Ethanol (95% solution)............................................................10.0mL
Preparation of Solution 6:  Add  DL- thioctic acid to 10.0mL of ethanol. Mix thoroughly. Store solution at −20°C.
Solution 7:
Composition per 10.0mL:
Coenzyme A...............................................................................0.01g
Na 2 S·5H 2 O (0.05% solution).....................................................0.2mL
Preparation of Solution 7:  Prepare Na 2 S·5H 2 O solution in freshly boiled distilled/deionized water. Add coenzyme A to 9.8mL of distilled/deionized water. Mix thoroughly. Add freshly prepared Na 2 S·5H 2 O solution. Mix thoroughly. Store the solution at −20°C.
Solution 8:
Composition per 100.0mL:
Oleic acid......................................................................................0.1g
Preparation of Solution 8: Add oleic acid to 50.0mL of distilled/deionized water. Adjust pH to 9.0 with NaOH. Gently heat until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5°C.
Preparation of Medium: Add components—except agar, potato starch, and solution 8—to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 6.5 with 20% KOH solution. Filter sterilize. In a separate flask, add potato starch to 50.0mL of distilled/deionized water. Add the starch solution to 450.0mL of boiling distilled/deionized water. Add 10.0mL of solution 8 and the agar. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 70°C. Aseptically combine the two sterile solutions. Pour into sterile Petri dishes or distribute into sterile tubes.
Use: For the cultivation and maintenance of Histoplasma capsulatum in the mycelial phase.