Hexamita Medium

[所属分类:培养基配方] [发布时间:2021-4-6] [发布人: ] [阅读次数:] [返回]

Hexamita Medium

培养基配方

Composition per liter:
TYGM-9 medium..................................................................250.0mL
Sonneborn's Paramecium medium ........................................750.0mL
TYGM-9 Medium:
Composition per liter:
NaCl..............................................................................................7.5g
K 2 HPO 4 .........................................................................................2.8g
Casein digest.................................................................................2.0g
Gastric mucin................................................................................2.0g
Yeast extract..................................................................................1.0g
KH 2 PO 4 .........................................................................................0.4g
Bovine serum, heat inactivated................................................30.0mL
Rice starch solution..................................................................30.0mL
Tween™ solution.......................................................................0.5mL
pH 7.4 ± 0.2 at 25°C
Tween™ Solution:
Composition per 100.0mL:
Tween™ 80..............................................................................10.0mL
Preparation of Tween™ Solution: Add Tween™ 80 to absolute ethanol and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.
Rice Starch Solution:
Composition per 100.0mL:
Rice starch.....................................................................................5.0g
Phosphate-buffered saline solution........................................100.0mL
Phosphate-Buffered Saline Solution:
Composition per liter:
NaCl..............................................................................................9.0g
Na 2 HPO 4 ·7H 2 O.........................................................................0.795g
KH 2 PO 4 .....................................................................................0.114g
Preparation of Phosphate-Buffered Saline Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.
Preparation of Rice Starch Solution: Heat sterilize rice starch at 150°C for 2 hr. Aseptically add 100.0mL of sterile phosphate-buffered saline solution. Mix thoroughly. Use immediately.
Preparation of TYGM-9 Medium: Add components, except rice starch solution, Tween™ solution, and bovine serum, to distilled/deionized water and bring volume to 939.5mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 30.0mL of sterile bovine serum, 30.0mL of sterile rice starch solution, and 0.5mL of sterile Tween™ solution. Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks.
Sonneborn's Paramecium Medium:
Composition per liter:
Solution 1......................................................................................1.0L
Klebsiella pneumoniae
cultured on solution 2.......................................................variable
Solution 1:
Composition per liter:
Rye grass cerophyll.......................................................................2.5g
Na 2 HPO 4 .......................................................................................0.5g
Source: Cerophyll can be obtained from Ward's Natural Science Establishment, Inc. Dairy Goat Nutrition distributes Grass Media Culture, which is equivalent. Cereal Leaf Product from Sigma Chemical is similar to cerophyll.
Preparation of Solution 1: Add cerophyll to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Boil for 5 min. Filter through Whatman #1 filter paper. Add 0.5g of Na 2 HPO 4 . Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Distribute 10.0mL volumes into tubes. Autoclave for 15 min at 15 psi pressure–121°C.
Solution 2:
Composition per liter:
Agar............................................................................................20.0g
Yeast extract..................................................................................4.0g
Glucose.......................................................................................0.16g
Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute 5.0mL into tubes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position.
Preparation of Sonneborn's Paramecium Medium: Inoculate the surface of agar slants of solution 2 with a culture of Klebsiella pneumoniae. Incubate at 37°C for 24–48 hr. Scrape cells from the surface of the agar slants and add to 10.0mL of solution 1. Incubate at 30°C for 24 hr.
Preparation of Medium: Aseptically combine 3.0mL of sterile TYGM-9 medium with 9.0mL of Sonneborn's Paramecium medium in 16 × 125mm screw-capped test tubes.
Use: For the cultivation of Hexamita inflata, Hexamita pusilla, Mastigamoeba invertens, and Trepomonas agilis.