Axenic Dimastigella Medium

[所属分类:培养基配方] [发布时间:2019-11-15] [发布人:] [阅读次数:] [返回]

山东拓普生物工程有限公司

Shandong Tuopu Biol-Engineering Co.,Ltd

Axenic Dimastigella Medium
Composition per liter:
Sonneborn's base Paramecium medium................................980.0mL
Vitamin solution.......................................................................10.0mL
Heat-killed bacterial suspension..............................................10.0mL
Sonneborn's Base Paramecium Medium:
Composition per liter:
Rye grass cerophyll.......................................................................2.5g
Na2HPO4 .......................................................................................0.5g
Preparation of Sonneborn's Base Paramecium Medium: Add
cerophyll to distilled/deionized water and bring volume to 1.0L. Mix
thoroughly. Gently heat and bring to boil. 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 vol-
umes into tubes. Autoclave for 15 min at 15 psi pressure–121°C.
Source: Cerophyll can be obtained from Ward's Natural Science Es-
tablishment, Inc. Dairy Goat Nutrition distributes Grass Media Cul-
ture, which is equivalent. Cereal Leaf Product from Sigma Chemical is
similar to cerophyll.
Vitamin Solution:
Composition per 100.0mL:
Calcium  D -(+)-pantothenate........................................................0.05g
Nicotinamide...............................................................................0.05g
Pyridoxal·HCl.............................................................................0.05g
Riboflavin ...................................................................................0.05g
Pyridoxamine·HCl ....................................................................0.025g
Folic acid...................................................................................0.025g
Thiamine·HCl .............................................................................0.15g
Biotin ...................................................................................0.0125mg
DL -Thioctic acid.........................................................................0.5mL
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize. For long-term storage, preserve under nitrogen at −20°C.
Heat-Killed Bacterial Suspension:
Composition per 100.0mL:
Heat-killed Klebsiella pneumoniae.......................................10 12 cells
Preparation of Heat-Killed Bacterial Suspension: Inoculate a
loopful of Klebsiella pneumoniae subsp. pneumoniae ATCC 27889
into 5.0mL of nutrient broth. Incubate at 35°C overnight. Transfer
0.5mL aliquots of nutrient broth with bacterial suspension to each of
ten 1.0L Erlenmeyer flasks, each containing 250.0mL of nutrient broth.
Incubate cultures at 35°C for 24 hr. Aseptically transfer bacterial sus-
pensions to 500.0mL sterilized screw-capped centrifuge bottles. Fill
bottles with a maximum of 400.0mL. Centrifuge in a refrigerated cen-
trifuge at 5000 rpm for 10 min. Decant supernatant and resuspend pel-
lets in Page's balanced salt solution. Pool all suspensions in a single
bottle. Centrifuge in a refrigerated centrifuge at 5000 rpm for 10 min.
Discard supernatant and resuspend pellet in Page's balanced salt solu-
tion. Final volume of cell suspension should be approximately
400.0mL. Decant supernatant and resuspend pellets in Page's balanced
salt solution. Centrifuge in a refrigerated centrifuge at 5000 rpm for 10
min. Discard supernatant and resuspend pellet in Page's balanced salt
solution. Decant supernatant and resuspend pellets in Page's balanced
salt solution. Final volume of cell suspension should be approximately
400.0mL. Centrifuge in a refrigerated centrifuge at 5000 rpm for 10
min. Decant supernatant and resuspend pellets in Page's balanced salt
solution. Final volume this time should only be 100.0mL. Agitate to
ensure that cells are thoroughly suspended. Transfer to a 125.0mL
screw-capped serum bottle and bring volume to 100.0mL with Page's
balanced salt solution. Serially dilute the suspension to a dilution of
10 −9 . Plate 0.1mL aliquots in triplicate from the 10 −7  to 10 −9 dilution
tubes. Place the aliquots in the center of 100.0mm Petri plates contain-
ing nutrient agar and spread evenly over the surfaces with a sterile glass
rod. Incubate plates at 35°C overnight. Place the 125.0mL screw-
capped serum bottle containing the bacterial suspension in 100.0mL of
Page's balanced salt solution into a 60°C water bath. Make sure that the
liquid level of the water bath is above that of the suspension in the bot-
tle. At 10-min intervals, swirl the bottle. Incubate for a total of 30 min.
Allow the bottle to cool to room temperature. This treatment should kill
all bacterial cells. Determine bacterial cell concentration from the seri-
al dilution plates. Adjust the concentration of the heat-killed bacteria to
10 10 cells per mL. As a check that the cells are not viable, add 3 drops
of the cell suspension prepared in step 10 to the edge of a 100.0mm Pe-
tri plate containing nutrient agar. Hold the plate vertically to allow the
drops to move to the opposite edge. Incubate plate at 35°C for 48 hr.
Nutrient Broth:
Composition per liter:
Pancreatic digest of gelatin...........................................................5.0g
Beef extract...................................................................................3.0g
Preparation of Nutrient Broth: Add components to distilled/de-
ionized water and bring volume to 1.0L. Mix thoroughly. Distribute
into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Page’s Balanced Salt Solution:
Composition per liter:
Solution 1...............................................................................500.0mL
Solution 2...............................................................................500.0mL
Solution 1:
Composition per 500.0mL:
Na2HPO4 .....................................................................................2.84g
KH2PO4 .......................................................................................2.72g
Preparation of Solution 1: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to 25°C.
Solution 2:
Composition per 500.0mL:
MgSO4 ·7H2O.............................................................................8.0mg
CaCl2 ·2H2O ...............................................................................8.0mg
NaCl............................................................................................0.24g
Preparation of Solution 2: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to 25°C.
Preparation of Page’s Balanced Salt Solution: Aseptically com-
bine 500.0mL of solution 1 with 500.0mL of solution 2.
Nutrient Agar:
Composition per liter:
Agar............................................................................................15.0g
Pancreatic digest of gelatin...........................................................5.0g
Beef extract...................................................................................3.0g
Preparation of Nutrient Agar:  Add components to distilled/de-
ionized water and bring volume to 1.0L. Mix thoroughly. Gently heat
and bring to boiling. Distribute into tubes or flasks. Autoclave for 15
min at 15 psi pressure–121°C. Pour into sterile Petri dishes.
Preparation of Medium: Aseptically add 10.0mL of the vitamin
solution to 980.0mL of Sonneborn's base Paramecium medium. Mix
thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
25°C. Aseptically distribute 10.0mL aliquots into T-25 tissue culture
flasks. Add 0.1mL of the heat-killed bacterial suspension to each flask.
Inoculate immediately with Dimastigella species.
Use: For the cultivation of Dimastigella trypaniformis and other
Dimastigella species.