Name: XTC-2 MEDIUM
Creator: MediaDive
License: http://creativecommons.org/licenses/by/4.0/

Vol.

Cultivation of Diplorickettsia massiliensis #

Equipment needed: Filter

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
1 Cultivation of XTC-2 (from Xenopus laevis) cells
Leibovitz's L-15 medium	93	ml	-	-
Fetal bovine serum	5	ml	-	-
Tryptose-phosphate	2	ml	-	-
Glutamine	2	mM	-	-
2 Filter-sterilize (0.2 µm) and keep no longer than 4 weeks. Store at room temperature to facilitate detection of contamination.
3 Prepare a 25 cm² flask and seed cells according to standard protocols. Incubate at 28 - 32°C in hermetic flasks. Cells are easily detached mechanically, no enzyme is necessary. Medium should be changed once a week. When a confluent layer has formed, infection can be carried out.

Infection medium: #

100

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Leibovitz's L-15 medium	96	ml	-	-
Fetal bovine serum	2	ml	-	-
Tryptose-phosphate	2	ml	-	-
Glutamine	2	mM	-	-
1 Infect cells with 1 ml of Diplorickettsia stock solution (thawed quickly to 37°C). Incubate for 1 h at room temperature. Cultivation temperature: 28°C.
2 Up to 100% of the cells should be infected after several days. The infection level is evaluated after 3 days by Cytospin and Gimenez staining. Cytopathic effects may be visible after 3-5 days. Specific PCRs can be performed to verify the presence of diplorickettsiae.

Compound

Conc. [g/L]

Conc. [mM]

Leibovitz's L-15 medium

Fetal bovine serum

Tryptose-phosphate

phyla	1	1 strain
temperature	mesophilic	1 strain