Name: Maintaining Wolbachia in Cell-free Medium (Video) | JoVE Journal
Uploaded: 2007-07-04T00:00:00.000+00:00
Duration: 13 min 33 s
Description: Learn to purify Wolbachia pipientis from Anopheles gambiae cells using glass bead lysis and differential centrifugation. This method enables cell-free culture and viability assessment for microbiology research.

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maintaining wolbachia in cell-free medium

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

My name is Courtney Ston. I work at the Johns Hopkins Bloomberg School of Public Health in the laboratory of Dr.Jason Rascon. Today we'll be doing bacterial purification from cultured insect cells.It's actually a very straightforward procedure. We start by harvesting our cells and then physically disrupting the eukaryotic host cell membranes using glass bead lysis. And then we use differential centrifugation to separate the bacteria from the host cell debris.And then we use a series of filtration steps to further remove host cell particles so that at the end we are left with just the purified bacterial pellet. This is actually one of the major requirements. It's a cell culture hood and it's used to keep everything sterile because cell culture is really easy to contaminate.We also need cell culture media, the cells themselves and we grow them in plastic flasks. We also need three millimeter blast beads, 50 ml conical tubes, and we need a centrifuge that is capable of a high speed centrifugation, approximately 18, 000 G to pellet our bacteria, but also capable of doing about a 3000 G or 2, 500 G to pellet our host cells. So the first thing we'll do is we'll start with Our cultured ce

maintaining-wolbachia-in-cell-free-medium

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Learn to purify Wolbachia pipientis from Anopheles gambiae cells using glass bead lysis and differential centrifugation. This method enables cell-free culture and viability assessment for microbiology research.

Maintaining Wolbachia in Cell-free Medium

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