Large-scale (up to 20L) Culture
The Intestinal Microbiology Core has large-scale bacterial culture and processing capability using the NewBrunswick Bioflow 4500 fermentor. 20L cultures of aerobes, anaerobes, or microaerophilic bacteria are readily produced. Our cell press, originally purchased for lysis of large-scale culture pellets to release cytoplasmic proteins, can also be used to process small batches when sonication is an undesirable option.
A Pellicon tangential flow filter apparatus is used for rapid harvest of bacteria from large culture volumes, and in parallel with ultrafiltration membranes, to reduce culture volumes. This rapidly concentrates culture supernatants from 20L to 500ml, facilitating purification of secreted proteins. The cell press and the Pellicon apparatus are available to scientists for either small- or large-scale cultures.
Scientists who wish to order large-scale cultures should contact the core laboratory facility director to discuss scheduling. Cultures>10L require prior approval from the Institutional Biosafety Review Committee. We try to deliver within two weeks following receipt of the strain. The culture can be delivered either as a cell pellet or as the supernatant following ultracentrifugation of the cell lysate. If the desired product is in the culture supernatant, we can reduce the volume to 500ml and wash with any buffer desired.
We require a plate with the organism of interest, IPTG if required for induction, and a protocol that should include:
- Growth conditions and medium to be used
- Information regarding antibiotic resistance
- Desired culture optical density prior to induction
- Duration of incubation post induction
- Buffer for wash, if desired
- Preferred lysis buffer, and protease inhibitors, if desired
Culture of Pathogens
The core laboratory in Tupper 822 contains all the instrumentation required for culture, including an autoclave, incubators, water bath shakers, and the 20L fermentor. The facility is certified at a P2/ large culture level of containment, and appropriate lab practices are strictly observed.
Organisms provided over the past five years include enterohemorrhagic E. coli, Vibrio cholera, Yersinia pseudotuberculosis, Legionella pneumophila, Clostridium difficile, Hemophilus influenzae, and Helicobacter pylori. The latter organism requires special chambers for the microaerophilic environment, and 10 such chambers are available in the core.
In addition, the core has the equipment and expertise to culture Giardia lamblia and Entamoeba histolytica in large culture volumes. Stocks of both these parasites are maintained in liquid nitrogen. Requests for culture should be made to the core laboratory facility director; the price is based on the cost of the medium used.
Storage and Retrieval
The core maintains a large collection of bacteria (frozen stocks) available as a resource for all investigators. This library is on a database on the core computer and can be sorted using a number of identification properties. The basic collection includes pathogenic bacteria, several standard host strains, and commonly used plasmid vectors. The core also maintains microorganisms for --these are the sole repository of bacteria for some laboratories, or are a second storage site to prevent loss of valuable strains.
Requests for bacteria to be delivered on plates can be made by phone or e-mail, and delivery is usually on the following day. For bacterial or plasmid storage we require a plate streaked for isolation. There is no charge for this service.