狼友必上 5 comments
狼友必上 6 comments
Picture 1: T25, T75 and T175 Tissue culture flask, Taken from www.wwmponline.com/cart/images/T/flasks-01.jpg
Before the required number of host cells is seeded into the flask, medium has to be first added into the tissues culture flask.
This volume of medium, is just enough to cover the surface of each flask:
T25 flask requires 5ml
T75 flask requires 10ml
T175 flask requires 18ml
Then the required volume of cells (depending on the concentration no. of cells/ml) is added to the medium in the flask. The flask is held horizontally and rocked back and forth at a gentle angle of about 30 degrees. It is rock for a few times to evenly spread the cells so that the cells covers the whole surface of the flask as it begins to attach to the treated surface of the flask. These flask are incubated for 1 hour (at 27 degrees celcius, 5% carbon dioxide incubator) for the cells to attach.
The cells are incubated at 27 degrees celcius because it is a non-mammalian cell.
The flask cap has a unique 0.22micrometers membrane, which allows carbon dioxide to diffuse into flask.
After the cells attach it looks like this:
This is just one small part of a T175 flask under 10x magnification
Picture 2: Virus Host cell (Taken with permission)
After the cells has attached to the flask treated surface, a certain volume of the medium is pipetted out to concentrate the cells in a smaller volume. This would allow the virus to attach to the cells more easily later, when the virus is added.
T25 leave behind 1ml
T75 leave behind 3ml
T175 leave behind 5ml
Add ~100microliters of the virus stock (the current one that we have) to the concentrated cells. The volume to add can vary, depending on how old the virus stock is. A older virus stock may have a lower virus titer (pfu/ml), so we can add more volume such as ~200microliters .
These flask are then put on a rotator (for 2 hours) which continuously rock back and forth to allow the virus to find and attach to the cells.
After the virus attach to the cells (assuming, because we can't see any changes yet), we add more medium to each flask to maintain the infected cells in the flask.
T25 add 5ml
T75 add 10ml
T175 add 18ml
The infected cells are incubated for 72 hours (at 27 degrees celcius, 5% carbon dioxide incubator), so that the cells are now in the very late infection cycle.
These are how the cells look like during the very late infection cycle: under 10x magnification with 1.6x aperture
Signs to take note of: Cells look swollen, has black dots in the centre of the cells, uneven surfaces of the cells and some cells have even fuse together.
Picture 3: Infected virus host cells after 72 hours (Taken with permission)
When these signs are observed in the very late infection cycle, we assume that majority of the viruses has bud out of the infected host cells, (virus are too small to be seen under our phase contrast microscope. It can only be seen unless a high power electron microscope is used) and it is very likely that the cells are going to lyse soon. Virus has to be collected before the cells lysed. When the cells lysed, enzymes released can cause break down of proteins found on the surface of the virus and lowers the virus titer greatly.
Since the cells are attached to the flask surface, as the viruses buds out of the infected cells, it is released into the medium. Therefore, virus can be collected by pouring the medium from the flask into a 50ml tube. The infected cells remains attached to the flask surface and are discarded.
This tube is spin down at 1000rcf for 10minutes to remove any cells debris (from a small proportion of infected host cells that may have detached). The supernatant is collected in a new 50ml tube without disturbing the pellet of cells debris. The new tube is wrapped with non-shiny side of the aluminium foil (because viruses are light sensitive), and stored at 4 degree celcius for future use.