Lab Bites">Lab Bites

As a rule of thumb: The lower the temperature the longer the storability. At 4 °C sera can be stored for 1-2 weeks. At -20 °C, one year; at -40 °C, two years; and at -80 °C three years is possible.

A quick indicator of quality of your FBS is hemoglobin content. Check the analysis spec sheet - this will be higher in lower quality FBS.

Do you spray your pipettes and other bits right before using them in your safety cabinet?
Ethanol actually works best over a longer period of time, so try spraying your items with ethanol about 20 minutes before you begin working, such as when you can put your media into the water bath.

Record down the contamination type to look for trends.

Try doing a gram stain:

Gram negative rods are likely to be water bourne pathogens, indicating a water-bath issue (or could be E.coli, which indicates bad aseptic technique, or if someone has food poisoning).

Gram positive rods or cocci may be environmental pathogens and therefore could have originated from clumsy handling.

Gram negative cocci include respiratory symptoms, meningitis, and sexually transmitted disease... Yikes!

Are you worried that your cells are getting cold when you work in your safety cabinet?
Place your cells onto a (clean) polystyrene lid in your safety cabinet, rather than on the cold metal surface so their temperature does not drop as sharply as they normally would.

Do you warm up your media in small aliquots of what you actually intend to use?
Rather than heating a whole bottle of media, heating up smaller amounts:
- Is faster
- Reduces media degradation
- Is safer from contamination
- And is easier to pipette from!

You can use a commercial stain.

Or you can save money and time by using food colouring liquid instead - add it to your cells for 20 mins, then wash once in water and view your cells under a microscope to see it's whole morphology... and in any food colour of your choice!  The stain comes off when you do extra washes with PBS and you can do other staining/immunolabelling protocols.

Your water bath is a common source of contamination.  Keep it clean and wash it weekly.  If you can't afford to use commercial cleaning products like Aqua Stabil that keep microbes away, you can add methanol to 1% or enough copper sulfate to make the water blue.

When using little petri dishes, it's much easier for you to carry them around when you put these petri dishes within larger ones.  They are also less likely to fall through the stage of your microscope when you want to view your cells!

Freezing cells down, but the cell-freezing box has gone for a walk?  Try this for plan B.  Cool cells in iced water in a fridge for 30 min, then place them into a closed cardboard or Styrofoam box to -80°C.  Or at least place them in a beaker with paper towels.

...Don't make a habit out of this technique though!

When seeding cells into a multiwell plate (like a 96-well plate), pipette your cell suspension into the middle of the well so it does not cause a vortex, which causes the cells to concentrate to certain parts of your well, such as the sides.  Don't shake your plate afterwards either, as it also creates a vortex.

Are plastic surfaces like your tissue culture flasks, adherent for cells?

To tell if the surface is good for adherent cells, add a drop of water and see if it sits and spreads out onto the surface.  If the surface isn't good for adherent cells, the water droplet will bead up like oil in water.

DMSO is often added to media to help freeze down cells.

But be careful!  Adding DMSO to media creates an exothermic reaction, heating up your media.

If you pre-heat your media to 37°C, your media could heat up to 40°C.  Remember that your cells are less tolerant to heat shocks than to cold shocks, so this could kill some of your cells even before they start getting frozen down!

So if you tend to pre-heat your media, try to offset this by giving it time to equilibrate back to 37°C.

The optimum temperature for cells is 37.5°C.

But it is safer to set your incubator at 37.0°C.  This is because cells are more likely to survive cold shocks rather than heat shocks.  For example, cells may survive for days at 30°C, but may die within hours at 40°C.

 When passaging cells, standard protocol tells you to wash your suspended cells with media to remove remaining drops of trypsin, then to spin the cells into a pellet and pour off the supernatant.  And then you resuspend this pellet to count your cells with a hemocytometer.

 

...Why don't you try taking a sample of cells out first before spinning your cells into a pellet and do a cell count then?

 This way you can already work out the number of cells you have before resuspending the pellet.

When staining your cells with fluorescent antibodies, sterile filter the diluted fluorescent antibodies to remove the non-specific “blobs” that show up.