Lab skills quiz – Tissue / Cell Culture

Why is CO2 often used in an incubator to culture mammalian cells?
to control cell growth
to control the pH
yes, most culture medias contain a bicarbonate buffering system that relies on CO2 to control pH. CO2 independent media does exist for occasions such as time-lapse microscopy when you may not have a CO2 source available
to control oxygen consumption
to control or prevent viral/bacterial contamination
Why should virkon NOT be left in the tissue culture hood for extended periods?
It is highly flammable
The hood will explode if you close the night door
It will contaminate the circulating air
It is corrosive and will damage the metal
Oh, no! One of your dishes of mammalian cells has bacterial contamination! What is the most likely reason?
You exposed the cells to trypsin for too long
You fed the cells too often
You touched the dish
You forgot to add penicillin and streptomycin to your media
Which of these cell types poses the greatest risk to a user?
MDCK – canine kidney epithelial cells (immortalised)               
primary human retinal epithelial cells
Yes, cells isolated from a human are more likely to be carrying infectious material that could harm humans than other cell types. Immortalised human cells are likely to have been in culture for a long time and so are slightly lower risk.
C2C12 – mouse myoblast immortalised line 
  hTCEpi – human corneal epithelial cell (immortalised line)
primary mouse myoblasts transduced with adenovirus 72 hours previously
You should treat these with care however, adenovirus are short-lived in culture and therefore there shouldn’t be infectious agents present at this time point. Most modern adenoviruses are designed to be replication deficient except in specific cell types. Treat with care, but there are more dangerous options on this list
Why could using too much Ethanol actually lead to increased probability for contamination?
EtOH is not effective at killing bacteria when used a too high a volume
Evaporation of the excess EtOH would lead to salt build up
Excess liquid would provide an entry route for microorganisms
EtOH doesn’t kill everything – anything it doesn’t kill would be able to use the excess liquid as an entry route. Don’t go crazy with spraying down everything!
It is corrosive

over typs

You are working with an epithelial cell line. Yesterday when you split your cells they looked good, today they look like the image above. What could have gone wrong?
They spent too long in trypsin
Yes, the cells haven’t reattached, they’ve stayed round. Trypsin eats away at cell surface proteins, if you leave it on for too long the cells may not be able to re-attach and for a lot of cell lines the cells will die i.e. will never spread out again. Easy done but hopefully you have kept some spares ‘cos these guys aren’t going to recover.
You used the wrong media
Probably not, likely the wrong media would either cause the cells to die or just not divide, these cells are round and floating, probably a different explanation is more likely.
You spun the cells down too fast
probably not, likely the cells would burst open if you did this and you would have lots of debris floating around.
You have introduced bacterial contamination
bacteria would be visible in a few other ways; as small, moving orgs. in addition to your cells, they would change the appearance of the media, the mammalian cells would either be dead or look relatively normal depending on which stage you caught the contamination at. Either way, there would probably be floating debris as well as cells.
Which of the following should not be used in most TC hoods? Select all that apply 
most hoods don’t have appropriate filters to protect the environment (i.e. the air that you are breathing!) from vapours. You need a fume hood or a tissue culture hood that has a charcoal filter
most hoods don’t have appropriate filters to protect the environment (i.e. the air that you are breathing!) from vapours. You need a fume hood or a tissue culture hood that has a charcoal filter
Ponceau S
  What is the difference between waste from genetically modified tissue culture and non-GM waste?
GM waste glows green under fluorescent lighting
General TC waste does not need to be autoclaved
Virkon does not kill GM material
GM tissue culture waste disposal requires additional labelling and other procedures
I’m trying to make this quiz as general as possible. In general waste from GM cultures is treated slightly differently. It will be covered by GMM risk assessment instead of general risk assessment and in that GMM assessment you will define the disposal routes. In our institute this involves additional labelling, and more of the waste going to incineration. Simple message; check your local procedures
GM waste is never liquid

What is the time required for virkon treatment to kill living material in most cultures (note some specific exceptions do apply)?
5 mins
20 mins
1 hour 
1 day 
The longer the better 

You need to pipette a 50μl from a 500ml stock bottle what should you do?
Use a 1000μl pipette with blue tip
1000μl pipettes aren’t accurate for this small a volume.
Use a 200μl pipette with yellow tip
This is risky. When you enter the stock bottle the probability of you touching the sides of the neck or elsewhere on the bottle with the barrel of your pipette are quite high. If you do that its likely you will contaminate the stock, and therefore any subsequent cultures
Use a stripette to remove small volume from the stock bottle into a small tube then use a 200μl Gilson with yellow tip
This will take marginally longer than going direct but is a safer option with respect to probability of introducing contamination.
Use a stripette to remove 1ml from the stock solution then add 1 drop to your plate


You count your cell solution and your average count in the red sized squares (1mm2) is 50. Your protocol calls for 200,000 cells per well to be plated and you will need 6 wells in a total volume of 12 ml. How much of your cell solution and how much media do you need?
2.4 ml cell solution, 9.6 ml media
First calculate your concentration of your cell solution.  You have 50 cells / 100 nl = 500 cells / μl = 500,000 cell/ml. Then work out how many cells total that you need. 200,000 x6 = 1,200,000. Divide the two. 12/5 = 2.4 ml. Then 12   2.4 = volume of media = 9.6 ml
400 μl cell solution, 11.6 ml media
6 ml cell solution, 6 ml media
  800 μl cell solution, 11.8 ml media
8.2 ml cell solution, 3.8 ml media
It’s Friday and you are splitting your stock cultures. On Monday you will need at least 9 million cells ready to plate for your experiments. You also want to have spare to continue your culture for the following week. When they are at optimal densities, your cells double approximately every 24 hours. A confluent flask can hold 3 million cells but your cells will only divide efficiently if they are between 5% and 80% confluent.   How should you plate up your cells?
5 flasks with 280,000 per flask
OK, let’s start by working out what the optimal concentration ranges we can work with are; 10% of 3 million = 150,000. 80% of 3 million = 2.4 million. We don’t want to go out of this range; above or below and our cells will divide very slowly, also if we go too high our cells will probably begin to be contact inhibited, may die off or differentiate. That could change our culture and ruin our experiment. With these numbers we can say that on Monday we need 9/2.4 million flasks minimum, rounding up = 4 flasks. This would give us 1 million spare. Working backward, for our cells to be plated on Friday and ready on Monday we would want 1.2 million per flask on Sunday, 600K on Saturday, so 300K on Friday. Therefore 4 flasks of 300K/flask would give us our 10 million on Monday. But we don’t want to risk going over our optimal conc so a fifth flask with 250K per flask, would give us enough plus some spares in case things progressed a little slower than planned. We could set up more flasks than this but it would be a waste of plastic (~£2 per flask) and media (varies, could be ~£10/flask)
4 flasks, 320,000 per flask
3 flasks, 120,000 per flask
5 flasks, 2,000,000 per flask
10 flasks, 200,000 per flask
You are designing an experiment where you will determine if there is a correlation between the age of the cell donor to the number of colonies formed from the isolated stem cells. What sort of statistical test would be appropriate for your power analysis?
linear regression
you are comparing one continuous variable (donor age) against another continuous variable (colonies formed) therefore a regression analysis is appropriate here
logistic regression
I always get this one mixed up too! Logistic regressions are for when the outcome variable has two measures (eg if you your measurement was i) do form colonies ii) don’t form colonies, rather than how many)
1 way ANOVA
This would be if you were comparing each donor against each other, you could do that but it wouldn’t really answer your question
T test
If you decided to break your donors into “old” and “young” then a t test would be the right choice, however, you would need a reason for why you chose the split point. Assuming you can get enough donors there would be a better option.


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