5
$\begingroup$

I am wondering about the logistics of a simple experiment with say 3 types of alcohols, and various concentrations of each, with a control(s)

I would like to research the effects of alcohol on mitosis. Do I need any expensive equipment or is it do-able with standard equipment? I am looking specifically at what I expect to be a negative impact on cell growth. Potential cell death and slowing of mitosis.

"We expect mitosis to slow and stop entirely or become corrupted as the amount of alcohol used increases. Of the three alcohols we will be testing, we expect some to be more damaging than others. The 3 chosen alcohols, listed in order of their hypothesised toxicity are Methanol, Ethanol and 2-methyl-2-butanol."

I am basically trying to see if tertiary alcohols are safer due to ethanols main metabolites acetaldehyde & acetic acid being more harmful/toxic? Since Tertiary alcohols for example, aren't metabolised into aldehydes. Which makes them less toxic than ethanol in theory.

Now I am unsure as to which cells to use to test upon, I'm also unsure as to the legitimacy of my hypothesis. Budget, time and a lack of knowledge is against me. I am researching as much as possible as quick as possible. Thankfully I have help available soon or I would be totally out my depth. This is me out of practice in the field trying to get a head start and help a friend out as best I can. I'll be his assistant and feel thats more than appropriate.

I realise I can't test for too many things. I need one clear goal. If we choose to test certain cells over others do you have any suggestions? and why?

The liver processes ethanol into acetaldehyde & acetic acid. So I would need these chemicals potentially in order to properly test ethanols effects on cells?

I am so out of my depth I feel right now I'm hoping for a prod in the right direction.

The 3 chosen alcohols, listed in order of their hypothesized toxicity are Methanol, Ethanol and 2-methyl-2-butanol. - concentrations of each yet to be considered.

I suffer from dyslexia and my wording is bad at the best of times. But please realize this is a very first draft / ideas on the table. Any help at all is appreciated, apologies for my lack of knowledge in the field. I'm trying to refresh my studies from 10 years ago in 2 days.

$\endgroup$
4
$\begingroup$

Great idea! It seems that this research has been done before and your hypothesis is correct, but testing a wide variety of alcohols and testing your hypothesis for each one of them would be a great project.

Now, to do this, I would expect you would need to check the growth of the cells after certain periods of time. How would you do this? Well, the best device would be a hemocytometer. It looks like the price varies from 10.00 - 400.00 which is pretty cheap. Of course, there would be mitosis if there is growth during periods of time, which means there would be more cells after certain periods of time.

What cells to choose? I would choose something easy to work with, such as E.coli. It is safe and easy to work with. It needs minimal resources. Here's a link to a simple culture you can probably make at home:

http://makezine.com/projects/how-to-make-bacterial-brothagar/

Later, however, when you get good data, you can use mammalian and human cells to show that the research is relevant to human beings. However, mammalian cells are harder to grow because mammalian cells grow slower than bacteria and fungi and moreover contamination can cause problems. You would also need biosafety cabinets or laminar flow hoods and learn of aseptic and sterile technique.

$\endgroup$
4
$\begingroup$

Based on T Abraham's answer, a hemocytometer would work. However, a hemocytometer requires a microscope, but if you are in a cell lab, you probably have access to a microscope. I would recommend using mammalian cells instead of bacteria, they're larger and easier to see, and more relevant to human health. If you're interested in liver toxicity in particular, try getting HepG2 cells. These are human liver cancer cells, but extra paperwork might be involved because they can potentially carry hepatitis virus.

Should also use trypan blue staining with your hemacytometer to mark live and dead cells. Dead cells can't export the dye and turn dark.

Alternatively, an MTT assay is a nice way to measure cell viability if you have access to 96 well plates and plate readers. Using a cuvette based spectrophotometer is possible with this assay if you can grow your cells in larger containers, such as 10cm dishes.

The hemacytometer assay can get you total cells and percent of cells that are alive. The MTT assay tells you number of live cells. To get percent of cells that are alive you need a control that represents 100% viable.

Neither of these can tell you much about mitosis specifically, just the number of cells and percent viability.

$\endgroup$
  • $\begingroup$ I didn't realize about dead cells... but would it matter? Sure, it you want a quantitative and really accurate measure on whether the cells are undergoing mitosis, then maybe it matters... But if you are just thinking about whether mitosis is occurring, I think we can still get a reasonable estimate without worrying about dead cells... $\endgroup$ – TanMath Dec 13 '14 at 0:13
  • $\begingroup$ The trypan blue staining just gives you some more options for collecting data. You can still count total cells, but with the stain you can differentiate between live and dead. I find it also makes seeing the live cells easier when you have a slightly darker background. $\endgroup$ – user137 Dec 13 '14 at 0:25
3
$\begingroup$

For assaying effect of alcohol on cell growth:

    Prokaryotic Cells

  • Take ~5ml medium (LB for E.coli) in test-tubes/plastic tubes and add appropriate concentration of alcohol(s) in these.
  • Inoculate 1% bacteria from a starter culture (OD~0.6)
  • After different time intervals or a fixed time point take some culture, dilute and spread plate it (for each tube).
  • Count colonies (CFU)

    Eukaryotic Cells

  • You can try this in a 6-well plate.
  • Seed the cells such that they attain 60% confluency in 24 hours
  • After 24 hours: Change the medium and add the medium with appropriate concentration of alcohol
  • Wait for at least one division time (varies for different types of cells). 24 hours would be a fine interval for the treatment.
  • As mentioned in the other answer, you can use MTT assay or Trypan blue to detect viable cells (It would be difficult to differentiate inviable cells from the viable ones by just looking in the haemocytometer without staining)
  • You can use indirect assays similar in principle to bacterial spread plating; split the cells (if the cells are adherent) and inoculate a small percentage of them into another flask/plate. Count the number of cells in the new flask after 24hours.
  • You can also do flow cytometry of cells stained with propidium iodide to identify cells in different stages of mitosis.
  • For yeast you can simply grow them as you would grow bacteria and assess cell viability by either colony based methods or flow cytometry
$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.