shirosands

Well the issue has been resolved but it wasn't effortless... I was not budging on it for safety reasons, and after much arguing she agreed to comply for legal reasons.

According to what I found, I believe that if someone wanted to sue her or the institution for not wearing gloves with tissue culture, they could.  To be in line with current Federal regulations, it is probably illegal to not wear gloves during tissue culture.  btw here is the legal justification, according to what I found:  


Here is where the current CDC BSL Guidelines are:  
http://www.cdc.gov/b...bl5_sect_iv.pdf

Which states, for both BSL1 and BSL2:  "Gloves must be worn to protect hands from exposure to hazardous materials."

Here is the ATCC MSDS for cell lines:  
https://www.atcc.org...69C6552299.ashx

which states:  "Various animal Cell Cultures at Biosafety Level 1 or 2 ... this product should be handle according to good lab practices, with proper personal protective equipment...Handle as a potentially biohazardous material...Universal Precautions according to 29 CFR 1910.1030 should be followed at all times when manipulating these cell lines."

29 CFR 1910.1030 are the Federal regulations concerning Bloodborne pathogens, found online at:

http://www.osha.gov/...ARDS&p_id=10051

Per federal US Dept. of Labor OSHA Regulations 29 CFR 1910.1030 (d)(3)(ii):

"The employer shall ensure that the employee uses appropriate personal protective equipment."

Per OSHA Regulations 29 CFR 1910.1030 (d)(3)(ix):

"Gloves shall be worn when it can be reasonably anticipated that the employee may have hand contact with potentially infectious materials."

So in the end, even if you consider something to be harmless enough to get on your skin, it must be treated as a POTENTIAL hazard no matter what.  The key word being "potentially" which as I see it is valid for all BSL1 and 2 organisms, and I also gave the reasoning that cancer cell lines derived from humans should still be treated as a bodily fluid, whether or not any real danger exists.  Anyway, there are a lot of other reasons to wear gloves of course, but we are finally in agreement here !

Of course she has entirely different tissue culture technique than I do and doesn't want me regulating her on that, which I will not, because probably no one will sue you about how you split your cells, but it's possible they could sue you over not wearing gloves.

yea she trained in the 80s back with mouth pipetting and all that, but to this day she insists it's BETTER to not use gloves...  unfortunately our small college has a completely ineffective safety officer, but I am trying to gang up on my PI with the department's "jack of all trades" tech, but she has a lot on her plate.  So really I just want to get some outside opinions about it to add to my own reasons to present to her before our lol-biosafety meeting.  

btw according to the CDC they say you must wear gloves even for BSL1 work:  http://www.cdc.gov/b...bl5_sect_iv.pdf

hanks contains glucose to help keep cells alive and happy for a longer time; in pbs alone they can become stressed and die if left for too long.  

for instance I used hanks was for washing freshly isolated primary cells which may be sensitive to dying if not treated well.  

if your solution isn't being used for live cells, you should use PBS for dilutions.  the glucose in the HBSS may affect experimental results and is only needed for live cells.

set up the experiment in a 96 well plate to reduce any pipetting error if possible,

and when removing mtt+media and before adding DMSO be very careful not to disturb the cell layer and alter results.  

you can also try to add the DMSO and then watch it, help dissolve by pipetting, and then as soon as you think it's all dissolved transfer it to the 96 well plate - don't wait 10 minutes.   the formazan should start dissolving right away and should be almost all dissolved within a minute I think; and perhaps spending too long in the DMSO is altering the results - I usually add DMSO, mix, and read, within 5 minutes total, and get very small errors.  

are the control cells 100% confluent after 5 days or not?  If not, perhaps you could plate all the cells at a higher density, so there are more of them at the end of the experiment for a greater total final reading.  

it could be you are just using too few cells per well to see the difference between treatments.  up the cell number to increase the final signal

Use DMSO and read immediately.  

in my lab we always use DMSO.  we use 200ul of DMSO per well of 96 well plate but I think 100ul will work just as well.  After I have added the DMSO to all the wells (you will see the formazan start to dissolve right away), I immediately set the multi-channel pipettor I just used to add the DMSO to half of the volume of DMSO and mix all wells, from lightest to darkest (I set my plate up so the lightest wells, PAO all dead control are at one end, and darkest untreated control most living at the other end, so I don't have to change tips as I go) mix by pipetting up and down 3-4 times per well.   Then read immediately.  Should get tight numbers unless something is very off.  Also I have noticed that if you do NOT read right away, the readings will start to vary a lot more.  It seems if you leave it very long the color intensity starts to vary in random ways, so read it immediately.  We also have a trick to remove any bubbles - to blow hot air on the samples with a hair dryer, immediately pops any bubbles.  But DMSO does not hold bubbles very long anyway, and I set it  up in the windy fume hood, so usually no problem having bubbles.  But you do want to make sure there are no bubbles in any samples as that can really alter the reading.

I've not used the SDS or any water-based method in my lab, and for our student who went to another lab for the summer and introduced using DMSO, the PI was greatly impressed by the much better results.