Contamination of cultured cells - (Nov/01/2007 )
I check the lab manual but I did not find any solution for my contamination. our incubator make is "SANYO O2/CO2 incubator, model no: MCO-175M " also, our biological safety cabinet make is "NUAIRE model no: NU:440-400E series 9" how can I get disinfection manuals for this type of incubator? thanks!!
I'm still unsure on what the contaminating organism was so this is a very general procedure.
Always disconnect the unit from the electrical service when cleaning.
Assure all volatile or flammable cleaners are evaporated and dry before
reconnecting the unit to the power supply.
If you use chlorine-based bleaches or abrasive cleaners this will modify the stainless steel interior finish so they must be followed with several rinses of distilled water to remove residual residue.
DO NOT USE hard tools such as metal wire brushes or steel wool. Use non-abrasive cleaners and soft tools or a lint free cloth.
Drain and refill the water jaket; Use only DISTILLED water in the water jacket (some like to add waterbath anti-fungal solution) and the humidity pan. DO NOT USE de-ionized water due to its reactivity with metal components.
All stainless steel parts (shelves, brackets, water tray) should be removed and cleaned seperately.
Clean the interior chamber with the selected detergents; Special care should be taken when cleaning around sensing heads to prevent damage. Remember to pay close attention to cleaning the grooves in the door seal. The interior door and gasket are often overlooked in cleaning and they are at high risk of carrying contamination due to condensation developing.
The 0.3um hepafilter on the interior air circulator should be replaced.
Choose your weapon:
Sporicidin; spray and sit for 15 minutes, smells horrible
70% EtOH; spray and soak the interior, wipe clean with a lint free cloth (sterile is good but cost is high)
Quat disinfection solution (3m quat); Follow label instructions
Parts could be autoclaved if available
Replace the water in the bottom of the chamber (or water pan); use distilled water
and add waterbath cleaning solution (anti-fungal or silver ion stick).
Turn it back on when all is dry and reassembled
Did it work?
Place an open TSA 100mm plate on each shelf monitor 6-8 hours of operation. Incubate 5-7 days at 37C.
-ethanol your gloves before you enter the hood and ethanol them every time you re-enter the hood, eg. after you've gone to get you media bottle from the fridge or something like that - hands are a nice easy source of contamination in your cell culture
-work in the back of the hood - the closer you are to the front of the hood, the closer you are to potential sources of contamination, i.e. air
-keep lids on bottles when not in use - some people leave their lids off in the hood assuming that the hood is completely devoid of microorganisms - keeping them on just helps reduce the chance of contamination
Good point! Also, the sleves of the lab coat are another source. Sleeve covers, although some dont like the feel, can also be sprayed with 70% ethanol.
One thing that always made my aseptic nerve raw was people working in the hood with a gap between the sleeve and the glove exposing their wrist. My choice is glove pulled over sleeve cover.
So today I went back to lab and found out my cell line was contaminated again. And I dont really know how to handle this situation. Coz this is already the second time in a roll ofcontamination, it happened as well last week. What do you guys usually do once your cell line was contaminated?Also, how to minimize the chance of contamination?
RS/Binder/Nuaire/Heracell have high temperature decontamination cycles which aid cleaning of the incubator. These are different temperature cycles: 120/180/90and 140/90 oC respectively.
This does not however solve the problem. As many have said USE GOOD ASEPTIC technique.
Have a rota for all things tissue culture:
Weekly changes of water in waterbaths/Incubators
Stocking of TC rooms
Cleaning of Floors and work surfaces in TC areas.
Have seperate side labs for TC work
Have seperate labs for different applications i.e. Viral transfection, primary, human samples, Mycoplasma negative and quarantine areas.
Cleaning of class II cabinets.
Making up IMS
Have seperate TC lab coats
Use quality TC ingredients
USE PLENTY OF IMS to wash gloved hands. Use IMS to clean TC flasks, in and out of the CO2 incubator.
GET TRAINED BY SOMEBODY WITH PLENTY OF EXPERIENCE.
CHUCK AWAY ALL CONTAMINATED CELLS......DO NOT TRY TO CLEAN THEM UP.
DO REGULAR MYCOPLASMA TESTING.
Some people are "luckier" than others with contamination..........these people always have better technique than those who are "unlucky".
And as Dominic rightly said.....these observations are from 30 years of experience.
Another issue I see often is technicians storing the pipets, tips, buffers, biohazard bags, and other commonly used items inside the BSC. It is never a good idea to store any items in the hood as this not only prevents effective cleaning but it also blocks the air flow inside the hood.
We have several Heracell incubators in our facility and the "sterilization cycle" is not intended to replace a monthly manual cleaning. If the cycle is run too often the seals on the sensors will go bad. Which is a serious issue with the water sensor at the bottom of the incubator because the water will leak out. But it is very helpful in preventing the spread of contamination.
[quote name='Tsunade Sai' date='Nov 7 2007, 06:02 AM' post='116187']
thanks for your advices! ter are really helpfull!!