The source of contamination - (Aug/16/2016 )

Hello!

I work with cell cultures. A few weeks ago I decided to control the reagents that we use in our laboratory and aseptic conditions in BSCII, incubator and laboratory room.

I made three 24well plates with reagents (Medium complete, only Medium, FBS, Medium + antibiotic and etс.). One plate I put in incubator, one I left on the table in BSCII and one I put on the table in laboratory room.

In 24 hours I checked plates and saw under the microscope:

- contamination in whole plate from BSCII (cocci)

- contamination in whole plate that stood on the table in the room

- from incubator plate was clean

So how is it possible? I worked in BSC with reagents and then in incubator plate stayed clean, but plate in BSC - contaminated. Then instruments, which lying in it must be contaminated too (I don't believe that UV ''save'' tips and tube during 1 hour before work if in BSC such contamination). Furthermore, in cells with only Medium (in plates from room table and BSC-table I saw something similar to a yeast. And I remember, in incubator whole plate was clean!

I made also 3 flask T-25 with complete medium and in 24 hours I saw, that flasks from tables were also contaminated. So I guess that, probably, the reagents are contaminated, because it's very difficult to imagine that something can get into flask through HEPA-filter.

But what is it the organism, that grows at room temperature and don't grow in incubator? Or, maybe, it's somethinge else.

Please, I'm waiting for your suggestions, because I don't understand, suorce of contamination.

There are a couple of options here:

1) that you set up the two contaminated plates after the non-contaminated and happened to contaminate in-between.

2)your medium or some of the components are contaminated

3)The hood is not properly maintained (filters need to be changed regularly) or improperly used (i.e. the airflow is disrupted be having pipettes/bottles/etc in the air path)

4) your sterile technique is poor -spray everything with ethanol before it goes into the hood, wear lab coat and gloves, ensure that lids of bottles are not placed on surfaces, don't wave your hands over open plates and bottles)

UV does not decontaminate those parts it can't reach. Most plastics are UV impermeable, so will not allow sterilization inside the tip boxes for example. UV will also not reach areas in the shadow of items left in the hood - clear the hood entirely after each use. UV does not replace wiping out the hood before and after each use - use 70% ethanol for this. Ensure all surfaces are clean.

Bob1, thank you for answer!

Unfortunately, contamination between filling the plates is impossible - I made the plate at the same time and then used theese reagents for other flasks.

Also I think, if the problem were in filters or some reagents, contamination would be in a number of cells in plate.

I guess, source of contamination is the outside. How do you think - may incorrect ventilation in laboratory be a reason of this situation? This is new room for cell culture and we have some difficulties with layout at the present time

I was assuming you made the plates at the same time, however, you presumably filled all the wells of each plate in order (i.e. didn't fill one well of one plate then move to one well of the next plate etc.)... is there some possibility there? I have kept plates in heavily contaminated (fungal) incubators and had single wells of a plate be contaminated before, without the infection spreading between wells or coming in from the incubator - all done in the absence of antibacterials and antimycotics, so the contamination showing on the bench and hood is most likely to be from something that happened when the plates were set up. Yes, there is air-flow into plates, but everyone transfers plates between the incubator and hood all the time with very few problems in most labs. Also you have contamination in the flasks; the 0.2 um filters on these should exclude gross contamination from external sources - reagents are contaminated or user has poor technique. Greater than 90% of contamination issues stem from user error, not the environment. 

Air flow around hoods can be a problem for maintaining containment of the samples, hoods need to be at least a couple of meters (maybe 3 m recommended?) apart if facing each-other, or could be positioned back-to-back, so as to not disrupt air-flow around the working space, but this will depend on the hood and where the external air flows go. Ceiling ventilation can also be a problem if it is bringing in a lot of particulates, but this can be fixed by placing simple filters over/inside the vents or making dispersers (basically plates placed over the vent to spread the air sideways rather than straight down. There is a possibility that you have contaminated air-conditioning ducting, with moisture allowing growth of bacteria on filters or surfaces inside the system, that is then steadily entering the lab via the vents. However, you can easily test for this by putting an open bacterial plate (LB or 2YT work well, you might want to try a yeast plate too) out on the bench for an hour or two. Most airborne plates of this type in a clean lab will show colonies in the 10-30 colony per plate range, most of which will not be readily identifiable as common bacterial species.

Thank you, Bob1!

I will check air-filters in laboratory room and ask other person made a plate for vetrification.

I hope, the problem will be solved.