Catherine- the problem is that your cell samples may get exposed to room air right before and after going through the FACS machine, so it is often difficult to tell whether a dirty machine is the culprit, or you are getting contamination from another source (ambient air, culture vessels, culture medium).

Try to find a FACS machine that has dedicated use for mammalian cells, or at least does not allow yeast or bacterial analysis. Especially with yeast, it can be difficult to completely sterilize the cytometers and their surrounding environments after use. Also confirm that your FACS technician bleaches the lines before your sort.

One useful control would be to reserve a small amount of your input sort material for subsequent culture; often you don't need to sort the entire cell suspension, and you could test any residual suspension that didn't pass through the machine for signs of contamination.

As far as I am aware infections can be spread from a sorter, it's less likely due to the cleaning regimes used during shut down which essentially involve running bleach through the system but it's not impossible. From my experience infections are more likely to come from culture technique or the outside environment, we recently had a spate of infections which coincided with some building work. I guess the only way to be sure is to test your cell culture technique using flasks containing only media, if these get infected then you can narrow down the causes, however if they don't you at least have some evidence to take to the technician. If he's willing you could try running counting beads through the system and culture those to test for infection? The other thing to ask is have other users been affected by infections after sorting?

I hope that helps

Catherine- the problem is that your cell samples may get exposed to room air right before and after going through the FACS machine, so it is often difficult to tell whether a dirty machine is the culprit, or you are getting contamination from another source (ambient air, culture vessels, culture medium).

Try to find a FACS machine that has dedicated use for mammalian cells, or at least does not allow yeast or bacterial analysis. Especially with yeast, it can be difficult to completely sterilize the cytometers and their surrounding environments after use. Also confirm that your FACS technician bleaches the lines before your sort.

One useful control would be to reserve a small amount of your input sort material for subsequent culture; often you don't need to sort the entire cell suspension, and you could test any residual suspension that didn't pass through the machine for signs of contamination.

You may ask your FACS technician to run bleach in front of you, and then the sheath and then PBS. After this you should be the first person to start with. Or look for a FACS machine which is dedicated for only mammalian cell line work. If still the problem persists as Mr. Daniel Cohen said please use the appropriate control

the only infections I ever had were when using cells coming out from a BD aria sorter....

for sure it is the sorter. put antibiotics in your media.

cheers

Another option may be to see who is on the Aria before and after you and see if they also get contamination?

Or maybe use magnetic beads to sort?

From spending a lot of time in a FACS sorting lab I can perhaps appreciate the technicians perspective. It is not uncommon for an angry scientist to storm into the flow room and accuse the operator or contaminating their cells... obviously your technician should work with you to solve your problem, it is rather unprofessional of them not to, but after a few years of getting yelled at by angry scientists the operators may get apathetic.

I have experienced contamination after sorting on MoFlo sorter. All other ways of contamination were excluded, because culture medium was clean and the cell culture which I used for sorting, was growing without any problems (I was continuously cultivating unsorted cells).

I see, Daniel Cohen also suggests this point and I absolutely agree with him.

The contamination can come from any number of sources, but the room air in which the sorter is located may well be the culprit, particularly, as mentioned, if there is construction work near by or the sorter is not in a deidcated room, but one where lots of poeple are coming in and out. As Daniel says, reserve some of your culture without sorting it. I might add a 3rd option, save some of your culture in a tissue culture hood (1), save some in the tube that was opended and used for the sort (2) and then your sorted cells (3). If something grows in tube 1, 2 and/or 3 you will know where the contamination is coming from. As the others have mentioned, good lab practice (mammalian cells only, sterile sorts 1st thing post clean) will help too.

The shut down procedure of the Aria is quite stringent and less likely to allow contamination of the samples.But then if the FACS Tech cuts corners on the cleaning procedure, that could be a potential source. If the source of the cells is e.g., mouse spleen or LN, blood, carefully re-examine the isolation prodecures they could be the source of infection. If antibiotics can be used in the downstream application of the cells then use them.

I think that it is not possible to rule out FACS sorter as the source of infection.

Contaminations on FACS sorters are a common problem and there are a couple of sources for contamination. Firstly, your 'ultraclean' water may be contaminated and we had a lot of problems before we autoclaved our sheath fluid. Secondly, your sample could be the issue. It is not sufficient to autoclave sheath fluid if you had contamination in previous sorts. You have to remove the filter of the sorter, purge the system with 70% ethanol, followed by autoclaved water and autoclaved sheath fluid and then exchange or sterilise the filter (better replace filter). This should resolve your contamination problem.

To find out the source of contamination, prepare two independent samples, sort one and leave the parallel sample untouched. Put both cells back into the incubator. This should convince you and your FACS technician.

Hey I understand your issue, I would like to give away my little input to you to get rid of this problem,

Answer me for these questions,

1. What FACS machine you are using?

2. Is the FACS machine is kept in side a LAF?

3. Is your operator doing a Aseptic cleaning before starting the sorting?

4. Fluidic start up and shut down will always help but not GOD'S WORD that it should help.

5. Did you ever tried collecting just the sheath out from the nozzle and put it into your culture media? This will tell you is the FACS is the culprit.

6. The room where the FACS could also be a reason, but it should not affect only you, so did you ever checked with other users who uses the sorters and the taking the sortouts to culture also facing the same issue.

7. If yes the contamination is also by common a microbe? (please try to know what is the contaminant)

8. As mentioned in the earlier post, you can always try adding antibiotics in to the collection media, or if you are already adding antibiotics try adding 1X extra Antibiotics (if you are sure that will not screw your cells).

I can tell you more once you come up with the answers for those questions.

Senthil.

Dear Cathrine. Even though your FACS technicians do the "prepare for aseptic sorting" procedure they cannot claim your sorter is aseptic. We do regularly (every 14th day) take sample from the stream and culture it on blood agar plates for two days at 37°C to verify the system is aseptic. We once had our AriaIII infected and found the infection was in the blue liquid lines connecting the sheath filter. After we once a week shut everyting down in 70% ethanol (including the sheath filter) we have not had problems since. You need though to do 3 repeated startups - on after the other - to get all the ethanol out of the sheath filter afterwards. Good luck.

Best regards, Charlotte Petersen

Dear Cathrine,

Check the sterility of the FACS as mentioned by Charlotte

Use antibiotics in the culture media, shut everyting down in 70% ethanol (including the sheath filter)

Thanks

It is not uncommon for samples to become infected following a sort. It is very important that the sorter is thoroughly cleaned and decontaminated prior to sorting. You can also add pen and strep or gentamicin to media but decontamination may be more effective. It may be worthwhile looking for another cytometer if problem is ongoing. Regards. Stephen

Dear Catherine, if your technician washes carefully the instrument between two sorting and at the beginning and end of the day with EtOH 70% it is difficult to have a contamination due to sorting. It is also important to have a clean tube support and flow chamber and you have to place attention in how your sorting tubes are handled during all the process since it does not occur in a sterile room. One simple experiment to be sure that your machine has not a contamination inside the fluidics components is to sort simply the sheat flow and culture it for one-two week in a complete medium. If something in the sorter is dirty a contamination will came out!

You need to consider a number of steps in order to find out weather the problem is from the sorter, from the air in the room environment or from your culturing technique and the sample.

- use a freshly made by you culture media.

- make sure that your working environment in the tissue culture room especially the hood you are using is sterile. and the same goes for the tubes and the incubator if your procedure includes incubating your samples.

-ask the FACS technician to bleach the the machine thoroughly including the nozzle.

-when taking your samples to the FACS try to run them ass soon as you are there as the delay could cause contamination.

-ask the technician kindely to keep your samples in a close distance from the machine to minimize the time that the sample is exposed to the air in the surrounding.

-as some people mentioned above, check with other users who have previously used the same machine iv the have had similar problem.

- if you have access to another sorter (which might be difficult) try it would indicate if the problem is with the sorter (or the sorter technician handling of the sample), or you sample is problematic.

- just try to be patient and kind to the FACS technician as much as you can to gain his cooperation and help.

Thanks

This happened to me last year. After I switched to another vender for antibiotics, the problem was solved.

Having worked in an HIV immunology lab, it is well-circulated knowledge that one cannot use a FACS-Aria to flow potentially HIV-positive WBC samples since the machine actually passes the sample through air after the sample has been split into the small droplets that allow any FACS to work. These particles are aerosols, and upon exposure to air, can come into contact with the mucous membranes of flow operators, if they are not in an isolation suit. Since HIV is infectious in aerosol form, this is a big no-no. Thus, we only use BD LSR-II blue to flow possibly HIV-infected or -containing samples, since the LSR-II blue does not pass the sample droplets through air prior to laser excitation & detection, and is indeed aerosol-tight, so to speak.

It would be pertinent to know which type of infection your lab workers are experiencing, and what sample(s) you are flowing. If the samples are known to harbor infectious particles (viruses), and the infections are viral in nature, and the known infectious agent is infectious in aerosols, your issue is most likely with the FACS-Aria, and you should switch to a FACS machine that does not expose the sample to air. If your samples are not known to harbor infectious particles, and are perhaps cultured cell lines, you may want to verify your cell line has not been infected with an aerosol-viable virus capable of symptomatic human infection since primary culture. Same goes for if you are using animal specimens - make sure the animals do not have viral zoonoses. I take it that you are using antibiotic in your culture, since that is common/standard practice, and that would make bacterial infection unlikely unless your antibiotic is ineffective for some reason (eg expired). If the infection is surely bacterial and your antibiotic is effective against the culprit pathogen, these infections may be coincidental, or related to the state of the flow machine (eg contaminated). Coincidence may be the case regardless of infectious etiology.

This situation certainly merits attention. Your biological safety officer should be informed of this - he/she should fill you in on the details of testing for these pathogens, although it will likely involve PCR.

Good luck, and my condolences on your lab workers' illnesses.

@Micheal, I think the originator of question is talking about contamination of cells once they have been sorted through the FACS machine

Catherine, this is more of a public relations problem in my books... Sure, the sorter can very well be the source of the problem, and if the technician is being adamant that it is not, then you need to work out a way of communication with him. Use your controls like Daniel said, so that you will have proof of what the problem is. I'd go a step further and tell you to get the control vial with you to the sorter, just don't sort it.

If it is indeed the sorting, talk to him about the facts without making him feel he is being accused. The problem is most probably the handling rather than the machine itself (I am going to assume that he cleans it well). So get an ethanol spray and go crazy with it, and put a suitable antibiotic in your cell culture.

And be patient, the "it's not my fault and I'm not discussing it" is one of the most annoying problems, but unfortunately a very, very common one. At the same time, be sure to avoid having the attitude yourself - check everything that you do with controls!

@Felix: Ah, my apologies, I've been in the clinical world for the past year. In that case, I agree with everyone else's answers. Run bleach through the machine no less than 5 minutes, rinse with PBS for another 5.

Simple experiment: culture a plate in parallel with the plate you use for FACS, keeping all procedures the same except once you get to the FACS, don't flow the parallel plate and just incubate (but take the parallel plate with you to the flow room, set it next to the other plate, everything exactly the same, just dont flow). If that doesnt tell you, spare one procedure at a time until no infection (eg leave the parallel plate in the incubator instead of bringing it to the flow room). That should tell you your source of infection. Could also be the whole batch of cells that are contaminated (eg cell cryopreservation technique). Or the incubator. And everything between. Good luck!

There is very few things to add after everything that has already been posted above. I tend to agree with most comments and find all of them very useful. However, a quick way of detecting if the contamination issues come from the cell sorter or not is to inquire about similar problems with other "customers" of this same instrument. So, is this instrument located in a facility? If so, it is probably used by other scientists with other experiments. Has anyone else complained about post-sorting contamination? This would be a very quick way of knowing if there is room for a deep-drive discussion about maintenance, cleaning and decontamination workflow with the instrument's technician. Take care and good luck!

I'm afraid I'm going to side with the technician here. I've been operating cell sorters for 15 years, and am pleased to say that I have never had a case where the instrument was the cause of contamination.

If the Aria operator is following the recommended (BD) start-up and shut down, and is conscientious in cleaning between sorts, there should be no reason for instrument derived contamination. Particularly if the instrument is run on a commercial sheath fluid such as FACSSheath, Isoton, or IsoFlow.

By all means, save an aliquot of your sample to check for prior contamination, and culture sheath fluid that has passed through the sorter, but only culture it for as long as it takes to see contamination in your sample.

On a well maintained instrument run on commercial sheath fluid the only area of contamination should be the sample line that takes the sample from the tube into the flow cell. I am assuming we are dealing with an Aria II or III here, the valve on the Aria 1 can be an added problem.

I will add a caution about bleach, you need at least 2x the volume/time with sheath after running bleach as you ran bleach for. Bleach is highly corrosive & can cause serious damage to the metal and plastic components of the instrument. For the record, I use Coulter Clenz instead of bleach, but will resort to bleach or even lyse solution for 'sticky' samples.

In fact when I first started sorting an a MoFlo back in 1998 we used to run 10% bleach for 1 minute, then 70% EtOH for 1 minute then (in those days home made) PBS for 1 minute. I once sorted and analysed 9 different samples in the same day (yeast, bacteria, macerated tissue, human & animal blood, etc) without cross contamination. If you are using home-made sheath, then all bets are off, unless it is freshly made and autoclaved, and used within a week. Unfortunately there are lots of places that attempt to save money with home-made PBS, but end up with contamination issues.

Dear Catherine, as you know FACS is a fluidics system. So, any device may be infected , independent from the model and an UV not always destroys all sources of pollution. But it is the theory, and you need the action. Therefore:

1 . Check sterility of cell culture.

2 . Before each samole running you need to use the general cleaning protocol (bleach 50 min, DDW 10 min. )

3 . Try to check air contamination in sorter lab.

4 . After each example tube place a tube with sterilePBS and wash out a sample needle (instead of water. Since, sometimes, between the tube reading, the drop of water gets to culture and destroys the cells. That can become an icontamination source).

I think that my offers will help you with your difficulty.

Dear Anna,

I must disagree, if you are running a commercial sheath that is bacteriastatic & fungistatic, then the contamination (assuming you are following the companies protocols) is most likely (99.99%) from the sample.

I'm sorry but 50 minutes of bleach followed by 10 mins of water is no good to the instrument and can potentially kill the sorted cells (excess bleach residue in the sample line). As a side note I currently have an analyser out of commission (the engineers are having to replace several parts 'eaten' away by bleach) because users have been too heavy handed with bleach without sufficient washing with water.

The sample line on any sorter is very fine, 1 minute with bleach (or Coulter Clenz or EtOH) followed by 2 minutes with sheath is more than adequate for all but the stickiest samples.

I routinely do 2-3 sorts/day, without cross-contamination, using the described short clean cycle.

Now I routinely sort with a machine in a biohazard hood, but for the past 15 years I have sorted in the open lab, without issue. It is my experience that it is cell prep that is the weak link when looking for contamination sources.

Dear Robert. My error was a slip about the bleach running time: 5 instead 50 min. To confirm the written above I attach a link to the FACS clean protocol.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046871

http://nerce.med.harvard.edu/_downloads/FACS%20hand%20outs.doc ( Shut down and Monthly Maintenance)

Dear Catherine!

I'd like to notice, that if there are such problems, why you not to execute a sorting with use of BD magnetic beads.This protocol will reduce risk of culture contamination, specificity of sorting is very high, the sorted cells then can be checked by FACS (beads are very small and don't disturb the analysis). Because of high vitality of cells after bead-sorting it is possible to use for future mol-bio application( microarray , NG deep seq ).Please, see link with our paper: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046871

Regards

Dear Anna,

I hope I didn't offend, but I do know of operators with very lengthy cleaning protocols.

Regards

Rob

Dear Robert,

The target of this entire conversation was to explain Catherine how to stop the cycle of multiple infections in her culture.

I'm using this protocol (5 minutes with bleach, 10 minutes with water) since 2000, after I graduated from the clinical and research FACS operators course in Dartmouth college,NH. So up until five years ago,I have ran two to three sortings per day in the clinical FACS unit in a hospital.

All of my runnings were without cross-contamination, but personally, I think that sorting by beads is easier,quicker and more applied to the downstream application.

But that is only my opinion, based on my experience,and it isn't related to the conversation about FACS cleaning.

Sincerely

Dear Catherine, are you the only one experiencing problems with infection following sorting on the FACS? If so, then it is quite likely your technician is correct. What type of infection are you getting? Bacterial, fungal, other? What are you sorting? Labelled, unlabeled?

Dear Catherine. If the cleaning process is done well, there is only a minor chance of infection caused by sorting procedure. Normally, you use antibacterial sheath fluid with azide. For sorting, you exchange it to sterile PBS (after cleaning procedure). There is also a big difference between Aria I and Aria II and III which have optimized fluidics and less possibility of contamination. But even with ARIA I you can ensure an efficient sterilization procedure. If you continue to have problems try to contact BD staff. They can help you. You can easily provide a control assays as well, i.e. cultivating of unsorted population vs. sorted cell gate. Best, Ludek

Dear Ludek,

Just as a point of clarification, most commercial sheath (at least from BD and Beckman Coulter) now contains Sodium Fluoride rather than azide. This makes the sheath bacteriostatic & fungistatic without adversely effecting the sorted cells.

If you are using commercial sheath & following the manufacturers recommended start-up, shutdown and cleaning procedures it is usually safe to assume the fluidics up to, but not necessarily including the flow are clean.

The "dirtiest" part of an Aria, and the hardest part to clean is the sample uptake tube.

On the Aria 1 the worst part of the instrument to clean is the sample valve, which tends to capture all sorts of rubbish. This valve was removed in the Aria II (& the Aria II upgrade), as part of the fluidics upgrade.

Dear Catherine, I've just seen the discussion on your problems with contamination and though instrument contribution should be low, I think it's a poor operator who would not take the time to do a few simple checks to verify the instruments aseptic status simply for themselves, if not for you!

A mock test sort collection of sheath into some sort tubes pre-loaded with some culture media ± antibiotics or a mock sort sample of sheath plated on a LB plates is a standard check I do regularly, especially after a service change of the fluidic line filters. This has identified at least one potential instrument contamination risk from filter replacements on my Aria I which was addressed before subsequent sorting.

I also put a couple of LB plates into the collection area to evaluate the air borne contamination level in the facility and I always recommend the inclusion of x2-5 pen/strep in collection medium, when compatible with the sorted cells to mitigate the opportunist contamination risk of sorting in the open

Likely scenarios in my experience can be:

1. It is very likely that the tubing in your FACS machine may be contaminated. If the tubes are old they will sustain cracks where yeast and bacterial can hid from weekly bleach runs. If they are more than 2 years old, I would replace the tubing. FACS aria, specially the first generation, is know to have contamination problems. In our facility this was sourced to excessively long tube usage in this machine. The next generation of the Aria machines have significantly shorter internal tubing which can be replaced easily.

2. handling of the samples by the sorter operator can also be a significant problems. The collection tubes in particular need to be handled with much care. Gloves much be worn at all times and changed often during the sort. The lids must only be removed inside the collection chamber and replace while the collection tube is still in the chamber.

3. Check the sterility of the cells before being sorted (as handling/preparation of cells for sorting may have contaminated them) and also collect some sheath fluid from the machine and place them in an incubator to determine if the fluid running through the FACS machine is contaminated. If possible, these tests should be done in an incubator where other cell cultures are not being carried just in the case that contamination is found.

In our facility we perform random sterility checks where sheath fluid from the storage tanks and the tubes are tested for contaminants at least 3 times a month. The storage tanks are metal and are autoclaved once a month.

I hope you find these suggestions helpful. Good luck!

Several years ago we faced a pseudomonas contamination of your Aria sorter. Took a lot of time, bleach and tubing exchange to get rid of it.

Most of the sorts were contaminated, and despite using the provided sheath fluid and following step-by-step every possible cleaning procedure to get rid of our "unknown bug" (at that time), contamination kept coming back.

We did a blank sort (sorting PBS in a steril collection tube) and handed it over to the bacteriology dept for identification. It showed up being pseudomonas. Pseudomonas has the lovely hability to be able to survive in some desinfectant, and create biofilms. Meaning that while you try to clean it away, you only remove a superficial layer of the colonies, and it keeps coming back.

Your operator may be honest thinking that he follows all cleaning procedures and that the machine should (in theory) be clean, but even then, contamination may occur.

A simple sheath fluid sort in culture medium (clean and sterile before hand, tube closed under a steril hood, and opened only before being installed in the sorter, should let you localize the source of the contamination you observe. Take another tube, do the exact same thing, place it in the sorter as well, but don't sort anything. Let it there for as long as another tube used for real sorting would stand in the machine... If both get contaminated, then the environment around the machine need to be cleaned, if only the tube where sheath was sorted gets contaminated, you will need to have a serious talk with your operator. And just to make sure, get a third tube, prepared at the same time as the other two, but don't do anything with it (no sorter encounter). Place the content of the three back in independant culture plate and see if (and where) anything is growing.

With our contamination, the only way we had to get rid of pseudomonas was to fill the whole tubing system with bleach and let it stands for 24-48 hours, before changing all the tubes which did not like this approach (long bleach treatment is not very tube-friendly) and run several cleaning procedure for the new installed tubing. Luckily, pseudomonas never came back.