In our lab we mainly work on E.coli. For the past 2 months we are getting contamination in our LB tubes (a thread like think is growing). We have contamination only in LB tubes, so we cleaned all the tubes and autoclaved it and we also fumigate our lab too. We also fumigated our shaking incubator too. But still we are getting some thread like contamination growing in our tubes. Even we add antibiotics for our resistant gene that tubes also had that type of growth.
I doubt your problem has anything to do with aseptic technique; I've seen people do unspeakable things in the lab and still get away with it when working with E. coli (using non-sterile material, picking a colony with a pipette tip held with bare hands & then throwing the tip into the culture, you name it...). Is there any chance you could have a case of phage contamination? Inconsistent, occasional unreliability and 'thread-like' things on the cultures does sound to me like contamination with E. coli phages.
Assuming you are following a strict aseptic technique, I'd say you have probably contaminated your stock, and so you will have to start from a fresh cell stock. But you say you have contamination only in LB tubes. Does this mean you have no contamination in other media? If so, then the problem is coming from your media. In my experience, you can waste an awful lot of time eliminating things one by one, and so it is always quicker to throw everything away and start again! Hope this helps.
First, you should check your autoclave and make sure that it is getting up to >121C and 15 psi. Whatever is contaminating is probably already in your powdered LB ingredients. You could try purchasing fresh ingredients. If it is growing in antibiotics, it is likely a fungal contaminant present as spores. You could try adding cyclohexamide, which inhibits eukaryotes.
Have you tried looking at the contaminant under the microscope? If the autoclave is working properly, it should kill spores. It's possible you have salts precipitating. I've had precipitates form interesting crystals in sterile media, and they can look like contamination. Looking at the contaminant under the microscope could help narrow down what you're dealing with.
Do you prepare this media in a specific laminar flow chamber? Is the HEPA filter ok?
Thread likes are probably fungus... How much time are you leaving the autoclave on?
All the above are correct and worse checking out.
Make sure the medium is sterile
autoclave working correctly
Laminar flow cabinet - hepa filters are not damaged? (are you getting these serviced frequently?)
What are the plugs on your tubes, are they sealing well? Autoclave twice if necessary.
Are you keeping them in a temperature room or incubator? A faulty cooling unit in a temperature room can also cause fungal contamination as this is blowing them around (you may have to use fungizides to decontaminate it!).
If you are not sure what the contamination is, try growing it on Cornmeal agar or Nutrient Agar and then examine under microscope.
But I would start again with new LB medium as mentioned above. LB medium is very rich in nutrients, that may be the reason that you see the contamination only in there and not in other media.
good luck!
I believe the above suggestion will be sufficient. However, you may also want to consider the loops you use to inoculate the media and regarding the shaker, if it is a water shaker, try to add disinfectants to the water also. And reconfirm all the sterility workflow, as mentioned.
I do agree with above suggestions. However, i will suggest to consider the followings that might cause contamination
1) Check your inoculum (might have contaminant)
2) Check the autoclave. Is it working properly at (121 C and 15 psi for upto 15-20 min?
3) If you are using glass vials due care must be given to the stopper sterilization that might cause contamination. If you are using cotton then better to replace it.
4) If you are using plastic tube better to replace them with sterilized disposable tubes that will help to understand the exact cause of contamination
5) As mentioned above growth medium might be the cause of contamination
6) Check your microscope lens (very important).
7) Check the functioning of Laminar air flow cabinet and its HEPA filter
If you will sort out all these things, i hope you will be able to point out the actual cause of contamination. Wish You the Best of Luck
I agree with Valerie. For classical E. coli work, Laminar flows and stuff should even not be necessary. A bunsen flame and good microbiological practices should do the trick. I would not trouble shoot too much, it will take time and money. Clear out old stocks of LB and cultures, make sure everything is clean/aseptic and start over. In the course of that troubleshoot: e.g. Bio-indicators in the autoclave (Bacillus stearothemophilus spores, commercially available). Check autoclave and sterilization ovens with independent temperature probes (can be done cheap with standard min max thermometers), put open nutrient agar plates in your labs (15 minutes opening should not yield more than a few colonies), ....
I doubt your problem has anything to do with aseptic technique; I've seen people do unspeakable things in the lab and still get away with it when working with E. coli (using non-sterile material, picking a colony with a pipette tip held with bare hands & then throwing the tip into the culture, you name it...). Is there any chance you could have a case of phage contamination? Inconsistent, occasional unreliability and 'thread-like' things on the cultures does sound to me like contamination with E. coli phages.
You can try sterilizing your empty tubes once in oven at 180 C for three hours, you may have some problem with your autoclave, to be sure you can try autoclaving contaminated LB tubes and leave them again in incubator and see if the contaminant will grow again
I Would also be looking at the moisture levels where you are storing the tubes. If the tubes are not completely dry when stored or stored in a completely dry area, you may get growth of filamentous microfungi (thread like things growing). Us here in the tropics of Australia have this problem all the time. Our labs are kept at 18 oC (continious) or dehumidifiers are used. Our tubes, after autoclaving, are placed in a dryer, cooled down to room temperature and then stored in dry cupboards.
All the above are true, I think you may need to test your autoclave and if the autoclave is well, you may need to increase your sterilization cycles or increase cycle time to 25 min instead of 15 min.
Best wishes
Just a thought, a friend of mine was getting a lot of fungal contamination (we think) from another room where fungi were being grown. A lot of our otherwise clean glassware was becoming contaminated as soon as it came out of the autoclave - might be worth making sure the ventilation in your lab is working as it should be (HEPA filters too) and if your room is negatively pressured, that it isn't drawing air straight from an adjacent room. It's unlikely, but might be worth checking.
Perhaps these ideas could be of any help:
How to decontaminate materials and work surfaces
There are several disinfectants available, all of which are characterised by their own specific mode of action. In general, there are two common modes of action:
1) by destroying the lipid membrane of a micro-organism which results in the leaking of cell material
2) by destroying proteins and enzymes necessary for the survival of the micro-organism Ethanol, quaternary ammonium salts and surfactants (a.o. detergents and soap) exploit the first mode of action. Strong oxidising agents like chlorine of hydrogen peroxide exploit the second mode of action. Phenolics like lysol work by destroying both proteins and the lipid membrane.
Some more information on specific disinfectants:
• Ethanol (alcohol)
Ethanol is highly effective against vegetative bacteria, fungi, and viruses surrounded by a lipid membrane, but not against spores. Its effectiveness against non-lipid viruses strongly varies. Ethanol should be used as a 70% solution. Mixtures of ethanol and other substances, such as ethanol mixed with 100 g formaldehyde per liter or 2 g chlorine per liter, are more effective than ethanol alone. Note that the degenerated alcohol used in laboratories is poisonous. Solutions of ethanol deteriorate over time, which is why they should be replaced at least once a month.
• Sodium hypochlorite (bleach)
The chlorine released is a strong oxidising agent, which is effective against all types of micro-organisms. Sodium hypochlorite must be used as a 20 ml/l to 100 ml/l solution, depending on how “dirty” the circumstances are, for instance a 1 in 10 solution of domestic bleach. Solutions of chlorine deteriorate quickly. They should be replaced at least once a week to ensure their decontaminating effect.
• Formaldehyde
Formaldehyde is a gas that is effective against all micro-organisms, provided that it is used at a temperature higher than 20°C and at a humidity of at least 70%. It is suited for the decontamination of fluids, safety cabinets and even whole laboratories, by means of atomisation. Note that formaldehyde is a suspected carcinogen. Moreover, it is an irritant and should not be inhaled. For this reason, formaldehyde is only used in exceptional cases, when a safety cabinet or the whole laboratory needs to be decontaminated.
Examples are an airtight L3 laboratory that has been contaminated, or an airtight animal house, the floor and walls of which may be covered with dangerous microorganisms.
Decontamination by means of formaldehyde is a time-consuming and dangerous procedure that can only be performed safely by specialists. It is therefore recommended that the safety and prevention service be consulted before such procedures are carried out.
• Hydrogen peroxide
Like hypochlorite, this substance is effective against all types of micro-organisms, owing to its oxidising characteristics. It is used as a 6% solution. Hydrogen peroxide is not suited for use on aluminium, copper, sink or bronze.
• Modern wide-spectrum disinfectants
There are many modern wide-spectrum disinfectants on the market. Mostly they combine different chemical substances with different modes of actions against micro-organisms.
They can contain oxidising chemicals, quaternary ammonium salts, surfactants and other substances. These disinfectants can be used for decontamination as well, provided that they are validated for the types of micro-organisms you work with. Do not only check the user manual of these disinfectants, but also check what substances in what concentration are present and what modes of actions are combined in the disinfectant.
Decontamination, especially the decontamination of surfaces, can never be 100% effective.
Decontamination can only result in a sharp reduction of the number of viable microorganisms
(a well-performed bacterial decontamination will lead to a 90% reduction, viral decontamination is often more effective). What is more, after the decontamination, the remains of micro-organisms often stay present. This is why it is important to use hot water and soap to clean the work surfaces, floors and door knobs during the weekly cleaning of the laboratory. You need to decontaminate twice in order to decontaminate successfully.
The effectiveness of different decontaminants bacteria lipid non-lipid
Inactivation Table 1
It is often quite difficult to destroy biological material. Bacterial spores, for instance, are resistant to temperatures of 100°C. Sterilisation - heating water under pressure at a temperature of 121°C for 20 minutes - is the recommended method in such cases. Sometimes the water is even heated at temperatures of 134°C. This is a very effective way to inactivate micro-organisms, provided that the steam can reach all areas in need of sterilisation. Air pockets are a well-known problem. Even animal cells are known to be able to survive in an air pocket during the sterilisation process. A sterilisation tank should be filled in a very careful manner, and all caps should be loosened. Sterilisation is the recommended method to kill bacteria, yeasts, and fungi. For other organisms, like plant and animal cells, and some viruses, simpler methods are available. Heating up to 80°C or exposing the cells to strong detergents is often sufficient to kill them. It is, however, highly important to check whether the method to be used is a validated one; in other words, whether the organisms will be effectively destroyed. Inactivation is much more effective in killing micro-organisms than superficial decontamination: if well-performed, inactivation can be 100 % effective.
When bacteria present in fluids need to be killed, sodium hypochlorite solutions are often used. It is important to make sure that the final level of chlorine in such solutions is high enough to kill the bacteria (20-100ml/l). It should be mentioned that sterilisation is still the most environmentally friendly method. In practice, the fluids containing chlorine are poured down the drain all too often. Principally, fluids containing substances that might affect the proper functioning of a water purification plant should not end up in the company’s waste water. For a decontaminant to be effective, it is also very important that it is in contact with the micro-organisms long enough, i.e. for at least 15 to 30 minutes in general.
Table 2
we finally found that this is a phage contamination.So we are planing to clean our lab with all the possible ways....this will be useful for future people.....
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology