I just started working at a mycology lab and I've been having trouble with contamination (i'm working with yeasts). Is there a cheap protocol I could apply to reduce the fungal concentration in the room?
Try the Fomaldehyde fumigation process which is effective and is a cheaper one.
Use the 1:1 mixture of water and 35%formaladehyde.
But for to do this you have to take utmost care and should not use the room for atleast three days completely and better try this in this weekend.
You have to completely seal the room so that its vapours will not come and you are leaving the doors of cabinets open during the process.
Strict precaustions should be followed as its vapours are dangerous and is a potential carcinogen at higher concentrations. You should put a big notice on the door so that no one enters the room during the process.
After three days leave open all the doors after closing the cabinet doors and leave the room open for ventillation for two to three hours and u can use it afterwards.
But be carefull with process and have a look at the precautions that you have to take while carrying out.
Good luck and it works good to avoid all the contaminations in the Microbial safety cabinets.
Try to clean the MSC filter before carrying out the above process, which will furhter helps you to reduce the contamination.
A room is too expensive to sterilize. It can be done with air inside the room at positive pressure. An alternative is to use a laminar airflow hood and perform all culture activities in there. Basic aseptic techniques, designed entry through double airlocks and regular disinfection with bleach is essential. Wipe your work bench with bleach. Once a week fumigation with formaldehyde is perhaps the best solution for your room. Regular maintenance is the key. Keep records of plate counts to monitor the microbial flora - Trypton-soya agar plates or Sabouraud's should work. Aim at zero yeast and mold counts for perfection . Good luck.
Try the Fomaldehyde fumigation process which is effective and is a cheaper one.
Use the 1:1 mixture of water and 35%formaladehyde.
But for to do this you have to take utmost care and should not use the room for atleast three days completely and better try this in this weekend.
You have to completely seal the room so that its vapours will not come and you are leaving the doors of cabinets open during the process.
Strict precaustions should be followed as its vapours are dangerous and is a potential carcinogen at higher concentrations. You should put a big notice on the door so that no one enters the room during the process.
After three days leave open all the doors after closing the cabinet doors and leave the room open for ventillation for two to three hours and u can use it afterwards.
But be carefull with process and have a look at the precautions that you have to take while carrying out.
Good luck and it works good to avoid all the contaminations in the Microbial safety cabinets.
Try to clean the MSC filter before carrying out the above process, which will furhter helps you to reduce the contamination.
Hire UV equipment for a weekend and illuminate the room . This will kill all organisms (so remove your bacteria or put them in the fridge). After that working clean should be enough to get rid of the fungi, unless they are present in your aircondition system.
use peracetic acid. It's cheap and absolutely safe. Just contact Matthias Seifert [email address deleted].
Perform fumigation with KMNO4 and formaldehyde. Use adequate precautions - avoid inhaling the fumes. You may also consider hiring a UV lamp or a Air purifier for sometime. May cost tad more though.
Strong formaldehyde fumigation process, will reduce the microbial contamination.
Try that one: http://www.bode-chemie.com/products/surfaces/bacillocid_rasant.php
and wash the surfaces afterwards with sterile, distilled water
UV lamp linked with a airpum (it is on equipement special build to clean air...)use it all the time long and fumigation with formaldehyde once or when you need.
Fumigation with kmno4 and 40%formalin. For 2.8cubic cm area 60g kmno4 + 120ml 40% formalin. Room must be sealed well during fumigation
.
I have attached link for standard and advanced methods for Room fumigation
http://www.swissbiosafety.ch/fileadmin/Event_2010_Kuemin.pdf
Based on many years of experience with germfree technology and working with the gnotobiotic animals (germfree isolator technique) peracetic acid (usually 2%) evaporated may be recommended as one of the most reliable sterilizing agent both for the initial inner space and surfaces sterilization. After 0,5 - 2 hr exposure (depending on the volume of inner space, surface properties etc.) followed by ventilation through air filter system. HEPA filter system in spite of good protection capability against of particulate contamination may not be always efficient against yeast and fungi depending on air humidity and time of using.
First you need to identify if the contamination comes from the room or the way you are handling lab materials and media. Be aware that smoking people with long hair are common source of fungal contamination when handling lab material. But, if you have short hair, clean hands, surfaces and lab coats, you may have other sources in the lab. Plants in vases, carbon boxes, dusty shelves and aquariums have no place in a lab. Clean filters in laminar flow is a must, they commonly need to be changed every year or two. If all those factors are looked into, you may consider install some UV lamps at the ceiling. You should have enough lamps to cover the whole room. However, you must have a special switch for these lamps. They should never be on when people are around. Before I go home, I switch mine on for 15 min and when I arrive in the next day, I do it again for another 15 min before starting working. Everything is contamination free. 70% ethanol amended with iodine or phenol is also always at hand for cleaning surfaces and hands before each procedure. Good luck.
Formalin (formaldehyde) tablet as says above is the best option available, but it must be deactivated at working hours.Keeping low contamination level at working place and inoculation under aseptic condition is best option available.
yes off course formaldehyde with KMNO4 is best and cheaper method for room sterilization. it works better than any other method
Combination of UV and antimicrobial treatment (as discussed by others in this group however, use of harsh chemical is highly discourse ) coupled with thermal stability (70-72°F) and Relative Humidity control (less than 60%) is ideal not only for addressing the room sterilization in a cost effective and environmental friendly manner but also in preventing and maintaining a room against microbial contamination. Use of MERV rated (12 or more) filter is helpful for the purpose, if room has artificial ventilation system.
Fumigation with formaldehyde (produced from heated 40% formalin) + KMnO4 and successive neutralization with fumigated ammoniak is the cheapest method. (There exist apparatuses for fumigation of both formalin and ammoniak). 15ml 40% formalin/ cubic meter room volume will suffice. Relative humidity must be above 70% and the temperature must not be below 18° C. Room must be sealed during fumigation and any mechanical ventilation must be put off. After room disinfection and neutralization all surfaces must be cleaned. Formalin tablets are NOT suggested as the liberation of formaldehyde from them can not be dosed.
I got this from the internet, so its available to all; just search!
here it is:
Good luck!
Fumigation Procedure
To sterilize the operation theatre formaldehyde gas (bactericidal & sporicidal) is widely employed (cost effective) for fumigation. Formoldehyde kills the microbes by alkylating the amino acids and sulfydral group of proteins and purine bases.
Step1: Preparation
1. Thoroughly clean windows, doors, floor, walls and all washable equipments with soap and water.
2. Close windows and ventilators tightly. If any openings found seal it with cellophane tape or other material.
3. Switch off all lights, A/C and other electrical & electronical items
4. Calculate the room size in cubic feet (L×B×H) and calculate the required amount of formaldehyde as given in step 3.
Step 2: Precaution
1. Adequate care must be taken by wearing cap, mask, foot cover, spectacle ect.,
2. Formaldehyde is irritant to eye & nose; and it has been recognized as a potential carcinogen.
3. So the fumigating employee must be provided with the personal protective equipments (PPE).
Step 3: Fumigation
1. Potassium Permanganate Method: For every 1000 cubic feet add 450gm of Potassium permanganate (KMnO4) to 500 ml of formaldehyde (40% solution). Take about 5 to 8 bowels (heat resistant; place it in various locations) with equally divided parts of formaldehyde and add equally divided KMnO4 to each bowel. This will cause auto boiling and generate fume.
2. After the initiation of formaldehyde vapor, immediately leave the room and seal it for at least 48 hours.
Step 4: Neutralization
1. After the fumigation process neutralize the formaldehyde vapor with ammonia solution. On inspection (or Surgery) day enter the operation theatre at 7 a.m with 150 ml of 10% ammonia (for 500ml of formaldehyde used, i.e., for 1000 cu. ft).
Place the ammonia solution in the center of the room and leave it for 3 hours to neutralize the formalin vapor.
Formaldehyde is pretty toxic and spreads out of any tiny crevice or opening in the room. If you choose to use this method, make sure the room is very well sealed before beginning and monitor surrounding spaces for escape of the gas. The humidity needs to be high - about 70% or more to minimize residue formation on surfaces. Even so, you will have to clean surfaces of residue afterward.
We switched to vaporized hydrogen peroxide years ago to avoid the many problems and hazards associated with using formaldehyde.
Dr. Mathew is right......... Avoid formaldehyde it will also spoil your equipments
Sterile technique is always a relative matter. The precautions required depend on the experimental situation, including the growth media used, the competitive abilities of the experimental organism, the duration of the experiment, and the intended use of the culture. For these experiments the most serious contamination problem is mold. Many common molds grow well on yeast media and compete effectively, over-growing the plates and obscuring those yeast that do manage to grow. Bacteria are less of a problem as most of them do not grow well on these media and, with a little experience, one can easily distinguish them from yeast. Other strains of yeast can be disastrous contaminants if they go undetected. In particular, a red yeast that sometimes shows up can be really confusing, but is distinguished from our red yeast because it doesn't require adenine.Storing media in the cold is actually counter-productive. It does not prevent contamination, but slows its growth. Consequently contamination may go undetected until the cultures are incubated. It is much better to store media at room temperature and detect contamination before the medium is used.
Sterilizing With Alcohol
The most effective way to sterilize objects is with ethanol. Either 95% or 70% will work. The latter is actually more effective, but the former is often more convenient. Of course the alcohol must be allowed to evaporate or be burned off before the object is used in contact with the yeast. Use a flame to burn off the alcohol, a candle is generally less expensive and safer than a gas burner. The alcohol, more than the heat, does the sterilizing, so just "light" the alcohol to minimize heating and speed up the process. Also, wipe the bench with alcohol before starting an experiment to remove mold-laden dust, the most common source of contamination. If your skin is not particularly sensitive, wipe your hands with a small amount of alcohol, too.
Keeping Sterile Things Sterile
The most common sources of contamination during an experiment are dust, from the bench top, from the air and from people. This dictates several obvious principles:
1) Keep things covered as much as possible.
2) Don't touch anything that will come in contact with the culture and if you do touch it sterilize it again before using it.
3) Wipe down the surface around the experiment with alcohol and minimize air turbulence.
4) Avoid talking, singing, whistling, coughing, or sneezing in the direction of things that should be sterile. Long hair, if not tied back, may be a source of contamination.
5) Maintain a suitable area for preparing, storing, and using sterile media. Unfortunately, house plants, animals, and other materials such as Drosophila media, commonly found in biology class rooms, are abundant sources of mold and must be kept far away from the area used for sterile procedures.
(6) Study Your Contamination to Avoid It Next Time
fumigation using formaldehyde is banned by WHO because of the carcinogenic nature of formaldehyde and also its toxic effects on respiratory tract. the best, easy and short term sterilization method is using BACILLOCID or any other alcohol based spray disinfectants. only 30 mts contact time is sufficient and this method will not have any side effects and is effective against the resistant spores and fungi.
There are in the market solutions that could help you, and are safe to use, one is the mix of peracetic acid-peroxides (companies like Ecolab, DIversay, etc...and is sure you have a local distributor or manufacturer) the supplier share you an equipment to spray the chemical in the lab, previous a deep cleaning, if you want you could check this article...http://www.cdc.gov/hicpac/Disinfection_Sterilization/13_06PeraceticAcidSterilization.html...greetings...and don't forget that more than sterilized in a microbiological lab is absolutly necessary a frequent cleaning and disinfetion programm.
Try 70 % ethanol as disinfectant it is safer comparatively. autoclave all equipments before use.
fumigation using formaldehyde is banned by WHO because of the carcinogenic nature of formaldehyde and also its toxic effects on respiratory tract.
Dear Efren, You have quite a few cheap options. Try a few and let us know which one works better for you
Try to trace the points at which the contamination takes place so that you can simply tackle it at those. There is every possibility that your entire room is not contaminated. Your research tools and equipment need to be checked. And if by chance you were able to trace it to your inoculation chamber, then turn on your UV light for 2hrs with the chamber closed.
fumigate your lab with formaldehyde and KMnO4 don't open your lab atleast one r two days
Before room sterilization close all windows of the room. Then add about 5g Potassium per manganate and 15ml formaldehyde in petri plate wit good luckhout lid. immediately close the room . entry in the room should be restricted for 2 days. place about 3 -4 plates of this mixture at different places in room.
Cheaper, efficace and easy to find and easy to apply: bleach! (sodium hypochlorite)
you can try to fumigate your lab with a disinfectant with sporicidal activity (apparently, ethanol wouldnt wanna be your choice). clean and wash all exposed surfaces, mop your floor with disinfectant and simply restrict entry or carriage of particle-generating materials to the room. to monitor whether you are in control of the contamination in your work area, always have a plate exposure to measure the fall-out of the air spora in your work area, the Bacteriological Analytical Manual suggests that you should have less than 1 CFU per plate exposed within 15 minutes if I am not mistaken.
if you happen to work in a laminar flowhood, make sure that you saturate and wipe your hood down with a disinfectant, turn on the UV lamp and keep the fan motor and air filters running for at least 30 minutes prior to your work proper.
Hope this helps!
Best regards,
Kris
PS. the way we used to do it in the lab is that we have to wipe all walls and mop all floors first with a liquid detergent, rinse with distilled water, then disinfect all walls and surfaces and floors and allot a contact time of at least 15 to 20 minutes prior to rinsing with distilled water. then we agar expose plates to measure the fall out rate of the air spora by virtue of gravity within 30 minutes.
i used to work in a pharmaceutical manufacturing before and that was one of our practices, even in our sterility testing rooms. if you happen to have a merck air sampler or MAS100, then that is a better alternative to measure the extent of contamination in your lab
Phenol disinfectant is better way to sterile your lab and after the usage of phenol 40% formaldehyde with potassium permanganate fumigation may give better results.
Better to use private cabin to isolate bacteria and fungi. This prevents contamination of the air samples from the laboratory. Prevent pollution. By air. Then use the phenol
You have 2 main options - either formaldehyde or hydrogen peroxide. Suggest you schedule exercise at the weekend to minimise exposure of other staff using the site.
Fumigation with Potassium permanganate and 40%formalin. This is found to be effective
With the help of powerful germicidal UV lamps fitted in the inoculation room you can reduce the fungal concentration in the room?
Use of formaldehyde is said to be carcinogenic please verify then use or use any other disinfectant that will give good results.
Dear, i suffered from this proplem for long time , first u can use UV-C lamb irradiation for 30-to60 min it will be effective in room steralization also ucan use dilute chlorine in room cleaning before irradiation, avoid obened doors and windows during inoculation or mycologial work, also we use 5% savlon in sanitation, with best weshes
Please be aware of using formaline as possible as it has alot of health proplems.
Extensive using of formalin as a disinfectant is not recommended due to health hazards particularly with eyes, besides the flame try to clean up the bench area, the instruments and your hand with ethanol, also usually keep the places in which you are working are closed (away from air current), u will achieve good results, best wishes.
You can use formalin, but you must have to assure 24 hours time of contact, minimum 14 degrees in Celsius and 70% relative humidity. After that, you can neutralize the indoor formalin using a double volume of an ammonium solution.
Ultraviolet C radiation is an alternative, but be sure that the emission lamp is not overused (or too old) and inactive. Clean all surfaces first using a face mask.
There is one disinfectant which is cheap, highly effective (bactericidal, fungicidal, sporocidal, viruzidal): peracetic acid ... and non-toxic, not carcinogenic, .... For more details: see www.kesla.de.
In earlier comments I suggested the use of Formaldehyde i used almost forty years ago. Later besides corrosion its carcinogenic effect came about and this cannot be ignored. I write this to withdraw my suggestion on use of formaldehyde for fumigation- it is hazardous. Perhaps peracetic acid as suggested might be a good substitute but positive airflow is the surest way to keep a room sterile when the inside is supported by UV lamps for initial decomposition. Bleach is nowadays used for disinfecting surfaces in sterile hoods and could be used but that too has corrosive long-term effect.
In our animal facilities, some of which harbor SPF animals, each stable is sterilzed by the use of formaline. However, you need to neutralize the formalin as soon as the necessary decontamination period has been reached. Ideally, you should leave the room for another 24 hrs after this, while ventilating the room in order to get all vapors out of the room. I would like to stress the necessity of neutralisation with ammonium and the following ventilating period.
I think the contamination is a result of your technique because fungi are ubiquitous. Improve you skills and use appropriate antibiotics in your media.
STERILIUM is an effective, safe alcohol based disinfectant . using inoculation hood / bsc cat 2 is the ideal equipment for handling fungal cultures and prevent contamination
I think Stephan suggested best option. In my openion also peracetic acid is best option.
You can also use Formaldehyde and Potassium permanganate mixture. But it is hazaardour.
Dear colleague
It is NOT cheap to buy a hydrogen-peroxide-vapour room decontomiantor, such as Glosair-R (previously known as "Sterinis-R"). But in Europe you can have a paid visit from companies to decontaminate a single room. It just might be more affordable than to arrange fumication? And certainly less toxic to the environment.
(I tried to look up this service in Mexico, but so far with no success :-(
There is a specific antifungal fumigation and desinfectant, Enilconazole, commericlly available in most countries.
For many years we are working with the simple protocol to alternate 3 types of solutions (Hipochlorite 5%, Ethyl Alcohol 80% and Phenol 5%). The dissinfection is clean the surfaces with cotton soak in the solution, for 2 weeks we disinfect the area, and then change the solution
Formaldehyde fumigation is the best one... But be careful while fumigating the laboratory.
After performing the room decontamination, I would recommend attaching a regular HVAC type air filter to the supply air ducts. We attached ours using elastic straps (bungee cords) to the duct diffuser. Although crude and somewhat ugly, this has finally cut down on our fungal contamination.
Clean all surfaces like Hilda suggested then use a UV lamp for minimum 30 min
Avoid air power fluxes, use antibacterial air filters, treat all surfaces with ethanol (70-80 %), then turn on UV lamps for 30 min at least.
Fumigation with Formaldehyde is the best and cheap method for room sterilization. Take formaldehyde 15 ml in a pertiplate and add small quantity of Potassium permanganate and close the room. Perform this exercise by the weekeds only. Leave the room atleast overnight with fumigation. Please take care that formaldehyde fumes will irritate the eyes. Based on the room size the no. of fumigation plates can be decided. Remove the plates on the next working day, switch on the Air conditioner and can do the work after 2-3 hours.
To prevent entry of outside air into the sterile area use air curtains.
There is only one cheap protocol: to be professional and to work cleanly/
Yes, is true. The best effectiveness cheap protocol starts with a professional & clean work; and these work must be based on protocols developed for each activity. All of ideas exposed here are good, but the best idea is the idea that is better for our purposes
I agree with Prof. Vladimir: Maintaining good level of cleanliness togetherwith observance of aseptic procedueres comprehensively will take care of the problem.
As suggested by Dr. Nalam-->The old school approach of using a formalin fog is very effective....it kills microbes not only on the surface of benches and walls, but also penetrates into seams of floors, walls and ceilings. It is very hazardous so you should check with your local Environmental Health Officer to make sure that you can use it as this treatment--and as suggested above, place a lot of signs to warn people so that they do not enter the room during fumigation or the days following (it takes several days to de-fumigate). Since this is for a mycology lab--the fog will kill fungal spores as well!
Hello, happy new year!
You can use the "anios" device.
It's an efficient diffuser.
You can choose the product to use with.
Some commercial products are available. You can also use H2O2; Avoid formol because it's very harmful!
ggod luck
I think you are already using autoclave for sterilizing the media,
if yes then
you have a problem in inoculation of yeast to your sterilize media.
Then you should use just a Bunsen burner,
Sterilize your needle or wire-loop on flame (until it red hot), cool near the flame (no more than 15cm away.
WORK NEAR THE FLAME (WITH PRECAUTIONS),
Your media and culture (to be inoculated) must be withing the limit of 15-20cm from the flame.
Remember that your culture (to be inoculated) should not be contaminated already.
Hello,
We use Dismozon to clean surfaces that produces peroxide evaporating into the air. If you use an excess of it (wiping large surfaces) it might help, but it has a very unpleasant smell for days.
Make extra effort to clean the lab, from top to bottom. You should also scrub the lab walls with disposable dry cleaning tissue papers once a week as part of the lab cleaning process. Many fungal spores like dust will stick to wall surface. Conduct all the sub culturing work in sterilized laminar air flow cabinet (UV sterilize for 15 minutes and wipe with 75% EtOH).
I recommend using light U.V. and after continuous use rotary hypoclorite 10%, phenol 5% and ethanol 70 % to avoid cross contamination.
Just you add the Phenol crystal with water and keep boiling with slight flame (Bunsen burner) around 4 to five hours with entire place closed, Subsequently boiled phenol with 1:20 ration of Distilled water was used as the surface cleaning..
Mixture of potassium permanganate and formaldehyde is a cheap protocol for room sterilization .
Use fumigation with ethanol 40% vol/vol in water at room temperature, do not exceed 40% because of the flammability limit of ethanol. Otherwise contact for 48-72h, over the WE is excellent. Close the premises, about 1l per 20 cubic meter. Maximise the surface of the aqueous solution for fumigation. Inexpensive, not harmful and efficient, about 5 log reduction for 72h contact with fungi (Dao et al, 2010. Journal Applied Microbiol, 109, 408-414)
Using formalin is a good idea, but it will leave marks on all the surfaces which are difficult to remove. Also formalin is considered as carcinogen and is regulated by institutions heavily. It is bad for the machinery in the room as well as culture hoods. If the institution allows then get a positive pressure room with a hepa air filteration system. Altenatively you can use hoods which circulates air after passing thru the hepa filters
I agree with Mr Nalam.......fumigation is the best way for removal of contamination.Use fumigation mixture as per size of the room.
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