Hi There,

I would make a dilute suspension and filter through a .45um filter. This should remove the bulk of fungal elements and spores. Then plate onto Nutrient agar with cycloheximide. Use a temperature that is Ok for the bacteria and sub-optimal for the fungus and subculture colonies as soon as they appear (a dissecting microscope is handy!).

hope this helps,

cheers,

Richard

The best would be a highly selective media for the respective bacteria and rapid subculture after start of visible growth of the bacteria. This can result in a pure bacterial culture again.

In principle from all bacteria of interest kryoconservation should be prefered to be able to get back to the stock culture if a contamination happens.

I agree with your Dr. Johannes. Thank you. 

If you have access to tools like a micromanipulator, you can use hanging drop method to get a pure strain back as long as there is no other bacterial contamination. It might be easier to pass it through a size selection filter as well. Or if you know the two strains well you can use selective media or try sub culturing.

Hi There,

I would make a dilute suspension and filter through a .45um filter. This should remove the bulk of fungal elements and spores. Then plate onto Nutrient agar with cycloheximide. Use a temperature that is Ok for the bacteria and sub-optimal for the fungus and subculture colonies as soon as they appear (a dissecting microscope is handy!).

hope this helps,

cheers,

Richard

Hi

The answer is very simple. Make a subculture to a selectrive media or a basal medium like NA, LBA and just after 12-14 hrs of incubation at 37+2 0 C , go for another subculture onto the same medium. As fungi take a longer time for growth and generation.

Always maintain Glycerol stocks of the isolates under Cryo conditins.

If you have specific strains of bacteria for which there available selective media then you can simply subculture the stock to this selective media and/or the specific growth conditions that are optima or selective for those strains, and try to keep the minimum time for incubation to avoid fungal growth, because several fungi can grow on selective media for bacteria. Alternatively, or if you don't have selective media you can dilute the semi solid stock with sterile water or sterile diluent (e.g. Maximum Recovery Diluent) and then filter it through sterile GF/C filters of 1,2 micrometers into a sterile flask. This way you can get read of the fungi which are over 2 micrometers in diameter, and then you can culture the filtrate with the bacteria onto a nutrient agar or plate count agar (if you don't have selective media)  

Make a  subculture on selective media under specific conditions  as temperature ,minimul time for incubation ,Ph and also use anti -fungal agent is one of the most best  way  to rid up from the contamination of fungi

One caution. The contaminating fungus may also be carrying other bacteria in its spores.

Selective media are useful.

If possible, spread to single colonies on agar plates and then spread three colonies to single colonies again and make stock cultures from these. The reason for the three colonies is (1) that bacterial isolates have a tendency to change their genetic characteristics during laboratory growth, particularly subjected to insults such as contamination and (2) if you have fungal contamination, it is natural to suspect other kinds of contaminants as well.

Combine this with one or more of the procedures that other have suggested if you can.  

If the bacterial isolate is a new one, you should watch out for mutational adaptation to laboratory conditions - such as shedding plasmids that are important for growth in the wild but costly to maintain. Variable colony size is a hint that this is going on. Therefore, if there is a chance of this happening, hang on to your old contaminated culture. 

You should retest your purified isolate in order to make sure it has the same properties as the original one. 

Likely, the best approach is combine several of the suggestions presented here:

• use a media that promotes a fast growth of your bacteria if it is available. Usually, fungi are slow growers.
• Subculture as soon as you get colonies, even if they are small, try it.
• If your bacteria can survive at 37° C or higher, try it. Many fungi, specially if they are from environmental samples cannot grow at such high temp, and if so, they are really slow.
• use a broad spectrum antifungal or a cocktail of antifungal agents. They may not kill but inhibit growth. It is still recommended to subculture a couple of times at least.
• Avoid moving the contaminated plates as much as possible to limit the release of spores.

In my experience, specific species of bacteria are usually more sensitive to variations of pH. So, unless you know that your isolate can grow in such extreme conditions, it could be more detrimental than beneficial. Salinity may help but unless it can grow very well with more than 2.5-3% NaCl I wouldn't change it, as it will also affect severely to the bacteria.

In any case, those last are general recommendations and very prone to change depending on the species of bacteria and/or origin of the isolate (don't know if it is an environmental or a culture collection bug).

The best way using selective media that contain 0.5 gram per liter cycloheximide (protein synthesis inhibitor in eukaryotes but not bacteria) it will inhibit most of the mold growth.

Hi, use PDA (potatoes dextrose agar) or SDA to culture the fungi