Dear Kunal,

You have no necessity to dissolve oil in the medium, in our Inst. we just add oil (any mineral or natural) to the liquid medium, add our microbes (which we hope to degrade oil) and after period of cultivation checking oil degradation. Or another way is to use agar mediums and in the center of the medium you made a well, you plate your microbes onto agar and after that into the well you add some oil of interest. Oil will migrate into agar and growth of some microbes will be decreased or inhibited, if no inhibition or even intensification of growth noticed that you find your oil resistant and oil-consuming bacteria. For sterilization of medium with oil we use three time 20min heating-cooling on the boiled water bath. We did not checked autoclavation, but you can check.

Best,

Sergei

I guess that you mean mineral oil, am I right? It has a wide range of chemical composition and physical properties, and I think you better to use oil as

1) fine emulsion (as suggested by Dr. Hoyles),

2) fine medium particles covered by oil (http://www.ncbi.nlm.nih.gov/pubmed/3148538), 3) oil absorbed by minerals (like calcium carbonate from Utah) or

4) by organic compaunds (http://www.biocleanenvironmental.com/content/product/bio_sorb/Brochure_BioSorb.pdf)

You may use catechol in place of crude oil. As catechol is an intermediate of many oil degradation pathways, organisms capable of utilizing it as sole source of carbon should also be capable of growing on oil.

To increase mixing of oil with media, you may add some surface active agent.

1) Attaching on particles. When I prepare petroleum-oil agar, oil was mixed well with silica powder. Then, added into prepared mineral media, autoclave and solidifying in petri dish.

2) Just put into liquid media. Degraders could grow at interface.

Dear Kunal,

You have no necessity to dissolve oil in the medium, in our Inst. we just add oil (any mineral or natural) to the liquid medium, add our microbes (which we hope to degrade oil) and after period of cultivation checking oil degradation. Or another way is to use agar mediums and in the center of the medium you made a well, you plate your microbes onto agar and after that into the well you add some oil of interest. Oil will migrate into agar and growth of some microbes will be decreased or inhibited, if no inhibition or even intensification of growth noticed that you find your oil resistant and oil-consuming bacteria. For sterilization of medium with oil we use three time 20min heating-cooling on the boiled water bath. We did not checked autoclavation, but you can check.

Best,

Sergei

For isolation of toluene utilising micro-organisms we developed a continuous flow technique which is usable for liquid water insoluble, volatile and gaseous substrates as well as for substrates with growth-inhibiting properties. See:

Mutafov S, Angelova B, Schmauder HP, Avramova T, Boyadjieva L. Titrostat: a reliable technique for continuous cultivation of microorganisms utilizing liquid or gaseous water insoluble substrates. Biotechnol Bioeng. 2003 Oct 20;84(2):160-9.

Have a nice day.

S.M.

Autoclave the agar medium without the oil (whichever oil you use), filter sterilize the oil and add it to the cooled (40-45°C) preinoculated agar medium. Stir heavily with help of a magnetic stirrer using a previously autoclaved magnetic bar. Immediately after stirring, pour plates and let agar solidify. Although some oil may form drops on the surface, it does not hinder the microbe from growing (see http://revistes.iec.cat/index.php/IM/article/viewFile/60590/pdf_275)

Alternatively, you can use DMSO as surface active agent. DMSO usually does not hinder or enhance microbial growth. Mix DMSO (maximum of 3 % final concentration in agar) and oil, filter sterilize the mixture and add under agitation (as above) to the precooled sterile agar medium and immediately pour plates. After plates are solidified, inoculate the agar (streak or spread inoculation).

If it is a highly volatile oil, you place some on the inside of the lid, use a carbon-poor agar, inoculate your agar and incubate the plate upside down. Obviously this doesn't work if your carbon source has a low vapor pressure.

You need use a surfactant, the surfactant gives you a media to disperse the oil in the liquid or solid if you make a mixture

No need to dissolve oil in medium, I am agreed with serge voychuk!

Please specify what do you mean by oil. Is it some petroleum product? if yes, it is difficult to isolate such degrading organisms but not impossible. Note if can not always dissolve oils in water, but definitely you can disperse it using a suitable wetting agent say Tween series of surfactants, or Teepol etc. It is not advisable to add oil to the medium and autoclave it. It may not get sterilized properly, Better to sterilize it separately (at a higher temperature) and add it to the medium before inoculation.

Surfactants may be more easily degraded than the oil, so be mindful that you are not isolating surfactant-degrading bacteria.

i got this very interesting am currently on it..cheers

before and after autoclaving

We are also working on the degradation of oil pollutants by bacteria. If you want to use a solid medium, then you should have to dissolve the oil in the medium using magnetic stirrer. But in a liquid medium, it is not necessary to dissolve the oil only incubate the culture in shaking incubator.

In solid medium there is no need to try to dissolve the oil (presumably hydrocarbon) using magnetic stirrer. Just add some inert emulsifier so that the insoluble oil will be dispersed as evenly as possible. Here if you use a magnetic stirrer, you may land up in breaking the emulsion.