Microbiology and Spectrophotometers

Does PAH stand for polycyclic aromatic hydrocarbons? As long as it is all completely dissolved, it should not affect the turbidity. The oils would tend to affect it if not dissolved. Any powder that does not dissolve will affect turbidity.

What kind of spectrophotometer? What wavelength(s) are you operating at?

Mike

Yes,polycyclic aromatic HC.I'm using used motor oil whereby it does not dissolve completely with media(mineral salt medium). And after inoculation,ill take the reading at 600nm with UV Spectro Libra S12.After one week,i got few negative results reading (means blank) and the graph od vs days are not linear. What can i do to avoid the turbidity due to PAH itself?

Have all the cells (bacteria) died?

If you are getting a negative effect after several days, this could indicate the particles are settling to the bottom of the cell. You could try a magnetic flea with very gentle stirring or use a continuous (recycling) flow cell. I would have thought powders would float on the surface and not become part of the solution unless they have dissolved. If you are not interested in the turbidity try filtration.

You might try to add something to improve the solubility of the motor oil. If your bugs can grow using PAH, they may be able to withstand a bit of something like ethanol? If you can get a more homogeneous solution, then more of the turbidity you measure should be related to microbial growth, and less due to oil droplets.

As a control, you would need to add the same amount of organic, without the motor oil, to see what effect it has on the bugs. (My guess is that they may use a simpler carbon source preferentially, then move to the PAH :( )

Does the non-soluble portion of the oil float on top of the solution? If so, you could also pipette the liquid from below the oil layer. Whether tht is valid will depend upon your experimental design

Hi ter!

I tried adding the ethanol to oil then introduce it to media with bacteria. Stil,some showed positive result (eg 0.330) and some with negative result (eg-0.0222). Why on the same (0 day) there's difference in reading??quite blur.

Are you sure that you bacterial spikes are consistent? You will need to have a culture, diluted so that you know how many cells you are adding. Otherwise, the difference could be entirely due to inconsistent spikes.

Were the bacteria added in both cases from the same culture and the same age?

How homgeneous is the Motor oil? Is it possible there was more of something inhibitory in one vial than in the other?

On the ethanol front, what happens to the turbidity with no ethanol, ethanol only to the culture, and ethanol/oil mix to the culture? What happens when you add sterile media to the ethanol/oil mix without the bacteria? Do you get additional turbidity, not related to growth

http://www.google.com/search?hl=en&rlz=1T4ADRA_enUS339US339&q=microbes+that+utilize+motor+oil&start=10&sa=N

looks like surfactant could also help you in getting a more consistent emulsion....

Nop.I didnt do any dilution. btw,this is how i carried out hte experiment:

I add 100ml MSM media,1ml used motor oil(mixed with ethanol) and 2ml indepedent bacterial culture that have been grown in Luria broth for 48hr.It was then incubated at room temperature on shaker at 160rpm. Every 2days,ill take the reading. The blank is medium and oil without inocuation and control is media with ethanol.

The control's reading on 1st day was -0.061 .

May i know what you mean with inconsistent bacterial spikes and bacteria from same culture? im using 6 different types of bacteria taken from gylcerol stock prepared by my fellow seniors.

Can you suggest me any reference book that would be helpful in my experiment entitled developing microbial consortia for use in bioremediation of hydrocarbon contaminated soil?Thank you.

From a quick internet search:

http://www.google.com/search?sourceid=navclient&ie=UTF-8&rlz=1T4ADRA_enUS339US339&q=Bacterial+bioremdiation+of+contaminated+soil

When you are looking at growth curves, to get consistent results, you need to know approximately how many bacteria you are adding to your experimental sample. (Too many can really skew things, as you will begin with high turbidity) If you don't control how long the cuture has grown prior to beginning the test, you can have wildly different cell numbers added, and inconsistent results between experiments. To get a starting number of cells in your culture, serially dillute your culture, and plate the dilutions until you get a countable number of colonies.

Also, what happens when you add the ethanol to the media/oil mix with no bacteria? If that still becomes turbid, it could be the cause of some of your problems. (This solution should be used as your blank, to compare with the same solution with bacteria added). That will help correct for your background turbidity (If it is not too high). If the blank is turbid, then you may need to do some lit searching to find out what growth media work well when oil is added to it.

If there is solid media left in your culture, that will contrubute to turbidity (particles scatter light)

Have you tried sonicating your initial solution? I would mix like you normlly would, take a reading, sonicate, and read again. Could make it worse, could make it better.

This sounds like a fun project, and will present some interesting challenges! If you look on the net, there appears to be a coule compnies that sell a bacterial bioremediation product- that could be a good control to run in combination with blanks, and your mixed culture-

Thank you very much for the suggestions and for all the replies. Will try to manipulate this.

Have fun! (That is the most important thing