Have you considered sequencing the 16S rRNA of this bacterium?

Indeed I ask you to proceed for 16S rRNA or 23S rRNA sequencing using universal primers and you can consider its metabolic and biochemical profiles (using BIOLOG or API50CHB strips) other you can prospect by SEM microscopy.

Where did you isolated it?

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The first step is 16S RNA analysis. After you received the bacterial identification you can do biochemical tests. You have to choose a biochemical tests kit according the 16SRNA results

Thanks everyone. I will proceed with sequencing before continuing my biochemical characterization. It was initially isolated from a Drosophila laboratory culture from a stock center that has persistent problems with the food spoiling (turning black and foul-smelling) and causing reduced numbers of adult flies. There also appears to be at least two different fungi in the mix that contribute to this persistent problem.

First, make sure that you have pure culture of bacterium without any fungi contamination. Then you have to identify the isolate using molecular (16S rRNA gene sequencing), biochemical and morphological methods. Sometimes, the various stocks is getting contaminated with different type of bacterial and fungal strains. It is necessary to find the origin of contamination to be able to remove them. Drosophila is relatively pestilence-free, but mites, fungi and bacteria can be problems in laboratory cultures. It is good practice to clean your bench top and fly pushing equipment regularly. This is particularly important if a problem is evident. Clean the bench top and all equipment that comes into contact with potentially contaminated stocks with 10% bleach, 70% ethanol or soap and water after use. Sharing pounding pads, CO2 pads, fly pushers and sorting plates can aid the spread of contaminants. If sharing is unavoidable, the need for cleanliness should be understood by all and enforced.

It seems you have found a psicrophilic bacterium rather than a mesophilic one. In my experience, I found aerobic, gram positive rods with brownish colonies growing at 25°C in mineral waters. In that case, it was Rodococcus erythropolis.

As they already said, you need at least a 16S RNA analysis. Alternatively, you can do biochemical tests (Biolog) and use the Bergey's Manual of Microbiology (even dichotomous keys worksheets) and try to identify the family or, if you are lucky, the genus of your bacterium. Have you noticed any endospore in old cultures?

Did you find mucous layer in the stocks? The most common problems are caused by mucus producing bacteria. Although not directly toxic, larvae, and to some extent adults, become trapped in the heavy layer of mucus that coats the surface of the food.

Large numbers of larvae overcome the effects of the bacteria in a healthy stock, but weak stocks or pair matings can be seriously compromised. A widespread bacterial problem may indicate that the pH of your medium is too high; try lowering the pH to about 5. Individual stocks can be treated with antibiotics for one generation. A quick approach that often works: add 100 µl of penicillin-streptomycin solution (10,000 u/ml and 10,000 µg/ml, respectively) to the surface of the medium in a vial and allow it be absorbed. Add a small amount of yeast and transfer flies to the treated medium. Discard adults before progeny eclose; subculture progeny on untreated medium. Other antibiotics may be tried if the contaminant proves to be resistant to penicillin and streptomycin.

If its something you would like to publish and know more about, I suggest you sequence the whole strain

This seems to be very specific in its temperature requirement and very likely to be associated with the intestine of some host which has also living in a habitat that is not subjected to large variations in temperature. Your Gram stain results seems to be inconclusive. If it is Gram positive rod, why did not you do Spore staining?

The best way to clear the confusion is to go for 16S rRNA gene sequencing.

The growth features and colony morphology points to a kind of LAB. However, LAB is catalase negative. But you can never discount the possibility of mutants.

Hi Rosa - the bacterium may be a psychrotroph but it does not seem to be a psychrophile as it does not proliferate at 4 deg C. I will be attempting sequencing in the near future. The biochemical tests were easy enough to do since the reagents were available.

Kambiz - thanks, we've tried all the standard antimicrobial measures you suggested with this culture but they haven't solved the problem permanently. This includes antibiotic treatment with pen-strep, ampicillin and kanamycin. There is no mucous layer on the food surface. Although the larvae survive the infection and many do eclose, the food starts to go bad shortly after L3s appear. Anyway, there is at least one yeast and an unidentified fungus associated with this bacterium (mycelia are visible in old cultures), so all three seem to contribute to the infestation.

Mohamed, the rods are very short and do not appear to be standard Bacillus species type rods, so I have not attempted the endospore stain yet, although I probably will in the future, after the sequencing has been done.

Thanks again everyone.

I realize I haven't updated this thread in a while, but the bacterium we determined is not the offender here. On its own, it causes the browning of the medium without visible effects on the flies. Problems begin when it is paired with what we previously believed to be a fungus, but 26S sequencing revealed this eukaryotic unknown to in fact be an alga that no longer has functional chloroplasts. Very curious, but we aim to publish this story in the near future.

Hi Mark, we are experiencing the same problem. How did you resolve the issue with alga?

thanks

My original plan was to use nystatin and/or amphotericin B to address the algal contamination, although ultimately we were able to reorder the affected stock. At least amphotericin B can have mutagenic effects on Drosophila, so the concentration must be kept low.