New data and more to come......

On Monday we started our carbon source utilization tests. We did this using the medium described by Ludeman (1968). Eugene had prepared 16 different carbon sources plus basal medium without an added carbon source (this will act as the control). We spotted 10ul of a cell suspension of each of our strains. The cells had been washed with 0.9% saline to remove any residual carbon source from the initial growth medium.

For the strains that had shown good growth at 10C we streaked these (from the stock plate – grown at 25C) on the medium they grow best on and incubated them at 4C. If these grow at 4C after 20 days we will reduce them to 2C.

The pH plates which had been incubated for 20 days at 25C were scored on Wednesday and we found that some strains grew through the range pH 4 to pH10. Others did not grow at either end of the range tested. We scored these as 1-4 + so as we can say what the optimum pH for growth was as well as the range. See a very nice example of the plates from our pH experiment below. Interestingly these strains did not grow at pH 4 or pH 5 but did grow at pH 6 through pH 10. Also of interest is the change in color of strain 69 for example (bottom right corner in pictures below) at different pH values. At pH6 strain 69 is black while at 8, 9 and 10 it is orange/tan.

We need to look in our strain excel sheet and see which soils samples these strains (67, 69, 70, 75 and 76) came from. Did they all come from the same sample and what was the pH of the original sample. We should measure the pH of some of the original soil samples which Rainey will have in his lab.

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9 pH 10

In a previous class we demonstrated that we could use the Geo specific primers (Salazar et al) to test if a strain is a member of the Geodematophilaceae. We used these on some strains from our strain collection as well as on strain we had isolated from the 3 soil samples we used in class. This week we extracted DNA from these same 3 soils (without radiation) using the MOBIO Soil DNA Extraction Kit. We used the kit that starts with 10g of soil. The reason for using 10g is that these desert soils have low numbers of CFUs/g and so will probably have a low DNA yield. We also attempted to maximize the DNA amount we recovered by doing the final step (the elution) using half the amount of MD5 (4ml instead of 8ml). Next week we will use this DNA in PCR using the Geo specific primers to demonstrate the presence of members of the family Geodermatophilaceae in an environmental sample without having to isolate the actual organisms.