Lab class at high altitude…..

Monday saw Rainey on Pico de Orizaba at some 4700m looking for Martians! Check out this link for a small explanation of “Why Pico de Orizaba” – http://www.hightreeline.blogspot.com/. Some above refer to Rainey catching some rays – and yes that was very true as the uv at such high altitudes is significantly higher than below sea level back in the swamp. The application of some very good sun screen is a must to protect against uv and wind burn on Pico de Orizaba. A number of strains that we are using in class came from this site they have the PO designation.

Monday was microscopy day in the Socolofsky Microscopy Center in the basement of Life Sciences Building. The plan was to look at 6 of our strains (2 strains per group) using light microscopy and Transmission Electron Microscopy (TEM). Reportedly there were some problems with staining the cells with DAPI and will getting the cells attached to the copper grids for TEM. These problems are most likely caused by the properties of the organisms and not by anything we were doing wrong.

Wed, temp repeats 10C plus new 45 and 50C. Since we had some that grew at 42 we had gone to 45 and 50. Those plates were incubated in plastic bags to stop them drying out. The 37C plates just had parafilm and some dried out. We sent our results to Rainey who will update the spread sheet on AirSet. More data on the temperature range for growth of these strains. Thsose strains that grew at 10C we will now try at a lower temperature like 4C (this is the temperature of the cold room). We will set these 4C tests up on strains that grew at 10C this Monday.

We also checked the results of xylan, Avicel and Granular Cleeulose degradation. The plates were stained as follows: Xylan –iodine, Avicell (crystalline cellulose) and Granular Cellulose – Congo red. In eth case of Congo red we applied it to the plates for 15 mins then poured it off and applied saline solution for 15 mins then observed the plates for zones of clearing around the colonies.

All of our strains tested –ve for hydrolysis of these compounds. This is not too surprising as this is the case for the type strains of the species of the genera to which they belong. There is still the option to test them using filter paper in liquid culture. The cellulose of filter is easier to digest than crystalline cellulose by some organisms.

We also ran out the PCR that we set up last Wednesday using the Geodermatophilaceae specific primers from the paper of Salazar et al 2006 (at AirSet). Some groups got strong positive bands for their strains from the class collection. For the isolates from the soil samples pre and post irradiation there were both positive and negative results. A positive result is a band around 550bp. There is some debate about the amount of DNA being recovered from the strains using the MOBIO kit. Some groups have good DNA while others seem to have little or no DNA.
Those isolates that gave a positive band with the Geo primer PCR will have their 16S rRNA gene sequence to determine which of the Geodermatophilaceae these isolates belong. We will set up 16S rRNA gene sequence PCR on those isolates next Monday.