Identification of subsurface microorganisms at Yucca Mountain. Quarterly report, July 1, 1995--September 30, 1995

More than 1100 bacterial isolates were obtained over a two year period from 31 springs in a region along the southern boarder of California and Nevada. Water samples were collected from 17 springs in Ash Meadows National Wildlife Refuge and 14 springs in Death Valley National Park. Bacteria isolated from these samples were subjected to extraction and gas chromatography to determine the cellular fatty acid profile of each isolate. Fatty acid methyl esters (FAME) extracted from cell membranes were separated and classified using the Hewlett Packard by gas chromatography. The FAME profiles of each isolate were then subjected to cluster analysis by the unweighted pair-group method using arithmetic averages. During this quarter the relatedness of FAME patterns of bacterial isolates were examined at the genus level by counting the number of clusters produced in a MIDI dendrogram at a Euclidian distance of 25. This information was then used to determine microbiological relationships among springs

Identification of subsurface microorganisms at Yucca Mountain; Third quarterly report, January 1, 1994--March 31, 1994

Bacteria isolated from ground water samples taken from 31 springs during 1993 were collected and processed according to procedures described in earlier reports. These procedures required aseptic collection of surface water samples in sterile screw-capped containers, transportation to the HRC microbiology laboratory, and culture by spread plating onto R2A medium. The isolates were further processed for identification using a gas chromatographic analysis of fatty acid methyl esters (FAME) extracted from cell membranes. This work generated a presumptive identification of 113 bacterial species distributed among 45 genera using a database obtained from Microbial ID, Inc., Newark, Delaware (MIDI). A preliminary examination of the FAME data was accomplished using cluster analysis and principal component analysis software obtained from MIDI. Typically, bacterial strains that cluster at less than 10 Euclidian distance units have fatty acid patterns consistent among members of the same species. Thus an organism obtained from one source can be recognized if it is isolated again from the same or any other source. This makes it possible to track the distribution of organisms and monitor environmental conditions or fluid transport mechanisms. Microorganisms are seldom found as monocultures in natural environments. They are more likely to be closely associated with other genera with complementary metabolic requirements. An understanding of the indigenous microorganism population is useful in understanding subtle changes in the environment. However, classification of environmental organisms using traditional methods is not ideal because differentiation of species with small variations or genera with very similar taxonomic characteristics is beyond the capabilities of traditional microbiological methods

Identification of subsurface microorganisms at Yucca Mountain; Second quarterly report, October 1, 1993--December 31, 1993

The primary effort of this past quarter was to develop a procedure where accumulated data files could be evaluated to determine the naming consistency and inter-relationships of the various species which have been identified by the Microbial Identification System (MIDI) system. This involved a series of steps, including the clustering of similarly named organisms in a dendrogram format to determine how closely similarly named isolates are related. The experience of other researchers using the MIDI system has shown that clusters which are joined at a Euclidian distance of 10 or less belong to the same species. Strains which are very similar cluster at less than 6 Euclidian units and clusters below two units have nearly identical fatty acid patterns. When the dendrograms derived from the springs were scrutinized, some organisms were found which did not match the pattern of their named group. Then a decision was made whether to rename the isolates and exclude them from the group or redefine the group. This decision was assisted by plotting the principal components derived from an analysis of the fatty acid composition of members of the genus. Each species can be examined by the same procedure to determine group homogeneity. In these 2-dimensional plots members of the same species are roughly bounded by a box of 100 squared units while closely related strains are grouped more tightly together. The 2-dimensional plot of isolates of Micrococcus luteus demonstrates the presence of three identifiable sub-species

Desiccation Resistance of Bacteria Isolated From An Air-Handling System Biofilm Determined Using a Simple Quantitative Membrane-Filter Method

Twelve strains of bacteria recovered from a biofilm growing on cooling coil fins in an air-handling system, representing recognized members of the coil fin biofilm community, were assessed for their desiccation resistance. A quantitative membrane filter method was used to assess desiccation resistance over a 24 h period. The method proved to be a reliable and inexpensive means of quantitatively assessing desiccation resistance in bacterial isolates. Five pink-pigmented budding rod (PPBR) isolates, related to Methylobacterium, were resistant to desiccation over the test period (47-100% of original viable cfu were recoverable on R3A agar after 24 h desiccation). Methylobacterium-like PPBRs represented the dominant culturable members of the coil fin biofilm community. An unidentified Gram-negative filamentous rod was also somewhat desiccation-resistant (45% of original viable cfu were recoverable or, R3A agar after 24 h desiccation). The remaining six strains tested, three Gram-negative isolates and three Gram-positive isolates, were sensitive to desiccation with only 0-11% of the original viable cfu being recoverable on R3A agar after 24 h desiccation. Since the coil fin biofilm is subjected to extended periods of desiccation, the results suggest that desiccation resistance is at least partly responsible for the dominance of the coil fin biofilm community by the Methylobacterium-like PPBR