Peter Febbroriello Research Support Group 847 Litchfield Street Torrington, CT 06790 [ contains wide page - refer to EMAIL version if not readable] The science of speleogenesis is old. The science of speleomicrobiology is in its infancy. The discussion of the physics and chemistry of cave formation would not be complete if this one field of research were ignored. Microorganisms have long been suspected of playing a role in the creation or shaping of cave formations. Bacteria have been cultured from moonmilk and fungi have been observed and cultured from stalactites and other formations. The biochemical processes used by these organisms can be studied in the laboratory and the results used to interpret observations made in caves, and to suggest others be made. Other information comes to light indirectly as a result of these studies. Management of caves for public recreation can be enriched by the knowledge generated. New tools for medical science, and antibiotics can be discovered as well. When it was learned that a dive trip (Bolt Penetration) was to take place in LWR, it seemed prudent to take the opportunity to make a collection for pure research. Samples were collected by the diver, Peter Bolt, carefully transported from the cave with the direction of Louise Hose and shipped to this investigator by Felix Hernandez on May 12, 1992 Louise also very carefully collected samples of the calcite rafts that were to be disturbed by the dive. A method of analysis was already in development that could be used to correlate collections from different areas. Some algorithms were developed and tested and first used on the LWR dataset. These programs are in constant revision, with improvements added on a regular basis. Individual studies of metabolites or biochemical reactions with regard to depth were immediately done: Glucose oxidation with production of acid, citrate metabolizing capability, catalase activity, oxidase activity, production of indoles, nitrate and nitrite reduction, and urease production. Slides were prepared and gram stained, and examined microscopically with cooperation of Dr. Dennis Koppel of the University of Connecticut Health Center, Farmington, CT. Biochemical comparison of two collections or more could now be done. Also, biodiversity of a collection and comparison with other locations could also be calculated. A useful byproduct of this study is the ability of the programs to assist in elimination of all organisms found in collection "A" from collection "B". This is the same concept as a "background subtraction" might be used to eliminate "noise" from a dataset. Theoretically, one could take samples from a virgin area and determine the degree of "contamination" from a variety of vectors. ________________________________________________________ Data reduction programs and methods An algorythm was devised that was successful in matching organisms by biochemical and morphometric test comparison, using a simple scoring system based on the number of positive matches on a test result divided by the the number of comparable tests. This technique differs somewhat from one method mentioned in Bergey's manual. In that case only positive matches were scored, and negative scores were not tallied at all. Using this method, closely allied strains would show up as very close matches to the nearest genera/species. It was even possible to compare organisms from field studies and compare them with published data. The system allows an investigator to scan the data with simple stand alone software that is readily available and of universal application. details A program called GRID written in GWBASIC and readily used on any system capable of interpreting generic BASIC code. GRID looks for a file called DIRDAT.LST that contains the names of data that will be analyzed. The data is maintained in a set of ascii files with the extension .DO Each .DO file contains data derived from laboratory analysis of bacteria, using standard microbiological morphological criteria, and chemical reactions with several key reagents and stains. A list of these tests is maintained in a file called TESTS.NDP TESTS.NDP contains (at this time) two columns of data, separated by a comma. The first field is the name of the test. The second field is a column number (as in a spreadsheet). GRID takes the .DO files in DIRDAT.LST and parses the data using TESTS.NDP as a selective filter, placing the data into the proper columns of a master file with uniform headers. This file has the extension .GRD, one .GRD file produced for each .DO file. Data is stored in .DO files. These are created on remote data gathering devices, portable computers, notebooks, library references, etc. The format of the .DO file is of two subtypes, TAGGED FIELDS, and CHART. The Tagged fields format is best for lab data entry. A technician can easily enter data at the lab bench by using a simple three or more character code, followed immediately by a + or- sign or possibly other type of result code, some with modifiers. CHART format follows the columnar field layout found in spreadsheets. One line of data is generated for each line of published material from a chart or table of test results, eg Bergy's manual. The data is typed in exactly as published in most cases, with commas delimiting columns. A minimum of allowable control codes are used to simplify learning the method of data entry. The # symbol is used as the first character of a control line. Control lines contain special commands or flags that can be ignored or used by the "compiler" of which GRID. BAS is the most recently written. The command #TAGS signifies that tagged fields are in use. #CHART or #TABLE are treated identically, and several others are parsed as per CHART format, including #MICROBE. The flag #NOTE is ignored by GRID, but is published to standard output at compile time to allow the operator to flag the process or maintain control over the database (editing, updating, documenting source material etc.). No flags will appear in the resulting .GRD files. All .GRD files are of uniform structure and can be compared with any other .GRD file. >GRD files can be combined using the DOS command COPY, or using a simple ascii text editor, or Word Perfect (eg) ascii output option. The program COMPARE.BAS will produce a dynamic display of the comparison of two .GRD files that can be redirected to a file ( gwbasic compare >file ) The program also produces a file CMP that contains the results of the entire comparison. The file "CMP" is formatted for simplicity of processing for the next module called CHOICE, which simply looks at CMP and selects the best match, always placing the highest scored match in the queue. Resulting output is sent to standard output and can be re-directed as usual. Additional modules can be written to carry out any number of schemes limited only by the investigator's imagination. _____________________________________________________________ lab analysis techniques culturing the sample Bacteria are suspended in natural media and pipetted or streaked via platinum or commercially sterilized plastic loops (Sigma). Media used for the Lake of the White Roses samples include malt agar, Sabaraud's agar, and trypticase soy agar. Penicillin was added to additional plates of malt and Sabaraud's. These agars are designated m, s, ts, pm, and ps respectively. Number designations are written on the cover of the dish identifying the sample as to depth collected. The full designation might look like: lwre1 where lwr is the location e1 is the designation chosen by the collector to indicate a surface sample of calcite rafts. transfer to tube media and biochemical tests The colonies are picked from the dish using the pointed end of the plastic loop or the tip of a platinum wire which has been flame sterilized. The plastic loops are disposable. Each colony is inoculated onto a tube of special media prepared according to published methods. See TESTS.NDP for a complete list of the tests in use. Not all available tests are used in every case, but an attempt is made to include as many as practicable. A special set of media were also used to search for sulfate and sulfide oxidizing organisms. ATCC Beggiatoa media was prepared in a Winogradsky column in order to determine if Beggiatoa were present. Several small tubes of media enriched with peptone were also prepared by filter sterilization of water from Movile cave. These would contain dissolved H2S in quantities optimal for floating mat type cultures as are found in Movile cave. A laminar flow hood using Laminair micron filter was used for all preparation and transfer steps. The hood could be operated in reverse isolation and forward isolation modes or both. Control media were often left open in the hood before and during preps and were always found to be clear and lacking in any growth. Contaminated cultures, when found could always be tracked to mishandling of vials and containers used in the field. The outer surfaces of these are now routinely treated with tincture of Zephiran spray prior to opening. Smaller vials could be placed in the hood's vent screen where they would be exposed to sterile air while drying. All cultures would be observed for growth of any kind, and immediate sub-culturing and isolation to axenic culture were carried out immediately. Each bacterial colony from any agar dish was cultured in analytical media or subjected to a number of tests and observations as listed in TESTS.NDP. Any remaining samples or specimens were stored for future analysis of hardy organisms. All fungi that continued growth to sporulate were allowed to dry in the tube for future re-culture from the spores, and DNA screening when available. Bacteria were also allowed to dry and be held for future lipid assay or DNA analysis. The database is continuously updated and maintained at http://www.well.com/user/peter/lwr.do additional files include the above-mentioned BASIC files and other databases for direct comparison. logging data from lab tests The database is then updated from these results. Each data line is given the name of the location as above, and appended to that is the specific information that identifies the culture, e.g. lwr 1 pm where the numeral 1 is the colony number picked from the dish ps is the agar, in this case penicillin treated malt. A comma is appended to separate the fields and following this the technician appends the results of the observations and tests, usually on a laptop or palmtop computer or data storage device near the microscope or lab bench. Interpretation of the entry: lwr10 6 pm,/white shiny,cat+,ure-,g-r,len.5,wid.25,?oval the / indicates the appearance of the tube culture (slant) cat+ indicates a positive reaction to hydrogen peroxide, generating gaseous oxygen (catalase positive) ure- indicates that the organism does not produce urease and cannot cleave urea to ammonia and carbon dioxide. g-r is the notation for gram negative rods length and width is .5 x .25 microns The shape is oval, and is in question. The program will ignore this. One would normally proceed by running the programs mentioned above and attempting to identify the genus by looking for the best match. running the programs (In the above example, the program COMPAR.BAS is given LWR.GRD and BACHAR.GRD to compare. Followed by running CHOICE.BAS, the result is a pointer to Shigella. Examination of the results of these program runs, one can see that Shigella holds a close biochemical similarity to many of the LWR samples, with some depth relatedness. Since the lwre series were made from pre-dive surface material, it is not a contaminant of Peter Bolt's wetsuit.--pf) By copying the following files from the system and placing them in a convenient working directory on his computer the user can carry out a direct comparison of Lake of White Roses heterotrophic bacteria with those from Movile Cave, Romania. GRID.BAS COMPARE.BAS CHOICE.BAS TESTS.NDP LWR.DO M12.DO [ WEB page readers can now download LWR.GRD and M12.GRD as well, skipping the gridding step.--pf] The user then creates a file on his system containing the following two lines: lwr m12 The file should be named DIRDAT.LST (directory of data) and placed in the same directory as the other files. By running the user's copy of BASIC he can run"grid" and see the resulting compilation of LWR.GRD and M12.GRD take place. Several NOTE lines and comments generally appear on the screen. Next step is to run COMPARE.BAS and enter lwr for file 1 and m12 for file 2. The program will generate a line by line match and print the result using dots, exclamation points and asterisks to display poor, medium or good-to-perfect match. The average for each iteration will appear next to the name of the strain as it is compared to each strain in the first named file. That is to say, if one of the M12 strains was biochemically similar to a large number of the lwr strains, this average would appear to be higher than others. ________________________________________________________ Biodiversity determinations Biodiversity of bacterial populations can be measured. If a population of organisms is compared with itself on a point to point basis, diversity might be observed as an overall lack of similarity. The program COMPARE.BAS can score similarities within a population by using the same filename for both files requested. In such a comparison, the resulting score could be treated as inversely proportional to the biodiversity of the population. Or, this score could also be treated with a simple mathematical transform that is customarily used to standardize test scores in academia. When the comparison score of a population under study is subtracted from the mean for a set of populations' scores and the result divided by the standard deviation of the set's mean, the signed result is now a relative index of diversity for these populations. This was attempted for the Lake of the White Roses bacterial results, as well as a group of caves in New York, Connecticut, Vermont, Massachusetts, Colorado, and Romania. Additional tests were done on bacterial populations found in different types of meteoric waters, some in calcitic aquifers, and others consisting on collected surface runoff. The cave comparison is presented here in order of decreasing diversity: Movile (early) RO 2.45 (Sulfur cycle - near virgin) Howe C. NY 1993 0.79 (Heavily touristed - stream conduit) Manitou Springs C. CO 0.63 (heavily mineralized) Mount Anthony C. VT 0.61 (surface stream insurgance) Howe C. NY 1992 0.33 (Heavily touristed - stream conduit) Surprise C. NY 0.12 (treated sewer runs through) LWR Lechuguilla C. NM -0.24 (Scuba penetration) Bass Smith Resurg NY -0.41 (unopened cave - possibly sewer) Bashful Lady C. CT -0.50 (swamp drainage runs through) Dolo C MA -0.54 (surface stream pirated at high flow) Schoharie caverns NY -0.77 (stream conduit - resurgence) Movile (late) RO -0.87 (sulfur cycle - after several scuba) Grahm Mountain C. NY -1.53 (bat colony) It would seem that isolation of a cave biome enhances biodiversity (Movile, early), while "contamination" by human and animal activity will cause both increases and decreases in the relative levels. (Howe and Grahm Mtn). The scores are relative to only the caves included in this grouping. It is too early in this research to draw specific conclusions as yet. Clearly, more collections need to be included in the study. ------- It is interesting to consider the possibility that the biodiversity within a sample could be measured across a second parameter, such as water depth. In the case of The Lake of White Roses data, samples were taken at the surface, 5 feet, 10 feet, and 20 feet in depth. If the data file is simply arranged with these samples in increasing order in depth, the resulting output can be re-directed into a file for later examination. Such a file was created, with a "." indicating an insignificant matching, a "!" indicating a mild similarity, and a "*" signifying a very close match. The identification and score for the organism is presented at the far right hand side of the page (screen). In this matrix, a diagonal line can be found indicating the same organism is compared to itself, with a perfect match. (The upper right corner of the matrix is therefore a mirror image of the bottom left, and can be ignored, but is maintained by default, and might prove of some convenience during study.) Each dot or other symbol represents one organism in the same collection as listed down the right side of the matrix, and in the same order going from left to right. [file: lwr.mtd ] [warning: wide screen required] *...!.!!..*..........!.!.....!.!.!.....!!...!.!..!..!......!!..!.!!.!.......!...!..!!.!!..!.!!..!!.!.!!.!. lwr vs lwre1 1 pm .6683963 .*......!........................!.............!.....!!.........!......!.................................. lwr vs lwre1 1 ps .4821138 ..*....!.........!.........!!!...!.!..........!.!!................!..........................!..!!..!!!.!. lwr vs lwre1 1 s .6133911 ..!*!.....!.!.!.!!..!......!...!!!....!.......!.!!.........!!..!.!!.............!......!.....!..!!..!.!.!. lwr vs lwre1 1 ts .6331191 !...*.!...!......!.....!...!...!!!.....!!...!.!..!..!.....!!!..!.!!.!.......!!..!..!!.!!..!.!!!.!!.!!!.... lwr vs lwre1 2 pm .664537 .....*..................................!...!..!.!..!......!....!...!.....!..!!!..!.!...........!.!!.!.... lwr vs lwre1 2 ps .5890118 ....!.*.!.!..!.........!.........!.....!...!..!!.....!!..!.!!...!......!...........!..!......!..........!. lwr vs lwre1 2 s .6141063 ....!..*.........!.........!.!.!.!.!..!..........!.........!!....!..............!......!.........!........ lwr vs lwre1 2 ts .6100202 .!....!.*....!.....!..!........!..........!!...!.....!!..!.......!.....!.....................!..........!. lwr vs lwre1 3 pm .5536309 .........*.......................!...............!............................!........................... lwr vs lwre1 3 ps .5385079 !...!.!...*............!.......!.!.....!!...!.!..!..!......!!..!.!!.!.......!...!..!!.!!..!.!!..!!.!.!!.!. lwr vs lwre1 3 s .6644655 !...!!....!*.........!.!!...!!.!!!....!.!...!.!..!!!!...!..!!..!!.!!!.......!.....!.!.!..!!!!...!!!!.!!.!. lwr vs lwre1 3 ts .6752406 ............*.!.....!..........!...........................!...!................!......!.........!...!.... lwr vs lwre1 4 pm .5705023 ......!.!....*.....!..!....................!...!.....*!..!......!......!...........!.........!..........!. lwr vs lwre1 4 ps .5750423 ..............*.!...*..!!!.................................!...!...........................!.............. lwr vs lwre1 4 s .597232 .......!.......*...........!.!.!.!...............!.........!.....!..............!......!.........!........ lwr vs lwre1 4 ts .5828594 ..............!.*!!.!..!.!..........................................................................!..... lwr vs lwre1 5 pm .5804938 .................*....!....!....................................!......!.................................. lwr vs lwre1 5 s .5614006 ..................*....................................................................................... lwr vs lwre1 5 ts .4938562 ........!....!...!.*.!!...................!!.!.!...!.!!.!!......!......!..........!.....!.!.!.!..........! lwr vs lwre1 6 pm .6215755 ..............*.!...*..!!!.................................!...!...........................!.............. lwr vs lwre1 6 s .597232 !...!.....!!.........*.!.....!.!........!.!.!!....!!!...!..!!!!!.!..!!..!!.!...!!.!!!...!!*!!!!...!!.!.... lwr vs lwre1 6 ts .6700281 ........!....!...!.!.!*.!.......!..........!!!.....!!!!.!!......!...!..!..........!.!...!.!.!.!.!.!......! lwr vs lwre1 7 pm .6297686 ....!.!.......!.....!..*!!.............!!.!.!!......!......!!..!!...!.!.............!.....!!.......!.!.... lwr vs lwre1 7 s .6300649 .......................!*........!.....!!...!!...!.!!......!....!...!.!...........!.!......!....!.!!.!.... lwr vs lwre1 8 pm .6298149 .......................!.*....!..!...............................!..............!......!.......!.!........ lwr vs lwre 1 m .5315861 ..........................*!................................!..............................!.............. lwr vs lwre 1 ps .3801645 ..........................!*.....................!........................................................ lwr vs lwre 1 s .5196067 ..!....!.!..............!.!.*!...!.!..!.!...!...!!.!!......!..!!....!....!..!...!.!.!..!!....!..!!!!.!!.!. lwr vs lwre 1 x .647275 ....!..!.......!.!...!.!...!!*.!.!.!..!.!...!....!..!......!!..!.!..!...........!...!..!..!......!.!.!.... lwr vs lwre 1 ts .6347628 .........................!....*..!............!.!................!!.............!.....!!........!!...!.... lwr vs lwre 2 m .5731018 .....................!.........*.!..!.....!........!....!...!.!..!.......!........!!..!...!.!!!..!........ lwr vs lwre 2 ps .5946723 !.!!!.....!!............!..!....*!..........!.!.!!.!!......!..!!...!!........!....!.!...!...!.!.*!!.!.!.!. lwr vs lwre 2 s .6520736 ....!.!!.!..............!!...!!!!*......!...!.!.!!.!!.....!!!.!..!!!!....!!..!..!.!.!.!!!...!!..!!!!!!.... lwr vs lwre 3 m .6606278 ...............................!..*.*!.!..!............!.........................!.........!.............. lwr vs lwre 3 ps .4651805 .......!.!..................!!.....*.............!..!......!..!.....!.......!.......!........!..!!!....... lwr vs lwre 3 s .6079508 ...............................!..!.*............................................!........................ lwr vs lwre 4 ps .4583029 ..................................!..*........................!..................!.........!.............. lwr vs lwre 5 ps .4421946 !.!....!.!!!................!!.....!..*.....!....!.........!...!.!..!...........!................!.!.!.... lwr vs lwre 6 ps .6150049 !.....!...!............!!.........!..!.*.....!.........!..............!..........!.!.......!.............. lwr vs lwr5 1 m .5688655 ....!!.....!.........!.!!....!...!......*...!....!!.!......!...!!.!.!.............!.!....!!.!...!!!!.!.... lwr vs lwr5 1 pm .6422718 .........................................*................................................................ lwr vs lwr5 1 ps .4836895 ...............................!..!.......*................................................!.............. lwr vs lwr5 1 s .5725627 ......!.!....!.....!..!....................*...!.....!!..!......!......!.....................!..........!. lwr vs lwr5 2 m .5486063 ....!!.....!.........!.!!....!...!......!...*....!!!!......!!.!!!.!!*.....!..!....!.!...!!!.!...!!!!.!.... lwr vs lwr5 2 pm .6601976 .....................!.!!..............!.....*.....!...!......!.....!.!........!..!.....!.!!......!....... lwr vs lwr5 2 s .6079068 !...!.!...!......................!............*..!..!...!..!......!................!!.......!!..!!........ lwr vs lwr5 3 m .6203049 .....!!.!....!.............................!...*.....!!.........!......!.............................!.... lwr vs lwr5 3 pm .5573735 ..!....!.........!.........!!!!..!..............*!..!.........!.....................!..!!.......!!..!.!.!. lwr vs lwr5 3 s .5727441 ....!..!...................!.!...!.!....!...!.!.!*.!!.....!!!.!...!.!....!..!.....!.!.!.!...!...!!!!!!.... lwr vs lwr5 4 m .6524225 ....!!.....!.........!.!!....!...!......!...!.!!.!*.!...!..!!..!!.!.!.!.........!...!....*!.!...!!!!.*.... lwr vs lwr5 4 pm .6550673 ...........!.........!.........!.................!.*!...!..!.!!!.!..!!..!!.!......*!!...!!!.!.!.!.!....... lwr vs lwr5 4 s .6413195 ....!......!.........!.!!....!...!......!...!.!..!.!*...!..!..!!....!.............!.*...!!!.!...!!!!.!.... lwr vs lwr5 5 m .6479616 ......!.!....*.....!..!....................!...!.....*!..!......!......!...........!.........!..........!. lwr vs lwr5 5 s .5773292 ......!.!....!.....!..!....................!...!.....!*..!......!......!.................................. lwr vs lwr5 6 pm .5165431 ..................................!..!.!.....!.....!...*..............!..........!!.!......!.............. lwr vs lwr5 6 s .5345436 ...........!.........!.........!..............!....!!...*..!.!!!.!...!..!..!......!!!...!!!.!!!...!....... lwr vs lwr5 7 pm .6325196 ......!.!....!.....!..!....................!.........!!..*......!......!...........!....!................. lwr vs lwr5 8 pm .5682232 !...!.!...!......................!.!....!.....!..!..!.....*!..!.....!....!..!...!.!!!.!.!!..!!!.*!!!!..... lwr vs lwr5 9 pm .6512076 ....!!!...!!.........!.!!....!.!.!......!...!.!..!!!!...!..*!..!!!!.!.......!...!.!!!.!!.!!!!...!!!!.!.... lwr vs lwr10 1 m. .6746327 ....!..!.......................!.!..........!....!.........!*.......!.....!.................!...!!.!.!.... lwr vs lwr10 1 pm .6242522 .....................!....................!........!....!...!*...!!..!..!..!......!!....!.!.!.!........... lwr vs lwr10 1 ps .608801 .....................!.........!...!.!..........!!.!!...!.!...*..!..!....!........!!!.!.!.!.!!!.!.!....... lwr vs lwr10 1 s .6360071 !...!!!...!!..!.....!!.!!...!!.!.!......!...!.!..!!!!...!..*!..*!!!.!.......!...!.!!!.!!.!!!!...!!!!.!!.!. lwr vs lwr10 2 m. .6841767 .....!!......!...!.!..!!!.!.............!..!!..!.....!!..!.!....*...!..*..!..!!!.....!.............!.!.... lwr vs lwr10 2 pm .584876 ....!...!.!..........!...!....!!.!....!...!........!....!..!!!!!.*...!..!!.!....!.!!..!!..!.!!!!.!........ lwr vs lwr10 2 ps .6415375 ....!............................!......!...!.!..!.........!.!....*.!!.....!..........!.!...!...!..!.!.... lwr vs lwr10 2 s .6090716 !...!.....!!............!.......!!.!....!...!.!.!!.!!.....!!!.!!..!*!....!!.!!....!.!.!.!...!...!!!!.!!.!. lwr vs lwr10 3 m. .6656304 ....!!.....!.........!.!!....!...!......!...!....!!!!.....!!!.!!!.!.*.............!.!...!!!.!...!!!!.!.... lwr vs lwr10 3 pm .6545448 .....................!....................!........!....!...!!...!!..*..!..!......!!....!.!.!.!........... lwr vs lwr10 3 ps .6101556 .......................!!..............!.....!.!..!....!..............*..........!.......!.!.........!.... lwr vs lwr10 3 s .5550216 ......!.!....!...!.!..!....................!...!.!..!!!..!......*...!..*............!...!.......!......... lwr vs lwr10 4 pm .6016127 .....................!....................!........!....!...!!...!!..*..*..!......!!....!.!.!.!.......!... lwr vs lwr10 4 ps .621694 .....................!.........!.!...............!.!......!...!..!.......*......!.!!..!.!.!.!!!.!!!....... lwr vs lwr10 4 s .617487 .....!...........................!..........!....!..........!...!...!.....*..!!.................!......... lwr vs lwr10 5 pm .5691768 .....................!.............................!....!....!...!!..!..!..*......!!....!.!.!.!........... lwr vs lwr10 5 ps .6041692 ....!..............................!..........!..!.........!.......!........*...............!!..!!........ lwr vs lwr10 5 s .6137901 .....!...........................!..........!....!..............!...!.....!..*!.................!......... lwr vs lwr10 6 pm .5762633 .....!...!....................................!!................!.!.......!..!*......................!.... lwr vs lwr10 7 pm .562725 .....!...............!.......................!.....!..........!.!...!..........*..!.....!.!.......!....... lwr vs lwr10 8 pm .5823813 ....!.!!..!.!..!.......!.!..!!!..!.!..!.!...!.!..!!.!.....!!!..!.!..!....!..!...*..!!.!*.!!.!!.!!*!!!!.... lwr vs lwr20 1 pm .6394775 ...................................!!!.!.!............................!..........*.........!.............. lwr vs lwr20 1 ps .5151427 ...........!.........!.........!........!........!.*!..!!.!!.!!!.!..!!..!!.!......*!!...!!!.!.!.!.!!...... lwr vs lwr20 1 s .6430906 !...!.!...!..!.......!.........!.......!..!...!....!.!..!!.!!!!!.!...!..!!.!....!.!*....!.!.!!!........... lwr vs lwr20 1 ts. .646386 ....!!.....!.........!.!!....!...!......!...!.!..!.!*...!.!!..!!....!.............!.*...!!!.!...!!!!.!.... lwr vs lwr20 10 s .6494563 .....................................................................................*.................... lwr vs lwr20 11 s .5017622 !...!.!...!!.........!.!!....!!!.!......!...!.!..!..!!..!..!..!!.!!.!....!......!..!!.*.!!!.!!..!!!!.!..!. lwr vs lwr20 12 s .6531556 ....!.!...!.!..........!.!..!!...!............!.....!......!...!.!..............*..!!..*..!..!...!...!.... lwr vs lwr20 2 pm .5675587 .....................!..........................!!.!!...!.!..!!...!.!!...!.!......!!!.!.*.!.!!!.!.!....... lwr vs lwr20 2 ps .6234993 ....!!.....!.........!.!!....!...!......!...!.!!.!*!!...!.!!!..!!.!.!.!.....!...!.!.!.!..*!!!!..!!!!.*.... lwr vs lwr20 2 s .6675482 ....!.....!!.........!.!.......!........!.!.!!....!!!...!..!!!!!.!..!!..!!.!...!!.!!!...!!*!!!!...!!.!.... lwr vs lwr20 2 ts. .6660973 ...........!..!.....!!.!!.!.......!..!.!..!..!.........!...!...!......!..........!.......!!*......!....... lwr vs lwr20 3 ps .581844 !.!!!!!!.!!!...!.!.!.!!!!..!!!.!!!.!....*...*!!!!!*!*...!.!*!!!!!!!!*!!!!!!!!!!!!.!!*.!.!*!.*.!.*!**!*!.!! lwr vs lwr20 3 s .7366326 !.....!.!.!..!.......!......!..!.!.!..........!......!..!.....!..!.......!..!...!..!..!.!.!..*!.........!. lwr vs lwr20 4 pm .5955003 .....................!.........!!..................!....!....!!..!...!..!!.!......!!....!.!.!!*.!......... lwr vs lwr20 4 s .6286308 ...............................................................................................*.........! lwr vs lwr20 5 pm .4823264 ....!...........................!!.!....!...!.!.!!.!!.....!!!.!...!.!....!..!!....!.!.!.!...!.!.*!!!!!.... lwr vs lwr20 5 s .6216345 ....!..!....!............!...!.!.!.!..!.!...!.!.!!..!......!!....!..!....!..!...!...!.!!........!*.!!!.... lwr vs lwr20 6 pm .587272 ....!!.....!.........!.!!....!...!.!....!...!.!..!!!*...!.!!!.!!!.!.*....!..!...!.!.*.!.!!!!!...!!*!.!.... lwr vs lwr20 6 s .6725273 ....!!.....!.........!.!!....!...!......!...!....!!.!......!!..!!.!.!.............!.!....!!.!.!.!!!*.!.... lwr vs lwr20 7 s .6436518 ...!.............................!...............!..............................................!!..*..... lwr vs lwr20 8 s .5909354 ....!!.....!.........!.!!....!...!......!...!.!!.!!.!...!..!!..!!.!.!.!.............!....!!.!...!!!!.*.... lwr vs lwr20 9 s .6249206 !.!.!..!..!.................!!..........!...!.!.!!..!......!!!.!!.!.!!.!!..!........!.......!...!..!!!**!. lwr vs lwr20 op ss 1 .6512239 ..........................................!........................!........!.................!.!.....**.. lwr vs lwr20 op ss 1 .358176 !.!.!.!!!.!..!..............!!...!......!..!!.!!!!..!!.....!!..!....!...............!.!.....!!..!!.!!!!.** lwr vs lwr20 op ss 2 .662398 ...........!.......!.!!!!..................................!...!.........................!!.!.!!!.!.....** lwr vs lwr20 op ss 2 .4267295 The organism "lwr20 3 s" stands out with an index of .737 with a preponderance of similar strains at 20 feet, tapering off towards the surface. LWR20 3 s is biochemically similar to Shigella dysenteriae. This result simply points out a similarity, and that many of the LWR organisms are similar as well, but is not a definitive identification. Too few tests are involved in this simple study for proper determination. In fact, an examination of the raw data file shows that only six measurements were made on this organism. ------ additional studies using simple dos programs Further consideration of contamination by possible fecal materials can be examined by looking for indole and urea metabolizers. in this case, one can use the UNIX command FGREP to screen the .DO files directly for ind+ and ure++. Doing so shows that no indole producers are found in the LWR samples (at least from those colonies actually picked from the agar).(More recent specimens from Movile cave, Romania had one colony out of the entire collection from the floating mat in air bell #3 on the expedition of 8/9/93.) Urine would contain urea and serve as a substrate for Proteus. This genus is commonly found near bat colonies and has been cultured from new York State bats hibernating in caves. Lake of White Roses data can be screened, and the organisms with ure++ can be examined for similarity. We see that two surface colonies (lwre 1 m and lwre 2 m)) contain ure++ organisms and one from a depth of 20 feet (lwr20 6 pm). They are not well matched to Proteus, however. This organism may indeed be within the genus, but has drifted genetically over eons. There is a possibility that it could pose the same health risks as Proteus. The surface colonies are dissimilar in appearance, one being yellow and the other white. The deep specimen, when compared to other specimens from 20 feet can be seen to be biochemically similar to two others (lwr20 2 pm and lwr20 1 pm), one difference being the rapidity of urease production. Only in the surface sample and one at 5 feet does this similarity re-appear, but with even less correlation. Of interest in direct activity of bacteria in limestone solution via acidic products is the possibility of direct acid production via metabolism of simple sugars as might be found in an enriched environment. The Methyl red test is an indicator of acidity. LWR organisms were tested using glucose, salicin, and arabinose as carbon source. The results are shown by percentage positive and by depth: glucose 13 mrd+ 30.23% 27 mrd- 62.79% 40 e1 7- 0% e 7+ 2- 77.78% 5 1+ 5- 16.67% 10 3+ 3- 50% 20 2+ 7- 22.22% salicin 14 sal+ 100.00% 0 sal- 14 e1 not tested e 3+ 0- 5 not tested 10 5+ 0- 20 6+ 0- arabinose 1 ara+ 6.25% 14 ara- 87.50% 15 e1 0+ 7- e not testd 5 0+ 5- 10 0+ 2- 20 1+ 0- some discussion It was found that populations of cave organisms contain a number of unique strains, and although some are similar to common organisms, have evolved to be different by one or more parameters from their surface counterparts. Many cannot be identified to genus. As everyone using the data and programs would find a use unique to their own interests, this investigator believes that this study points out the need to collect from locations that would make possible a microbe map of the cave. Species and strain distribution maps would paint a clear picture of biological activity in Lechuguilla cave providing guidance in management activities, conservation measures and future exploration plans. A good baseline is now in hand to assist in judging collections from sulfur deposits for degree of human contamination. It seems intuitive that the principle organisms introduced to these environments (LWR, Voids etc) one might find would be from meteoric waters or trapped airborne organisms that adhere to moist formations when moisture condenses from the air. Others, referred to by some researchers as "contaminants" might be from human activity. However, it pays to remember that animal activities are documented in some areas of the cave and have somehow influenced the distribution of microbes over the centuries. Future studies will no doubt be able to trace the flow of biodiversity through the cave. Organisms suspected of generating sufficient acid to dissolve limestone can be demonstrated from this collection. It is not proven, however, that sufficient carbon source is available in the "medium" to actually produce this acidity. Another phenomenon that is hinted at by the distribution of organisms found in this study is the possibility that organisms are ordered preferentially by depth, and at some point after sufficient collections are made with suitable apparatus, it will be found that organisms are found in distinct layers, determined by a gradient of oxygen, carbon dioxide or some other parameter. No attempt to identify to species is made in this investigation. The programs will only provide a starting point in this direction. More useful towards the purpose of answering questions pertaining to speleogenesis is the elucidation of biochemical pathways in use by the organisms as a "matrix". Since multiple symbiotic relationships are assumed to be in operation, one should expect to find "source:sink" relationships that can be defined and measured. One should also investigate the source of acidity that will eventually come to dissolve limestone, releasing calcium and magnesium and other trace elements into the aqueous media. In addition, defense mechanisms used within the colonies found in situ may yield antibiotics of use to mankind. Additional screening of axenic cultures are now to be routinely done by a collaborating laboratory. Future collections based on speleogenesis research Several collections from diverse cave ecosystems are planned and some are indeed underway. Sulfur Void samples especially are desired in order to carry out a study of sulfur metabolizing organisms. These will hopefully bear similarity to published organisms, but it is suspected that a numerical analysis will be necessary to make the required determinations. There have been reports that an organism may be responsible for unusual coloration of the sulfur deposits. This observation fits with the possibility that a purple sulfur bacterium is involved. Since these organisms are photosynthetic, it would be of great interest to see what they are doing in the cave. It is more likely that they are changed from an original organism that actively involved itself in the metabolism of hydrogen sulfide. It has either evolved, (via mutation) or simply changed it's food preference. The color may be something other than photopigmentation. It would be useful to chromatograph a sufficient number of organisms to make this determination. Desulfovibrio is also a target organism. Again, similarity indices will be prepared, and special differential media is planned. it should be possible to make the required determination with the tools described above. When organisms are found that fit into these categories, an attempt will be made to map out a metabolic flow chart showing the progression of organisms and changes that occurred as the shift from sulfide to oxygen environment took place. It is believed that a possible intermediate stage, which I am calling a "matrix" formed which may have been characterized as a pellicle, or gelatinous surface layer. Such can be found in the acidic tea mushroom known as "Kombucha". Several bacteria, yeasts and possibly filamentous fungi are closely allied within a gelatinous pellicle that floats on the surface of an air/water interface. The pellicle may consist of a variety of secreted substances which serve to protect the organisms within from environmental stresses such as would be found during the conversion from H2S rich to oxygen rich gas tensions. Collections from Villa Luz will possibly be matrices of this nature. Movile Cave in Romania is such an example, where It is hoped that such a matrix can be reproduced by re-introducing the conditions under which they were originally formed. This has been done successfully with samples from Movile cave, however, these organisms are still heavily invested in the H2S process, and were relatively easy to deal with. But, hopefully it has already been partially shown that organisms from LWR can be induced to grow using water from Movile cave. A filter sterilized Movile water sample, rich in H2S was used to successfully cause growth of fine filaments in a lab culture from the 20 foot level of LWR. The culture has not been disturbed for five years. _________________ As usual, all results of investigations by this investigator are immediatel posted and made public domain. Agreements are in effect with certain commercial organizations investigating antibiotic activities, as well as anti-cancer and other medicinal substances produced by cave organisms. A cooperative arrangement of sample sharing and resource pooling is in effect. [portions of above are from a paper in preparation--pf]