COUNT 2.41 (Macintosh) OBJECTIVES This program was created to assist in the enumeration of differentially repetitive items and to maintain the integrity of data transfer between the physical count and data manipulation. The experienced user of this program will find that the time spent counting samples is greatly reduced compared to the traditional methods of tally bars or pencil tics. Once be- coming touch sensitive with the 10-keypad, the user will be able to maintain continuous eye contact with the sample, reducing the time spent moving and refocussing their eyes. The risk of loosing track of what was or was not counted is also substantially reduced. The time spent on data transfer is essentially reduced to zero as the output from this program is directly importable to any major brand of spreadsheet (Excel¨, Lotus 123¨, MacWorks¨, etc.) . Furthermore, this importability reduces transfer error rates to zero. GENERAL INFORMATION This program has been created using the True Basicª (Kemeny and Kurtz) programming language. It has been compiled and bound into a free-standing application that needs no other software support in order to run. The program uses a master file containing taxon names as row titles, sample names as col- umn titles, and their associated counts. This format is common for data spreadsheets. The program assigns each taxon a numeric integer equivalent. This integer is entered on the computer keyboard and used to score and record the numbers of a taxon observed in a sample. The samples are referenced by their names. The program is also capable of tracking two levels of area observed, for instance the number of random microscopic fields observed and the number of transects across a microscope slide observed. The initial mas- ter file for a batch of samples must be created using a spreadsheet application. The master file is then called, manipulated, and saved by the counting program until the batch is com- plete. The program also creates a small backup file for each individual sample containing only counts without taxon names. The count program calculates a Shannon Diversity Index* (H') on demand, when the mas- ter file is saved, or when the program is exited. The maximum possible diversity value (Hmax) is also calculated as is species eveness (J'). A small rudimentary graph of change in diversity value is displayed after a sample's second diversity calculation during a particu- lar session. *· (pi log2 pi), where pi = ni / N CREATING THE MASTER FILE As mentioned above, the master file must be initially created using a spreadsheet applica- tion. The first cell of the first line must define the size of the data matrix, that is the number of taxa and samples you wish to declare. It must be in the form such as 50X25, where 50 is the number of declared taxa and 25 is the number of samples (the 'X' must be upper case with no spaces between the numbers). The first cell of the next line should contain the title of the file. The following cells in that line should have the names of the samples for that batch. The first cell in each line below the file title should contain the name of a known or anticipated taxon. Extra rows should be allocated to accomodate unknown or new taxa found in the samples. These extra allocations must have some "generic" code assigned to them, i.e A, B, C, D. Once created, "generic" rows can later be assigned a proper name from within the spreadsheet application. Six additional lines should be created after the last declared taxon. They should be titled; "Total Units", "# of Fields", "# of Strips", "H'", "Hmax", and "J'". All empty cells in the matrix bounded by the last sample name and J' should then be filled with a 0 (zero). The file must be saved as a tab delimited ASCII text file. See APPENDIX I for an example of the master file in spreadsheet form. GETTING STARTED Following a short introduction you will be presented with the standard 'Macintosh' open box, proceed as normal and the program will load your file into memory. You can not edit the master file taxon names or sample names from within the counting program, they must be edited externally as described above. You will then be prompted to define a multiple unit key. This key will alow you to enter multiple units of a particular taxon. This non-numeric key should the one above the 'enter' key associated with the 10-key portion of your keyboard. You are then presented with an on screen menu of your sample names (the program can ac- commodate 70 samples in a batch). Point and single click on the box associated with the sample you wish to count. The program loads the sample and displays pertinent informa- tion about that sample, including; the number of declared taxa available, the total number of cells counted to date, the number of fields counted, the number of strips counted and the sample name. You should have a hard copy reference list of all taxon numbers and their assigned name, either specific or "generic" before you begin counting. When you assign specific names to "generic" taxon numbers you can record them on this reference list and update the master file using a spreadsheet application later. COUNTING SINGLE ENTRY OPTION: A taxon is enumerated by entering the number associated with that taxon and will increase the cumulative count for the taxon by one unit. If you attempt to enter a positive integer greater than your last allocated taxon number you will be warned with a unique sound and asked to reenter a taxon number within the allocated range. A similar result occurs if you attempt to enter a fractional taxon number, i.e. 12.6 or 0.3. Upon successfully scoring one taxon number you will hear one short beep. The pitch of that beep is unique for that taxon. The program then displays the taxon name scored, the taxon number, the cumulative count for that taxon, and the cumulative count for all taxa. MULTIPLE UNIT ENTRIES: If you have a field of view containing a filament, a colony, or multiple cells of one taxon, you can score multiple units of one taxon by hitting the key you defined as the multiple unit key within 0.25 seconds of entering the taxon number. Entering the taxon number scores the first unit of the group, you then enter the number of additional units you wish to score and are returned to the 'Enter the Taxon Number to Score' prompt. You may also subtract through this subroutine by preceeding the number of units with a '-' sign or enter fractional units (remember to account for the unit scored to get into the subroutine). SUBTRACTION: Immediately after scoring one unit of a taxon using the single entry subroutine you may subtract that unit by entering '0' (zero). It may be that you changed your identification of that unit or miskeyed the entry number. After you have scored the next taxon the '0' sub- traction option applies to the new taxon. SCORING FIELDS OF VIEW: Any negative number, except '-1' and '-11', will add one field of view to the cumulative total of fields. SCORING STRIPS OF VIEW: By entering '-1' you will add one strip of view to the cumulative total of strips. Entering '-11' subtracts one strip OPTIONS MENU: When you enter '999' the program displays an on screen options menu. By pointing and single clicking you can calculate a Shannon Diversity Index, save your work, start a new count, save your work and quit the program, or exit the options menu back to the current count. When you save your work you will get the 'MAC' open box. If your satisfied with the count overwrite the old master file. If you suspect you have input some kind of erro- neous information during the current count, change the title and save. The old master file, prior to the current count, will remain intact and you can exit the program and redo the cur- rent count using the untouched old master file. The program creates a small backup file for that sample in addition to updating the master file whenever you save your data, start a new count, or quit the program. This file will be titled by the sample name, it will not contain taxon or sample names, but rather contain the number counted for each taxon in order. Additionally, the total units, fields, strips, and diversity information are included in the backup file. DATA TRANSFER When all samples in a master file are completed the data is ready to be transferred to a spreadsheet application. To transfer the data, open the spreadsheet application, define tab delimitation (if necessary) to the application, and open the master file into the spreadsheet. The data should then be manipulatible to your needs. ADDITIONAL INFO Should you run out of allocated taxa space before a batch of samples are complete you may add taxa directly to the master file by opening it from within a spreadsheet application. Insert and name as many taxa rows as you feel you need, enter a 0 in all blank cells, return to the top line of the file and change the number before the 'X' to reflect the number of additional taxa, and save the file as an ASCII text file. Several 'run time errors' may occur, particularly when opening the master file after some external modification. These may include; "Too many input items", "Too few input items", "Reading past the end of the file", or "File is the wrong type". Check to insure the addi- tional lines are formatted properly, that you have entered a 0 to fill the matrix, that you have changed the first cell to define the number of taxa and samples in the matrix, and that the file is saved as ASCII text. Although I have diligently attempted to protect this program and your data from miskeys and illegal entries, I am human and may not have anticipated some scenarios. I therefore cannot be responsible for lost data. To make any data loss minimal, I suggest you make a duplicate of the master file (throwing away the old duplicate) after every couple of samples. If you somehow destroy the master file from within the counting program I would appre- ciate knowing about it and how it happened so that I can correct the problem for you and future users. I would also appreciate receiving general ideas as to how the program might be improved. If you would like a copy of the source code to modify for your particular needs I would be happy to supply if for an additional $25. Please feel free to contact me for any reason. David W. Johnson Dept. of Biological Sciences BGSU Bowling Green, OH 43403 LAB (419)372-8562 FAX (419)372-2024 e-MAIL IN%"djohnso@opie.bgsu.edu" APPENDIX I SAMPLE MASTER FILE (as viewed using EXCELª) 20X12 DARBY 1 C1 C2 C3 N1 N2 N3 P1 P2 P3 NP1 NP2 NP3 Achnanthes 0 0 0 0 0 0 0 0 0 0 0 0 Amphora 0 0 0 0 0 0 0 0 0 0 0 0 Cocconeis 0 0 0 0 0 0 0 0 0 0 0 0 Gomphonema 0 0 0 0 0 0 0 0 0 0 0 0 Fragilaria 0 0 0 0 0 0 0 0 0 0 0 0 Hantzschia 0 0 0 0 0 0 0 0 0 0 0 0 Melosira 0 0 0 0 0 0 0 0 0 0 0 0 Navicula 1 0 0 0 0 0 0 0 0 0 0 0 0 Navicula 2 0 0 0 0 0 0 0 0 0 0 0 0 Nitzschia 1 0 0 0 0 0 0 0 0 0 0 0 0 Nitzschia 2 0 0 0 0 0 0 0 0 0 0 0 0 Rhoicosphenia 0 0 0 0 0 0 0 0 0 0 0 0 Surirella 0 0 0 0 0 0 0 0 0 0 0 0 AA 0 0 0 0 0 0 0 0 0 0 0 0 BB 0 0 0 0 0 0 0 0 0 0 0 0 CC 0 0 0 0 0 0 0 0 0 0 0 0 DD 0 0 0 0 0 0 0 0 0 0 0 0 EE 0 0 0 0 0 0 0 0 0 0 0 0 FF 0 0 0 0 0 0 0 0 0 0 0 0 GG 0 0 0 0 0 0 0 0 0 0 0 0 TOT UNITS 0 0 0 0 0 0 0 0 0 0 0 0 # of FIELDS 0 0 0 0 0 0 0 0 0 0 0 0 # of STRIPS 0 0 0 0 0 0 0 0 0 0 0 0 H' 0 0 0 0 0 0 0 0 0 0 0 0 Hmax 0 0 0 0 0 0 0 0 0 0 0 0 J' 0 0 0 0 0 0 0 0 0 0 0 0 APPENDIX III NUMERIC ENTRIES for THE COUNTING PROGRAM 1) Any integer between 1 and your last allocated taxa number, inclusive, scores one unit to that taxon number. 2) Zero (0) subtracts one unit from the taxon just scored. 3) -1 adds one strip. 4) -11 subtracts one strip 5) Any negative integer except -1, -11, and -999 scores one field of view. 6) 999 displays the options menu a) Calculate a Shannon Diversity Index b) Save your work c) Start a new count d) Quit the program e) Exit the options menu (return to your current count)