COUNT 2.41 (DOS) 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 becoming 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 (i.e. Excel). Furthermore, this importability reduces transfer error rates to zero. GENERAL INFORMATION This program has been created using the True Basic (Kemeny and Kurtz) programming language. It has been compiled and bound into a free-standing application that needs no other software support. Files to be edited must be opened from within another application (i.e. spreadsheet). These files must be saved as an tab delimited ASCII text file. The program uses a master file containing taxon names as row titles, sample names as column 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 master file for a batch of samples must be created as a tab delimited spreadsheet and saved as ASCII text. The master file is then called, manipulated, and saved from within the counting program until the batch is complete. 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 master 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 particular session. *sum(pi log2 pi), where pi = ni/N THE MASTER FILE When creating a master file for a batch of samples, space for more taxa than are known or are anticipated should be allocated. This will accomodate unknown or new taxa found in the samples. The extra allocations must have some "generic" code assigned to them, i.e A, B, C, D. Six extra lines for summary information should be included after your last taxon (see APPENDIX I). Each non-sample name or taxon name cell should be filled with a 0 (zero) before you begin your first count. Once created, "generic" rows can be assigned a proper name in the master file from within a spreadsheet application. Once opened by another application, the file must be saved as an ASCII text file. See APPENDIX I for an example of the master file in spreadsheet form. The program reads the first line of the master file (i.e. 50.25) to tell it how many taxa and samples are in the file. In this case there will be 50 taxa and 25 samples. It is important to note that these two numbers be separated by a '.' (period). You can add more taxa or samples by using your spreadsheet program, but be sure to change the appropriate number in the header. Its been reported that Lotus 123 does not write a text file format that my program can use. GETTING STARTED The first question the program will ask is to name the file to open. Do so and the program will load it into memory. 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 nearest the enter key associated with the 10-key portion of your keyboard. You'll also be asked to hit your 'tab' key. This is because the numeric code for that key is different on different PC clones. COUNTING You will be presented with an on screen menu of your sample names (the program can accommodate 70 samples in a batch). Point and click on the box associated with the sample you wish to count. The program loads the sample and displays pertinent information 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, the master file name (if any is given at this point), 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. 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. 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 above 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 (remember to subtract one addtional unit to account for the first unit scored to get you into the subroutine) as well as enter fractional units. SUBTRACTION: Immediately after scoring one unit of a taxon 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' subtraction 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. At this point you may 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 be prompted to enter a file name. If this is the initial save for the master file you should entitle it at this time, or else overwrite the old master file. If you suspect you have input some kind of erroneous 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 current 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 names, but rather contain the count 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 are 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. Then insert and name as many taxa rows as you feel you need, enter a 0, return to the top line of the file and change the number before the '.' 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 additional lines are formatted properly, that you have entered a 0 for every sample, that you have changed the first number in the top line of the file, and that the file is saved as ASCII text. If a problem occurs while loading a sample or taxon names file, check to insure that each item is on its own line, and that a carriage return follows the last character of the line, except the last line of the file. 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 appreciate 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. 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 djohnso@opie.bgsu.edu APPENDIX I SAMPLE MASTER FILE (as viewed using EXCEL) 20.9 DARBY1 C1 C2 C3 N1 N2 N3 P1 P2 P3 Achnanthes 0 0 0 0 0 0 0 0 0 Amphora 0 0 0 0 0 0 0 0 0 Cocconeis 0 0 0 0 0 0 0 0 0 Gompho 0 0 0 0 0 0 0 0 0 Fragilaria 0 0 0 0 0 0 0 0 0 Hantzschia 0 0 0 0 0 0 0 0 0 Melosira 0 0 0 0 0 0 0 0 0 Navicula1 0 0 0 0 0 0 0 0 0 Navicula2 0 0 0 0 0 0 0 0 0 Nitzschia1 0 0 0 0 0 0 0 0 0 Nitzschia2 0 0 0 0 0 0 0 0 0 Rhoico 0 0 0 0 0 0 0 0 0 Surirella 0 0 0 0 0 0 0 0 0 AA 0 0 0 0 0 0 0 0 0 BB 0 0 0 0 0 0 0 0 0 CC 0 0 0 0 0 0 0 0 0 DD 0 0 0 0 0 0 0 0 0 EE 0 0 0 0 0 0 0 0 0 FF 0 0 0 0 0 0 0 0 0 GG 0 0 0 0 0 0 0 0 0 TOT UNITS 0 0 0 0 0 0 0 0 0 # of FIELDS 0 0 0 0 0 0 0 0 0 # of STRIPS 0 0 0 0 0 0 0 0 0 H' 0 0 0 0 0 0 0 0 0 Hmax 0 0 0 0 0 0 0 0 0 J' 0 0 0 0 0 0 0 0 0 APPENDIX II 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)