-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Welcome to Bacterial Identification Program (B.I.P.) version (3.0) for DOS 1997, Murat AYDIN Ph.D. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= BIP helps to correct and faster identification of an unknown microorganism. Usually, a microbiologist is able to use this program. Mostly, each menu of BIP contains a self-explanatory words or messages. This program does not entail windows, mouse, special card, color monitor, external device or any other. It was planned that this program must serve to scientific purposes with a minimal configuration of computer. Further information can be provided from me or the folowing literature: Aydn M., Gnay ., Kksal F., Serin MS. Taxometri and computerization in bacterial identification Mikrobiyol Bul. 1996. 30(3). P 281-7. (British Library Document Number: EN043266410,IS:0374-9096) *** INDEX *** 1. Installation of the BIP 2. Introduction 3. BIP menus 3.1 Jotter Why jotter is necessary ? Which type of data must be present in jotter files? 3.1.1 Read 3.1.2 Edit 3.2 Utility 3.2.1 Add new one 3.2.2 Input data 3.3. Identification To describe a threshold (cut-off) Exit to DOS 4. Further identification Why necessary to delete a bacterial specimen from the list? 5. To save data 6. To load data 7. An example session for bacterial identification 8. Appendix-A 9. References ----------------------------------------------------------------------------- 1. INSTALLATION of THE BIP : 1.Step: Create a directory preferable in harddisk,( although, BIP can run in a floppy diskette too, but slower than that in HDD.) 2.Step: Decompress the original zip file into the same directory with use PKUNZIP. Those files must be present : BIP.EXE <- Main prg. README.TXT <- This file BAK.BAK <- Identification tables of bacteria INDEX <- Names&numbers bacteria present IDENT <- 62 of standard physiological and biochemical test pattern EDITOR.COM <- Creates&edits jotter SINIR <- Cut-off value identification procedure OKUMA.COM <- Shows only, do not edit an ascii file sort.exe <- YOU must add this one 3.Step: Copy the current version of SORT.EXE into the same directory 2. INTRODUCTION: To identify a microorganism with genetically, is safest method. It bases on stationary molecular architecture of bacterial cell instead of transient adaptational motives. However, It takes much more time, effort, instrument and money than that phenotypic identification procedures. Usually, there is no time for both physician and patient, particularly while any hard infection threatening in life. To make FASTER identification of a pathogenic microorganism is necessary for early control of infection. For make a bacterial identification with BIP; a pure and young culture of Suscepted Bacterial Sample (SBS) is prepared. Some physiologic and biochemical tests (see Appendix-A) are performed on the SBS. For true identification, the tests must be a number of as much as possible . Although, tests can be distinguished as simple and fast resulted ones according to the individual laboratory conditions. Then, the test results are given to the BIP with type on keyboard. BIP will prepare a list of bacteria which are possible. At this point, user (microbiologist) can decide which bacterial specimen is pathogenic, or he/she can request to make a further identification from the BIP. In result, BIP gives a report from screen and/or printer about identity of the SBS. Notice that, this program can be used for educational purposes too, However, when I wrote this program, I had not directly aimed to this target. (for instance, to find some bacteria which are catalase positive and oxidase negative rods, or to find which bacteria are lactose negative but urease positive and nonmotil and anaerobic but encapsulated?). 3. BIP MENUs: 3.1. JOTTER: It consists of some practical informations and laboratorial clues about a selected bacterium. This jotter is empty when BIP was first installed, because every user may desire to input individual data. More important that, any different language can be used in the individual text file (e.g., french, finnish, german, spanish..). Why jotter is necessary ?: Color, niff, bulk and shape of bacterial colonies are very useful clues of its identity. These observations and other subjective inspections could NOT be easy translated to a computer language. On the other hand, some times, these informations are highly descriptive for make a bacterial identification. For this reason, this menu must be ready while the program running. Which type of data must be present in jotter files There is never an obligation!. Contents of these files are fully individual. Some examples: .It is smelling as lucky flower .There is some greenish on Endo agar, but not on blood agar .Colonies are reflecting the sun-light and they are slimy .Colonies are giving fluorescence under U.V. but in first 1/2 hour .Don't believe the catalase test, because last four were catalase variable .Colonies are looking like Actinobacillus suis, but some times Staph. .If this found, give a message to Dr......., he is interesting with this .For confirmation of this specimen, make latex fixation, but careful that some species can give false positive cross-reaction with of P. penneri .If this found, Carbenicillin can be more potent! etc. 3.1.1. READ: All bacteria will be alphabetically listed in a window. User selects one of bacteria listed. All practical data (if was input before by user!) will be shown on the screen, user can print these with use of

key. These files can be called and read also from the identification menu (see `further identification'). 3.1.2. EDIT: This is used when edit/create jotter file of a selected bacterium. All of bacteria will be alphabetically listed in a window. User selects one of them. Thus, the BIP jumps to an ASCII editor (editor.com) which is present in the original package. The current file name will be automatically adjusted by the BIP to number of selected bacterial specimen. During the editor program is running, user may press for learn which keys are active. The user may replace any ascii editor program instead of this one (Editor V1.3B, Venetek). 3.2. UTILITY: 3.2.1. ADD NEW ONE: The name of new bacterium which will be added to the BIP, is asked to user. User inputs name of bacterium, then, the BIP automatically check whether or not it is already exist. If it is exist, BIP will alert the user and re-run, otherwise the name will be recorded into the BIP. End of this operation, user must immediately input phenotypic profile of that new bacterium. (see next sub-menu). There is not an upper limit for input new bacteria to the BIP. Total number of bacteria present are 440 in current Ver 2.0. However, they can be enlarged if any user input new bacteria. The program is not static. It is dynamic and very flexible. 3.2.2. INPUT DATA: In the BIP, 62 of standard physiologic and biochemical tests were based. Test results of each bacterial specimens which are currently present in BIP, were taken from the safety sources (see references). Despite to this fact, an user may wish to add (or change) test results. Because of this, there are not satisfactory informations in literatures about particular test results of some bacteria. I have not input if it is an unpublished test result. Thus, some test results of some bacteria were left as blank. With this sub-menu, user can easy revize the data present. For make this, user marks the bacterial specimen through this sub-menu, test results of the bacterium will be showed to user and allowed to change them. In 'input data' option, those keys are acceptable: <+> character as a positive test result (90%-100%), <-> character as a negative test result (0%-10%), <*> character as an undecided test result (11%-89%), for unknown test results or for input a numeric data. As you see, user is free input a numerical data. For instance, DNase activity of Fusobacterium naviforme is 44. This is mean, 44% of species of this bacteria have a DNase activity. Pres , while the bar is waiting on the DNase test line, a new window will be open for input a numeric data, type 44 and pres , this value will be accepted to be result of DNase test for F. naviforme. Also, this number will be showed to the user at upper-right corner of the window while the highlighted bar is on the DNase test line. However, in the `identification menu', it will never be permitted to any numerical input. (See below) 3.3 IDENTIFICATION: Makes bacterial identification. When this menu activated, two windows will be opened. The first, at right, identification window (IW), it consists of test pattern, a high-lighted bar stops on the first test (gram stain). The second, at left, it is called Possible Bacteria Window (PBW), but it is empty yet. At IW, Type the results of the tests which you have performed in laboratory one-by-one. It is not prerequisite to perform and input all of tests. At this point, some lines may be left as blank if the test was not treated on the SBS despite to fact that test pattern of BIP contains 62 of applicable laboratory trials. In this section, only those keys are welcome; <+> key as a positive test result (exact mean is 100%), <-> key as a negative test result (exact mean is 0%), <*> key as an undecided test result (exact mean is 50%) or represents the test was not treated on the SBS However, user CAN NOT TYPE ANY NUMERIC DATA in this step. This is important. Some microbiologists said that: "We wish to input a numerical test result for SBS. Exactly no. Any of standard physiological and biochemical test may be resulted positive, negative or, rarely, may be undecidable. Any gradual value can not be prompted for one standard test. (such as 33.08% or 79.2% positive) To describe a threshold (cut-off): User can describe a cut-off value between 1 and 99 for preparing the list of suscepted bacteria. When the cut-off value was described as to be 80 (defaultly), BIP will assort the possible bacteria which their similarity percentage is equal or greater than 80%. If a higher value was chosen as a cut-off by the user, for instance 90, the BIP will give lesser number of bacteria but more similar to the SBS. So that, current identification will continue in a selectable magnitude of perspective by user. This specificity is not present in many computer supported microbiologic devices even at similar softwares. After a threshold was described, press . BIP will prepare a list of possible bacteria in the PBW. A bacterial specimen can be distinguished from the PBW as pathogenic microorganism by microbiologist. User gives a report to clinician as soon. In this step, user may request to make a further identification if he/she could not decide yet. EXIT TO DOS: User leaves the BIP. 4. FURTHER IDENTIFICATION: For further identification, user selects a bacterial specimen in the PBW with the high-lighted bar, then, user invites F1 function. BIP calculates their taxometric distances between selected and other bacteria listed. At this point, user can reach the jotter menu of a selected bacterial specimen if he/she uses the key instead of . I am advice that, before further identification, impossible bacteria (if any) must be removed from the list by use key. For example, if aerobic incubation was made, you must exclude all strickly anaerobic specimens listed. Simply, for make this, the high-lighted bar is transferred to the PBW with use of key. With use of cursor-movement keys, the bar is placed on bacterial specimen has lowest possibility and key is pressed. To remove operation does never reflect complete deletion the bacterial specimen from the BIP, but does not assess that bacterium only. Why necessary to delete a bacterial specimen from the list?: Some times, bacterial specimens may have one or more `phenon's. For instance, the both, Streptococcus faecalis and Mistuokella multiacidus are highly different bacteria. However, despite to they have very-very different gentypes, unfortunately, the tests of fermentations of glucose, sucrose, lactose, mannose (and some others) are resulted as to be positive by the both. S. faecalis is facultative, Gram-positive, short cocci chain. The second is strictly anaerobic, Gram-negative and it has a rod shape. A microbiologist is able to easy understand which strain is wrong in the list. If user did not input the results of those 3 tests; 'gram stain', 'anaerobic' and 'coccus', the BIP includes the both bacteria into the same list. Already, BIP will exactly advise to perform of above three tests for further identification under these conditions. For this reason, user easy decides that one or more bacterial specimen(s) which are listed, must be deleted from the list of possible bacterial specimens. BIP will design a report from screen or/and printer. This report includes: i) Which test(s) is more specific in order to make an exact differantion of the SBS from the others?. All tests are checked, one by one, and tests which are particular identic, will be advised to user. ii) What is identification power of each of advised tests?, iii) In final, gives a list for each test (not only advised ones) are assorted according to their identification parameters from biggest to less. User must choice one or more of tests which was advised by the BIP. And, he/she must perform them in his/her own laboratory on the SBS. (These specificities are not present too in many of similar softwares and computer supported microbiologic devices.) So that, indispensebality and spontaneously, the user will establish a true laboratory strategy for true identification. Further more, lavishness time, to spend of chemical substances and also money can be prevented by this way. During the calculation of relationships between bacteria, BIP uses hundreds or thousands mathematical operations. For example: (where ; n = number of tests (1..62); deney, result of the test (0..100); OTU, Operational Taxonomic Unit (0..100); r(), test result of suscepted bacterial specimen; rr(), test result of converse one; t, total similarity of the both; d, taxonomic distance of two bacteria (0..1).) if r(n) and rr(n) then deney = deney + 1: t = t + 100 - ABS (r(n) - rr(n)) OTU = Int (t * deney^-1) end if d^2 = 1 - (OTU x 10^2) ('d' parameter is calculated by the BIP for assortment of bacteria, but not shown on the screen, it is unnecessary for user) 5. TO SAVE DATA: At the end of identification, user presses key and gives a protocol number. This number will be asked by the BIP when necessary. 'Only' numeric data is acceptable as a file name. DON'T INPUT patient-name!. The current information on the SBS will be saved in the current directory with very specific format. The file name will be .HST. If user wants to save a data in an already exist file, he/she must type <@:> before the protocol number (e.g., @:2381). Otherwise, the BIP will reject any overwrite operation. 6. TO LOAD DATA: For loading an old data, user comes to 'identification' menu, and uses key. BIP asks protocol number for load it. These informations should be used for statistical purposes. However, there is not a statistical menu in this version of BIP. 7. AN EXAMPLE SESSION FOR BACTERIAL IDENTIFICATION: Suppose that, a material was taken from throat of a boy who is 8 ages old. White membranes are present on tonsillar tissue tend to spread over oro-pharynx. A fever in 40-42 C is fluctuating since 2 days. Non-sporing, non-motile, gram positive rods were dominant in direct stain of fresh material. The material was inoculated to BHI and blood agar, incubated aerobically. Usually, after 18 hours, bacterial colonies appear. One type of colony was apparently dominant on the blood agar plate. They were hemolytic and gram positive rods. One loopful material was taken from that colony, inoculated in BHI broth in order to perform tests. While incubation period, early clues which we have about this specimen were given to the BIPfrom its IDENTIFICATION menu. These are: hemolysis, +; coccus, -; spore, -; motility,-; anaerobic, and gram stain, +. One-hundred-eight (108) of possible bacteria were listed in the PBW by the BIP. This was normal. Folowings were removed from the list possible bacteria (PBW) with key: Three of Eubacterium, five of Bifidobacterium, eight of Actinomyces, two of Propinobacterium, seven of Peptostreptococcus, fourteen of Streptococcus, fifty-two of Lactobacillus, two of Haemophylus and one of Actinobacillus. Because their cell shape, clinic symptoms were missing. Whereby, seven of Corynebacterium and one of Nocardia were remained in the list. In fact, Nocardia should be removed, but some species of Nocardia may seem like a diphtheroid. Corynebacterium diphtheria is a quite forbidding specimen in this bacterial population. The bar was placed on the C. diphtheria, and F1 key was pressed. BIP advised that; it is necessary to perform the fermentation of rhamnose, raffinose, maltose, arabinose, trehalose, sucrose and urease activity, and also NO3->NO2 tests. Each of above tests were performed on young and pure culture of the bacteria while another plate was reincubated. It was found: rhamnose -; raffinose, -; urease, +; NO3->NO2, -; maltose, -; arabinose, -; trehalose, - and sucrose, -. These results were given to the BIP. In first bench, Corynebacterium ulcerans was placed with similarity of 86 OTU, then, Corynebacterium renale (80 OTU), Corynebacterium cystitidis (80 OTU) and some anaerobic bacterial specimens. Corynebacterium ulcerans was reported to clinic together with its antibiotic susceptibility test result. -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 8. Appendix-A: ----------- Standard physiologic and biochemical tests used in BIP and their standardizations: _________________ * Gram stain (koopler's modification for anaerobes), * catalase production (3% for facultatives, 15% for anaerobes), * oxidase, coagulase (with rabbit plasma), * sporulation (with spore stain), * capsule forming (with india ink), * flagellar motility (neither twitching nor gliding, only flagellar), * hemolysis (on sheep blood agar), * indole production from tryptophan, * methyl red, * Voges-Proskauer (acetyl-methyl-carbinol production), * citrate utilization (as a carbon source), * acid and gas production from glucose, sucrose, lactose, mannose, * fermentation of mannitol, * hydrogen sulphide production, * urease activity, * anaerobic? (strictly anaerobic, but neither microaerophilic nor capnophylic), * dextrose fermentation, * NO3>NO2 (nitrate reduction), * gelatinase activity, * growth in KCN, (15 ml of KCN 5% at 1 liter), * growth bile, (tolerance of 40% bile), * lipase activity, * glycerol fermentation, * trehalose fermentation, * abide production from maltose, * gas production from maltose, * fermentations of arabinose,raffinose, cellobiose, melibiose, rhamnose, xylose, dulcitol, adonitol, sorbitol, erythritol, salicin, myo-inositol, * coccus, (is cellular shape a coccus?), * tyrosine pellucidation or melanin production, * -methyl glycoside, * ornithine decarboxylation, * lysin decarboxylation, * aesculin hydrolysis, (not fermentation), * -galactosidase production (ONPG), * phenyl alanine de-amination, * arginine hydrolysis, * DNase activity, (on DNase agar), * fermentation of mucate and malonate, * growth at 42C, 22C and 5C, respectively. Standardization of above tests is according to bellowing references: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 9. References: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Ballows A, Hausler WJ, Herrman KL, Isenbirg HD, Shadomy HJ(ed.). Manual of Clinic Microbiology, 4.th ed., American Society for Microbiology, Washington D.C. 1991. Baltimore, Williams & Wilkins, 1980 Steel KJ. The oxidase reaction as a taxonomic tool. J Gen Microbiol 25 : 297-306, 1961. Barritt MM. The intensification of the Voges-Proskauer reaction by the addition of -naphthol. J. Pathol Bacteriol 42 : 441-454, 1936 Barry AL et al: Improved 18-hour methyl red test. Appl Microbiol 20 : 866-870, 1970. Barry AL, Feeney KL. Two quick methods for Voges-Proskauer test. Appl Microbiol 15 : 1138-1141, 1967. BBL Manual of Products and Laboratory Procedures, 5th ed,pp 115-138. Cockeysville,MD, BioQuest, 1968 Blazevic DJ, Ederer GM. Principles of Biochemical Tests in Diagnostic Microbiology, pp29-36 New York, John Wiley&Sons, 1975. Carlquist PR. A biochemical test for separating paracolon groups. J Bacteriol 71 : 339-341, 1956. Christensen WB. Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other and from Salmonella and Shigella types. J Bacteriol 52 : 461-466, 1946. Clark WM, Lubs HA. The differentiation of bacteria of the colon aerogenes family by the use of indicators. J Infect Dis 17 160, 1915. Elmer WK, Stephen DA, William MJ, Paul CS, Washington CW (eds). Color atlas and textbook of diagnostic microbiology. 4.th ed, Lippincott JB Company, Philadelphia, 1992. Falkow S. Activity of lysine decarboxylase as an aid in the identification of Salmonellae and Shigellae. Am J Clin Pathol 29 : 598-600 , 1958. Finegold SM, Martin WJ, Scoot EG. Bailey and Scoott's Diagnostic Microbiology, 5th ed, p490. St.Louis, CV Mosby, 1978. Gale EF. The bacterial amino acid decarboxylases. In Nord FF(ed), Advances in Enzymology and Related Subjects of Biochemistry Vol.6, New York, Interscience Publishers, 1946. Gordon J, McLeod JW. The practial application of the direct oxidase reaction in bacteriology. J Pathol Bacteriol 31 : 185-190, 1928. Hendriksen SD. A comparison of the phenylpyruvic acid reaction and urease test in the differentiation of Proteus from other enteric organisms. J Bacteriol 60 : 225-231, 1950. Hendriksen SD, Closs K: The production of phenylpyruvic acid by bacteria. Acta Pathol Microbiol Scand 15 : 101-113, 1938. Isenberg HD, Sundheim LH: Indole reactions in bacteria. J Bacteriol 75 : 682-690, 1958. Koser SA. Utilization of the salts of organic acids by the colon-aerogenes groups. J Bacteriol 8 : 493-520, 1923. Lennette EH, Balows A, Hausler WJ Jr, Shadomy EJ(eds). Manual of Clinical Microbiology, 4th ed. Washington, DC, American Society for Microbiology, 1985. MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria, 2nd ed, pp 78-93. Baltimore, Williams&Wilkins, 1980. Miller JM, Wright JW. Spot indole test: Evaluation of four reagents. J Clin Microbiol 15 : 589-592, 1982. Moeller V. Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Pathol Microbiol Scand 36 : 158-172, 1955. Noel RK, John GH. Bergey's Manual of Systematic Bacteriology. Edited by Barbara Tansill, Vol.1,2,3 1984. Prosser JI. Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiology 140 : 5-17, 1994. Shaw C, Clarke PH. Biochemical classification of Proteus and Providencia cultures. J Gen Microbiol 13 : 155-161, 1955. Stuart CA, Van Stratum E, Rustigian R. Further studies on urease production by Proteus and related organisms. J Bacteriol 49 : 437-444, 1945. Vracko R, Sherris JC. Indole-spot test in bacteriology. Am J Clin Pathol 39 : 429-432, 1963. Voges O Proskauer. Beitrag zur Ernhrungsphysiologie und zur Differential-diagnose der Bakterien der hmorrhagischen Septicamia. Z. Hyg 28 : 20-32, 1898. Wallace GI, Neave SL. The nitrite test as applied to bacterial cultures. J Bacteriol 14 : 377-384, 1927. Weaver DK, Lee EKH , Leahy MS. Comparison of reagent impregnated paper strips and conventional methods for identification of Enterobacteriaceae. Am J Clin Pathol 49 : 494-499, 1968. - O - =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= This program can be distributed by free. I hope, BIP will expedite laboratory jobs of microbiologists. All comments and suggestions are welcome. Please send your comments and suggestions to the address below or e-mail them to: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Murat AYDIN, Ph. D. Kurtulus mah 298 sok 5/1 Adana-Turkiye E-mail: muratay@pamuk.cc.cu.edu.tr =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=