
        -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=  
                            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 <P> 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
<F1> 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%),
        <space>     for unknown test results or
        <ent>       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 <ent>, 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 <ent>, 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
        <space> 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): <F2>
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 <enter>. 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 <ent> key instead of <F1>. I am advice that,
before further identification, impossible bacteria (if any) must be removed
from the list by use <del> 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 <tab> key.
With use of cursor-movement keys, the bar is placed on bacterial specimen
has lowest possibility and <del> 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 <esc> 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 <protocolnumber>.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 <F1> 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
<del> 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

       =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=



