Patent Publication Number: US-9422588-B2

Title: Bacteria measuring apparatuses

Description:
RELATED APPLICATIONS 
     This application is a continuation application of U.S. patent application. Ser. No. 10/821,732, filed Apr. 8, 2004, now U.S. Pat. No. 8,669,097, which claims priority under 35 U.S.C. §119 to Japanese Pat. Appl. No. 2003-106569, filed Apr. 10, 2003, the entire contents of which are incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to apparatuses, methods, and programs for automatically detecting bacteria in a sample and, more particularly, to apparatuses, methods, and programs for automatically detecting a  bacillus  or  coccus  bacterium in a sample. 
     BACKGROUND 
     Detection of bacteria contained in a sample and determination of the type of bacteria are often performed in clinical examinations and food sanitation examinations. The types of bacteria are typically classified by Gram stainability of the bacteria (e.g., Gram positive or Gram negative), and by shape (e.g.,  bacillus  or  coccus ). Gram-negative  bacillus  and Gram-positive  coccus  frequently produce adverse effects on the human body. 
     The agar culture method is the most common method for classifying bacteria. This method involves culturing a sample in an agar medium for a predetermined time, either staining or not staining colonies formed in the culture, and having an observer classify the bacteria using a microscope. However, the agar culture method is a difficult process inasmuch as it is essentially a manual method. Furthermore, considerable time must elapse before the type of bacterium can be determined since culturing is required. 
     In recent years, methods have been tried which automatically measure bacteria using a particle analyzer, such as a flow cytometer or the like. This technology involves the following aspects: (1) a method and apparatus for measuring microorganisms, which respectively measure pre-culture and post-culture samples to prevent measurement errors due to impurities in the sample by determining the difference between the two measurement results (e.g., see: U.S. Pat. No. 6,165,740); and (2) a method for counting bacteria in samples containing impurities, which separates the bacteria from the impurities, counts the bacteria by adding a cationic surfactant to a sample containing bacteria so as to increase the colorant transmission of the bacteria, and stains the bacteria through the action of the colorant (e.g., see: European Patent Publication No. 1136563 A2). 
     Measurement can be accomplished in a relatively short time when the method automatically measures the bacteria by a particle measuring apparatus such as a flow cytometer or the like. However, among such methods, no technique has yet been proposed for differentiating  bacillus  and  coccus  with greater accuracy. 
     SUMMARY 
     The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary. 
     A method for measuring bacteria embodying features of the present invention includes (a) fluorescently staining bacteria in a sample; (b) detecting size information from the bacteria in the sample, and fluorescence information expressing intensity of fluorescent light emitted by the bacteria; (c) creating a scattergram representing a distribution of the bacteria based on the size information and the fluorescence information detected; (d) analyzing the distribution of the bacteria in the scattergram; and (e) determining whether the bacteria in the sample is  bacillus  or  coccus  based on a result of the analyzing. 
     A bacteria measuring apparatus embodying features of the present invention includes: (a) a sampling device for sampling a sample containing fluorescently stained bacteria; (b) a first detector for detecting size information from each bacterium in the sample; (c) a second detector for detecting fluorescence information expressing intensity of fluorescent light emitted from each bacterium in the sample; and (d) a control unit configured for creating a scattergram of the bacteria using the size information and the fluorescence information as parameters, for analyzing distribution of the bacteria in the scattergram, and for determining whether the bacteria in the sample is  bacillus  or  coccus  based on an analysis result. 
     A computer-executable program for analyzing bacteria embodying features of the present invention includes: (a) obtaining size information of bacteria and fluorescence information expressing intensity of fluorescent light emitted by the bacteria; (b) creating a scattergram representing a distribution of the bacteria based on the size information and the fluorescence information; (c) analyzing the distribution of the bacteria in the scattergram; and (d) determining whether the bacteria is  bacillus  or  coccus  based on a result of the analyzing. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 2  is a perspective view of the sample preparation unit of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 3  is an illustration of the optical system and flow system of the detection unit of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 4  is a block diagram of the analysis control unit of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 5  is a flow chart showing the overall control of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 6  is a flow chart showing the operation of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 7  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 8  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 9  illustrates the unit vector used in the analysis process performed by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 10  shows an example of a screen displayed by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 11  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 12  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 13  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 14  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 15  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 16  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 17  shows an example of a scattergram prepared by an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 18  is a flow chart of the operation of an automated bacteria measuring apparatus embodying features of the present invention. 
         FIG. 19  shows an example of a screen displayed by an automated bacteria measuring apparatus embodying features of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention provides techniques, apparatuses, and computer readable programs for rapidly and accurately determining whether the type of bacteria in a sample is  bacillus  or  coccus.    
     The methods for measuring bacteria in accordance with the present invention include (a) preparing a sample by fluorescently staining the bacteria in a specimen; (b) detecting size information of the bacteria and fluorescence information expressing the intensity of fluorescent light emitted by the bacteria of each type in a prepared sample; (c) creating a scattergram using the detected size information and fluorescence information as parameters; (d) analyzing the distribution of the bacteria in the scattergram; and (e) determining whether the type of bacteria in the sample is  bacillus  or  coccus  based on the analysis result. 
     A fluorescent dye for bonding to components of the bacteria and emitting fluorescent light is used in the bacteria fluorescent stain. For example, bacteria in a specimen may be uniquely stained using a nucleic acid staining dye which uniquely bonds with intracellular DNA and RNA of the bacteria. By way of example, polymethene dyes having structures (1) through (11) below may be used:
     (1) Thizole orange   

     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         (10) Compounds having the general formula: 
       
    
     
       
         
         
             
             
         
       
     
     wherein, R 1  represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R 2  and R 3  represent hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, or alkoxy groups having 1 to 3 carbon atoms; R 4  represents a hydrogen atom, acyl group, or alkyl group having 1 to 3 carbon atoms; R 5  represents a hydrogen atom or alkyl group having 1 to 3 carbons atoms which may optionally be substituted; Z represents a sulfur atom, oxygen atom or carbon atom substituted by two alkyl groups having 1 to 3 carbon atoms; n represents 1 or 2; and X represents an anion.
     (11) Compounds having the general formula:   

     
       
         
         
             
             
         
       
     
     wherein, R 6  represents a hydrogen atom or alkyl group having 1 to 18 carbon atoms; R 2  and R 8  represent hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, or alkoxy groups having 1 to 3 carbon atoms; R 9  represents a hydrogen atom, acyl group, or alkyl group having 1 to 18 carbon atoms; Z represents a sulfur atom, oxygen atom or carbon atom substituted by two alkyl groups having 1 to 3 carbon atoms; n represents 0, 1, or 2; and X represents an anion. 
     Among these dyes, (1) is commercially available, (2) and (3) are available from Japan Photosensitive Dye Research Center, and (5) through (9) are available from Molecular Probes, Inc. Dye ( 10 ) may be manufactured by the method described in U.S. Pat. No. 5,821,127. Dye ( 11 ) may be manufactured by the method described in U.S. Pat. No. 6,004,816. 
     Among the dyes represented by the general formula (10), the dye shown below is particularly desirable at present: 
     
       
         
         
             
             
         
       
     
     Bacteria having a specific level of fluorescent intensity may be distinguished from impurities which substantially lack fluorescent intensity if fluorescent information (fluorescent intensity) is detected from each particle in a sample subjected to a fluorescent staining process using the above-mentioned fluorescent dyes. 
     Electrical resistance methods, which use a detector having a member comprising a pore for passing through bacteria and first and second electrodes, detect the size of particles from the change in electrical resistance of a sample over time when the prepared sample passes through the pore. These methods may be used for detecting particle size information. Methods which detect scattered light pulse width or scattered light intensity emitted by particles using flow cytometry may also be used for detecting particle size information. In these methods, particle size information includes information reflecting particle diameter, particle width in a direction perpendicular to the particle diameter, particle volume or the like. The value of the change in electrical resistance detected by the electrical resistance method, and the pulse width and intensity of the scattered light signal detected by the flow cytometry method may be used as the size information. 
     Methods for detecting the intensity of fluorescent light emitted by fluorescently stained particles via flow cytometry may be used as the method for detecting fluorescence information. 
     Flow cytometry is a method in which laser light is used to irradiate a flowing sample fluid which contains target particles such as bacteria and cells. Optical information concerning scattered light and fluorescent light generated when the particles pass through the laser irradiation area is detected. Subsequently, the particles are analyzed based on the detected optical information. The various optical information is detected as pulse-like electrical signals by photoelectric conversion elements, such as photodiodes, photomultiplier tubes, and the like. Using these signals, signal intensity may be obtained based on the pulse peak height, and the light emission time may be obtained based on the pulse width. In general, the forward scattered light signal reflects the size of the particle, and the fluorescent light signal reflects the degree of staining of the particle which was fluorescently stained beforehand. 
     A scattergram is composed of dots corresponding to individual particles based on a plurality of particle information detected from each particle on a coordinate space having as its axes a plurality of types of information (e.g., size information expressing the size of the particle, and fluorescent light information), which reflect the characteristics of the particle. When the particle properties differ, differences occur in the distribution of dots on the scattergram. The present inventors have discovered that when  bacillus -containing samples and  coccus -containing samples were compared, the size information of both are substantially identical but the obtained fluorescent light information tends to be greater for the  bacillus  than for the  coccus , which results in differences in the dot distribution on the scattergram. Thus, in accordance with the present invention, a determination as to whether bacteria in a sample is  bacillus  or  coccus  is based on the differences in this distribution. 
     Differences in the distribution of dots in a  bacillus  specimen and  coccus  specimen are expressed in the slopes of the distributions. The slopes of the distributions are described below.  FIG. 7  shows an example of a scattergram obtained from a specimen containing  bacillus , and  FIG. 8  shows an example of a scattergram obtained from a specimen containing  coccus , wherein the fluorescent intensity (FL) is plotted on the X axis, and the forward scattered light intensity (Fsc) is plotted on the Y axis. Region BCT in the drawings is regarded as the region in which dots corresponding to bacteria appear. The population of dots corresponding to bacteria in the scattergrams is distributed so as to extend in a fixed direction (from lower left to upper right). In the coordinate space of the scattergrams, the slope having this “fixed direction” is the slope of the distribution. When  FIGS. 7 and 8  are compared, the slope relative to the X axis in the direction in which the population extends to the upper right (i.e., slope of the distribution) is larger in the  coccus -containing specimen ( FIG. 8 ) than in the  bacillus -containing specimen ( FIG. 7 ). From this fact, in accordance with the present invention, a determination as to whether bacteria in a specimen is  bacillus  or  coccus  is based on the slope of the distribution. The slope of the distribution is determined in the direction of maximum variance of the dots representing the bacteria, and the slope of the distribution may be determined by determining the slope in the maximum variance direction. A slope of approximate expression calculated from the dots representing the bacteria may also be used as the slope of the distribution. 
     An embodiment of the automated bacteria measuring apparatus of the present invention is described below. In  FIG. 1 , an external view of an automated bacteria measuring apparatus  1  is indicated by the solid lines, and the internal structure is briefly indicated by the dashed lines. The foremost surface of the apparatus is provided with an output part (e.g. a liquid crystal touch panel  11  for inputting various types of settings and displaying measurement results), specimen cover  12 , reagent cover  13 , and start switch  14 . When the specimen cover  12  is opened, a specimen may be set in the specimen holding unit provided within the apparatus. When the reagent cover  13  is opened, reagent may be set in the reagent holding unit provided within the apparatus. The specimen holding unit and the reagent holding unit are further described below. 
     In the internal structure of the apparatus indicated by the dashed lines, the top part accommodates an analysis control unit  400 , which includes a microcomputer and various types of circuits, and the like. The bottom part nearest the front side accommodates a sample preparation unit  200  for preparing sample fluids. The bottom part on the back side accommodates a detection unit  300  for detecting signals from the bacteria in the sample fluid. 
     Sample Preparation Unit 
       FIG. 2  shows the sample preparation unit  200  of the automated bacteria measuring apparatus  1 . The sample preparation unit  200  is provided with a specimen holding unit  201 , reagent holding unit  202 , incubator  204  as a reaction unit, and dispenser  205 . A receptacle  201   a  for accepting a specimen is placed in the specimen holding unit  201 . In the automated bacteria measuring apparatus  1 , a dilution fluid and staining fluid are used as measurement reagents, and a receptacle  202   a  accommodating a dilution fluid and a receptacle  202   b  accommodating a staining fluid are respectively set in the reagent holding unit  202 . A receptacle  204   a  for reacting the specimen and the reagent is placed in the incubator  204 . A dispenser  205  is movable vertically, forward and back, and side to side via a drive device, and suctions and discharges a set amount of fluid. The dispenser  205  respectively suctions predetermined amounts of specimen in the specimen receptacle  201   a  set in the specimen holding unit  201 , and dilution fluid in the dilution receptacle  202   a  or staining fluid in the receptacle  202   b  set in the reagent holding unit  202 . The dispenser  205  discharges the fluid to the receptacle  204   a  set in the incubator  204 . The incubator  204  maintains a predetermined temperature, and reacts the specimen and reagent to prepare a sample. The prepared sample is sampled by the dispenser  205 , and supplied to the sample receptacle  112 . Details of the operation of the sample preparation unit  200  are further described below. 
     Detection Unit 
       FIG. 3  illustrates the optical system and flow system of the detection unit  300 . A sheath flow cell  107  is used to flow the sample fluid supplied from the sample receptacle  112  of the sample preparation unit  200 , and is connected to the sample receptacle  112 . Furthermore, the sheath flow cell  107  is provided with a nozzle  113 , which discharges sample fluid toward an orifice  111 , a sheath fluid supply port  110 , and drainage port  114 . Near the sheath flow cell  107  are disposed a laser light source  117  for irradiating a laser beam on a sample fluid flowing in the sheath flow cell  107 , and various types of optical components (condenser lens  118 , beam stopper  119 , collector lens  120 , shield plate  130  having a pin-hole  121 , dichroic mirror  122 , and filter  123 ) for condensing the fluorescent light and forward scattered light emitted from the particles in the sample fluid irradiated by the laser light. Furthermore, a photomultiplier tube  124  is provided as a detection device for detecting condensed fluorescent light, and a photodiode  125  is provided as a detection device for detecting forward scattered light. 
     A sheath fluid receptacle  109 , which is pressurized by a positive pressure pump  147 , is connected to the sheath fluid supply port  110  through a valve  105 . The drainage port  114  is connected to a waste fluid chamber (not shown). The nozzle  113  is connected to the sample receptacle  112  through a valve  101 , and to a negative pressure pump  148  through a flow path  139  and valve  102 . A syringe pump  133  is connected on the valve  102  side of the flow path  139 . 
     The detection unit  300  of the above-described structure detects forward scattered light signals and fluorescent light signals from particles of bacteria and the like included in a sample prepared in the sample preparation unit  200  as it flows through the sheath flow cell  107 . The detected signals are transmitted to an analysis control unit  400 . Detailed operation of the detection device  300  is described below. 
     Analysis Control Unit 
       FIG. 4  is a block diagram showing the structure of the analysis control unit  400 . The analysis control unit  400  includes a computer incorporating a CPU, RAM, ROM, hard disk, and the like, and various types of circuits, and functions as an information processor  134  and a drive circuit  137 . As shown in  FIG. 4 , the information processor  134  is provided with an analyzer  141 , memory  145 , and controller  146 , and the analyzer  141  is provided with a scattergram generator  142 , analysis unit  143 , and determining unit  144 . 
     The memory  145  stores (a) analysis programs for analyzing, via the analyzer  141 , the signals obtained from particles in the sample fluid by the detecting unit  300 , and (b) control programs for controlling the operation of each part of the apparatus. The controller  146  controls the drive circuit  137  based on the control program. The drive circuit  137  drives the dispenser  205  shown in  FIG. 2 , syringe pump  133  shown in  FIG. 3 , valves  101 ,  102 ,  105 , positive pressure pump  147 , negative pressure pump  148 , and laser light source  117  based on the control of the controller  146 . 
     General Controls 
       FIG. 5  is a flow chart of the general control of the automated bacteria measuring apparatus  1  accomplished via the control program. After the receptacle  201   a  accommodating a specimen is placed in the specimen holding unit  201 , the receptacle  202   a  accommodating a dilution fluid and receptacle  202   b  accommodating a staining fluid are placed in the reagent holding unit  202 . The receptacle  204   a  for reacting the specimen and reagent (which is empty at this time) is placed in the incubator  204  of the sample preparation unit  200  ( FIG. 2 ). When the start switch  14  is pressed, the control program starts, and step A (sample preparation unit control), step B (detection unit control), and step C (analyzer control) are sequentially executed. In this way, the sample preparation unit  200 , detection unit  300 , and analyzer  141  are controlled, and the series of operations of the automated bacteria measuring apparatus  1  are automatically executed. Details of the operation of each part of the apparatus in steps A, B, and C are described below. 
     Step A Control of the Sample Preparation Unit 
     The operation of the sample preparation unit  200  by the sample preparation unit control is described below in reference to  FIG. 2 . First, the dispenser  205  measures a fixed quantity of the bacteria-containing specimen from the receptacle  201   a  of the sample holding unit  201 , and dispenses 50 μl into the receptacle  204   a  of the incubator  204 . Next, the dispenser  205  measured a fixed quantity of dilution solution from the receptacle  202   a  of the reagent holding unit  202 , and dispenses 340 μl into the sample-containing receptacle  204   a . The incubator  204  mixes the sample and the specimen and the dilution fluid for 10 seconds while maintaining a temperature of 42° C. Then, the dispenser  205  measures a fixed quantity of staining fluid from the receptacle  202   b  of the reagent holding unit  202 , and dispenses 10 μl into the receptacle  204   a . The incubator  204  shakes and mixes the fluids to induce reaction of the staining fluid while maintaining the receptacle  204   a  at 42° C. so as to prepare a sample. Approximately 400 μl of the prepared sample is retrieved by the dispenser  205 , and supplied to the sample receptacle  112 . The sample in the sample receptacle  112  flows to the sheath flow cell  107  of the detection unit  300 , as described below. 
     Step B (Control of the Detection Unit) 
     The operation of the detection unit  300  by the detection unit control is described below in reference to  FIG. 3 . When the sample prepared by the sample preparation unit  200  is accommodated in the sample receptacle  112 , the negative pressure pump  148  is actuated. Valves  101  and  102  are simultaneously opened, and the sample fills the flow path  139  between the valves  101  and  102  due to the negative pressure. Thereafter, the valves  101  and  102  are closed. 
     Next, the syringe pump  133  forces a fixed quantity of the sample in the flow path  139  into the nozzle  113 , and the sample is discharged from the nozzle  113  into the sheath flow cell  107 . Then, a sheath fluid is supplied from the sheath fluid container  109  to the sheath flow cell  107  by opening the valve  105  simultaneously with the discharge. 
     In this manner, the sample is surrounded by the sheath fluid, and the flow is constricted by the orifice  111 . By constricting the flow of the sample, the particles contained in the sample fluid flow along in a linear alignment. When the sample passes through the orifice  111 , the sheath fluid is discharged to the drain port  114 . 
     A laser beam emitted from the laser light source  117  and constricted by the condenser lens  118  irradiates the sample flow  126  flowing through the orifice  111 . 
     The laser light transmitted through the sheath flow cell  107  without irradiating the particles in the sample is blocked by the beam splitter  119 . The forward scattered light and fluorescent light emitted from the particles irradiated by the laser beam are condensed by the collector lens  120 , and pass through pin hole  121  of the light shield  130 . This light then impinges the dichroic mirror  122 . 
     The fluorescent light, which has a longer wavelength than the forward scattered light, is transmitted directly through the dichroic mirror  122 , detected by the photomultiplier tube  124  after the forward scattered light has been removed by the filter  123 , and output as a fluorescent light signal  127  (a pulse-like electrical signal). 
     Furthermore, the forward scattered light is reflected by the dichroic mirror  122 , received by the photodiode  125 , and output as a forward scattered light signal  128  (a pulse-like electrical signal). Then, the fluorescent light signal  127  and the forward scattered light signal  128  are input to an information processor  134  shown in  FIG. 4 . 
     Step C Control of the Analyzer 
     When the fluorescent light signal  127  and forward scattered light signal  128  from the detection unit  300  are input to the information processor  134 , the signals are analyzed by the analyzer  141 . This forms step C (control of the analyzer) in the general control of the automated bacteria measuring apparatus  1 . The operation of the analyzer  141  via the analyzer control is described below in reference to the flow chart of  FIG. 6 . Although the program representing this operation sequence is stored beforehand in the memory  145  together with other programs, this program also may be supplied from an external memory medium or communication network. 
     First, the fluorescent light signal  127  and the forward scattered light signal  128  detected by the detection unit  300  are input to the scattergram generator  142  of the analyzer  141  (S 1 ). 
     The scattergram generator  142  calculates the forward scattered light intensity Fsc from the maximum peak value of the input forward scattered light signal  128  as particle size information. Similarly, the fluorescent light intensity FL is calculated from the fluorescent light signal  127  as fluorescent light information. Then, a two-dimensional scattergram is created by plotting the obtained FL on the X axis, and the Fsc on the Y axis (S 2 ). 
     The analysis unit  143  discriminates the dots corresponding to bacteria from the dots appearing on the generated two-dimensional scattergram, and counts the particles identified as bacteria (S 3 ).  FIG. 7  shows an example of a two-dimensional scattergram generated from a  bacillus  bacteria-containing specimen. As shown in  FIG. 7 , the region BCT in which the bacteria are focused is set beforehand by discriminating the bacteria from the other particles. In this manner, the particles appearing within the BCT region are regarded as bacteria, and the particles within the region BCT are counted as bacteria. 
     The analysis unit  143  determines the variance of the dots of particles within the BCT region in the X-Y two-dimensional space, and determines the directional vector E in which there is maximum variance through the center of the variance. The determined directional vector E is shown in  FIG. 7 . The determined directional vector E is then converted to a unit vector (a vector having a length of 1), as shown in  FIG. 9 . The unit vector is broken down into a component in the X axis direction and a component in the Y axis direction, and the magnitude of the component in the Y axis direction is designated as P (S 4 ). P is a value representing the degree of slope of the directional vector relative to the X axis. 
       FIG. 8  shows an example of a two-dimensional scattergram generated from a  coccus -containing specimen, and indicates the determined directional vector similar to  FIG. 7 . A comparison of  FIGS. 7 and 8  clearly shows that the degree of slope of the directional vector relative to the X axis is greater in the  coccus -containing specimen ( FIG. 8 ) than in the  bacillus -containing specimen ( FIG. 7 ). Thus, the value of P is also greater in the  coccus -containing specimen ( FIG. 8 ) than in the  bacillus -containing specimen ( FIG. 7 ). The determining unit  144  compares the value of P calculated by the analysis unit  143  to a predetermined value A (in this case, A=0.68) (S 5 ). When P≧A, the bacteria included in the specimen is determined to be  coccus  (S 6  and S 7 ), whereas when P&lt;A, the bacteria is determined to be  bacillus  (S 6  and S 8 ). 
     The result of the determination by the determining unit  144  is combined with the scattergram created in S 2  and the bacteria count calculated in S 3  and output to the output part (e.g. the liquid crystal touch panel  11 ) (S 9 ). An example of the screen output to liquid crystal touch panel  11  is shown in  FIG. 10 . The scattergram, bacteria determination result, and bacteria count are displayed. In the spaces indicating the determination result for the type of bacteria, a mark is displayed in the [ Bacillus ] category among the [ Bacillus ] and [ Coccus ] categories to indicate that the determination result is  bacillus.    
     Specimen Measurement Result Examples 
     The measurement of a specimen using the automated bacteria measuring apparatus  1  described above and the bacteria type determination results are described below. 
     Specimens 
     Specimens (a) through (g) described below were used. 
     (a) Human urine containing  E. aerogenes  ( bacillus ). 
     (b) Human urine containing  E. coli  ( bacillus ). 
     (c) Human urine containing  S. aureus  ( coccus ). 
     (d) Human urine containing  S. epidermis  ( coccus ). 
     (e) Heart infusion broth mixed with  E. coli  ( bacillus ) (bacteria count approximately 6×10 5 /mL. 
     (f) Heart infusion broth mixed with  P. aeruginosa  (small type  bacillus ) (bacteria count approximately 6×10 5 /mL). 
     (g) Heart infusion broth mixed with  S. aureus  ( coccus ) (bacteria count approximately 6×10 5 /mL). 
     Assay Reagents 
     The specimens were processed using the dilution fluids and staining fluids listed below as reagents in preparing assay samples. 
     Dilution Fluids 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric acid 
                 100 mM 
               
               
                   
                 Sodium sulfate 
                  90 mM 
               
               
                   
                 Amidosulfuric acid 
                 100 mM 
               
               
                   
                 NaOH 
                 enough to attain pH 2.5 
               
               
                   
                 Tetra decyltrimethylammoniun bromide 
                  1 g 
               
               
                   
                 Purified water 
                  1 liter 
               
               
                   
                   
               
            
           
         
       
     
     Staining Fluids 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Pigment having the structural formula below 
                 40  
                 mg 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
                   
               
               
                   
               
               
                 Ethylene glycol 
                 1  
                 liter 
               
               
                   
               
            
           
         
       
     
     The two-dimensional scattergrams obtained from the measurement results of specimens (a), (b), (c), and (d) are shown in  FIGS. 11, 12, 13, and 14 , respectively. In all cases, FL is plotted on the X axis (horizontal axis), and Fsc is plotted on the Y axis (vertical axis). The values of P calculated from the two-dimensional scattergrams in  FIGS. 11 through 14 , and the bacteria type determination results based on the flow charts are shown in Table 1. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                 Determination 
               
               
                 Specimen 
                 Bacteria type 
                 P value 
                 result 
               
               
                   
               
             
            
               
                 (a) 
                   E. aerogenes  ( bacillus ) 
                 0.27 
                 
                   Bacillus 
                 
               
               
                 (b) 
                   E. coli  ( bacillus ) 
                 0.23 
                 
                   Bacillus 
                 
               
               
                 (c) 
                   S. aureus  ( coccus ) 
                 0.83 
                 
                   Coccus 
                 
               
               
                 (d) 
                   S. epidermidis  ( coccus ) 
                 0.77 
                 
                   Coccus 
                 
               
               
                   
               
            
           
         
       
     
     As shown in Table 1, the P values calculated from the  bacillus -containing specimens are smaller than the P values calculated from the  coccus -containing specimens. Furthermore, the bacteria type determination results match the actual bacteria types based on the result of the comparisons of the P value obtained from each specimen and the predetermined value A (in this case A=0.68). 
     The two-dimensional scattergrams obtained from the measurement results of specimens (e), (f), and (g) are shown in  FIGS. 15, 16, and 17 , respectively. In all cases, FL is plotted on the X axis (horizontal axis), and Fsc is plotted on the Y axis (vertical axis). The values of P calculated from the two-dimensional scattergrams in  FIGS. 15 through 17 , and the bacteria type determination results based on the flow charts are shown in Table 2. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 Determination 
               
               
                 Specimen 
                 Bacteria type 
                 P value 
                 result 
               
               
                   
               
             
            
               
                 (e) 
                   E. coli  ( bacillus ) 
                 0.35 
                 
                   Bacillus 
                 
               
               
                 (f) 
                   E. aeruginosa  ( bacillus ) 
                 0.21 
                 
                   Bacillus 
                 
               
               
                 (g) 
                   S. aureus  ( coccus ) 
                 0.74 
                 
                   Coccus 
                 
               
               
                   
               
            
           
         
       
     
     As shown in Table 2, the P values calculated from the  bacillus -containing specimens are smaller than the P values calculated from the  coccus -containing specimens. Furthermore, the bacteria type determination results match the actual bacteria types based on the result of the comparisons of the P value obtained from each specimen and the predetermined value A (in this case A=0.68). When the large-type  bacillus E. coli  and the small-type  bacillus P. aeruginosa  are compared, the  P. aeruginosa  often appears at a position of lower value for Fsc in the two-dimensional scattergram, so that a difference arises in the state of distribution. However, both  E. coli  and  P. aeruginosa  may be accurately determined by the bacteria type determination based on the P value. 
     Determination Accuracy 
     Specimens of human urine which were bacteria typed by the plate agar culture method were measured using the automated bacteria measuring apparatus  1 , and the bacteria typing determination accuracy is shown in Table 3. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 
                   Bacillus- 
                 
                   
                   
               
               
                   
                 containing 
                   Coccus -containing 
               
               
                   
                 specimens 
                 specimens 
                 Total 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Number of 
                 53 
                 26 
                 79 
               
               
                   
                 specimens 
               
               
                   
                 Number of 
                 47 
                 21 
                 68 
               
               
                   
                 matches 
               
               
                   
                 Accuracy (%) 
                 88.7 
                 80.1 
                 86.1 
               
               
                   
                   
               
            
           
         
       
     
     As shown in Table 3, 47 matches (accuracy 88.7%) were obtained among the  bacillus -containing specimens, and 21 matches (accuracy 80.1%) were obtained among the  coccus -containing specimens. Furthermore, there were 68 matches (accuracy 86.1%) among a total of 79  bacillus -containing specimens and  coccus -containing specimens. Accordingly, the bacteria measuring apparatus of the present embodiment has a high determination accuracy greater than 80%. 
       FIG. 18  shows a modification of the sequence of the flow chart shown in  FIG. 6 . This example only modifies the sequence when P&lt;A in S 6 , and is otherwise identical to  FIG. 6 . 
     In the flow chart of  FIG. 18 , the determination unit  144  compares the parameter P and a predetermined value B, which is less than A (e.g., B=0.60) when P&lt;A (S 6   a ). When P&lt;B (S 6   b ), the particles contained in the specimens are determined to be  bacillus  (S 8 ), and when A&gt;P≧B, determination is deemed difficult (S 6   b  and S 6   c ). 
     Screens corresponding to these determination results are output to the output part (e.g. the liquid crystal touch panel  11 ) (S 9 ). Furthermore, the scattergram created in S 2 , and the bacteria count calculated in S 3  are combined and output to the liquid crystal touch panel  11 . An example of the screen output on the liquid crystal touch panel  11  is shown in  FIG. 19 . The two-dimensional scattergram, bacteria typing determination result, and bacteria count are displayed. In the spaces indicating the determination result for the type of bacteria, a mark is displayed in the [Determination Difficult] category among the [ Bacillus], [Coccus ], and [Determination Difficult] categories to indicate that the bacteria typing determination result for this specimen is difficult. 
     When A&gt;P≧B in S 6   b  of the flow chart in  FIG. 18 , and determination is difficult, an indication that determination is difficult is output as shown in  FIG. 19  without determining whether the bacteria type is  bacillus  or  coccus . However, the presence of bacteria whether  bacillus  or  coccus  may be suggested, or the possibility of the presence of bacteria may be indicated without suggesting the type. 
     Furthermore, when outputting the bacteria typing determination result, the degree of reliability of the determination result may also be output. For example, when bacteria in a specimen is determined to be either  bacillus  or  coccus , the degree of dissociation of the calculated P value and the predetermined value A may be calculated and output, as in the flow chart of  FIG. 6 . The larger the degree of dissociation, the greater the reliability of the determination result. When either [ Bacillus], [Coccus ], or [Determination Difficult] is determined as in the flow chart of  FIG. 18 , when  coccus  is determined (P&gt;A), the degree of dissociation between the P value and the predetermined value A is calculated. The larger the degree of dissociation, the higher the reliability of a result determining the bacteria is  coccus . When  bacillus  is determined (P&lt;B), the degree of dissociation between the P value and the predetermined value B is calculated. The greater the degree of dissociation, the higher the reliability of the result determining the bacteria is  bacillus.    
     Although the present invention has been described above in terms of examples of presently preferred embodiments, the present invention is not limited to these examples. The present invention easily determines whether bacteria in a specimen is  bacillus  or  coccus  from the distribution of a scattergram created by bacteria size information and fluorescent light information. Since bacteria cultures are unnecessary in the present invention, it is possible to determine the bacteria type extremely quickly and efficiently. 
     The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.