Patent Publication Number: US-2005124013-A1

Title: On-line apparatus and method for determining endotoxin levels

Description:
RELATED APPLICATION  
      This application claims benefit under 37 CFR §1.78 of provisional application 60/518,003, filed Nov. 7, 2003. The full disclosure of the application is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION  
      An apparatus and method for determining trace endotoxin levels within a fluid, more particularly, an apparatus for positioning in fluid communication with a fluid line and method for on-line determinations of endotoxin levels in fluids.  
     BACKGROUND OF THE INVENTION  
      Bacterial endotoxin is a potentially widespread contaminant of a variety of materials, such as water, food, pharmaceutical products, and parenteral preparations. Bacterial endotoxins (lipopolysaccharides) are released from the outer cell membranes of Gram-negative bacteria during early stages of growth, phagocytic digestion, or autolysis of bacterial cells. Lipopolysaccharides are water-soluble stable molecules that have both hydrophobic and hydrophilic regions. The latter are composed of repeating oligosaccharide side chains attached to a polysaccharide core.  
      There is considerable variation in the details of the structure of endotoxins derived from different bacteria. While the polysaccharide moiety is responsible for the immunogenic properties of endotoxins, their toxicity is elicited by the hydrophobic part (called ‘lipid A,’ which is virtually invariant in composition across different bacterial species). Even in small doses, the introduction of endotoxins into the circulatory system of either humans or animals is capable of causing a wide spectrum of nonspecific pathophysiological changes, e.g., fever, increased erythrocyte counts, disseminated intravascular coagulation, hypotension, shock, cell death, etc. In large doses, it causes death in most mammals. Early-life exposure to endotoxins exerts long-term effects on endocrine and central nervous system development and increases predisposition to inflammatory diseases. Shanks et al.,  Proc. Natl. Acad. Sci.  97, 5645-50, 2000; see also Pearson III, in P YROGENS : E NDOTOXINS , LAL T ESTING, AND  D EPYROGENATION , Pearson III, ed., Marcel Dekker, Inc., NY, 1985, pp. 11-19; URL address http file type, www host server, domain name “bact.wisc.edu,” file name “Bact330/lectureendo/.” 
      Given current concerns regarding bioterrorism, it is useful to note that inhalation of high concentration of endotoxins causes dry cough and shortness of breath, accompanied by a decrease in lung function and fever. Rylander, in O RGANIC  D USTS : E XPOSURE , E FFECTS AND  P REVENTION , Rylander &amp; Jaccobs, eds., Lewis Publishers, Boca Raton, Fla.,  1994 ; Heederik &amp; Douwes,  Ann. Agric. Environ. Med.  4, 17-19, 1997. Epidemiological and animal studies show that chronic respiratory exposure to endotoxins may lead to chronic bronchitis and reduced lung function. Rylander,  Scand. J Work Environ. Health  11, 199-206, 1985.  
      It is thus essential to ensure that the endotoxin contents of parenterally administered drugs or other fluids remain below permissible levels (in the US, this is set by the US Food and Drug Administration). Sterile water for injection or irrigation, for example, has a maximum permissible limit of 0.25 Endotoxin Units (EU)/mL (for endotoxin derived from  E. coli,  1 EU is approximately 75-200 pg). See the URL address: http file type, www host server, domain name “fda.gov,” file type “ora/inspect_ref/itg/itg40.html”; United States Pharmacopeia, USP 24-NF 19, Suppl. 2, 2761-62; Jul. 1, 2000.  
      Measurement of Endotoxins  
      The rabbit pyrogen test (fever induction in a rabbit) was introduced in the U.S. Pharmacopoeia in 1942 for the general testing of pyrogens, which include bacterial endotoxins. The test is slow and qualitative and has largely been replaced by some form of the  Limulus amebocyte  lysate (LAL) test. In 1964, Levin and Bang discovered that bacterial endotoxins can greatly accelerate the rate of clotting of blood from the horseshoe crab  Limulus polyphemus . Levin &amp; Bang,  Bull. Johns Hopkins Hosp.  115, 265-74, 1964; see also the URL address: http file type, www host server, domain name “dnr.state.md.us,” file type “fisheries/education/horseshoe/horseshoefacts.html.” By  1987 , the US Food and Drug Administration (FDA) published guidelines for the validation of the LAL test as an alternative to the USP Rabbit Pyrogen Test. The superiority of the LAL based assay over the rabbit test has been known for some time. See Levin, in E NDOTOXINS AND  T HEIR  D ETECTION WITH THE  L IMULUS  A MEBOCYTE  L YSATE  T EST , Watson et al., eds., Alan R. Liss, Inc., NY, 1982, 7-24. Berzofsky U.S. Pat. No. 5,310,657 clearly showed that the LAL test is two orders of magnitude more sensitive than the rabbit test and also less expensive, less time consuming, and easier to perform.  
      LAL contains several protease enzymes responsible for endotoxin induced gel/clot formation. Through a series of cascade reactions, the primary protein component sensitive to endotoxins activates the proclotting enzyme to form the clotting enzyme. Berzofsky &amp; McCullough in I MMUNOLOGY OF  I NSECTS AND OTHER  A RTHROPODS , Gupta, ed., CRC Press, Boca Raton, Fla., 1991, pp. 429-48; Morita et al.,  Haemostasis  7, 53-64, 1978. The clotting enzyme then transforms coagulogen to coagulin, which self-associates to form a gel.  
      Presently there are three major versions of LAL tests: the gel-clot assay (Levin &amp; Bang, 1964; Levin, 1982; U.S. Pat. No. 5,310,657), the turbidimetric assay (Levin et al.,  J. Lab. Clin. Med.  75, 903-11, 1970; Cooper et al.,  J. Lab. Clin. Med.  78, 138-48, 1971; Pearson &amp; Weary,  J. Lab. Clin. Med.  78, 65-77, 1971); and the colorimetric assay (Teller &amp; Kelly, in B IOMEDICAL  A PPLICATION OF THE  H ORSE  S HOE  C RAB  ( LIMULIDAE ), Cohen, ed., Alan R. Liss Inc., NY,  1979 ,  423 - 34 ; Ditter et al.,  J. Lab. Clin. Med.  78, 65-77, 385-92, 1971; Dubczak et al.,  Haemostasis  7, 403-14, 1978; Novitsky &amp; Roslansky, in B ACTERIAL  E NDOTOXINS : S TRUCTURE , B IOMEDICAL  S IGNIFICANCE, AND  D ETECTION WITH THE  L IMULUS  A MEBOCYTE  L YSATE  T EST , Cate et al., eds., Alan R. Liss, Inc., NY, 1985, 181-93; Sturk et al.,  Haemostasis  7, 117-36, 1978; Iwanaga et al.,  Haemostasis  7, 183-88, 1978; Tsuji &amp; Martin,  Haemostasis  7, 151-66, 1978; Tsuji et al.,  Appl. Env. Microbiol.  48, 550-55, 1984).  
      Turbidimetric assays measure turbidity due to gel formation; apparent turbidity is somewhat affected by the size and the number of particles, etc. but this problem can be largely overcome. Ohki et al.,  FEBS Lett.  120, 217-20, 1980. Turbidity measurement is generally unaffected by color present in the sample. A quartz oscillator has been used to measure the viscosity change that occurs during gelation; this technique allows turbid samples to be analyzed. Novitsky et al., in D ETECTION OF  B ACTERIAL  E NDOTOXINS WITH THE  L IMULUS  A MEBOCYTE  L YSATE  T EST , Watson et al., eds., Alan R. Liss, Inc., NY, 1987, pp 189-96.  
      In a colorimetric assay, a synthetic chromogenic peptide is hydrolyzed by the clotting enzyme to release the terminal colored chromogenic moiety. It provides better quantitation and is less laborious than clotting based methods. It is also more sensitive because the amount of enzyme needed for the hydrolysis of the chromogenic substrate is less than the amount needed to form a clot. Friberger et al., in E NDOTOXINS AND  T HEIR  D ETECTION WITH THE  L IMULUS  A MEBOCYTE  L YSATE  T EST , pp 195-206.  
      Turbidimetric and colorimetric assays can be practiced in two modes. In the endpoint mode, turbidity or color is measured after a fixed incubation period. In the kinetic assay mode, which offers greater dynamic range, the turbidity or color development is measured continuously as a function of time. In the end point assay mode, a calorimetric reaction can be stopped by adding acid or a surfactant solution (e.g., SDS), and the absorbance can be measured at any time thereafter. In a turbidimetric assay this is not possible; addition of acid also destroys the turbidity.  
      Automation  
      A degree of automation of the turbidimetric end point assay has been achieved with a commercially available system (Muramatsu et al.,  Anal. Chim. Acta  215, 91-98, 1988; Homma et al.,  Anal. Biochem.  204, 398-404, 1992); however, poor correlation with other methods and generally higher results have been observed (Tsuji &amp; Martin, 1978).  
      For some time now, the chromogenic LAL test is the most widely used. Jorgensen &amp; Alexander,  Appl. Environ. Microbiol.  41, 1316-20, 1981; Novitsky et al.,  Parenteral. Sci. Technol.  36, 11-16, 1982.  
      A robotic automated system has been developed for the chromogenic test. Tsuji &amp; Martin, 1978. This early system and its subsequent commercial counterparts has impressive capabilities but the overall cost is very high. See Bussey &amp; Tsuji,  J. Parenter. Sci. Technol.  38, 228-33, 1984; Martin et al.,  J. Parenter. Sci. Technol.  40, 61-66, 1986. In fact, the cost is prohibitive for deployment at each point of use, as is necessary, for example, in sterile water testing applications. Rather, most users utilize microplate reader based instrumentation where 96-well plates are manually loaded with samples, standards, and reagents. See the URL address: http file type, www host server, domain name “Cambrex.com,” file name “biosciences/lal/b-EndotoxinDPS-instrument.htm# 1.” 
      It is known in the art to use flow injection analysis or sequential injection analysis when attempting to detect the presence of a species. Conventional sequential injection analysis involves the use of a system comprising, typically, a rotary, multi-position selection valve around which multiple liquid solutions including samples and reagents are arranged. A bi-directional pump is used to draw up volumes of these samples and reagents through respective ports of the selection valve and into a holding coil where the samples and reagents are stacked and then delivered to a detector for analysis. This process causes mixing of the sample and reagent segments leading to chemistry that forms a detectable species before reaching the detector. The detector is typically attached to one port of the rotary valve via which the stacked segments can be made to flow by the pump. Stacking is the process of providing a plurality of aliquots, slugs or segments of fluids in a single conduit, either discrete and apart one slug or aliquot from another or adjacent to one another. Conventional systems can involve the use of a single pump (syringe or peristaltic) and a single rotary selection valve. Conventional multi-position selection valves permit random access of the ports that are connected to the samples, the reagents and the detector. Conventional selection valves that are usable in sequential injection analysis systems are can have between six and twenty-eight ports. Commonly, the section valves have between eight and ten ports. An electronic actuator that, in some instances, moves through the ports in both clockwise and counter-clockwise directions controls the operation of the selection valve. Typically, only one port is accessed at any time. When compared to flow injection analysis, sequential injection analysis systems have the advantage of being able to access an increased number of solutions with just one pump. However, these types of sequential injection analysis systems have not been used to determine the presence of the endotoxins due, at least in part, to the difficulties in cleaning the system between different test samples.  
      There is, therefore, a need in the art for an affordable, sensitive, and fully automated (“on-line”) endotoxin determination system that can be used for point of use endotoxin determinations with a fluid line.  
     SUMMARY OF THE INVENTION  
      Aspects of the present invention include an apparatus and method for on-line testing for the presence of an endotoxin within a fluid sample from a fluid line. The sampling and analysis can occur while the fluid line is in operation. Also, the testing can occur by diverting part of the fluid in the line without having to shutdown or interrupt the operation of the fluid line.  
      The apparatus is positioned in fluid communication with a fluid line to perform the on-line fluid testing for the presence of at least one endotoxin. The apparatus can include a housing and a fluid sampling system positioned in fluid communication with the fluid line. The fluid sampling system can comprise a valve for controlling the fluid flow from the fluid line into the fluid sampling system. A fluid flow well is positioned within the housing and in fluid communication with the fluid sampling system. A removable assembly can also be secured within the housing. The removable assembly comprises a plurality of wells for receiving used and unused fluid carrying members that can receive samples from the fluid flow well, a plurality of fluid sample receiving wells, and a plurality of vessel retention positions comprising recesses for securely receiving portions of respective fluid vessels. A detecting system is provided for testing a control sample and a sample of the fluid from the fluid line. The results of these tests are compared in order to determine if the fluid sample is carrying any endotoxins. In an embodiment, fluorescence testing of the sample is compared to that of the control in order to determine if the sample includes an endotoxin.  
      The method for performing on-line detection of an endotoxin within the fluid carried by the fluid line can include the steps of positioning an endotoxin testing apparatus within the fluid line of the fluid system and directing fluid from the fluid line into the testing apparatus. The method can also include sampling the directed fluid and delivering the sample to a receiving well. Additionally, the method can include the steps of obtaining an endotoxin identifying agent, introducing the agent into the receiving well containing the fluid sample and detecting the presence of any endotoxin within the sample. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       FIG. 1  is a schematic drawing of a fluid line system including an on-line endotoxin detecting apparatus according to aspects of the present invention;  
       FIG. 2  is a perspective view of the endotoxin detecting apparatus illustrated in  FIG. 1 ;  
       FIG. 3  illustrates a fluid sampling system that forms a portion of the apparatus illustrated in  FIG. 2 ;  
       FIG. 4  illustrates an alternative embodiment of the fluid sampling system illustrated in  FIG. 2 ;  
       FIG. 5  is an exploded view of a fluid sampling system illustrated in  FIG. 4 ;  
       FIG. 6  illustrates a removable assembly shown in  FIG. 2 ;  
       FIG. 7  is a partial illustration of a detecting system that forms a portion of the apparatus illustrated in  FIG. 2 ;  
       FIG. 8  illustrates a sensor arrangement of the detecting system of  FIG. 7 ;  
       FIG. 9  is a partially broken view of a portion of the assembly of  FIG. 6 ;  
       FIG. 10  is an exploded view of a cartridge illustrated in  FIG. 6 ;  
       FIG. 11  illustrates a portion of the cartridge for securely retaining fluid vessels;  
       FIG. 12  is a partially broken view of a cap retention portion and a vial retention portion of the cartridge;  
       FIG. 13  is a cross section of a vessel retaining region of the cartridge illustrated in  FIG. 11 ;  
       FIG. 14  illustrates the cartridge including a cover;  
       FIGS. 15 and 16  illustrate portions of the undersurface of the cartridge;  
       FIG. 17  illustrates the cartridge assembly, positioning system and detecting system illustrated in  FIG. 2 ;  
       FIG. 18  is a partial isometric view of the positioning system;  
       FIGS. 19A and 19B  are isometric views of portions of the positioning system;  
       FIG. 20  is an isometric view of a portion of the positioning system;  
       FIGS. 21-23  illustrates steps for removing a cap form a fluid vessel according to an aspect of the present invention;  
       FIGS. 24 and 25  are isometric views of a system for obtaining and ejecting fluid carrying members;  
       FIG. 26  is an isometric view of the detecting system of  FIG. 2 ; and  
       FIGS. 27-29  are isometric views of the heating and cooling portions of the cartridge assembly illustrated in  FIG. 2 . 
    
    
     DETAILED DESCRIPTION  
      The invention provides automated endotoxin detection systems (i.e., automated “on-line” flow analysis systems) that can perform a  Limulus  amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins. The systems can be used to test fluid samples from production lines to detect the presence of endotoxin during the preparation of, for example, water, food, drink, pharmaceutical products (including those for animal and human health), and parenteral preparations.  
      In systems of the invention, a test fluid sample is mixed with agent(s), such as a chromogenic substrate and an LAL reagent in a well to form an assay mixture at the point of use. Assay mixtures are then tested to detect the presence of an endotoxin and its level of concentration. An automated system of the invention determines endotoxin concentration with good accuracy and reproducibility in the range of 0.01 to 10 endotoxin units (EU)/mL (r 2 ≧0.99). The automated systems of the present invention have performed using a standard curve from 0.05 EU/mL to 5 EU/mL. A manual system according to the present invention determines endotoxin concentrations with good accuracy and reproducibility in the range of 0.005 to 50 endotoxin units (EU)/mL (r 2 ≧0.99). Based on three times the standard deviation of a blank and the slope of a calibration curve, systems of the invention can detect endotoxin concentrations of 0.003 EU/mL or lower. The variability of the assay method is less than 20% (n=10). Analysis time required for a 0.05 EU/mL standard typically is less than 100 minutes. For example, the analysis time can be about 60 minutes.  
      LAL Reagent and Chromogenic Substrate  
      “LAL reagent” as used herein refers both to amebocyte lysates obtained from horseshoe crabs (e.g.,  Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleudus tridentata , or  Tachypleudus gigas ) and to “synthetic” LAL reagents. Synthetic LAL reagents include, for example, purified horseshoe crab Factor C protein (naturally occurring or recombinant) and, optionally, a surfactant, as described in WO 03/002976. One such reagent, “PyroGene™,” is available from Cambrex Bio Science Walkersville, Inc. Reagents such as that discussed in U.S. patent application Ser. No. 10/183,992, published as U.S. Patent Publication No. 20030054432, can be used herein. LAL reagents preferably are obtained from Cambrex Bio Science Walkersville, Inc. Lyophilized LAL reagent can be reconstituted with 1.4 mL of LAL reagent water (endotoxin-free water) and kept refrigerated until use.  
      Any chromogenic substrate that can be used to detect an active serine protease (thrombin, trypsin, etc.) (i.e., has the sequence “Arg-chromogenic substrate) can be used in the automated systems disclosed herein. Such substrates are well-known and are commercially available. For example, the buffered chromogenic substrate (p-nitroaniline terminated pentapeptide (Ac-Ile-Glu-Ala-Arg-pNA, S50-640) is suitable and can be reconstituted with LAL reagent water and stored under refrigeration until use. Fluorogenic substrates having the sequence “Arg-fluorogenic substrate” also can be used and are encompassed within the term “chromogenic substrate.” 
       E. coli  055:B5 lyophilized endotoxin obtained from Cambrex Bio Science Walkersville, Inc. can be used to generate standard curves. Typically, lyophilized endotoxin is reconstituted with endotoxin-free water (LAL reagent water, Cambrex Bio Science Walkersville, Inc.) and vortexed for at least five minutes to yield a concentration of 50 EU/mL. Refrigerated reconstituted endotoxin is stable for at least one month. For the preparation of working standards, the stock solution is warmed to room temperature, vortexed for 5 minutes, diluted with LAL reagent water, and vortexed again before use.  
      Lysate-substrate reagents for use in chromogenic assays typically consist of a mixture of amebocyte lysate and substrate, which is supplied as a co-lyophilized solid in sterile containers. Immediately before use, the user or a robotic system reconstitutes the lysate-reagent by adding a prescribed amount of endotoxin-free reagent water. Equal amounts of the reconstituted reagent and a test sample are pipetted into microplate wells using standard sterile techniques, and the absorbance is monitored as a function of time. A plot of the logarithm of the time t for the starting absorbance to increase by a fixed amount (typically 0.2 AU) vs. log [endotoxin] is linear with a negative slope (color develops faster as the endotoxin concentration increases). The endotoxin concentration of a sample is determined by reference to a calibration curve generated with endotoxin standards and the same reagent batch, usually on the same microplate.  
      In systems such as those disclosed herein, the LAL reagent and chromogenic substrate should be reasonably stable. Preferably, these components are kept in separate vessels until their combination at the point of use increases stability of these components.  
     pH and Temperature for the Chromogenic LAL Assay  
      According to the literature, the optimum pH for the activation of the LAL reagent is 7.5, while that for the enzymatic cleavage of pNA from the substrate is 8.2-8.5 (Tsuji et al.,  Appl. Env. Microbiol.  48, 550-55, 1984; Bussey &amp; Tsuji,  J. Parenter. Sci. Technol.  38, 228-33, 1984; Dunér,  J. Biochem. Biophy. Meth.,  26, 131-42, 1993). In a single mixed solution, the optimum pH is 7.7-7.8; the sensitivity is constant in this region (Dunér, 1993). The optimum temperature for the chromogenic LAL assay has been investigated by several researchers and reported to be 37° C. (Bussey &amp; Tsuji 1984; Dunér, 1993). We found that these reported optima apply to the systems disclosed herein as well.  
     DESCRIPTION OF THE FIGURES  
      Aspects of the present invention relate to a method and an automated apparatus  10  for performing on-line testing of a fluid to determine endotoxin concentrations. In an embodiment shown in  FIG. 1 , the fluid tested is water within a process loop  2  such as a WFI or high purity water system  1 . In an embodiment, the automated apparatus  10  monitors endotoxins within the water using agents, such as LAL or a recombinant endotoxin moiety, through the use of a chromogenic or fluorogenic detection scheme.  
      As shown in  FIG. 2 , the apparatus for determining endotoxin concentrations  10  comprises a housing  11  with an opening  12  for receiving water within the fluid line of the fluid process loop  2  of the water system  1 . The housing  11  can include a door  5  that is latched to a frame portion  6  of the housing  11 . The door  5  and frame portion  6  can include an optic or contact sensor to determine if the door  5  is properly closed and locked. Also, electronic controls  8  for the apparatus  10  can be positioned on the housing  11  and spaced from the door  5  so that the electronic controls  8  are easily accessed when the door  5  is open.  
      As shown in  FIG. 4 , a flow path  16  extends between the opening  12  and a fluid sampling delivery system  20 . The flow path  16  can include a rigid or flexible fluid delivery tube  17  or other type of conventional fluid delivery conduit, such as a pipe. In an embodiment, the flow rate with the flow path  16  is adjustable between about 0 ml/min to about 100 ml/min.  
      The fluid sampling delivery systems  20 ′ and  20 , shown in  FIGS. 3-5 , each includes a solenoid valve  22  that opens and closes to control the flow of water into a fluid storage tank  26 . The solenoid valve  22  has a preset lower limit at which it opens and a preset upper limit at which it closes. The upper limit of the solenoid valve  22  can be set between about 10 psi and 80 psi. In another embodiment, the upper limit can be set between about 15 psi and 55 psi. In a further embodiment, the upper limit at which the solenoid valve  22  opens can be set at about 16 psi. The lower limit of the solenoid valve  22  can be set between about 1 psi and 20 psi. In another embodiment, the solenoid valve  22  can have a lower limit between about 5 and 15 psi. In yet another embodiment, the lower limit at which the solenoid valve  22  closes is set at about 6 psi. The system is relatively insensitive to the fluid pressure within the water loop  2 . Sampling system  20  can be used with water loops having pressures up to, or in excess of, eighty psi.  
      The fluid storage tank  26  that will contain fluid entering sampling system  20  is positioned downstream from the solenoid valve  22 , as shown in  FIG. 4 . The fluid storage tank  26  can have Teflon or other types of lining materials that prevent the endotoxins from binding to the inner surface of the tank  26 . As shown in  FIG. 5 , the tank  26  includes an outer tank  121 , an inner tank  122  and a cover  123  that is positioned over the top of the inner and outer tanks  121  and  122 . The cover  123  includes a plurality of input and output ports. As shown in  FIG. 4 , the cover  123  can include three input/output ports  124 ,  125  and  126 . In other embodiments, the cover  125  can include less than three ports or more than three ports. In another embodiment, illustrated in  FIG. 3 , the tank  26  includes an input opening  127  at its upper end and an output opening  128  at its lower, downstream end.  
       FIG. 4  illustrates a metering fluid control valve  28  is located downstream from the tank  26 . In one embodiment, the fluid flow control valve  28  is a pinch valve. The fluid control valve  28  is set to control the flow out of the tank  26  so that a substantially continuous flow exits the tank  26  and flows into a fluid delivery conduit  29  for delivering the fluid sample to a fluid flow well  30 . The fluid conduit  29  can include a pipe, tube or other known fluid carrying conduit. The fluid flow control valve  28  is set at a pressure that is lower than the lower limit of the solenoid valve  22  so that the pressure within the tank  26  is always greater than pressure maintained by valve  28 . As a result, fluid will substantially continuously flow from the tank  26  and into the well  30 . In an embodiment, the terminal, downstream end of the fluid conduit  29  is spaced above the opening of the well  30  so that the liquid, such as water, exiting the downstream end of the fluid conduit  29  drops into the well  30 . A gauge shutoff valve  94  can be positioned in the flow path at any point between the opening  12  and the well  30 .  
      In operation, the fluid sample received from the fluid line of loop  2  will move into the flow path  16  ( FIG. 4 ). The solenoid valve  22  will remain closed until the lower limit pressure of the solenoid valve  22  is reached within the tank  26 . This pressure will be lower than the pressure within the water line  2  of the system  1  being tested. When the lower pressure limit of the solenoid valve  22  is reached, the solenoid valve  22  opens and fluid from within the flow path  16  moves into the tank  26 . Then when pressure in the tank  26  reaches the upper limit of the solenoid valve  22 , the solenoid valve  22  closes until the pressure within the tank  26  reaches the lower limit as a result of fluid passing out of the downstream end of the tank  26  and past the fluid control valve  28  into the well  30 . As will be understood, the pressure within the fluid delivery conduit  29  created by the fluid control valve  28  is less than the lower limit of the solenoid valve  22  so that continuous flow occurs through the fluid delivery conduit  29  when the tank  26  is draining and being filled.  
      The portions of the embodiments of the above-discussed fluid sample delivery system  20  that contact the water to be tested can be covered or lined along at least their inner surfaces with a Teflon or PE material in order to prevent the binding of the endotoxins from attaching to the wetted surfaces of the parts of the flow path within the system  20 .  
      As shown in  FIG. 6 , the fluid flow well  30  is positioned within a replaceable cartridge assembly  40  of the apparatus  10 . The cartridge assembly  40  is removably and replaceably positioned within a moveable drawer  450  ( FIG. 2 ) so that new cartridge assemblies  40  can be positioned within the drawer when a carried cartridge assembly is spent. The drawer  450  is slidably positioned within the housing  11  as illustrated in  FIG. 2 . The housing  11  can also include an optic or contact sensor  452  ( FIG. 8 ) to determine if the removable drawer  450  is closed. The housing  11  also includes a magnetic detent  454  that permits the accurate and repeatable positioning of the drawer  450  during closure ( FIG. 7 ). A solenoid lock  456  ( FIG. 8 ) can be included for preventing the drawer  450  from unintentionally opening during the operation of the assembly  10 . In the embodiment illustrated in  FIG. 7 , the housing  11  includes at least one dampening member  458  that dampens the movement of the drawer  450  as it moves into and assumes the proper closed position when, for example, the cartridge assembly  40  has been replaced.  
      The cartridge assembly  40  includes a cartridge housing  42  that has a plurality of openings for removably receiving a plurality of members that can be used during the testing procedures including packaging reagents, pipette tips, microplates and a disposable water sampling well. In an embodiment, the cartridge assembly  40  can be formed of a disposable plastic package.  
      As shown in  FIG. 9 , the cartridge housing  42  has a first opening  32  that defines an outer fluid well opening of the well  30  through which the fluid being tested enters the well  30 . The well  30  also includes an inner trough  33  and an outer trough  37 . The inner trough  33  has a fluid receiving interior  34  that receives the water exiting the fluid delivery conduit  29 . As shown in  FIG. 9 , the inner trough  33  has a sidewall  34  that has a first upper edge portion  35  that is vertically higher than an opposing, second upper edge portion  36  so that the received fluid that enters the well  30  will spill in a predetermined direction (directed spill) into the outer trough  37  for draining into an overflow drain  38  and into a drainage tube  39  that carries the overflow fluid to a waste container or returns it to the original fluid loop  2 . In a first embodiment, the second upper edge portion  36  can be formed or cut so that it is lower than the first upper edge portion  35 . In another embodiment, the inner trough  33  can be angularly oriented within the outer trough  37  so that the second upper edge portion  36  is positioned further from the upper edge of the outer trough  37  than the first upper edge portion  35 . In either embodiment, the water overflows the inner trough  33  through a gravity-induced crossflow. When fluid, such as water, samples are taken from within the well  30  as discussed below, these samples are taken from the fluid residing within the inner trough  33  at the time of sampling. A splash guard  31  can extend upward and form an upper portion of the outer trough that prevents water from spilling out of the outer trough  37 . In an embodiment, the inner trough  33  can be securely attached to the lower surface of the outer trough  37 . In another embodiment, the inner trough can be removably secured to an inner surface of the outer trough as shown in  FIG. 9 . The inner trough  33  can be lined with, or formed of, TEFLON or other materials, such as polyethylene (PE), to prevent endotoxins from binding to the inner surface of the inner trough  33 .  
      The cartridge housing  42  also has a plurality of openings for receiving other parts of the assembly  40  as shown in  FIGS. 6 and 10 . For example, the housing  42  includes at least one opening  43  for receiving at least one well plate  50 . The illustrated cartridge housing  42 , for example, includes at least three openings  43  that each receives a respective well plate  50 . The well plates  50  can be snap-locked into the cartridge housing  42  so that they are removably secured to the cartridge housing  42 . Resilient locking members carrying protrusions can extend through openings in the cartridge housing  42  to lock the well plates  50  to the cartridge housing  42 . Other known removable securing members can be used to secure the well plates  50  to the cartridge housing  42 .  
      Each well plate  50  includes a plurality of fluid receiving members, such as wells  52 . The well plates  50  illustrated in  FIG. 10  each includes ninety-six wells  52 . However, well plates  50  can include greater or fewer wells  52  than the illustrated  96  wells. For example, the well plates  50  could each include between 100 and 400 wells per plate. As discussed below, the wells  52  receive fluids used in the water testing process.  
      The cartridge housing  42  also includes at least one opening  45  that can receive a respective well housing  46  for fluid carrying members, as shown in FIGS.  6  and  10 . In the illustrated embodiment, the assembly  40  includes four openings  45  that each receives a respective tip well housing  46 . Each tip well housing  46  includes a plurality of wells  47  that receive and hold new pipette tips  48  before they are used and contaminated pipette tips  48  after they have been used to deliver a fluid to one of the wells  52 . These tip well housings  46  can each include about thirty wells  47 . However, each tip well housing  46  can include greater or fewer than thirty wells  47 . The number of wells  47  per cartridge housing  42  should provide a buffer of at least two empty rows of wells  47  between the used and the unused tips  48 . It is possible to have none or only one empty row of empty wells  47  between the used and the unused tips  48 . However, it is preferred that the cartridge housing  42  include at least two rows of empty wells between the used and unused tips  48 . Each tip well housing  46  can be removably secured to the cartridge housing  42 . The illustrated embodiment can carry about one hundred-five new and used tips  48 .  
      The cartridge housing  42  also includes a plurality of rows  60  of vessel retention positions  61  that are arranged to receive fluid containing vessels  70  as shown in  FIGS. 6 and 10 . In the embodiment illustrated in  FIG. 10 , the cartridge housing  42  has three rows  60  of vessel retention positions  61  spaced from each other along the cartridge housing  42 . In other embodiments, the cartridge housing  42  can have two rows  60 , four rows  60  or greater than four rows  60  of vessel retention positions  61  for receiving fluid containing vessels  70 . The number of rows  60  will depend on the number of fluid containing vessels  70  that are intended to be positioned within the cartridge housing  42 .  
      As shown in  FIGS. 6 and 10 , each row  60  of vessel retention positions  61  includes a plurality of openings  64  for receiving and supporting the fluid containing vessels  70 . As shown in  FIG. 14 , each fluid containing vessel  70  has an elongated body  71 . An upper end of each elongated body  71  has a radially protruding head  72  that is spaced from a radially protruding shoulder  73  by an elongated, vertically extending neck  74 . In an embodiment, the fluid containing vessels  70  include vials. The terms “vessel” and “vial” does not limit the fluid containing vessels  70  to any particular shape or size. Instead, the vessels can be of any known shape or size that will fit within the rows  60  and can be engaged by securing members  65  to securely hold the vessels  70  with their respective rows  60 .  FIG. 13  shows an exemplary embodiment of the fluid containing vessels  70  according to the present invention. As shown in  FIG. 13 , the neck  74  has a smaller outer diameter when compared to the head  72  (above it) and the shoulder  73  (below it).  
      Each adjacent vessel retention position  61  includes a keyhole  63  through which the vessel  70  is introduced into the row  60  and a cooperating retention opening  64  in which a vessel  70  is securely retained. As shown in  FIG. 10 , a first end of each row  60  has an enlarged keyhole opening  63  into which a fluid containing vessel  70  can be introduced for then being positioned in the first opening  64  as shown in  FIG. 10 . Similarly, each adjacent vessel retention position  61  has its own associated larger keyhole opening  63  and smaller diameter retention opening  64 . As a result, during production of the removable assembly  40 , all of the vessels  70  may be inserted simultaneously through their respective keyholes  63  into the cartridge housing  42  and the entire cartridge housing  42  can be shifted horizontally to move the vessels  70  into position in their retention openings  64 . The keyhole opening  63  has a greater diameter than the diameter of the fluid containing vessel  70 . As a result, the fluid containing vessel  70  can be easily received and vertically positioned within a respective one of the rows  60 . In an alternative embodiment, the openings  64  have a diameter that is substantially the same size as the diameter of the keyhole opening  63 .  
      In either embodiment of the openings  64 , securing members  65  extend into the openings  64  and engage the fluid containing vessels  70 . As illustrated in  FIGS. 10, 11  and  13 , the securing members  65  included molded, projecting portions of the cartridge housing  42  that protrude into the open rows  60  and deflect sufficiently as the vessels  70  are being snap-fitted into the openings  64  so that the vessels  70  are removably received with the openings  64 . The securing members  65  do not deflect enough to permit the removal of a vessel  70  as the positioning system  200  manipulates the vessel  70  and its cover  80 . In another embodiment, the securing members  65  can be biased into engagement with the vessels  70  by a spring. Well known materials that will deflect enough to receive the vessel  70  and not break either the vessel  70  or the securing member  65  include well-known plastics.  
      As shown in  FIG. 13 , the ends of the securing members  65  are shaped to engage the outer surface of the neck  74  of the vessel  70  and abut against the head  72  and shoulder  73  when the vessels  70  are vertically moved within the cartridge housing  42 . The positioning of the securing members  65  prevents vertical movement of the vessels  70  in both directions, while also preventing horizontal/lateral movement of the vials  70  within the rows  60 . As understood, “horizontal” relates to the directions that are parallel with a plane in which an upper surface of the cartridge housing  42  lies that is parallel to the length of the rows  60 . “Vertical”, on the other hand, is a direction that extends parallel to the height of the cartridge housing  42 .  
      The fluid containing vessels  70  can carry a fluid used to test the fluid samples taken from within the inner trough  33  (sample well), contained within the well  30 . In an embodiment illustrated in  FIG. 10 , a first set of vials  75  carry an enzyme for delivering to the wells  52 . In an embodiment, four vials  75  can each have an internal fluid capacity of about 5 cc to 10 cc and carry a total fill volume of about 1.2 cc or greater of an enzyme. The enzymes that can be contained in the vials  76  include those discussed herein including “PytoGene™”. At least one vial  76  can include an endotoxin. In an embodiment, the vial  76  has an internal volume of about 10 cc and contains about 7 cc of the endotoxin. Endotoxins carried by vial  76  can include  E. coli . The fluid containing vessels  70  can also include three vials  77  for carrying a substrate. Each of the three illustrated vials  77  has an internal capacity of about 10 cc. The three vials  77  have carry a total volume of about 6 cc of a preferred substrate. Substrates useable with the present invention include any known chromogenic or fluorogenic substrate that can identify the presence of an endotoxin. An additional set of vials  78  can carry any conventional buffer including those discussed herein. Each of the illustrated vials  78  has an internal capacity of about 10 cc and they carry a total combined fill volume of about to 5 cc of a buffer. Another set of vials  79  can carry clean control water. In the illustrated embodiment, the assembly  40  includes four 10 cc vials that hold a total of about 11.5 cc of water. In any of the above embodiments, the vials can have a greater internal volume than the volume mentioned above. Similarly, the fluid containing vessels  70  can be filled to include more or less of their respective fluids. Additionally, the number of vials carrying each liquid can be greater or less than mentioned above. Furthermore, the buffer and substrate may be combined to form one liquid reagent. Similarly, the buffer, substrate and recombinant enzyme may also be combined and lyophilized to form one freeze dried reagent.  
      The cartridge housing  42  also includes a plurality of slotted openings  84  for receiving covers  80  from the vessels  70 , as illustrated in  FIGS. 10 and 12 , while the vessel  70  is being accessed and fluids within the vessel  70  are being taken. The covers  80  include a flange  81  with a lower surface  82  and a plug portion  83  for positioning within an opening of one of the vessels  70 . Each opening  84  includes a keyhole  85  with a first diameter and a retaining hole  86  with a second diameter. As seen in  FIG. 11 , the diameter of the keyhole  85  is greater than the diameter of the retaining hole  86 . As a result, the cover  80  can be introduced into the keyhole  85  vertically, as discussed below, and then slid horizontally into the retaining hole  86 . The retaining hole  86  receives the cover  80  as shown in  FIG. 12 . An upper flange  87  extending around a portion of the retaining hole  86  engages the lower, under surface  82  of the flange  81  and supports the cover  80  within the retaining hole  86 . As seen in  FIG. 12 , the under surface  82  of the flange  81  is the only surface contacted by a portion of the cartridge housing  42  (Flange  87 ). The plug portion  83  that extends into the vessel  70  does not come into contact with the cartridge housing  42  as it is being introduced into the keyhole  85  and slid into the retaining hole  86 . As a result, any fluid or materials on the plug portion  83  of the cover  80  do not come in contact with and contaminate the cartridge housing  42 . Similarly, the covers  80  of different vessels  70  will not be contaminated by the cartridge housing  42 .  
      The replaceable cartridge assembly  40  can also include a cover  90  that is removably secured over the cartridge housing  42  ( FIG. 14 ). In an embodiment, the cover  90  could include a rubber serum stopper or a lyophilization stopper. The cover  90  protects the contents of the original or a replacement cartridge assembly  40  prior to the cartridge assembly  40  being placed within the housing  11 . The cover  90  includes posts  92  ( FIGS. 15 and 16 ) that extend into openings  94  in the cartridge housing  42 . Each post  92  includes at least one securing protrusion  96  that is received within an opening  94  in the cartridge housing  42  and/or the keyhole opening  63  so that the post  92  and cover  90  are secured to the cartridge housing  42  until the assembly  40  is ready to placed into the housing  11 . Each securing protrusion  96  can include at least one tooth or other member that can releasably engage with the cartridge housing  42  to prevent the unintentional removal of the cover  90  from the cartridge housing  42 . Prior to, or after insertion of the assembly  40  into the housing  11 , the cover  90  can be removed from the cartridge housing  42  by deflecting the posts  92  and their respective teeth  96  away from engagement with the cartridge housing  42 . Prior to being removed, the posts  92  and their respective teeth  96  can abut against one or more of the vessels  70  and secure them against movement relative to the cartridge housing  42 . Similarly, a plurality of the posts  92  and their respective securing protrusions  96  can also secure the well plates  50  and the tip well housings  46  against movement when they are covered by the attached cover  90 .  
      The apparatus  10  also includes a motorized positioning system  200  ( FIG. 17 ) positioned within housing  11 . In an embodiment, the positioning system  200  can include the illustrated motorized robotic arm. The motorized positioning system  200  carries and manipulates an integrated, articulatable head assembly  300  along X, Y and Z axes as shown in  FIG. 18 . As discussed below, the head assembly  300  can remove the covers  80  from the vessels  70 , retrieve tips  48 , obtain fluids from within the vessels  70  and the well  30 , deliver the fluids to the wells  52  in well plates  50  and test for the presence of endotoxins in the fluid containing wells  52 . The positioning system  200  can also position a fluorescent detection assembly  610  and/or a fiber optic fluorescent reader ( FIGS. 19A and 26 ) (carried by the head assembly  300  or separate from the head assembly  300 ) over the fluid containing wells  52  in the well plates  50  and move the head assembly  300  so that it removes a cover  80  from an opening  84  and returns it to, and in, its respective vessel  70 .  
      As shown in  FIGS. 17 and 18 , the positioning system  200  includes vertical mounts  210  that are secured to mounting plate  211  positioned in the housing  11 . First and second linear guiding and supporting rails  214  extend between the vertical mounts in a direction along (parallel to) the X-axis to provide support and stiffness to the positioning system. During the operation of the assembly  10 , the head assembly  300  can travel along the length of the rails  214  when an X-axis drive system  215  including a linear motion motor system  220  and a plurality of travel sensors  216  is operated. The travel sensors  216  limit the length of travel of the head assembly  300  along the rails  214 . The travel sensors discussed herein can be sensors that are activated by contact, by breaking a light beam emitted by the sensors or by causing motion within each sensors predetermined field of view. The travel sensors  216  can include a home sensor  217  and a limit sensor  218 . The travel sensors  216  can be any known motion limiting sensor that determines the linear movement of a member and controls a motor accordingly.  
      In the illustrated embodiment, the linear motion motor system  220  includes a housing  221 , an endless toothed belt  222 , a driven toothed pulley  224  and a follower pulley  226 . The driven toothed gear  224  is driven and powered by a conventional rotary stepper motor (not shown) within housing  221 . As will be understood, the teeth of the pulleys  224 ,  226  engage the teeth of the belt  222  in order to drive the head assembly  300  along the rails  214 . When the head assembly  300  activates either travel sensor  216 , the operation of the motor can be stopped and the direction of motion of the motor and the driven pulley  224  can be reversed so that the head assembly  300  travels in a direction away from the activated sensor  216 . In other embodiments (not shown), the pulley  224  can be driven by a conventional linear variable reluctance motor or a powered rack and pinion. The positioning system  200  can also include a cable guide  228  as known in the art. Also, the positioning system  200  can have a half-stepping resolution of about 0.006 inch.  
      The positioning system  200  can also move the head assembly along the Y-axis, illustrated in  FIG. 17 . Y-axis motion is created by the operation of a Y-axis drive system  240  including a linear motor system  242  and a plurality of motion limiting sensors  247  ( FIG. 19B ). The linear motor system  242  includes a rotary motor  243  and a lead screw  244  that extends at least the entire Y-axis travel distance. The rotary motor  243  drives the lead screw  244  as it operates. Any other known linear motion system, including those discussed above, can be used Y-axis drive system  240 .  
      As illustrated in  FIG. 19B , the head assembly  300  is operatively secured to a mounting platform  246  having an opening  247  through which the lead screw  244  extends. An internal surface of the opening  247  includes threads that mesh with and operatively engage the lead screw  244  so that the head assembly  300  moves along the length of the lead screw  244  into a predetermined position as the lead screw  244  rotates relative to the rails  214 . The platform  246  is secured to a support bracket  247  that includes projections that travel within grooved tracks  248  of a support member  249  secured to the housing  221  so that head assembly  300  secured to the platform  246  moves with the housing  221 . As illustrated in  FIG. 20B , the support member  249  can include one or more elongated, grooved rails extending below a slide  241 . The Y-axis drive system  240  has a half-stepping resolution of about 0.0005 inch.  
      As shown in  FIGS. 19A and 20 , the positioning system  200  also includes a Z-axis drive system  260  for moving the head assembly  300  along the vertical Z-axis. The Z-axis drive system  260  includes a rotary motor  262  and lead screw  264  that cooperate to drive a sliding member  310  of the head assembly  300  along a grooved linear slide  268 . The Z-axis drive system  260  operates in a substantially similar manner as the Y-axis drive system  240 . As show in  FIG. 20 , the lead screw  264  extends through a threaded opening  312  in a portion  314  of the sliding member  310 . As the rotary motor  262  turns, the lead screw  264  is driven in one of the two rotary directions. This rotation of the lead screw  264  causes the sliding member  310  and the head assembly  300  to move vertically either in the direction of the cartridge housing  42  or away from the cartridge housing  42 . Travel limiting sensors  269  prevent the sliding member  310  from moving beyond predetermined locations along the lead screw  264 . As with the sensors used with the Y-axis system, the travel limiting sensors  269  can be any known sensor including those discussed above with respect to the X-axis drive system  215 .  
      In addition to the sliding member  310 , the head assembly  300  also includes a system  320  for engaging and removing the covers  80  from the vessels  70 , as shown in  FIGS. 20-23 . The system  320  includes a housing  322  secured to the sliding member  310 . As illustrated, the housing  322  can be secured to the sliding member  310  proximate the portion  314  that threadably receives the lead screw  264 . The housing  322  has a lower portion that forms a lifting fork  324  for removing the covers  80  from the vessels  70 , positioning the covers  80  in the openings  84  and returning the covers  80  to their respective vessels  70 . In the illustrated embodiment, the lifting fork  324  includes a pair of spaced fork members  326  that have tapered forward ends for being introduced under a cover  80  ( FIG. 21 ). The fork members  326  are spaced from each other by a gap that is sized to receive the plug portion  83  of the cover  80 . The gap between the fork members  326  is sized greater than the diameter of the plug portion  83  so that the gap receives the plug portion  83  without engaging and being contaminated by the plug portion  83 . The lifting fork  324  includes an upper retaining member  328  that extends over the fork members  326  as illustrated so that a cover receiving space  327  is formed between the lifting forks and the lower surface  329  of the retaining member  328 . The cover member  328  holds the cover  80  of the vessel  30  within the fork members  326 . As a result, the lifting fork  324  is able to manipulate the cover  80  as it removes it from the vessel  70 , places it within an opening  84 , retrieves it from within hole  84  and returns the cover  80  to the vessel  70 .  
      As illustrated in  FIGS. 20, 24  and  25 , the head assembly  300  further includes a tip coupling member  340  and a tip ejector  360 . The tip coupling member  340  can be formed as a portion of the housing  322 , as illustrated, or it can be separate from the housing  322 . In either embodiment, the tip coupling member  340  is vertically moveable along the Z-axis. In the illustrated embodiment, the tip coupling member  340  includes an elongated, tapered member that is sized to be introduced into the hollow interior of a tip  48  as the tip coupling member  340  moves in a downward direction into engagement with one of the unused tips  48 . The tip coupling member  340  is introduced into and positioned within a tip  48  as the sliding member  310  and housing  322  move vertically downward toward the tips  48 . The tip coupling member  340  will frictionally engage the inner surface of a hollow tip  48  and remove it from its tip well  46  as it moves vertically upward away from the cartridge housing  42 .  
      In order to separate the tip  48  from the tip coupling member  340  and eject the used tip  48  into a tip well  46 , a lower surface  362  of a forked portion  364  of the ejector  360  engages the used tip  48  that has been positioned within a tip well  46 . The ejector  360  is brought into engagement with the tip  48  to be removed by a solenoid switch  366  that activates a plunger or piston rod  367  that is driven into contact with a portion of the ejector  360  ( FIG. 25 ). The rod  367  can be driven by any known drive source. After the rod  367  contacts the ejector  360 , the ejector  360  is rotated into engagement with the held tip  48 . While the ejector  360  remains stationary and the lower surface  362  is engaged with the tip  48 , the sliding member  310  and housing  322  are moved vertically upward in a direction away from the forked portion  364  of the ejector  360 . The forked portion  364  prevents the tip  48  from moving as the tip coupling member  340  is raised away from the respective tip well  46 . As a result, the tip  48  is separated from the tip coupling member  340  and left in a respective tip well  46 .  
      The head assembly  300  also includes a position sensing system  550  ( FIG. 20 ) for detecting the position of a cover  80  with respect to its vessel  70  and the position of a tip  48  with respect to a respective tip well  46 . The position sensing system  550  is particularly useful for determining the position of the cover  80  after removal from opening  84  or the position of a tip  48  after it has been used. The sensing system  550  includes a sensor  551  that can determine when the sliding member  310  is encountering resistance to its motion along the Y-axis in the direction of the cartridge housing  42  as a result of completing a vertical throw and either picking up or returning a cover  80  or tip  48 . When the sensor  551  has determined that the sliding member  310  is encountering resistance and that the cover  80  has been returned to the vessel  70 , the sensor  551  causes a switch to turn off the Y-axis drive motor.  
      In a first embodiment, the sensor  551  can be positioned proximate the portion  314  of the sliding member  310  that receives lead screw  264 . As a result, the sensor  551  will be engaged by the portion  314  as the portion  314  deflects in response to the stopping of the motion of the forward portion of the sliding member  310  and the continued rotation of the lead screw  264 . Alternatively, the sensor  551  activates a switch that stops the operation of the Y-axis motor when a spring loaded member is deflected into contact with the sensor  551  or the spring loaded member is deflect across a beam or into the vision of the sensor  551 . When the spring loaded member contacts the sensor  551  in response to the stopping of the sliding member  310 , the assembly  10  understands that the sliding member  310  has completed a vertical throw and either picked up or returned a cover  80  or tip  48 . This length of the vertical distance traveled by the sliding member  310  can also provide information to the processor and control system of the assembly  10  regarding the height at which horizontal motion of head assembly  300  takes place, thereby making the motion of the assembly more efficient.  
      As shown in  FIG. 26 , the assembly  10  can also include a system  600  that provides a chromogenic or flurogenic detection scheme for determining the presence of trace levels of endotoxins within the tested water from well  30 . The system  600  includes a detection assembly  610  that moves along the X-axis and the Y-axis. The detection assembly  610  includes a first grooved rail  620  that extends along the X-axis and a second grooved rail  625  that extends along the Y-axis. A detector head  630  is secured to the rails  620 ,  625  by brackets  622  and  627 , respectively. The brackets  622  and  627  are secured to each other by mounting plates  628 . The detection assembly  610  moves along the X-axis and the Y-axis via the operation of the X-axis and Y-axis drive systems  215 ,  240  used to drive the head assembly  300 . The detector head  630  can be operatively secured to the positioning system  200  so that it moves along the X-axis and Y-axis when the head assembly  300  moves along these axes or when the control processor of the assembly  10  activates the positioning system  200  to move the detection assembly without regard for the position of the head assembly  300 .  
      As shown in  FIG. 26 , the detector head  630  includes a U-shaped member  632  that has a recess  633  in which the well plates  50  are received. As can be seen, a lower arm  634  of the U-shaped member  632  is positioned beneath the well plates  50  as the detector head moves along the cartridge housing  42 . An upper arm  636  of the U-shaped member  632  extends over the well plates  50  as the detector head moves. The lower arm  634  has a plurality of passageways  640  that carry an LED  642  and a lens  644  positioned above the LED  642 . The lens  644  covers the aperture  646  at the upper end of the passageway  640 . A feedback detector  647  is positioned within a passageway  648  that extends within the lower arm  634  at an angle to the LED  642 . The feedback detector  647  provides information to the operating system of the assembly  10 . The upper arm  636  includes a slot carrying a conventional filter  652 , such as a solid state detector (photodiode) for absorbance assays, and a conventional photomultiplier detector  654  for fluorescence assays, such as those used in the industry, for example by Bio-Tek Instruments. The upper arm  636  also includes apertures within its outer surfaces for receiving light transmissions as is understood in the art. In an alternative embodiment, the upper arm  636  does not include the filter  652 . Also, the detector head  630  can have any shape that allows a first portion to extend under the well plates  50  and another portion to extend above the well plates  50 . In another embodiment, the head assembly  300  carries a fiberoptic fluorescent reader that will move over the well plates  50  and take the appropriate readings as the head assembly  300  moves over the cartridge housing (See  FIG. 17 ). In each of the above-discussed embodiments, light from the LED can be transmitted through a transparent and/or translucent lower surface of each well  52 . Also, light can be delivered to the LED and data can be transmitted from the detectors using fiberoptics. The illustrated U-shaped assembly that reads through the microplate wells is a preferred embodiment for assays using absorbance detection, including endotoxin detection and more commonplace assays such as enzyme-linked immunosorbant assays (ELISAs). This detector head  630  enables the use of the head assembly  300  for pipetting and moving materials as well as coupling light through a microplate well  52  with the detachable optics assembly  638 . The use of a single bundled fiber optic ( FIG. 20 ) on the head assembly  300  can be used in a preferred embodiment with fluorescence assays.  
      As shown in  FIGS. 27-29 , the assembly  10  can also include a heating system  400  for warming the fluid well plates  50  and a cooling system  420  for maintaining cool temperatures around the vessels  70 . The heating system  400  can include a heating element  410  that is positioned under the well plates  50  when the removable and replaceable cartridge housing  42  is positioned within housing  11 . In the illustrated embodiment, the heating element  410  includes a resistive heating element. Alternative known heating elements may also be used. A heat sink  415  can line the exterior vertical walls of the heating element to prevent heat from being radiated along the X-axis or the Y-axis. The cooling system  420  can include a source of cold air or refrigeration that cooperates with a plurality of cooling fins  422  on a lower surface of the cartridge housing  42  beneath the rows  60  carrying the vessels  70 . The cooling system  420  uses an electronic cooling device. In an embodiment, the cooling device includes a PELTIER thermoelectric cooler. A blower fan  425  is used to draw air into the housing  11  and across the heat sink  422  in order to remove heat from the cooling block within the cartridge housing  42 . An additional fan  429  positioned in the top of the internal housing transfers air from the upper chamber (electronics bay) to the low chamber (robot housing) through a high-efficiency (HEPA-type) filter that reduces the introduction of airborne contaminants into the chamber carrying the positioning system  200 . The fan  429  also creates positive pressure in the housing  11 .  
      The assembly  10  further includes a syringe pump  700  that is in fluid connection with the head assembly  300  ( FIG. 4 ). The head assembly  300  includes a vacuum port that creates a vacuum in the tips  48  and draws fluids from well  30  and vessels  70  into the tips  48  in response to the intake stroke of the piston of the syringe pump  700 . As the syringe pump  700  returns to rest, the vacuum within the carried tip  48  is released and the fluid is expelled into its respective well  52  in one of the well plates  50 .  
      In operation, the assembly  10  will receive and test a water sample from the loop  2  as previously discussed. In the manner discussed above, water from the loop  2  enters the flow path  16  and passes through the fluid delivery system  20  and into the well  30  in the manner discussed above. The positioning system  200  moves the head assembly along the X-axis and/or Y-axis until the tip coupling member  340  is positioned over a tip  48  within a tip well  46 . The sliding member  310  is then moved along the Z-axis until it engages a tip  48  and the sensor  551  is activated. When this occurs, the stroke of the sliding member  310  is reversed so that the tip  48  is removed from the tip well  46 . The processor and control system of the apparatus  10  then cause the positioning system  200  to locate the carried tip  48  over the inner trough  33  of the well  30 . Once the tip  48  is positioned over the trough  33 , the sliding member  310  is then driven vertically downward until the tip  48  engages the fluid within the inner trough  33 . The syringe pump  700  is then activated so that fluid to be tested is drawn up from the inner trough  33  into the tip  48 .  
      The fluid carry tip  48  is then moved by the positioning system  200  until it is positioned over a well  52  of the well plates  50 . The fluid carrying tip  48  is then driven toward the well  52  by the Z-axis drive system  260 . Upon reaching a predetermined height, an amount of the carried fluid for testing is released into a first well  52  by the operation of the syringe pump  700  as discussed above. The method of the present invention can include duplicating each test in a plurality of separate wells  52 . As a result, before the fluid within the tip  48  is released, the fluid carrying tip  48  can be moved to a second well  52  and the step of releasing the carried fluid into a well  52  can be repeated. After the carried fluid is released into the two wells  52 , the used tip  48  is located over an empty tip well  47  by the positioning system  200 . The empty tip well  47  is preferably spaced from the unused tips  48  by a space comprising at least one row of tip wells  47 , as discussed above. Once the used tip  48  is positioned within the tip well  47 , the sensing system  550  determines when the tip  48  has been fully inserted into its well  47  as discussed above and the tip ejector  360  separates the used tip  48  from the tip coupling member  340  in the manner discussed above. Then, the head assembly  300  is moved by the positioning system  200  along the cartridge frame  42  toward the vessels  70 .  
      Upon reaching the vessels  70 , the lifting fork  324  is moved vertically along the Z-axis into position proximate a cover  80  of the vials  79  carrying the control water ( FIG. 21 ). The lifting fork  324  is then moved horizontally so that the fork members  326  are positioned between the underside  82  of the cover  80  and the head  72  of the vessel  70  ( FIG. 22 ). Once the cover  80  is received and positioned in the cover receiving space  327 , the cover  80  is removed from its vessel  70  ( FIG. 23 ) by lifting the lifting fork  324  vertically away from the vessel  70 . The positioning system  200  then places the cover  80  over the keyhole  85  of the opening  84  and lowers the cover  80  to the opening  84  so that the plug  83  is positioned within the keyhole  85 . The introduced cover  80  slides horizontally into its retaining hole  86  in response to the movement of the positioning system  200 . The lifting fork  324  separates from the retained cover  80  and the head assembly  300  returns to the tip wells  46 .  
      Upon returning to the tip wells  46 , the tip coupling member  340  obtains another tip  48  in the manner discussed above and moves this tip  48  into position over the open vial  79  of the control water. The positioning system  200  then moves the sliding member  310  along the Z-axis and the carried tip  48  into the open vessel  79 . The syringe pump  700  then operates to withdraw the control water from the vial  79  and into the tip  48 . The control water carrying tip  48  moves into position over the well plates  50  as discussed above with respect to the water from trough  33  and releases the control water into at least two wells  52 . In a preferred embodiment, the control water is released into at least four wells  52 . The positioning system  10  then locates the used tip  48  over one of the empty tip wells  47  and the used tip  48  is ejected into the empty tip well  47  as previously discussed.  
      After the used tip  48  that carried the control water is positioned within the tip well  47 , the positioning system  200  then positions the lifting fork  324  proximate the cover  80  located in the hole  86 . The sliding member  310  moves along the Z-axis and brings the lifting fork  324  to the level of the cover  80 . The positioning system  200  causes the lifting fork  234  to engage the cover  80  and move the cover into the keyhole  85 , where the cover is then removed from the opening  84  and returned to its vessel  70 . After the lifting fork  324  has returned the cover  80  to its vial  79 , the head assembly  300  returns to the vessels  70  in preparation for removing the cover  80  from another of the vessels  70 . The steps of removing a cover  80 , securely placing the cover  80  within the opening  84 , obtaining a tip  48  from a tip well  47 , obtaining a fluid from the open vessel  70 , introducing the obtained fluid into appropriate wells  52 , ejecting the used tip  48  and returning the removed cover  80  to the open vessel  70  are done for each of the other fluids in the vials  70  in the manner discussed above. However, the endotoxin from vial  76  is only positioned in one of the wells containing the control water if only three wells  52  are being used in the test. In an embodiment in which the test is being duplicated and at least six wells  52  are being used, the endotoxin is introduced into two, or half, of the wells  52  containing the control water.  
      In an embodiment of the method, the system  320  for removing and positioning the covers  80  removes the cover  80  from one of the substrate vials  77  and the buffer vials  78  before obtaining an unused tip  48  so that the substrate vial  77  and the buffer vial are open at the same time. In this embodiment, the same tip  48  can be used to obtain and deliver the substrate and the buffer to each of the wells  52  containing the water to be tested and each of the wells  52  containing the control water. A different tip  48  from that used to deliver any of the other fluids receives and delivers the enzyme from vessel  75  to the wells  52  containing the water to be tested and the wells  52  containing the control water. The fluids from the vials  70  and the fluid to be tested, such as water, can be introduced into the wells  52  in any order. The order of delivering fluids to the wells  52  discussed above is not limiting on the method of the present invention.  
      Once the fluids from the vials  75 - 79  and the water to be tested have been positioned in their appropriate wells  52 , the detection system  600  including the detection assembly  610  and/or the fluorescent reader positioned on the head assembly  300  are passed over the fluid containing wells  52 . The detection system  600  determines either the optical density of the fluid containing wells  52  in the chromogenic or turbidimetric methods, or the relative fluorescent intensity of the fluid containing wells  52  in the fluorescent method. The detection system  600  then compares the results from its scan of the fluid containing wells and identifies if an endotoxin is present in the tested water.  
      All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference.  
      The above discussions do not limit the invention. Although the disclosure describes and illustrates preferred embodiments of the invention, it is to be understood that the invention is not limited to these particular embodiments. Many variations and modifications will now occur to those skilled in the art.