Abstract:
A test device provides an enclosed gas entrapment system for the safe determination of both the presence and rate of occurrence of fermentation gases where the targeted microorganisms were both present and active. The gas entrapment device has a density greater than the liquid culture medium within the chamber and therefore settles to the base of the chamber and includes slots in the vertical walls of the device which allow the passage of fermentative microorganisms into and out of the device during the period of culture and examination. When there has been the generation of gases entrapped within the device, the density of the device becomes compromised forcing the device to elevate in the liquid fermenting medium to form an indication that gases have been produced in the sample by the microorganisms and that organisms are present.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates generally to a method and apparatus for performing microbiological analysis, and is more particularly concerned with an appropriately sequenced cultural method and apparatus for the screening of a sample to determine the quantifiable presence of fermentative activity generating gases in the presence of the organism of choice.  
         DISCUSSION OF THE PRIOR ART  
         [0002]    Recent advances in microbiology have engendered many techniques useful to the engineer, microbiologist and various health specialists. A large number of these tests are conducted daily both in the field and in the laboratory. With the increasing awareness of the greater diversity in sources of microbially driven compromises of systems and causes of infections, there is an accelerating demand for the effective screening of substances, notably water and pathologic material to ensure accurate determination of cause for a given economically significant compromise or clinically important infection. However, due to the time required for these tests, and the cost of such tests, complete testing is not economically feasible so that the tests may only be run on substances under suspicion because of inferential evidence.  
           [0003]    Those skilled in the art will realize that an incipient problem may not manifest itself in the natural state for one or more of a variety of reasons; the contamination may become obvious after some inferences in the natural system. By way of example, bacteria may be present in small quantities, but may not be readily detectable through some understood biochemical or cultural function through the inadequate ability of the conventional cultural devices to display some form or other of affirmative occurrence in a manner that appropriately allows a quantitative determination of the event.  
           [0004]    The prior art includes cultural systems primarily utilizing liquid media within which a fermentative function is detected by the production of gaseous products. These gaseous products of fermentation were determined to be significant to the determination of the nature of the dissimilatory processes possessed by some of the important groups of microorganisms that has since become embodied into the standard cultural methodologies for the determination of the nature of fermentative microorganisms. Today the diagnosis of the enteric bacteria in samples is achieved with the aid of determinative procedures for the detection of gaseous end products of fermentation processes within specific forms of culturing devices. A variety of entrapment devices were developed to detect these gases and it was the Durham&#39;s tube that has become the most widely accepted. The concept was generated in the early years of the twentieth century and involved the simple placement of an inverted glass test tube in the liquid cultural medium that also filled this inverted tube. The presence of gas could be visually determined by a growing pocket of gas entrapped in the top region of the tube. The prior art leans on the simple nature of the Durham&#39;s tube to detect fermentation gases and act as the primary diagnostic system for the qualitative detection of fermenting microorganisms of concern. This art has been developed in particular to detect the coliform bacteria in samples since these bacteria are considered to be an acceptable indicator of health risks associated with the sample under test.  
           [0005]    Traditionally the method for the qualitative confirmation of selected fermentative microorganisms was by the application of various organic substrates with the confirmation being achieved by the appropriate demonstration of gas production in specific media. Classical science commonly directed the use of an inverted glass tube in the medium to entrap some of the gaseous products within a gas filled pocket that could be viewed as confirmatory of the fermentative activities. This device became known as the Durham&#39;s tube and has been adapted to quantitative studies through the determination of which of various dilutions generated observable gas production (therein considered positive indicators). The distribution of these said indicators could now be statistically interpreted to project a most probable population estimate for the number of targeted fermentative microorganisms within the original sample.  
           [0006]    Attempts have been made to improve the Durham&#39;s tube including one method that involved replacing the glass used in the traditional tube with a semi-permeable material that allowed the freer movement of microbes and chemicals between the inverted tube and the surrounding medium. This modification had the advantages of the gas entrapment causing a decline in density of the device leading to an elevation resulting from increased buoyancy generated by these gases. The time to such an elevation was further found to link in a direct manner to the size of the active population involved in generating said gases. This invention represents a new method of control for the operation of the U.S. Pat. No. 5,187,072 described above but involves a unique and separate method of control.  
           [0007]    Information disclosing prior art can be found in the following articles:  
           [0008]    PASTEUR, L (1857) Mémoire sur la fermentation appelée lactique. Comptes rendus de l&#39;Académie des sciences, Volume 45, 913-916.  
           [0009]    KLUYVER, A. J (1924) Eenheid en verscheidenheid in do strofwisseling der microbien&#39;. Chemisch Weekblad, Volume 21, 266  
           [0010]    CULLIMORE, D. R (2000) Practical Atlas for Bacterial Identification, Lewis Publishers, Boca Raton Fla. pp 209.  
           [0011]    TORTORA, G. J., FUNKE, B. R. and C. L. CASE (2001) Microbiology, an introduction, 7 th . Edition, Benjamin Cummings, Addison Wesley Longman Inc. New York.  
         SUMMARY OF THE INVENTION  
         [0012]    The present invention provides a method whereby the detection of the fermentation gases resulting from microbial activities within a culture vessel can be detected with greater qualitative convenience than the present Durham&#39;s tube method and also, where the time lag to the observation of confirmed gas entrapment, to determine quantitatively the size of the active targeted microbial population within the sample. Determination of the time lag may be based on either the presence of the gases within the present device changing the characteristics of the device in a definable manner or causing the relocation of the device as the density of the device declines with the admission of gases to the internal body of the device. Detection may be visual, spectrophotometric or involve some form of electromagnetic anomaly to the field in, and around, the device.  
           [0013]    The present device provides the form of an inverted tube similar in form to the Durham&#39;s tube to allow the entrapment of the fermentation gases. This device differs from the said tube in that it is constructed of a density adjusted material that causes the device to sink in liquid culture media when the device is evacuated of air. A second difference with the said tube is that there are vertical openings (slots) in the lower parts of the wall of the inverted device that allows the free access and release of microorganism and chemicals between the inside void of the device and the surrounding volume of culture medium containing the sample under examination. These slots act in the manner of allowing the two environments within, and without, the device to more freely exchange and equilibrate to the betterment of the fermentative processes occurring within the culture vessel containing the present device.  
           [0014]    The device described and claimed as a part of this invention above functions as a result of a set of circumstances that result in the elevation of the device as a result of the increasing buoyancy of the device due to the gases accumulating within the head space of the device. Elevation would, however, remain dependent upon the density of the device and the density of the surrounding medium and would occur at that point when the accumulating gases reduce the density of the device to less than that of the surrounding medium. Precision therefore becomes in part a product of the differences between the density of the device and density of the surrounding medium wherein the device should initially have a greater density than the medium at the start of the test. A critical set of interactive factors are the density of the liquid medium and the thimble, the population of microorganisms that are active and able to ferment the selective medium at the incubation temperature with the production of accumulative gases, and the relationship of the total volume (TV) of the medium to the volume of the medium entrapped within the voids (VV) in device confined at the top and by the sides. It may be surmised, by those familiar with the art that the amount of gas that may be entrapped by the device will increase where the VV approaches that of the TV. Such an event could cause a faster elevation of the device due to the greater entrapment of gases. This in turn has the potential to affect the time lag length prior to elevation and influence the precision with which the time lag could be quantitatively converted to a total enteric population (TEP). Two key elements to improve the precision would be to set a suitable range for the TV:VV ratio that would result in a common ability to entrap the fermented gases, and set a suitable tolerance that the density of the device will be greater than the liquid medium within which the device is sunk. For the purposes of an example the TV:VV ratio should be set at between 1:0.2±0.05 and the density of the device should be within the range of 0.05±0.02 g/mL above the ambient density of the liquid medium at room temperature.  
           [0015]    Given that sample can have differing densities then the device has the option to be constructed of greater or less dense materials to meet the needs for testing the specific sample such as fresh-water and sea-water and allow a similar time lag to the elevation of the present invention having compensated for density. To maintain position within the culture vessel, the present invention may have a series of extensions that may take the form of vertical vanes to assure that the device moves in a specific manner within the culture vessel. To allow spectrophotometric or electromagnetic monitoring of the position of the present invention as it moves through buoyancy created by the entrapment of gases within said device, the material for the construction of the device may be modified to significantly detect changes in the characteristics of light or electromagnetic fields that would be associated with the movement of the device resulting from the entrapment of fermentation gases. It should also be noted that the form of the culture vessel within which the determination is being undertaken may be modified to improve the determination of this event. By the exploitation of the time lag information generated by the entrapment of gases it is possible through to comparative studies to determine the population of the microorganisms that are of interest and are able to generate fermentative gases under the cultural conditions applied. 
       
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       [0016]    These and other features and advantages of the present invention will become apparent from consideration of the following specification when taken in conjunction with the accompanying drawings in which:  
         [0017]    [0017]FIG. 1 is a vertical diametric cross-section of a culture chamber having a test device therein made in accordance with the present invention, and including the final volume of medium presented as a liquid broth.  
         [0018]    [0018]FIG. 2 is a modified form of the device shown in FIG. 1 showing the effect of fermented gases have entered and caused, by way of an example, the vertical relocation of the present invention.  
         [0019]    [0019]FIG. 3 is a vertical side-view of the device as shown in FIG. 1 but illustrating two forms in which positioning vanes can be placed on the present invention to allow more precise positioning within the culture chamber and to act as ballasting where greater density of the material composing the present invention are required to assure the detection of a determinable quantity of gas.  
         [0020]    [0020]FIG. 4 is a modified version of FIG. 2 in which the typical light pathways are shown within an apparatus to allow the detection of the movement of the present invention as a result of shifts in buoyancy due to gas entrapment.  
         [0021]    [0021]FIG. 5 is similar to FIG. 4 except that the detection of the movement of the present invention as a result of shifts in buoyancy due to gas entrapment is recorded by shifts in the electromagnetic fields induced in, and around, the present invention.  
         [0022]    [0022]FIG. 6 is a composite of, and similar to FIG. 2 in which floatable device has been incorporated into the present invention positioned within the device to improve detection by any of the herein stated methodologies. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    Referring now more particularly to the drawings, and to those embodiments of the invention here presented by way of illustration, the embodiments of the invention shown in FIG. 1 includes the test device  10  within a culture chamber  14 . The physical arrangement is such that the test device  10  is set vertically filled with the culture medium  15  is sunk by virtue of the higher inherent density of the device  10  than the medium  15 . A critical feature of the test device  10  includes the admission of one or more slots  13  in the lower part of the vertical walls of the device and a closed top  11  that retains any gases generated within the device during the test period. To assure that microorganisms from the culture medium  15  can enter the device to cause gas formation, the under side of the device  12  is open as well as the slots  13  on the lower wall of the device. The device  10  can be saturated with the medium  15  by either steam sterilizing the culture chamber  14  when capped in which case the air entrained within the device  10  is replaced by steam that condenses to fill device  10 ; or the culture chamber can be inverted which leads to the entrained air bubbling out of device  10  and being replaced with the culture medium  15 . It should be noted that the slot  13  has a width that is large enough to allow microorganisms to enter and leave the test device  10  at the start, and during the early stages, of the test but does, however, become plugged with microbial biomass where an aggressive occlusive population of bacteria are present and active in the sample. Under such circumstances the whole length of the test device can become filled with fermentation gases adding to the buoyancy of the test device  10 .  
         [0024]    Where the culture medium  15  includes a sample containing microorganisms that can generate gases by fermentation then the manner in which these gases are detected is shown in FIG. 2. Here, the test device  10  becomes filled from the top down to create a gas-filled zone  16  where the culture medium is being displaced downwards ( 17 ) until the surplus gases begin to escape  18  through the slots  13 . This fermentative activity is commonly accompanied with clouding as a result of the growth in the biomass and the culture medium now becomes clouded  19  and the gas, where seated in a device that is transparent, such as a glass Durham&#39;s tube, becomes difficult to determine visually.  
         [0025]    In cases where the culture chamber  14  has a much larger diameter  20  than the test device  10  then there is a risk that the test device  10  may lean out of vertical sufficiently to become jammed. FIG. 3 illustrates a methodology to prevent this happening to the test device  10  through application of a single full length vane  21  connected to the body of the test device  10  by a suitable means  22  that will retain the position of the vane with respect to the said vane. Advantages in controlling the density or positioning of the test device  10  may be better achieved by the use of several partial length vanes  23 . These vanes may be arranged vertically along the sides of the test device in at least three positions vertically to assure that the said test device is centrally positioned. In either event, these central extensions of the test device  10  in the form of vanes allow the said device to occupy a suitable position within the culture chamber  14  such as at the central axis. In order to reduce the risk of the test device  10  becoming biologically attached to the floor of the culture chamber  14  the invention may also include extensions downwards  24  on the lower side of the vanes that are likely to come into contact with the floor of the said culture chamber.  
         [0026]    The nature of the clouding event  19  as a result of biological activity renders it difficult on some occasions to observe the presence of gases in the traditional sunken glass Durham&#39;s tube and even of the test device  10  itself unless it is constructed with either a distinct and contrasting color to the that of the clouded medium  19 . As an example black may provide a suitable distinguishing color for visual determination of the test device  10  under such circumstances.  
         [0027]    [0027]FIG. 4 illustrates the manner in which the relocation of the test device  10  as a result of gas entrapment and elevation  16  can be examined using two lateral light pathways  25  one of which is set in the upper region where the elevating device will intercede and block the light as the said test device rises while the other lateral light pathway is lower down and is blocked by the test device  10  at the start of the testing period but is not blocked when the said device has elevated as a result of gas entrapment. Both light pathways are served by light emitters  26  and the blockage of the light pathways are each determined by separate detectors  27  that record the light intensity being received and allows intelligent systems to determine the time lag to any recognized movement in the test device  10  that would indicate gas entrapment and an increase in the buoyancy for the test device. Under normal operating conditions the most likely characteristics for the light being emitted (from the emitters  26 ) would be in the red or infra red wave bands. In the event that it is not practical to use these forms of light then FIG. 5 illustrates an alternative sensing system in which the test device  10  is constructed using electrically responsive materials such as iron filings. When the test device elevates  16  then there is a change in the electromagnetic fields  29  being emitted by a single or multiple source of electromagnetic force  28  and detected on the far side of the culture vessel  14  by two sensors (upper  30  and lower  31 ) that respond to the changes in the field as a result of the shifting shadow where the test device is situated at that time. The time lag to this event as defined by those experienced in the art would be used to quantify the microbial activity.  
         [0028]    [0028]FIG. 6 illustrates an alternative system for the detection of entrained gas  16  through the admission of a small geometrically suitable low density device  32  into the test device  10 . This device  32  would float at the medium: gas interface  17  and would enhance the determination of the volume of the gas entrapped. Such a floating device  32  would allow a more precise determination of the gas production using the light pathway  25  or electromagnetic sensing  29 . In order to avoid the risk of the floating device  32  being lost from the test device  10  by the actions of agitation or gravity, the lower rim of said test device would be extended inwards  33  to retain the floatable device  32  within the test device  10 .  
         [0029]    It will be recognized by those familiar with the art that the present invention may be utilized in a number of manners, each of which will utilize some of the unique features which form a part thereof. Below are some examples of alternative protocols that may be accomplished using the culture chamber.  
         [0030]    In the first example of an alternative protocol, the test format is as shown in FIG. 1 except that culture medium  15  only that it is triple concentrated and fills only one third of the defined volume to be eventually be occupied by the liquid medium in combination with the sample in the prepared test format. Another divergence is that the test device  10  would operate within a larger culture chamber  14  that would require the use the extended vanes shown in FIG. 3 as full length vertical vanes  21  extended downwards  24  to keep the test device  10  off the floor of the culture chamber  14 . In this example the culture medium  15  is 50 ml of triple strength brilliant green bile growth that is selective for the fermentative activities of the species  Escherichia coli , the common indicator organism for health risks in water. This medium is diluted by the addition of 100 ml of the water sample to be tested and the combination of medium and water sample bring the liquid volume contained within the culture chamber  14  in FIG. 1 to an acceptable level above the sunken test device  10  so that the device is fully submerged. At this time when the liquid sample is applied to the said culture chamber, the test device  10  may float up to the surface due to reduced buoyancy resulting from entrained air. This may be corrected by slowly inverting the test device  10  within the sealed culture chamber  14  cap to prevent leakage from the culture chamber during inversion. Such a maneuver of inversion would cause the releases of entrained air from the test device  10  so that the density of said device now exceeds that of the surrounding medium  15 . Once the test device  10  has sunk within the liquid medium then the culture chamber  14  is incubated at blood heat for 24 hours. In the event of the fermentative activities of  Escherichia coli , the result would follow the form shown in FIG. 2 where the test device  10  has elevated due to entrapment of fermentation gases within the test device  10 . Where it is necessary to determine the numbers of  Escherichia coli  in the water sample then the time lag preceding said elevation can be quantified using either the shift in the interruption of the light pathways  25  illustrated in FIG. 4; or the movement of the shadow imposed by the test device  10  on the electromagnetic forces generated  28  illustrated in FIG. 5. Under normal conditions when testing said water samples the time lag would range from 14,400 seconds for an excessively large population of  Escherichia coli  related to the presence of billions of cells in the 100 ml water sample down to a single cell if the time lag was 86,400 seconds. By comparative studies against the standard recognized methodologies, a robust and valid methodology can be determined that this relationship exists between the time lag to test device  10  through its elevation in FIG. 2 as a result of the entrapment of fermentation gases  16 . This example therefore provides a novel and scientifically defensible method for the determination of  Escherichia coli  and, through modifying the culture medium  15 , other related enteric bacteria that could under, some circumstances pose significant health risks and can generate fermentation gases during incubation, could be detected.  
         [0031]    A second example relates to the need to determine the ability of a particular strain of a fermentative bacterium to ferment a range of carbon substrates as a necessary part of the identification of that bacterial strain. To undertake this survey there has to be a multiple of culture chambers of the type shown in FIG. 1 equivalent to the numbers of the said substrates that are to be investigated. Each individual culture chamber  14  contains a test device  10  and the culture medium  15  that is different for each of the substrates to be tested but has the volume illustrated in the FIG. 1. Each medium has a unique but standard formulation for the determination of the range of substrates that would be examined for the generation of detectable fermentation gases. An example of the such a set of fermentation results typical for a strain of  Escherichia coli  is (bracket shows G for gas detected by test device  10  elevating as a result of fermentation gas accumulation  16 ); and ND indicates that gas fermentation was not detected): Glucose, G; Lactose, G; Sorbitol, G; Adonitol, ND; and Inositol, ND. After the incubation period it is possible to view the position of the test devices  10  in each of the culture chambers  14  and determine the fermentative ability of the bacterial strain being tested as an aid to the identification of that strain of bacterium.  
         [0032]    As can be seen from the foregoing description, the present invention involves a number of stages in the conductance of the test procedure which generates advantages both from the perspective of user convenience and also from the perspective of improving the resolution of the impact of the selective culture medium  15  on the targeted organism. For the user, the procedure using the devices described above, these inventions can allow the examination of the given sample through incubation to generate clear and distinct visual evidence of the activities of the organism being determined by the physical relocation of the test device  10  and the time lag to that event after the start of the incubation of the inoculated culture chamber allows the quantification of the numbers of the targeted organism in the sample under examination. Where the user desires to determine the range of substrates that may be fermented with the production of entrapped gases recorded by the elevation of the test device  10  with a culture chamber  14  where at least one said cultural apparatus is devoted to each of the substrates for examination using a suitable culture medium  15  then there is the potential to use the fermentation of gases to aid in the determination of the organism.  
         [0033]    By way of a quantitative example of the use of the culture chamber  14  containing the test device  10  in a manner appropriate to the descriptions of this invention, the application of the technique will be described for the determination of the presence or absence of total enteric bacteria in a given water sample. For this example the culture vessel would have a sufficient capacity to hold 100 ml of the water sample for examination and 50 ml of triple strength Lauryl Tryptose broth to determine the fermentative activities of any total coliform bacteria present within the said sample. To assure evidence of entrapped fermentation gases within the test device  10 , the sealed culture chamber  14  needs to be inverted after the sample has been added to the medium to evacuate any headspace air from the device so that the said device will sink. For effective determinations of any fermentative activities generating gases, the test device  10  should have a height less than two thirds of the height of the liquids added to the culture vessel  14 . Incubation would be at blood heat (37±1° C.) and a positive detection of the total coliform bacteria would be the elevation of the test device  10  within the liquid medium  15  to create an image similar to that shown in FIG. 2 the time of which can now be reported by direct visual observation or by the use of spectrophotometric techniques (FIG. 4) or electromagnetic inferences (FIG. 5). By a calculation of the time lag as being that difference between the start of the incubation period of the investigation and the time to the first observation of the elevation of the test device  10 . An inverse relationship exists between the length of the time lag commonly measured in seconds and the population of total coliform bacteria measured in total coliform bacterial cells per 100 ml. One such equation, by nature of an example that would link the time lag (TL) to the total enteric bacterial population (TEP), is: 
         TEP= A*B   
         [0034]    Where A is (Power  10 (75,600)−TL) and B is (0.0001389+(0.00000002273*(TL−32,400). It will of course be understood by those familiar with the art that the particular embodiment of the invention presented are by way of illustration only, and are meant to be in no way restrictive; therefore, numerous changes and modifications may be made, and the full use of equivalents reported to, without departing from the spirit of the scope of the invention as outlined in the appended claims.