Patent Publication Number: US-10316346-B2

Title: Method and apparatus for dispersion of microbes in a liquid suspension

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. § 119 to Chinese Provisional Applications No. 201410503351.4, filed on Sep. 28, 2014, the entire contents of which are incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates generally to a method and apparatus for dispersing microbes in a liquid suspension sample. 
     BACKGROUND 
     During the processing of biological samples, microorganisms, such as  Mycobacterium tuberculosis , often cluster and therefore need to be dispersed before sample preparation. Existing conventional dispersion methods can involve mechanical grinding, agitation using a bacteria flask, or pipette blending, etc. Mechanical grinding is generally performed manually and the resulting dispersion is poor, non-uniform, and time consuming. The process of grinding and dilution is carried out in an open container, introducing undesirable opportunities for contact with the skin and/or respiratory systems and exposing the laboratory or hospital personnel to potential hidden danger of infection if the sample is an infectious or toxic microorganism. 
     After being dispersed, microbial suspensions samples can be analyzed. Some tests, such as turbidity tests, have strict requirements concerning the transparency of the vessel holding the sample. In traditional dispersion methods, sample grinding process generates liquid residue that gets dispersed on the vessel walls, and thus can interfere with the vessel&#39;s transparency. 
     Therefore, an improved way of dispersing microbial samples in liquid suspension is desired. 
     SUMMARY 
     According to an embodiment, an apparatus for dispersing microbes in a liquid suspension sample and measuring turbidity of the liquid suspension is disclosed. The apparatus comprises: a transparent container for holding a quantity of the liquid suspension in a sealed chamber, the container having a closed bottom end and an open top end, wherein the open top end is closable with a cap; an ultrasonic transducer having a transducer head portion that generates ultrasonic vibrations; a container-receiving guide block for holding the container in an operating configuration in which the container&#39;s bottom end and the transducer head are in direct contact, wherein the container-receiving guide block has a top end and a bottom end and a sidewall defining a longitudinally oriented bore extending from the top end to the bottom end of the container-receiving guide block for receiving the container, wherein the bottom end aligns the closed bottom end of the container with the transducer head, and wherein the container-receiving guide block provides a light source for measuring the turbidity of the liquid suspension; a lid assembly, movable between an open position and a closed position, that is configured and adapted for engaging the top end of the container and urge the container toward the transducer head when the container is being held by the container-receiving guide block and the lid assembly is in the closed position, so that the container bottom surface is in contact with the transducer head and the ultrasonic vibrations generated by the transducer head is transmitted through the container wall to the liquid suspension and disperse the microbes in the liquid suspension; a photo diode for measuring the suspension&#39;s turbidity; and an opening provided on the sidewall of the container-receiving guide block, the opening providing a viewing access to the container for the photo diode for measuring the liquid suspension&#39;s turbidity. 
     According to another embodiment, the apparatus for dispersing microbes in a liquid suspension sample and measuring turbidity of the liquid suspension comprises: an ultrasonic transducer having a transducer head portion that generates ultrasonic vibrations; a container-receiving guide block for holding a container of microbes in a liquid suspension in an operating configuration in which the container&#39;s bottom end and the transducer head come in direct contact, wherein the container-receiving guide block has a top end and a bottom end and a sidewall defining a longitudinally oriented bore extending from the top end to the bottom end of the container-receiving guide block for receiving the container, wherein the bottom end aligns the closed bottom end of the container with the transducer head, and wherein the container-receiving guide block provides a light source for measuring the turbidity of the liquid suspension; a lid assembly, movable between an open position and a closed position, that is configured and adapted for engaging the container and urge the container toward the transducer head when the container is being held by the container-receiving guide block and the lid assembly is in the closed position, so that the container bottom surface is in direct contact with the transducer head and the ultrasonic vibrations generated by the transducer head is transmitted through the container&#39;s wall to the liquid suspension and disperse the microbes in the liquid suspension; a photo diode for measuring the suspension&#39;s turbidity; and an opening provided on the sidewall of the container-receiving guide block, the opening providing a viewing access to the container for the photo diode for measuring the liquid suspension&#39;s turbidity. 
     According to another aspect of the present disclosure, a method for dispersing microbes in a liquid suspension and measuring the liquid&#39;s turbidity is also disclosed. The method comprising:
         (a) providing the liquid suspension sample in a container, wherein the container has a sidewall, closed bottom end, and a closeable top end;   (b) placing the container in direct contact with an ultrasonic transducer, wherein the ultrasonic energy generated by the transducer passes to the microbes via the sidewall of the container, thereby dispersing the microbes in the liquid suspension sample; and   (c) measuring the turbidity of the dispersed liquid suspension in the sample. In some embodiments, the step (c) is conducted while the ultrasonic transducer is dispersing the liquid suspension.       

     The method and apparatus for dispersing microbes in a liquid suspension described herein generally does not disperse liquid droplets to the container wall and thus avoids interference in measuring the turbidity from such surface liquid residue. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a cross-sectional view of the apparatus according to the present disclosure. 
         FIG. 2A  is a detailed cross-sectional view of the transducer head region of the apparatus including the bottom end of the container  11  showing the direct contact between the transducer head  9   a  and the bottom end surface of the container holding the liquid suspension of microbes. 
         FIG. 2B  is another detailed cross-sectional view of the transducer head  9   a  showing the recess  9   b  and its structure. 
         FIG. 3  is a perspective view of the transducer showing the physical arrangement of the container and the transducer in their operational configuration in the apparatus of the present disclosure. 
         FIG. 4  is an illustrative embodiment of the apparatus embodiment described herein (e.g. BACspreader/microbe dispersion couunter). 
         FIG. 5  is an illustrative embodiment of exemplary ultrasonic waves creating a ‘cavitation’ effect in the liquid, dispersing aggregates into their constituent particles. 
         FIGS. 6A, 6B, and 6C  is graphic representation of the light scattering-detection configuration that allows accurate measurements of turbidity obtained using a 90° light scattering constant source of illumination. Under proper light intensities, the turbidity of the solution and the 90° scattered light intensity are positively correlated.  FIG. 6A  represents 1/10,  FIG. 6B  represents 1/10 to 1/4, and  FIG. 6C  represents 1/4. Prior to detection, the instrument is calibrated using McFarland standard solutions. The turbidity of one exemplary McFarland standard unit is 3×108 CFU/mL. This example illustrates accurate synchronization between McFarland turbidity measurements and automatic dilution volume calculations. 
         FIGS. 7A and 7B  show the efficacy of an exemplary ultrasonic dispersion apparatus embodiment of the present invention (before and after). 10 μl inoculum of ( M. smegmatis ) and 2 ml saline are innoculated per tube (8 ml and 5 ml shown in  FIG. 7A ). Microbes settle at the bottom of the tube before ultrasound dispersion. After 30 s ultrasound dispersion, the microbes are uniformly dispersed (close up  FIG. 7B ). 
         FIGS. 8A and 8B  showed the comparison of dispersion using the exemplary BACspreader apparatus vs. traditional manual grinding.  Mycobacterium smegmatis  samples, dispersed either by using the BACspreader ( FIG. 8A ) or by traditional grinding ( FIG. 8B ), were diluted to the same McFarland turbidity value. The same volume of sample was then compared by microscopy. Results show that ultrasonic dispersion ( FIG. 8A ) gives more complete and uniform dispersion than manual grinding ( FIG. 8B ). 
         FIG. 9  shows a comparison of dispersion using the exemplary BACspreader apparatus and traditional manual grinding.  Mycobacterium smegmatis  samples were dispersed using BACspreader or by traditional grinding, diluted to the same McFarland turbidity value, then plated and incubated. Colony counts were compared. Results indicated that the BACspreader does not adversely affect the integrity and viability/activity of the microbes in the sample. 
         FIG. 10  shows the demonstration of the accuracy of turbidity measurements. After calibrating the instrument using McFarland standard solutions 0 and 6, the turbidity of six McFarland standard solutions (0, 0.5, 0.75, 1.5, 3.0, and 6.0) was determined. Turbidity measurements were recorded and the relationship between the recorded value and the true value was analyzed. Results indicate that the exemplary BACspreader apparatus measures turbidity accurately with an error rate of &lt;2%. 
         FIGS. 11A and 11B  are safety data demonstrating that there is no significant difference between the aerosol generated while dispersing  M. tuberculosis  and the levels of aerosol generated from a blank control.  FIG. 11A  shows ultrasound disperation using a press cap lid, and  FIG. 11B  shows ultrasound dispersion using a screw-top lid. Thus, these results show that using exemplary BACspreader apparatus greatly reduces user exposure the bacterial aerosols. 
         FIG. 12  shows the relationship between standing time and McFarland turbidity value. After dispersion, as standing time increases, bacteria settle and McFarland turbidity measurements gradually decrease. However, exemplary BACspreader apparatus of the present invention can give instant measurements of the turbidity of bacterial suspensions and can thus provide objective measurements that reflect true bacterial concentrations. 
     
    
    
     The features shown in the above referenced drawings are illustrated schematically and are not intended to be drawn to scale nor are they intended to be shown in precise positional relationship. Like reference numbers indicate like elements. 
     DETAILED DESCRIPTION 
     In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings which form a part hereof, and in which are shown by way of illustration of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. 
     To address the shortcomings of the conventional microbial dispersion method, the invention disclosed herein provides an improved microbial dispersion method which utilizes ultrasonic energy. The method provides a process that is highly efficient, biologically safe, and easy to use. Ultrasonic energy is transmitted through a transducer to the walls of the container holding the liquid suspension of microbes and serve to disperse microbes. The ultrasonic waves pass directly from the wall into the liquid suspension where they serve to disperse the microbes. The ultrasonic waves achieve the effect of dispersing microbe clumps by ultrasonic cavitation (see Figures). This dispersion process occurs in a sealed container and prevents the dispersion of fluid on the container walls that could affect subsequent operations. The ultrasonic waves are dispersed uniformly through the walls and the bottom of the vessel. 
     Compared to the conventional dispersion methods, the microbial dispersion methods disclosed herein are greatly improved. The ultrasonic waves are dispersed more uniformly to the walls and the bottom of the container and its surrounding area via the ultrasonic transducer. 
     In certain general aspects, the exemplary dispersion/detection apparatus of the present disclosure (e.g. BACspreader) can solves the unmet need of dispersing infectious microbes which tend to cluster, thus eliminating the need for traditional manual grinding of microbial colonies. The apparatus and methods disclosed herein are particularly suitable for the preparation of bacteria such as  M. tuberculosis  for drug sensitivity and other tests; thus providing an integrated solution for microbe dispersion and turbidity measurements. In certain embodiments, the exemplary apparatus can combine microbial dispersion functionality with real-time accurate McFarland turbidity measurements. In certain embodiments, the exemplary apparatus described herein can provide automatic dilution volume calculations. In certain embodiments, the exemplary apparatus described herein can perform dispersion and detection/assaying within a closed system, thus greatly reducing user&#39;s exposure to biohazards. 
     In certain aspects, the exemplary apparatus described herein can have instrument characteristics that includes:
         1) operating frequency of about 20˜40 kHz, power of about 5˜200 W;   2) ability to operate for long periods of time, thus, providing a platform to meet flexible diagnostic/laboratory requirements;   3) adaptably configured to work with replacement tubes: 75 mm&lt;L&lt;110 mm, 10 mm&lt;Φ&lt;13 mm;   4) ability to automatically perform closing and locking with a single key opening;   5) has emergency unlocking device; and   6) has working interface that is user-friendly and easy to operate.       

     In certain other aspects. The exemplary apparatus has the following working characteristics:
         1) well designed to integrate microbe dispersion and turbidity measurements;   2) can perform function within the same closed system, thus greatly reducing user exposure to biohazards; and   Ability to synchronizes accurate McFarland turbidity measurements and automatic dilution volume calculations.       

     Exemplary microorganisms that can be dispersed using the method and apparatus disclosed herein include  Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus albus  and  Aspergillus niger . In certain embodiments, any infectious, bacterial/microbial samples with tendency for aggregation can be dispersed using the methods and apparatus of the present disclosure. 
     When the microbe to be dispersed is  Mycobacterium tuberculosis , the suitable frequency of the ultrasonic transducer ranges from about 20 KHz to about 80 KHz, the power from about 5 W to about 200 W, and the duration for applying the ultrasonic energy can range from about 15 sec˜120 sec or more.  Mycobacterium tuberculosis  can be effectively dispersed using these conditions, and dispersion has no effect on bacterial viability and/or activity. 
     The present invention also describes a microbial dispersion instrument. The ultrasound dispersion instrument includes an ultrasonic wave generator and an ultrasonic wave transducer, and an electrical connection between the ultrasonic generator and ultrasonic transducer. The described dispersion device can also include a container which serves in microbe dispersion. The bottom end of the container is in close contact with the face of the ultrasonic transducer. The container can be of different sizes, is easily replaced, and the size of the area of close contact between the container and the transducer can be adjusted according to the desired dispersion effect. 
     The bottom of the vessel is in close contact with the ultrasonic transducer. The hollow in the face of the ultrasonic transducer is compatible with the bottom of the vessel. The close contact between the bottom of the container and the face of the ultrasonic wave transducer improves the dispersion of ultrasonic waves in the area surrounding the bottom of the container. 
     Referring to  FIG. 1 , an apparatus  100  for dispersing microbes in a liquid suspension and measuring the turbidity of the liquid suspension is disclosed. The apparatus  100  comprises a transparent vessel/container  11  for holding a quantity of the liquid suspension  50  in a sealed chamber formed by the walls of the container  11 . The container  11  has a closed bottom end  11   a  and an open top end  11   b . The open top end is closable with a cap  11   c . The open end  11   b  and the cap  11   c  are preferably provided with screw threads so that the the cap  11   c  can be closed tightly to prevent the liquid suspension of microbes from spilling out during the operation of the apparatus. The apparatus is equipped with an ultrasonic transducer  9  that generates ultrasonic vibrations. The transducer  9  has a transducer head portion  9   a  that is configured and adapted for directly contacting and engaging the bottom end  11   a  of the container  11 . 
     Referring to  FIGS. 2A and 2B , in a preferred embodiment, the transducer head  9   a  is provided with a recess (i.e., a blind hole)  9   b  having a chamfered edge  9   c  around the annular edge of the recess. The recess  9   b  allows the transducer head  9   a  to receive the bottom end  11   a  of the container which generally has a curved contour as shown. The chamfered edge  9   c  of the recess  9   b  also allows more intimate direct contact between the transducer head  9   a  and the container&#39;s bottom end  11   a  for efficient transfer of ultrasonic vibration from the transducer head to the container  11 . The chamfered edge  9   c  has a width of 0.75 mm and a chamfer angle of 45°. 
     The ultrasonic transducer  9  in the dispersion instrument described operates at a frequency in the range of about 20 KHz to about 80 KHz, a power range of about 5 W to about 200 W. Microbial dispersion under these conditions is effective, and does not influence the activity of the sample. 
     By changing the mode of action of ultrasonic waves, the ultrasonic waves pass through the transducer and are dispersed evenly via the walls of the container to disperse the microbe, with no need for a liquid medium between the transducer and the container, thus realizing the purpose of dispersion. The instrument is efficient and convenient, and is easy to operate. 
     The apparatus  100  also includes a container-receiving guide block  8  for holding the container  11  in an operating configuration. In the operating configuration, the container is in an upright orientation as shown and the container&#39;s bottom end  11   a  and the transducer head  9   a  are in direct contact. The container-receiving guide block  8  has a top end  8   a  and a bottom end  8   b  and a sidewall  8   c  defining a longitudinally oriented bore  8   f  extending from the top end  8   a  to the bottom end  8   b  of the container-receiving guide block for receiving the container  11 . The bottom end  8   b  aligns the closed bottom end  11   a  of the container with the transducer head  9   a  to enable dispersion of the microbes in the liquid suspension  50  by ultrasonic energy. 
     The apparatus  100  also includes a lid assembly  2 , movable between an open position and a closed position. The position of the lid assembly  2  shown in  FIG. 1  is the closed position and the open position is represented by the dotted outline  2   a . The arrow A shows the hinged open and closing motion of the lid assembly  2 . The lid assembly  2  is configured and adapted for engaging the top end  11   b  of the container and urge the container toward the transducer head  9   a  when the container  11  is inserted in the container-receiving guide block  8  and the lid assembly is in the closed position as shown. The urging by the lid assembly ensures that the container&#39;s bottom surface is in direct contact with the transducer head  9   a  and the ultrasonic vibrations generated by the transducer head is transmitted through the container wall to the liquid suspension  50  and disperse the microbes in the liquid suspension as desired. 
     Turbidity of a liquid suspension is measured by considering the propensity of particles suspended in the liquid to scatter a light beam shining on them. To measure turbidity this way requires a light source and a detector set up to the side of the light beam, i.e. 90° to the light beam. The more particles there are in the liquid, more of the source light will be scattered by the particles and reach the detector positioned 90° to the light beam. These measurements can be quantified to certain standardized units well known in the art. 
     In the apparatus  100  of the present disclosure, the container-receiving guide block  8  is provided with a light source  7  for measuring the turbidity of the liquid suspension  50  in the container  11 . The light source  7  is provided near the top end  8   a  of the container-receiving guide block so that the light shines downward into the liquid suspension  50 . The light source  7  is provided with a hole  7   a , which is aligned with the longitudinally oriented bore  8   f  of the container-receiving guide block  8 , for receiving the container  11  therethrough. A photo diode  70  is provided for detecting the light scattered by the microbes in the liquid suspension  50  so that the suspension&#39;s turbidity can be measured. An opening or a window  30  is provided on the sidewall  8   c  of the container-receiving guide block  8  to accommodate the photo diode  70  so that the photo diode  70  can detect the scattered light coming from the liquid suspension  50 . 
     In one embodiment, the apparatus can be configured with an appropriate controller or programmed with an appropriate controlling processor so that the dispersion of the microbes in the liquid suspension by the ultrasonic energy and the turbidity measurement of thus properly dispersed liquid suspension can be conducted in serial sequence or simultaneously. The two modes may be selected depending on the particular microbes that are being sampled so that more optimal processing sequence can be chosen. 
     Referring to  FIGS. 2A and 2B , in some embodiments, the container  11  has a cylindrical shape and the recess  9   b  provided in the transducer head  9   a  has a circular opening having a chamfered edge  9   c . The recess  9   b  allows the transducer head  9   a  to accommodate the various shapes that may be presented by the bottom end  11   a  of the container  11 . The chamfered edge  9   c  makes the contact with the container along the annular edge of the recess  9   b  for providing a close contact between the container bottom surface and the transducer head for efficient transfer of ultrasonic vibration from the transducer head to the container. In the illustrated example, the bottom end  11   a  has a spherical contour. 
     In some embodiments, the lid assembly  2  is provided with a spring-loaded mechanism for engaging the top end  11   b  of the container and urge the container  11  toward the transducer head  9   a . The spring-loaded mechanism comprises a receptacle  4  for receiving the top end  11   b  of the container including the screw cap  11   c . The receptacle  4  is nested inside a hub  5 . As can be seen in  FIG. 1 , the hub  5  is provided with an opening through which the receptacle  4  fits in slidable (in longitudinal direction) manner. Thus, the receptacle  4  is situated within the hub  5  and can slide up and down. The structures of the receptacle  4  and the hub  5  are configured to form a chamber  5   a  between the sidewalls of the receptacle  4  and the hub  5  and a coil spring  6  is captured inside the chamber  5   a . The configuration is such that the coil spring  6  is under compression and urges against the receptacle  4  and the hub  5 . The coil spring  6  pushes the hub  5  upward and it pushes the receptacle  4  downward in the orientation of  FIG. 1 . The hub  5  is affixed to the lid assembly  2  so that when the lid assembly  2  is in closed position and the latch  1  locks the lid assembly  2  into a stationary position, the hub  5  is not movable. However, the receptacle  4  will be movable up and down against the resistance of the coil spring  6 . Thus, when the lid assembly  2  is in the closed position as shown and the container  11  is in place, the spring-loaded receptacle  4  will provide a downward force on the top end  11   b  of the container and urge the container toward the transducer head  9   a.    
     According to another aspect of the present disclosure, the following apparatus  100  for dispersing microbes in a liquid suspension and measuring turbidity of the liquid suspension described above can be manufactured and provided to the end users without the containers for the liquid suspension of microbes. The containers can be supplied to the end users separate from the apparatus  100 . The containers can be formed in a variety of sizes and shapes as long as the closed bottom end of the container is appropriately shaped to make the proper direct contact with the recess  9   b  of the transducer head  9   a.    
     The ultrasonic transducer  9  is driven by an appropriate ultrasonic signal generator which provides high-frequency driving current to the ultrasonic transducer  9 . The high-frequency driving current drives the ultrasonic transducer to generate high-frequency, ultrasonic vibrations which is transmitted quickly through the walls of the container  11  and to the liquid suspension inside. The ultrasonic energy creates ultrasonic waves in the liquid suspension which cause cavitation in the liquid, dispersing any clusters of microbes into their constituent microbial particles. 
     The electronics and control circuits that are used to drive and control the active components of the apparatus  100 , such as the ultrasonic transducer  9 , the light source  7 , and the photo diode  70  are well known in the art. Therefore one of ordinary skill in the art would know how to power these components to make them operational. 
     When the microorganism is  Mycobacterium tuberculosis , the optimal frequency of the ultrasonic transducer is about 20 KHz to about 80 KHz, the power is about 5 W to about 200 W, and the reaction time is about 15 s˜120 s. The present method does not destroy the viability and/or integrity of the microbes. 
     The shape of the ultrasound transducer used has no special restrictions, and can be selected to match the shape of the containers used. The frequency and power of the transducer can also be varied depending on the type and weight of the sample. For example, when the sample is  Mycobacterium tuberculosis , the frequency of the ultrasonic transducer is about 20 KHz-about 80 KHz, and the preferred power is about 5 W-200 W. 
     In addition, there is a fixture for fixing the vessel in the ultrasound dispersion instrument to ensure that the vessel is in close contact with the face of the ultrasound transducer. 
     The various embodiments disclosed herein represent examples that fall within the scope of the invention but does not necessarily define the full scope of the invention. The full scope of the invention is defined by the claims provided herein.