Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Definitions

• Figures 3A-3B are graphs showing the correlation between CFU/ml count obtained by conventional method (Standard CFU/ml) and a CFU equivalent obtained in accordance with an embodiment of the invention (Fig. 3A) as well as the correlation between each value (CFU equivalent and conventional CFU/ml) per tested sample (Fig. 3B). .
• luminescent emitting moieties that can be used in accordance with the invention comprise, without being limited thereto, bioluminescents including luciferin based agents (e.g. 6-O-beta-galactopyranosyl luciferin), fluorescents including members of the Alexa Fluor family (Invitrogen), PromoFluor Dyes (PromoKine), HiLyte Fluors (AnaSpec), DyLight Fluors (Pierce, Thermo Fisher Scientific), and the ATTO Dye series (ATTO-TEC and Sigma-Aldrich).
• bioluminescents including luciferin based agents (e.g. 6-O-beta-galactopyranosyl luciferin), fluorescents including members of the Alexa Fluor family (Invitrogen), PromoFluor Dyes (PromoKine), HiLyte Fluors (AnaSpec), DyLight Fluors (Pierce, Thermo Fisher Scientific), and the ATTO
• the invention also allows for obtaining a cell count of a specific type of culturable microbial cell in a sample even having a mixture of microorganisms. This may be achieved using various specific signal emitting agents, either alone or in combination with a non-specific signal emitting agent.
• specific or specificity in the context of the term “specific signal emitting agent” is used to denote that the agent has affinity and/or selective binding to the membrane or to an intracellular component of a specific cell type (a cell, the detection of which in the sample is desired).
• a specific signal emitting agent may comprise a targeting entity i.e. a ligand having binding specificity to an extracellular component of the cell.
• the targeted signal emitting agent is such that it can internalize into the culturable cell, thereby allowing detection of only those that have internalized the signal emitting agent.
• the agent can constantly emit a signal or the signal can be generated as a result of a stimuli, such as an enzymatic process taking place within the intracellular compartment, the enzymatic reaction manipulating the agent to emit the signal or the enzymatic degradation product emits a signal (as discussed above), a radiation stimuli, etc.
• a stimuli such as an enzymatic process taking place within the intracellular compartment
• the enzymatic reaction manipulating the agent to emit the signal or the enzymatic degradation product emits a signal (as discussed above), a radiation stimuli, etc.
• the signal emitting agent associates with the cells' membrane.
• Cells having associated thereto a signal emitting agent are detected as signal emitting objects.
• the signal emitting agent may also associate with cell components or with other artifacts in the sample that is not of intact culturable cells and may provide a false signal.
• the signal may be from aggregated signal emitting agent (i.e. that was not sufficiently dissolved in the sample).
• the method provides a tool for detecting and selecting only those signal emitting objects that originate from cells having associated thereto and internalized therein the agent.
• the term "signal emitting objecf denotes any optical spot from the tested sample that emits a signal.
• the signal emitting object does not necessary have the size of a cell, and in fact, may be larger, due to a halo around the cell formed from the signal emitted from the cell, especially when the imaged cell is out of focus ("circle of confusion"). This is caused by a cone of light rays from a lens not coming to a perfect focus when imaging a signal emitting cell
• T2 This time period, represents the time window during which the signal emitted from the signal emitting object is retained at the plateau, essentially in a steady state.
• the selection criteria may require that the signal emitting objects have a predetermined morphology.
• cell morphology is generally characteristic of a given bacterial species and such cells come in a wide variety of morphologies. These may include essentially rounded (e.g. Coci), essentially elongated (e.g. Bacilli), rod-like morphologies etc.
• the signal emitting object would typically have a shape corresponding to (resembling) that of the cell from which it is emitted. For example, an elongated signal emitting object would typically originate from an elongated microbial cell. The detection of the object's shape allows not only the discarding of non-microbial signal emitting objects (i.e. the artifacts) but may also facilitate in identifying the type of cell from which the signal is emitted.
• At least two parameters preferably the intensity parameter and the size parameter need to be fulfilled for identifying signal emitting objects.
• these objects are selected and a quantitative value is deduced thereform.
• their mean root square value of intensity is summarized; this summarized intensity being preferably normalized with a predetermined normalizing factor (e.g. by using a predetermined equation (see below Materials And Methods)), to obtain a quantitative value indicative of the number of culturable microbial cells in the sample (i.e. a CFU equivalent).
• the normalizing factor is specific to a cell type or for a group of cells, per the type of tested sample (i.e.
• a normaltization factor (equation) of 0.025x + 0.3375 was determined for a test sample comprising a mixture of E. CoIi and Ps.aeruginosa.
• the invention also provides a kit for determining a quantitative value equivalent to the number of culturable microbial cells in a tested sample, the kit comprising:
• the kit comprises a plurality of signal emitting agents.
• the plurality of agents being characteristic for an application, i.e. a kit dedicated for testing quality of municipal water for home use which will thus include agents specific for microbial cells that typically are found in municipal water.
• the invention also provides a system for determining a quantitative value indicative of the number of culturable microbial cells in a tested sample, the system comprising a carrier the for holding a sample and for permitting contacting of the sample with one or more signal emitting agents.
• a detector for detecting signal emitting objects within the sample, and outputing data corresponding thereto;
• a memory unit comprising a database with predetermined selection parameters and one or more predetermined cell specific normalizing factors, each selection parameter each normalizing factor being specific for a microbial cell or a group of microbial cells;
• a processing unit for receiving the output data from the detector, and for receiving from said memory unit selection parameters and said normalizing factors specific for a microbial cell or a group of microbial cells and processing said output data with said parameters and said normalizing factor, to determine therefrom the quantitative value equivalent to the number of said culturable microbial cells in a sample.
• the carrier may be any receptacle that can hold biological cells.
• the carrier comprises a filter for filtering out at least a portion of liquid from said sample and holding the semi-dried or dried sample.
• the detector in accordance with the invention typically comprises a luminescent imaging system, such as a camera being capable of capturing one or more images of luminescent signals emitted from the tested sample.
• a luminescent imaging system such as a camera being capable of capturing one or more images of luminescent signals emitted from the tested sample.
• Non-limiting examples of cameras that can be used in accordance with the invention include charge-coupled device (CCD), CMOS detector, photodiode (PD) detector,, photomultiplier tube (PMT), gamma counters, scintillation counters or any other signal capturing device known in the art imaging of luminescent objects.
• the image processing unit is configured to receive one or more images emitted from the tested sample and identify therefrom, based on the selection parameters of signal emitting objects, the number of culturable cells in the sample.
• the term '"''processing unit' denotes any data processing and analyzing utility preprogrammed to collect measured signal parameters from the signal emitting objects in the sample and carry out data analysis consisting of selecting signal parameters according to predefined conditions and output a quantitative value based on the selected selection parameters.
• the processing unit carries a computer based program configured to carry out the analysis.
• the image processing unit is configured to select among the whole signal emitting objects, those that have a signal intensity within a predetermined range, a size within a predetermined range and a pre-determined morphology; and determined for the selected signal emitting objects the mean root square value intensity.
• the processing unit is further configured to output a quantitative value based on the mean root square value intensity emitted from the selected objects.
• the image processing unit is configured to normalize the quantitative value with a normalizing factor retrieved from the database to obtain a normalized value that is equivalent to CFU/ml count.
• a signal parameter includes one or more parameters.
• the term “comprising” is intended to mean that the methods, kits and systems include the recited elements, but not excluding others.
• “consisting essentially of” is used to define methods kits and systems include the recited elements but exclude other elements that may have an essential significance on the performance of the invention.
• Consisting of shall mean excluding more than trace elements of other elements. Embodiments defined by each of these transition terms are within the scope of this invention.
• samples comprising municipal water (tap water) mixed with predetermined concentrations of bacteria were used for determining the efficiency of the method of the present invention.
• the amount of viable reproducing microorganisms was quantified making use of the method of the invention as well as the standard CFU count (Standard Methods for Examination of Water & Wastewater (Lenore S. Clescerl (Editor), Arnold E. Greenberg (Editor), Andrew D. Eaton (Editor). 18-th Edition, 2002.).
• E. coli Native forms of E. coli (1 st preparation) and Ps. aeruginosa (2 nd preparation) were isolated from tap water, using for E. Coli, Tryptone Bile X-Glucuronide (TBX) Medium (Promega production) and for Ps. aeruginosa Cetrimide agar (Promega production).
• a 3 rd preparation contained regular tap water and is referred to as the microbial mix preparation (as it typically would contain microbial mixute).
• the cultured and isolated E.Coli and Ps. aeruginosa and the tap water preparations were each transferred into broth growing media and incubated by shaking using Lysogeny broth (LB) medium (Promega Cat.# 7290A).
• LB Lysogeny broth
• the cultured preparations were triple rinsed by centrifugation (6,000 RPM for 5 min) using Iso-normal PBS. At this stage microbial concentrations, as determined by microbial filtration CFU count, were determined to be about:
• HPC Heterotrophic Plate Count
• Ps.aeruginosa 3O 0 C for 48 Hr.
• Bacterial mix 3O 0 C for 72 Hr.
• FM1-43 styryl dye (Nishikawa S. Sasaki F. Internalization of styryl dye FM 1-43 in the hair cells of lateral line organs in Xenopus larvae, Histochem Cytochem, 1996 44(7):733-41) was used at a concentration of 1 ⁇ g/ml in PBS.
• Fluorescent staining of the microbial cell preparations was conducted at room temperature (about 25°C) using FM 1-43 styryl dye. Times of staining and working time window were determined using a monolayer preparation of the cells and it was determined that 2 minutes are required for the signal to reach the signal plateau (Tl, see Determination of Tl and T2 below) and the plateau remained at steady state for a period of time of about 600 seconds (Tl, see Determination of Tl and T2 below). At end of the staining the cultures were triple rinsed with iso-normal PBS. It is noted that the same staining method was used for filters surface adhered cultures or for suspended cultures (i.e. this is not specific for monolayer preparations).
• Fluorescent images of the signal emitting samples were obtained using Axiovert 200 Microscope (Karl Zeiss), 1.3NA Plan Neofluor XlO objective (BP 450-490 excitation filter (Excitation: 450-490 run; Beam splitter: FT 510 nm; Emission: 515-565 nm; Karl Zeiss). Image Capturing:
• the images were captured making use of a standard Sensicam qe (Cooke Corp.) 512 x 512 CCD.
• Image processing and data collection were performed using ImagePro+ software (2002).
• Tl three samples were prepared from the stock preparations, i.e. a tap water sample comprising a bacterial mix naturally existing in the tap water (after being cultured on a culturing medium and washed with PBS as described above); a second sample of isolated E.coli (after being cultured, and washed with PBS, as described above) and a third sample of isolated Ps. aeruginosa (after being cultured, and washed with PBS, as described above).
• Each sample was then diluted with PBS to obtain samples of about lOOOCFU/ml concentration.
• Each sample was then stained with FMl -43 and imaged when in monolayer (as described above) every 10 seconds until the signal emitted from the monolayer reached a plateau. The time at which the signal reached a plateau was determined as Tl.
• a total of three samples of tap water from three different geographical locations were collected and used. Preparations from each location were prepared as described above with respect to the determination of Tl, to obtain isolated E. CoIi and Ps. aeruginosa samples and a bacterial mix sample. For each location the concentration of each bacterial cell or the total bacterial count for each preparation was a priori determined based on HPC methodology (i.e. to obtain TBC before dilution) and then samples from the bacterial mix preparations (i.e.
• each of the 12 samples was analyzed according to HPC methodology and also according to the method of the invention, hi the latter case, each sample was stained with FM 1-43 as described above, washed after 120 seconds with sterilized water (i.e. at the beginning of the Plateau) and immediately imaged (at about 150-180 seconds). The image was processed according to signal selection parameters. Specifically, once image were captured, objects satisfying selection parameters were numbered, and their mean square root intensity determined. The results of the selection are provided in Tables IA- IB below.
• a total of 48 microbial test samples were prepared from the three different locations as described above (4 samples from each location, each sample in triplicate). Specifically, preparations from each location (with unknown cell concentration) either diluted with sterilized water xlO, xlOO, or non-diluted. Also, from each location, one sample of stock preparation was sterilized by filtration with a 0.22 ⁇ m mesh filter (Nitrocellulose, Milipore production). Each test sample was then stained with FM 1-43, washed with sterilized water and analyzed using the conventional HPC method or the method of the invention as described above. The results are shown in Table 2 below.
• a microbial signal emitting object i.e. an object to be counted for
• the samples were imaged 150 to 180 seconds following staining (i.e. shortly after the 2 minutes following staining). Further, exposure time was calibrated for maximal fluorescent intensity in "microbial objects" to be 254 gray levels (or between 60-254 GL). Exposure time was adjusted to 0.8 seconds.
• the invention is based on the understanding that reproducing (culturable) microbial cells can internalize lipid fractions of extracellular cell wall and concentrate the same inside the cell for a period of time (faster as compared to non-culturable microbial cells).
• the membrane trafficking rate and its intracellular concentration level is a function of cell activity, very high in reproducible microorganisms. Thus, it has been envisaged by the inventors that this phenomena can be utilized in order to distinguish and quantify the amount of viable, reproducible cells in a sample.
• Figure 1 shows that accumulation of FM 1-43 until the signal reached a plateau was at about 120 seconds following staining. This time point was thus marked as Tl. Stable intracellular dye accumulation was maintained steady in a time window of from 115 to 550 seconds from the beginning of the plateau. In other words, the fluorescent dye accumulation at the intracellular compartment of the mixture of cells became stable after 115 seconds and remained as such for at least 550 seconds. It has thus been determined that for quantitative determination E. CoIi and/or Sp. Aeruginosa in a liquid sample, a time window of from about 115 to about 550 seconds is an effective measurement window T2.
• predetermined selection criteria i.e. sizes 0.8-1.5 ⁇ m when visualized by light microscope corresponding to 12-28 pixels, maximal intensity of 254 gray levels.
• the determined mean square root values of intensity for the selected objects are included in Tables 1A-1B together with the CFU/ml counts obtained by the conventional HPC methodology.
• the approximate concentration was determined based on the CFU/ml count obtained for the stock preparations.
• the different samples are identified by location (A, B, or C) and sample number per location (Xl, X2 or X3).
• Table IB Analysis of E.Coli or Ps. Aeruginosa
• Table IA shows a correlation between CFU/ml values obtained by standard HPC method and the quantitative values obtained by the method of the invention.
• the CFU equivalent according to the invention provides means for a fast, and almost real time quantification of microorganisms in a liquid sample. Not only that the CFU equivalent is less time consuming as compared to standard CFU count methods, the time to receive the results in almost real time (between 5 to 10 minute, as compared to days when using the standard CFU count.

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