AU2017202998A1 - Mixed microbial standards - Google Patents

Abstract

MIXED MICROBIAL STANDARDS Abstract A microbial standard containing two bacterial species in defined numbers produced by counting and collecting the bacteria using flow cytometry from a liquid sample containing the two bacterial species.

Description

MIXED MICROBIAL STANDARDS

Related Application [001] This application claims priority to Australian provisional patent application No 2016901671 filed 6 May 2016 which is herein incorporated by reference in its entirety.

Technical Field [002] The technology relates to microbial standards having defined numbers of at least two different bacterial species and methods for their production. The standards can be used to validate the viability/specificity and selectivity performances of selective chromogenic media.

Background [003] Bioball™ technology (BTF Pty Ltd) is a patented product and process that was designed to provide high precision microbiology standards for quality control (QC) in food, pharmaceutical and cosmetic industries. One feature of Bioball™ standards relies on a unique flow cytometry-based technology for its production but also in the fine tuning of growth conditions in order to induce the highest level of resistance of the microorganism of interest to harsh conditions such as freeze-drying. Consequently, a maximum recovery of the cells and an unprecedented precision in viable counts are guaranteed after resuspension and plating. Common applications of Bioball™ technology relate to growth promotion testing, sterility assurance testing, food pathogen/non-pathogen testing or antimicrobial/preservative efficacy testing where low, medium to high numbers of organisms per standard are needed according to the requirements of the United States, European and Japanese Pharmacopoeias regulations. Therefore, a large panel of compendia strains are commercially available in Bioball™ format at various concentrations that are adapted to different OC tests. A custom service that can provide Bioball™ standards for any customer strains at requested concentrations is also available. In addition to standard microbiology testing, other applications of the Bioball™ technology have been suggested such as quantitative controls of PCR reactions or GFP-modified bacteria in Bioball™ format for fluorescence-based detection.

[004] Regarding the quality control of selective and/or chromogenic media, growth and/or inhibition of collection strains need to be investigated in order to validate the performances for viability, specificity and selectivity (CLSI M22-A3, EP 2.6.13). The standard procedure for performing those tests consist of plating collection strains at specific concentrations, usually between 10 and 100 colony forming unit (CFU) for viability and specificity testing and > 100 CFU for selectivity testing. While currently available Bioball™ standards could efficiently be used to perform such tests, a minimum of two to three different Bioball™ standards (one for each species) would be needed per media. For example, the validation of a mannitol-salt agar (MSA) would require a Staphylococcus aureus Bioball™ standard of 10 to 100 CFU for viability testing and an Escherichia coli Bioball™ standard at > 100 CFU for selectivity testing (EP 2.6.13 Table 2.6.13.1).

[005] The present inventors have developed a process for producing a microbial standard that can contain at least two different species of bacteria at specific ratios with high precision CFU counts.

Disclosure [006] In a first aspect, there is provided a microbial standard containing two bacterial species in defined numbers produced by counting and collecting the bacteria using flow cytometry from a liquid sample containing the two bacterial species.

[007] In an embodiment the microbial standard is substantially solid.

[008] The bacteria can be selected from two or more species selected from Escherichia, Legionella, Salmonella, Clostridium, Vibrio, Pseudomonas, Bacillus, Streptomyces, Staphylococcus, Campylobacter, Enterobacter, Enterococcus, Listeria, Salmonella, Pseudomonas, Lactobacillus, Citrobacter, Proteus, Lactococcus, Klebsiella, Aeromonas, Acinetobacter, Serratia, Edwardsiella, Rhodococcus, Yersinia, Methylobacterium, Haemophilus, Gardnerella, Mycobacteria, Bordetalla, Haemophilus, Shigella, Kluyvera, Spirochaeta, Rhizobium, Rhizobacter, Brucella, Neisseria, Rickettsias, or Chlamidia.

[009] In one embodiment the microbial standard can contain the following combinations of bacteria:

Campylobacter jejuni and Escherichia coli Neisseria gonorrhoeae and Proteus mirabilis Neisseria gonorrhoeae and Staphylococcus epidermidis Bacteroides fragilis and Peptostreptococcus anaerobius Bacteroides fragilis and Proteus mirabilis Bacteroides fragilis and Enterococcus faecalis Staphylococcus aureus MRSA and Staphylococcus aureus Staphylococcus aureus MRSA and Escherichia coli

Staphylococcus aureus MRSA and Pseudomonas aeruginosa

Enterococcus faecalis WDCM 00085 and Enterococcus faecalis WDCM 00087

Enterococcus faecalis WDCM 00085 and Staphylococcus aureus

Enterococcus faecalis WDCM 00085 and Escherichia coli

Enterococcus faecium and Enterococcus faecalis WDCM 00087

Enterococcus faecium and Staphylococcus aureus

Enterococcus faecium and Escherichia coli

Yersinia enterocolitica and Escherichia coli

Yersinia enterocolitica and Enterococcus faecalis

Yersinia enterocolitica and Pseudomonas aeruginosa

Clostridium difficile and Enterococcus faecalis

Clostridium difficile and Proteus mirabilis

Haemophilus influenza and Streptococcus pyogenes

Legionella pneumophila and Escherichia coli

Legionella pneumophila and Staphylococcus aureus

Legionella longbeachae and Escherichia coli

Legionella longbeachae and Staphylococcus aureus

Escherichia coli and Staphylococcus aureus

Escherichia coli and Proteus mirabilis

Staphylococcus aureus and Escherichia coli

Staphylococcus epidermidis and Escherichia coli

Neisseria gonorrhoeae and Proteus mirabilis

Neisseria gonorrhoeae and Enterococcus faecalis

Salmonella typhimurium and Escherichia coli

Salmonella typhimurium and Enterococcus faecalis

Shigella flexneri and Escherichia coli

Shigella flexneri and Enterococcus faecalis

Vibrio parahaemolyticus and Escherichia coli

Vibrio fumisii and Escherichia coli

Proteus mirabilis and Escherichia coli [010] Further examples of bacteria used to test culture media can be found in Guidelines : M22-A3 - Quality control for commercially prepared Microbiological Culture Media;

Approved standard - Third edition; and The Australian Society of Microbiology - Guidelines for the Quality Assurance of Medical Microbiological Culture Media, 2nd edition July 2012 (http://www.theasm.org.au/assets/ASM-Society/Guidelines-for-the-Quality-Assurance-of-Medical-Microbiological-culture-media-2nd-edition-July-2012.pdf).

[011] In tests for performance of culture media, the first listed bacterium above can be used as the test growth organism and the second listed bacterium can be used as the control as it should not be able to grow on that medium if it was made or is performing correctly. It will be appreciated that other mixtures of bacteria can be prepared by the present technology.

[012] In one embodiment the microbial standard contains Escherichia coli and Staphylococcus aureus.

[013] In one embodiment the microbial standard contains at least two bacterial species. The microbial standard may contain three bacterial species or four bacterial species or more, wherein each species is in a defined number.

[014] In one embodiment the defined number of each bacterium is small and can be from 10 to 1000 CFU. In some embodiments the small defined number of each bacterium is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[015] In some embodiments the defined number of each bacterial species is up to 1000000 CFU such as 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 or 1000000.

[016] In one embodiment the number of bacteria of each bacterial species is the same.

[017] In one embodiment the number of bacteria of each bacterial species is different.

The number between the two bacterial species can be in a ratio from about 1:1 to about 1:250. The number between the two bacterial species can be in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250 and any ratio in-between. The ratio above can be assigned to either of the two different bacterial species in the microbial standard.

[018] In one embodiment there is provided a microbial standard with ranges of between about 10 to 200 bacteria of one species and about 10 to 200 bacteria of the other species.

In one embodiment the microbial standard has a total of about 180, 200 or 250 bacteria. It will be appreciated that the total number can be anywhere between about 10 to about 1000.

[019] For microbial standards useful to assay performance of selective or chromogenic culture media such as agar plates, 10 to about 250 CFU of each bacterial species is useful.

[020] Typically, the microbial standards according to the technology are made in batches of 10 or more, or 100 or more, and packaged in smaller sets of 10 or more, or 20 or more, for use. Typically, products containing 10, 20, 50, 70 or 100 microbial standards are useful for consumers. It will be appreciated that the sets of microbial standards can contain any suitable number of microbial standards as required.

[021] The degree of error can be determined by analysing samples from a set of microbial standards and calculating the mean and the standard deviation. The number of samples that are analysed are typically between 3% and 10% of the entire set of the microbial standards. The analysis of the samples can be performed by any means that gives an accurate measurement of how many bacteria are in each sample.

[022] The degree of error is typically defined as the percentage coefficient of variance. This is calculated by dividing the standard deviation by the mean and multiplying by 100.

[023] In an embodiment where the microbial standard is substantially solid, the product can be formed by snap-freezing a volume of liquid containing the defined number of bacteria and then drying the frozen body to form the substantially solid product. The snapfreezing can be carried out by placing the volume containing the defined number of bacteria into a cryogenic liquid. The cryogenic liquid can be selected from liquid nitrogen, liquid helium and liquid oxygen.

[024] In an embodiment the cryogenic liquid is liquid nitrogen.

[025] In an embodiment the cryogenic liquid is placed in a container, a droplet containing the defined number of bacteria is placed in the container to form the frozen body, and the container holding the frozen body is then subjected to freeze-drying to form a substantially dry solid product in the container.

[026] After drying, the container can be capped or sealed for storage and transport of the product.

[027] In an embodiment the substantially solid product is a small roundish mass in the form of a ball or sphere.

[028] In a second aspect, there is provided a process for obtaining a microbial standard containing defined numbers of two bacterial species, the process comprising: providing a liquid sample containing two bacterial species; counting a desired number of each bacterial species from the sample using a flow cytometer; collecting the counted bacteria in a drop; and freezing the drop containing the two species of bacteria to form the microbial standard.

[029] In a third aspect there is provided a process for obtaining a microbial standard containing defined numbers of two bacterial species, the process comprising: mixing cells from two bacterial species in a single liquid sample at a desired cell ratio; using a cytometer to define a sort region for each bacteria; sorting a desired number of bacteria from the two sort regions; collecting the counted bacteria in a drop; and freezing the drop containing the two species of bacteria to form the microbial standard.

[030] The sorting can use an OR sort logic.

[031] In a fourth aspect, there is provided a microbial standard containing defined numbers of two bacterial species produced by the process according to the second aspect.

[032] In a fifth aspect, there is provided use of a microbial standard containing defined numbers of two bacterial species according to the first or third aspects is an assay to assay performance of selective or chromogenic culture media.

[033] In one embodiment the counting and collecting steps are repeated to form a set of microbial standards wherein the defined number of each bacterial species is within a degree of error of 20% or less between each standard of the set.

[034] It is difficult to prepare microbial standards containing accurate numbers of just one species of bacteria. Developing technology in order to produce microbial standard containing two bacterial species in defined numbers has been difficult.

[035] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[036] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

[037] In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings [038] Figure 1. Sort region-dependent variations of sort rates. This figure illustrates the type of variations that can occur in sort rate values depending on the location and/or size of the sort regions on the population. The scattering profile of a microbial population is shown in grey. Four sort regions representing three different sizes are represented, R3 and R4 > R2 > R1. The sort rate values corresponding to the sort regions are shown, x-axis: side scatter (SSC), y-axis (FSC).

[039] Figure 2. Schematic representation of the scattering profile of calibration beads. (A) Flow cytometry scattering profile of a solution containing bead 1. (B) Flow cytometry scattering profile of a solution containing bead 2. (C) Flow cytometry scattering profile of a mixture containing both bead 1 and bead 2. The populations of beads are represented as plain dots and the sort regions R1 and R2 shown as circles were created to select the populations corresponding to bead 1 and bead 2, respectively, x-axis: side scatter (SSC), y-axis (FSC). Estimated sort rates and microscopic aspects of the beads are also shown.

[040] Figure 3. Schematic representation of the scattering profile of bacterial populations. (A) Scattering profile of S. aureus. (B) Scattering profile of E. coli. (C) Scattering profile of S. aureus/E. coli mixture. The sort regions R1 and R2 shown as circles were created to select non-overlapping populations of S. aureus and E. coli, respectively, x-axis: side scatter (SSC), y-axis: (FSC).

Description of Embodiments Flow Cytometry [041] The flow cytometry technology used in this study was previously described in US 6,780,581 and US 7,372,566. Briefly, a Becton Dickinson FACScalibur™ flow cytometer was modified in order to capture the desired number of cells in a small drop of approximately 30 pi. One of the major changes operated on the flow cytometer instrument is the insertion of a hypodermic tube that connects the capture tube to the droplet nozzle where the sorted cells are harvested in a small volume. Another hypodermic tube conveys the lyoprotectant solution to the droplet nozzle where it is mixed with the sorted cells during droplet formation. These modifications are supplemented with a vacuum nozzle that is held near the droplet nozzle exit point to ensured that all liquids are sent to waste before the formation of the drop begins. The sequential sorting of the cells and removal of the vacuum nozzle to allow drop formation is well synchronized and repeated in cycles. Therefore, this droplet control system takes into account the time of the sorting process, the travel time of the cells in the hypodermic tube towards the droplet nozzle and the time for droplet formation to ensure that all the sorted cells are contained in the forming drop before it breaks loose from the droplet nozzle. The cell-containing drop falls into liquid nitrogen to form a frozen ball that is further freeze-dried under specific conditions to give a microbial standard product.

[042] Cells enter the flow cytometer through the sample tube. Sheath fluid enters the fluidic system of the flow cytometer and surrounds a single file stream of bacterial cells. Laser beam scatters on each cell, allowing the detection and selection of the gated population required for sorting. The capture tube, with the hypodermic tube inserted, is activated by the piezo solenoid to move in and out of the stream of cells to collect the selected cells. When the selected cells enter the capture tube the cells travel down the hypodermic tube to the droplet nozzle. Waste sheath fluid and waste cells exit the cytometer. Lyoprotectant is injected into the droplet nozzle through a secondary injection hypodermic tube. The cells and lyoprotectant exit the cytometer at the droplet nozzle while being mixed by the mixer wire. A pneumatic cylinder holds the vacuum nozzle to collect the waste droplets. When deactivated, the vacuum nozzle pulls back and a droplet is allowed to form. The droplet containing the sorted cells falls from the droplet nozzle and is frozen in liquid nitrogen.

[043] The production of the microbial standards can be performed using two different sorting modes on the flow cytometer defined as “normal sort” and “high speed sort”. In the “normal sort” mode the capture tube is moved in and out of the stream of cells to collect individual cells. In the “high speed sort” mode the capture tube is positioned in to the stream of cells and left in place until the desired number of cells have entered the capture tube. The capture tube is then removed from the stream of cells.

[044] Regarding the setup of the sorting process in the “normal sort” mode, several parameters may need to be considered:

The sort rate which can be defined as the estimated sorting speed (expressed in events/second) in the sort region selected. This sort rate is highly dependent on the size and the relative location of the sort region (Figure 1). The sort rate preferably does not reach the maximum sorting speed supported by the FACScalibur instrument which is theoretically 300 events/sec and more practically around 250 events/sec.

The abort rate can be defined as the number of coincidence per second that occurs when two consecutive particles pass through the laser beam light within approximately 20 ms of each other. As a result, particles that are not separated by a delay of more than 20 ms delay cannot be sorted as separate events and are flagged accordingly as “abort signals”. The abort rate is directly depended on the concentration of the sample. For a same sort region, the abort rate will logically decrease if the concentration of the sample is lowered down as the distance between the cells will increase.

The sorting time which is dependent on both the sort rate and the abort rate, should be compatible with the droplet control system detailed above. If the sorting time is too long, the last cells sorted will not have time to reach the droplet nozzle before the drop is released. While it takes approximately one second or less for a drop to form and break, the sorting time should be added to the time of travel nozzle of the last cell sorted to reach the droplet remain below one second. In order to obtain the maximum purity and accuracy in microbial standards, the sorting is performed using the “single cell” mode. This means that if an abort signal is detected, the corresponding cells are not sorted whether they are target cells (within the sort region) or not. Therefore, a higher abort rate will further increase the sorting time needed in order to obtain the desired number of cells.

[045] While the production of 30 CFU microbial standards are easily achievable using the normal sort system, microbial standards with higher CFU counts are more challenging due to the limitations mentioned above. For example, with a sort rate of 250 events/sec and an abort rate of 50 counts/second more than 200 cells would not be expected to be sorted in a single drop. Therefore, the challenge in producing high CFU microbial standards relies in defining the appropriate concentration of the sample and selecting the right sort region in order to obtain a high sort rate with the lowest possible abort rate. Another challenge is that the sort rate and the abort rate can only be observed in real time during acquisition and no mean values are obtained from the instrument. Therefore, a given sort rate of 250 events/sec is only a subjective estimation based on a few seconds of observation. This approximation is not an issue when producing low CFU microbial standards because the number of cells sorted is significantly lower than the sort rate and the sorting time still stays relatively short and compatible with our droplet control system. However, for higher CFU microbial standards, variability that can occur in the estimation of the sort rate and abort rate values could impact the final count in the drop as we are reaching the limit of the system.

[046] Sorting logic is used in the “normal sort” mode to program the cytometer how to sort particles. If two regions (R1 and R2) have been defined then there are a number of options as to how the cytometer sorts from these regions. Setting the sorting logic for these two regions as R1*R2 tells the cytometer to only sort cells that are in both of the regions. Using R1+R2 tells the cytometer to sort cells that are in either of the regions.

[047] The preferred sorting logic for this invention is R1+R2. The two cell types are mixed at the desired ratio and then sorted using R1+R2.

[048] An alternative method for sorting would involve selecting sorting region R1, sorting the desired number of cells and then changing the sort region to R2 and sorting the desired number of cells from this region.

[049] Regarding the “high speed sort” mode, the process is less complex. In this mode, the capture tube is moved in the stream of cells and remains there until the desired number of cells have entered the capture tube. Cells are counted as they enter the capture tube and regions are defined to specify which cells to count. Typically there are no sort rate or abort rate constraints used. However, the sample concentration should be adjusted to increase as much as possible the distance between two consecutive particles while keeping a sorting time compatible with the droplet control system. In the microbial standard production process, the growth conditions can be optimized for each species to ensure that the sorting is performed on an homogenous population of cells which further contributes to the low level of variability in the counts after re-suspension and plating. In the “normal sort” mode a highly homogenous portion of a population can be selected by the sort region. In the “high speed sort” mode, the sort number is chosen based on experimentally defined loss that occur during the process or due to microbial standards containing a more heterogeneous population of cells. This sorting mode will tend to be less accurate in the counts than the “normal sort” mode but the %CV obtained remains acceptable in a higher number of cells such 550 CFU and 10 000 CFU microbial standards.

[050] Once the different sorting parameters are set up, three controls are usually performed in order validate the different steps of the microbial standard production process:

The five-drop control: The sorting is activated off-cycle (vacuum nozzle pulled back) and five consecutive drops are harvested on the same agar plate and dispersed before overnight incubation for colony counting. The sorted cells are supposed to be in these first 5 drops. Therefore the number of colonies counted should be equal or close to the sort number set up on the flow cytometer. The aim of this control is to validate the accuracy of the sorting module.

The one-drop control: The sorting is activated with the cycle on and one drop is harvested on an agar plate for colony counting after overnight incubation. The counts should be equal or close to that of the 5 drops controls if the sorting setups are in accordance with the timing of the droplet control system (as discussed above).

The microbial standard quality control: The sorting is activated with the cycle on and the microbial standards are produced as previously described (Morgan CA, Bigeni P, Herman N, Gauci M, White PA, et al. (2004) Production of precise microbiology standards using flow cytometry and freeze drying. Cytometry A 62: 162-168). A microbial standard is dropped on the surface of an agar plate, dissolved in 100 μΙ of 0.9% NaCI solution and spread before incubation and colony counting. The counts should be equal or close to that of the 1 drop controls. This control ensures that the steps downstream the sorting such as snap-freezing in liquid nitrogen, freeze-drying or rehydration do not affect the viability of the cells.

[051] For statistic relevance, several replicates of the different controls are usually performed.

Freezing and freeze-drying the droplets [052] Droplets from the cytometer that contained the desired number of bacteria were collected into test tubes that contained liquid nitrogen. After collection of the droplets, the tubes were placed in a Telstar Lyobeta freeze dryer and dried overnight..

[053] The next day, the freeze-dried samples were removed from the freeze drier and stored in suitable containers.

EXAMPLES

[054] The main goal of this study was to develop microbial standards containing a mixture of species that could be used to facilitate quality control tests such as those performed on selective chromogenic media. The method was first developed using calibrated beads and further extended the study to bacterial populations.

Detailed Protocol

Analysis of viability and/or specificity [055] To control the viability and specificity of a solid selective media, two microbial species of interest plated together on the tested media should grow optimally and be easily recognizable based on size and/or morphology and/or color depending on media composition. Therefore, the aim was to design a mixed microbial standard containing two different microbial species at equal numbers. For the proof of concept, E. coli and S. aureus were chosen as they are easily recognizable by size, morphology and color on standard non-selective agar plate.

Experiment 1: Simultaneous sorting of two different calibration beads at equal ratio [056] In order to verify the potential of sorting mixed bacterial populations using the modified flow cytometer instrument calibrated beads that can be well discriminated by their scattering properties were used. The sorting parameters shown in Figure 1C suggest that a total of 100 hundred beads (SN=100) will be sorted using both regions (Gate = R1 + R2). This suggests that if the sort rate is identical in both regions and if the beads solutions are mixed at equal ratios, it should be possible to simultaneously sort 50 beads from R1 and 50 beads from R2. The experiments was performed as follows: [057] Each stock solution of beads was diluted and analyzed separately on the flow cytometer in order to have a sort rate of approximately 120 events/sec for each region R1 and R2 (Figure 2A and 2B). The abort rate observed in each region was insignificant.

[058] Equal volumes of the adjusted bead solutions were mixed and analyzed. The sort rate measured for the combination of R1 and R2 was confirmed at approximately 120 events/sec.

[059] The sorting was activated and 5 drops were harvested at the droplet nozzle on a filter membrane placed on a vacuum system in order to suck down the liquid while keeping the beads on the surface. As mentioned above, the majority if not all of the sorted beads are supposed to be in the first drop when the droplet control system is activated for microbial standard production. The aim of this experiment was to evaluate the variability that could be due to subjective sort rate estimation or pipetting errors for mixture solution preparation. Therefore, 5 consecutives drops were harvested to make sure that all the sorted beads were recovered independently of the sorting cycle.

[060] The filter membrane was placed on a microscope slide and analyzed using a UV fluorescent microscope (x100). The beads were easily recognizable as they appeared big and green or small and orange for bead 1 and bead 2, respectively.

[061] After sorting and harvesting 5 drops, a total of 100 beads were counted on the filter membrane with 48 counts for bead 1 and 52 counts for bead 2. Regarding the subjective evaluation of the sort rates and the potential pipetting errors that could have occurred during mixing, the ratio was perfectly respected.

Experiment 2: Mixed microbial standards of E. coli and S. aureus at equal ratios [062] Having confirmed the ability to sort two calibrated beads at the desired ratio, the same method was applied to bacterial populations. The experiment was performed as follows: a. Preparation of the bacterial cultures E. coli NCTC 9001 was grown for 24 h in a 2xYT medium (Oxoid). S. aureus NCTC 10788 was grown for 22 h in 2xYT medium.

After growth, the culture was sonicated for 2 min in a sonication bath to break bacterial clumps. b. Flow cytometry analysis and sorting parameters 1 ul of each culture was diluted in 1 ml of PBS and analyzed separately. For each population, a non-overlapping region was selected (Figure 3). After concentration adjustment of the samples and selection of the sort regions, the recorded values were as follows:

Sort rate of 150 event/sec and abort rate of 30 counts/sec for S. aureus

Sort rate of 150 event/sec and abort rate of 20 counts/sec for E. coli

Equal volumes of the cultures were mixed and analyzed. The sort rate measured for the combination of R1 and R2 was confirmed at approximately 150 events/sec for an abort rate of approximately 32 counts/sec (Figure 3) A sort number of 72 was set up and the sorting was performed on the mixture. In theory, we would expect to have mixed microbial standards containing 36 CFU of E. coli and 36 CFU of S. aureus. It was expected to have to the same number of E. coli and S. aureus because the level of recovery of both species after microbial standard re-suspension was found to be the same according to previous experiments. If two species having different level of recovery were used, this difference would have been taken into account in the volume ratios. c. Quality control

Five-drop control: five replicates One-drop control: five replicates Microbial standard quality control: ten replicates

All three controls were performed on nutrient agar and horse blood agar with 24h incubation at 37°C d. Results and discussion

Table 1. Colony counts on nutrient agar of E. coli: S. aureus mixed microbial standard at ratio (1:1)

Table 2. Colony counts on horse blood agar of E. coli: S. aureus mixed microbial standard at ratio (1:1)

[063] As seen in Figure 3, the scattering profile of the populations are more dispersed than calibrated beads. Therefore, careful attention had to be put on the selection of the sort regions to avoid any overlapping.

[064] Regarding the total counts, a drop of 9 to 10% was seen between the one-drop control and the microbial standards. This 10% loss is usually observed due to some mortality that can occur during snap-freezing, freeze-drying, rehydration and also loss of cells during plating. This loss can easily be anticipated by increasing the value of the sort number. A drop of 16 to 18% was seen between the five-drop controls and the one-drop controls. While some cells can be lost in the process, this relatively high difference is most certainly due to a long sorting time. Indeed, all the sorted cells might not have reached the drop before it broke from the nozzle. This can be fixed by either increasing the concentration of the samples or selecting larger sort regions to increase the sort rates while keeping relatively low abort rates.

[065] Overall, the ratio between S. aureus and E. coli are well respected in the mixed microbial standards and the variability observed in the counts are low with a CV < 20% in the worst case scenario.

Analysis of viability and selectivity

Experiment 3: Mixed microbial standard of E. coli and S. aureus at different ratios [066] In an effort to produce a microbial standard that could be used to validate both the viability and inhibitory properties of a media, mixed microbial standard of E. coli and S. aureus at different ratios were performed. As mentioned above, the species of interest could be in the range of 10 to 100 CFU while the species inhibited by the media could be at >100 CFU in order to show a selectivity factor index of 2 log10.

[067] This experiment was performed as follows: [068] The selection of the sort regions were done as shown in Figure 3. After concentration adjustment of the samples and selection of the sort regions, the recorded values for both populations were a sort rate of 250 events/sec and an abort rate of 50 counts/sec.

[069] The bacterial samples were mixed at an E. coli: S. aureus ratio of 4:1 (240 μΙ of E. coli sample + 60 μΙ of S. aureus sample). The recorded values for the mixture were a sort rate of 240 events/sec and an abort rate of 60 counts/sec.

[070] A sort number of 180 was set up and the sorting was performed on the mixture. In theory, it would be expected to have mixed microbial standard containing 144 CFU of E. coli and 36 CFU of S. aureus.

[071] The quality controls were performed as described in experiment 2, but only horse blood agar was used.

Results and discussion

Table 3. Colony counts on horse blood agar of E. coli: S. aureus mixed microbial standard at ratio (4:1) '

[072] A total mean of 121 CFU was obtained in the microbial standard with a mean of 99 CFU for E. coli and 22 CFU for S. aureus. Therefore, the 4:1 ratio was well respected.

[073] Regarding the total counts, a drop of 16% was seen between the one-drop control and the microbial standard. The explanation for this loss is detailed above. A drop of less than 10% was seen between the five-drop controls and the one-drop controls which is what is usually observed in normal situations. This suggests that the sort setups (sort regions, sort rates and abort rates) were compatible with a sorting time that was short enough to allow the majority of the sorted cells to reach the drop before it broke from the nozzle.

[074] Regarding E. coli counts, an acceptable level of variability was observed with a %CV of 7.5. Using the same sort settings it can expect to have a higher number of CFU. For instance, extrapolating from the results obtained, a sort number of 200 instead of 180 would give mixed microbial standard containing E. coli at 110 CFU and S. aureus at 24 CFU which would fit with simultaneous investigation of viability and selectivity performance of a media.

[075] Regarding S. aureus counts, a higher variability was observed with a %CV of 23.6. This variability is not an issue for viability testing as the range of CFU is still between 10 and 100. However, it may be an issue for standardization between different lots or labs. Therefore, a more precise selection of the sort region for the S. aureus population could be carried out. Although growth conditions were finely adjusted in order to have an homogenous population, there is still some inflexible variability in the physiological state and scattering profile of a microbial population. Therefore, the sort region selected on the population needs to be small enough to maximize the sorting of cells sharing the same characteristics especially for the production of microbial standards with low CFU counts where a tight variability range is desired. This is easily done when producing single species-containing microbial standard but more complicated for mixed microbial standard where overlapping should be avoided. A specific optimized media is routinely used for growth of a species before microbial standard production. However, other media that would show the same level a viability of cells in the microbial standard can also be developed. Knowing that the growth conditions of strains could directly reflect their scattering profile, a different selection of growth media can be considered in order to reduce the overlapping of two populations in a mixture. This should allow more flexibility in the choice of the sort regions to sort cells with enhanced homogeneity without the risk of overlapping. In any case it should reasonably be expected to have a CV of less than 20% for the species present at low CFU in the mixed microbial standard.

[076] Overall, this experiment shows the great ability of our technology to produce mixed microbial standard at desired ratios with a relatively good precision.

[077] The present technology is suitable for producing microbial standards with other combinations of two bacterial species at any ratio.

[078] The present technology is suitable for producing microbial standards with more than two bacterial species.

[079] The present technology is suitable for producing microbial standards with higher CFU counts.

[080] Regarding the production of mixed microbial standards in the “normal sort” mode, concentration of the samples are adjusted but a parameter to consider is the selection of the sort regions in order to have the desired sort rates. If the sort rates are identical in the sort regions representing two microbial populations in a mixture then the volume ratio is respected in the CFU counts.

[081] Regarding the production of mixed microbial standards in the “high speed sort” mode, no sort region-dependent sort rates can be adjusted. Therefore, the adjustment of sample concentrations can be used. In order to have a specific ratio in CFU counts between two species then volumes of two samples can be mixed to have same concentrations. Since cell concentrations in overnight microbial cultures are highly variable, it can be difficult to predict an exact dilution in order to reach a precise concentration of bacteria in the samples. One option would be to adjust the sample concentration using a separate system equipped with an absolute volumetric count prior to volume mixing and sorting. Existing systems such as the Cyflow Space flow cytometer from Partec could be used for this purpose.

[082] One advantage of using the “high speed” sort mode to produce mixed microbial standard is that population overlaps are not an issue. Another advantage would be the ability to produce a single microbial standard for several tests as currently used for the 550 CFU format. A mixed microbial standard containing 550 CFU of one species and 1650 CFU of the other species could be produced. When re-suspended in a volume of 1.1 ml, 10 tests can be performed by plating 100 pi volumes containing 50 CFU of bacterial strain 1 for viability validation and 150 CFU of bacterial strain 2 for selectivity validation.

[083] The present technology is suitable for producing microbial standards containing several species of bacteria for use as internal qualitative molecular biology standards for complex biological fluids.

[084] The benefits of the technology include use of one single microbial standard instead of two or three standards for viability, specificity and selectivity performance validation of selective chromogenic media; time saving on handling and performing OC tests; cost saving as the price of one mixed microbial standard could be lower than the price of two or three different individual microbial standards; cost saving as the number of media plates used for QC would be reduced; performance evaluation in conditions reflecting reality as it would take into account potential inter-species competition (current QC tests are made by plating single species on different plates).

[085] Table 4 contains a list of bacteria that are suitable as standards according to the present technology. It will be appreciated, however, that this list is not exhaustive.

Table 4. Suitable bacteria

[086] Table 5 shows examples of combinations of microorganisms used to test microbial media. It will be appreciated that the present technology cam provide microbial standards for such use.

Table 5. Combinations of microorganisms*

* adapted from The Australian Society of Microbiology - Guidelines for the Quality Assurance of Medical Microbiological Culture Media, 2nd edition July 2012 [087] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (15)

1. Claims:

   1. A microbial standard containing two bacterial species in defined numbers produced by counting and collecting the bacteria using flow cytometry from a liquid sample containing the two bacterial species.
2. 2. The microbial standard of claim 1 wherein the standard is substantially solid.
3. 3. The microbial standard of claims 1 or 2 wherein the bacteria are two or more species selected from Escherichia, Legionella, Salmonella, Clostridium, Vibrio, Pseudomonas, Bacillus, Streptomyces, Staphylococcus, Campylobacter, Enterobacter, Enterococcus, Listeria, Salmonella, Pseudomonas, Lactobacillus, Citrobacter, Proteus, Lactococcus, Klebsiella, Aeromonas, Acinetobacter, Serratia, Edwardsiella, Rhodococcus, Yersinia, Methylobacterium, Haemophilus, Gardnerella, Mycobacteria, Bordetalla, Haemophilus, Shigella, Kluyvera, Spirochaeta, Rhizobium, Rhizobacter, Brucella, Neisseria, Rickettsias, or Chlamidia.
4. 4. The microbial standard of any one of claims 1 to 3 wherein the standard contains at least two bacterial species wherein each species is in a defined number.
5. 5. The microbial standard of any one of claims 1 to 4 wherein the defined number of each bacterium is from 10 to 1000 CFU.
6. 6. The microbial standard of claim 5 wherein the defined number of each bacterium is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 CFU.
7. 7. The microbial standard of any one of claims 1 to 4 wherein the defined number of each bacterium is 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000,40000,50000,60000,70000,80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 or 1000000 CFU.
8. 8. The microbial standard of any one of claims 1 to 7 wherein the number of bacteria of each bacterial species is the same.
9. 9. The microbial standard of any one of claims 1 to 7 wherein the number of bacteria is in a ratio from about 1:1 to about 1:250.
10. 10. The microbial standard of any one of claims 1 to 9 wherein the standard comprises between about 10 to 200 bacteria of one species and about 10 to 200 bacteria of the other species.
11. 11. A process for obtaining a microbial standard containing defined numbers of two bacterial species, the process comprising: providing a liquid sample containing two bacterial species; counting a desired number of each bacterial species from the sample using a flow cytometer; collecting the counted bacteria in a drop; and freezing the drop containing the two species of bacteria to form the microbial standard.
12. 12. A process for obtaining a microbial standard containing defined numbers of two bacterial species, the process comprising: mixing cells from two bacterial species in a single liquid sample at a desired cell ratio; using a cytometer to define a sort region for each bacteria; sorting a desired number of bacteria from the two sort regions; collecting the counted bacteria in a drop; and freezing the drop containing the two species of bacteria to form the microbial standard.
13. 13. The process of claim 12 wherein the sorting and collecting steps are repeated to form a set of microbial standards wherein the defined number of each bacterial species is within a degree of error of 20% or less between each standard of the set.
14. 14. A microbial standard containing defined numbers of two bacterial species produced by the process according to claim 11, 12 or 13.
15. 15. Use of a microbial standard containing defined numbers of two bacterial species according to any one of claims 1 to 11 in an assay to assay performance of selective or chromogenic culture media.