WO2025103575A1

WO2025103575A1 - Method for monitoring cells

Info

Publication number: WO2025103575A1
Application number: PCT/EP2023/081729
Authority: WO
Inventors: Konstanze SCHIESSL; Samuel Mathias WÜTHRICH; Michael BESMER
Original assignee: Oncyt Microbiology Ag
Current assignee: Oncyt Microbiology Ag
Priority date: 2023-11-14
Filing date: 2023-11-14
Publication date: 2025-05-22
Anticipated expiration: 2026-05-14

Abstract

The invention relates to a method for monitoring cells, in particular bacteria, in a fluid system (1) by a monitoring device. The method comprises the following steps: collecting a first fluid sample by a sample collecting device (2,3,5) and diluting the first fluid sample with a first dilution ratio by a dilution device (2,3,6). After these steps, the cell concentration of the first fluid sample is measured by a measurement device (11), in particular a flow cytometer. Subsequently, it is determined, if the measured cell concentration lies within a valid concentration range. If the measured cell concentration lies outside the valid concentration range, the control device (13) automatically repeats steps the sample preparation and the measurement with a second fluid sample, wherein the second fluid sample is diluted with a second dilution ratio, which is different to the first dilution ratio.

Description

Method for monitoring cells

Technical Field

The invention relates to a method for monitoring cells , in particular microbial cells , in particular bacteria, microalgae or yeast , in a fluid system by a monitoring device . The method comprises the steps of collecting a first fluid sample by a sample collecting device and diluting the first fluid sample with a first dilution ratio by a dilution device . A cell concentration of the first fluid sample is measured by a measurement device , in particular by a flow cytometer .

Background Art

Dyeing with fluorescent stains coupled with flow cytometry is often used for monitoring, quanti fication and characteri zation of bacteria in engineered and environmental aquatic ecosystems including seawater, freshwater, drinking water, wastewater, and industrial bioreactors . Devices with full automation of sampling, staining, measurement and data analysis are known . The full automation allows automated measurements at 15-min intervals during several consecutive days . Daily and hourly variation of the measured ecosystem can be monitored by such large data sets , which would not be possible by infrequent sampling .

Small microorganisms , such as small environmental bacteria, need to be dyed with a fluorescent dye before measurement in order to distinguish them from background and/or to detect speci fic cellular features .

Disclosure of the Invention

The problem to be solved by the present invention is to provide a method for full automated monitoring of cells , in particular microbial cells , in particular bacteria, microalgae or yeast , in a fluid sample for a wide range of applications .

This problem is solved by the method of the independent claims . According to this , it is a method for monitoring cells , in particular microbial cells , in particular bacteria, microalgae or yeast , in a fluid system by a monitoring device . The method comprises the following steps which are controlled, in particular automatically controlled, by a control device : a ) collecting a first fluid sample by a sample collecting device . b ) diluting the first fluid sample with a first dilution ratio by a dilution device . The diluent could be a buf fer . Diluting the first fluid sample allows do automatically monitor fluid systems with a high concentration of bacterias . c ) measuring a cell concentration in the first sample by a measurement device , in particular a flow cytometer . The measurement device could be a BD Ac- curi C6 flow cytometer, equipped with a 20 mW solid state laser emitting light at di f ferent wavelengths . In particular, steps a ) to c ) or at least steps b ) to c ) are automatically performed by the control device .

Advantageously, the method does not comprise a step of dyeing the first fluid sample . Alternatively, the method comprises a step of dyeing the first fluid sample by a dyeing device , wherein the step of dyeing and the step of diluting the first fluid sample are separate steps .

Commonly used dyes are SYBR Green I and Pro- pidium Iodide , and particularly their combination . Certain dyeing procedures allow to distinguish between membrane compromised ( damaged) cells , which are considered to be dead, from intact cells , which are often described as potentially viable cells . The fluid sample can be dyed before the dilution or it can be dyed after the dilution .

The di f ferent steps of the described method are controlled by a common control device . Such an automatically working system can be applied in various applications since a separate dilution step allows to monitor di f ferent fluid systems with a wide range of di f ferent concentrations .

In particular, step b ) of diluting the first fluid sample comprises at least two serial dilution steps , wherein a serial dilution is the stepwise dilution of a substance in a solution .

Advantageously, the user can define and communicate the dilution ratio applied in step b ) to the control device . A flexible application of the monitoring device is possible .

In a preferred embodiment , the method further comprises the following steps controlled by the control device : d) After having measured the cell concentration, the control device automatically determines i f the measured cell concentration lies within a valid concentration range . In particular, the valid concentration range is defined by the capabilities of the measurement device , in particular the flow cytometer . I f the measured cell concentration lies outside the valid concentration range , the measurement could be inaccurate . e ) i f the measured cell concentration lies outside the valid concentration range , the measurement has to be repeated . Steps a ) to e ) are automatically repeated by the control device . A second fluid sample is collected and diluted with a second dilution ratio . The second dilution ratio is di f ferent to the first dilution ratio . I f the cell concentration of the first measurement was too high, a higher dilution ratio is used for the second measurement in order to move the measured cell concentration into the valid concentration range . I f the cell concentration of the first measurement was too low, a lower dilution ratio is used for the second measurement .

The second fluid sample can be collected from the same sample taken from the fluid system as the first fluid sample or the first fluid sample and the second fluid sample are di f ferent samples taken from the fluid system at di f ferent times or from di f ferent locations inside the fluid system .

I f the first fluid sample and the second fluid sample are diluted with a di f ferent dilution ratio , the measured cell concentration will be di f ferent . With other words , the measured cell concentration is the cell concentration of the diluted sample-dye mixture and not the cell concentration of the fluid sample taken from the fluid system . In order to determine the actual cell concentration of the fluid sample , the measured cell concentration has to be re-converted by the dilution ratio .

In particular, the steps a ) to e ) are automatically repeated by the control device until the measured cell concentration lies inside the valid concentration range , completing a valid measurement and providing valid measurement data .

The automatically working monitoring method has the advantage to be applicable for fluid samples with a wide range of cell concentrations . Up to now, monitoring devices for measuring cell concentrations were not equipped with an automatic, sequential dilution . Known monitoring devices have been limited to a predefined dilution ratio . Only fluid samples from a small concentration range could be measured due to limitations of the flow cytometer .

Applying the monitoring method for fluid samples with a wide range of cell concentrations means that the monitoring device can be used for measuring fluid samples in very di f ferent environments , e . g . , in a paper mill , in aquaculture systems , in cooling water systems , in bioprocessing, in particular in a probiotics production system, in a food factory, for fermentation of drinks , in hydroponic or aquaponic systems , in a biofuel factory etc .

In a cooling water system, the monitoring can be applied to check stability of microbial concentrations , to optimise or dynamically operate the treatment or disinfection regime . In a paper factory, the monitoring system can be applied to monitor microbial concentrations in process water, to optimise or dynamically operate the production, the treatment or disinfection regime . In ef fluent systems , the monitoring system can monitor the stability of biological treatment steps . It can assess the ef fect of disinfection steps on microbial concentrations or it can assess the microbial quality of effluent for water recycling purposes . In aquaculture , the monitoring system can continuously monitor the treatment ef ficiency and raise warning in case of issues in a recirculating aquaculture system . Additionally, the monitoring system can be applied in production systems for producing probiotics in order to quanti fy the growth performance and viability of the probiotical culture and to optimi ze growth conditions and harvest time , as well as to detect contaminants .

A fully automatic monitoring device can perform steps a ) to e ) in 15 or more minutes . I f two or more iterations with di f ferent dilution ratios are necessary to reach the valid concentration range , the procedure might take between 30 and 60 minutes . This allows measurement series with a measurement frequency of at least 1 measurement per 60 minutes , in particular per 40 minutes , in particular per 30 minutes between the measurement of two samples taken from the fluid system . One single measurement device can monitor a fluid system with a very high time resolution . A detailed monitoring of the fluid system is possible . In particular, the monitoring system is capable of automatically measuring the total cell concentration and the intact cell concentration for every fluid sample taken from the fluid system . Advantageously, the total cell concentration and the intact cell concentration are measured by two subsequent measurements . Subsequent means directly one after the other or with a delay of a maximum of 15 minutes .

Additionally, the monitoring device can be equipped to measure a cell concentration of probiotics inside the fluid sample . This allows to determine the ratio between probiotics and other cells , in particular other intact cells . Such a monitoring device can be used to monitor a fluid system to which probiotics are added .

In particular, at least the dilution device , the dyeing device and the flow cytometer are cleaned before repeating steps a ) to e ) .

Advantageously, the sample-dye mixture is incubated by an incubator before performing step d) . Consistent measurements over time are only possible i f every measurement is performed under equal condition . For example , the sample-dye mixture could be incubated for 10 minutes at 37 ° C .

In a preferred method, the dilution ratio can be varied within a range of at least 10 , in particular of at least 10³, in particular of at least 10⁵, in particular within a range of at least 10⁷ . High dilution ratios are achieved by serial dilution . A serial dilution can automatically be performed by the monitoring device .

In particular, the monitoring device is a small , portable device with a casing . The sample collecting device , the dilution device , the dyeing device and the control device are arranged inside said casing . The casing might have a si ze of less than 1 m³ .

In a series of measurements , the control device performs step b ) by using the dilution ratio of the last measurement which led to a measurement in the valid concentration range . This increases the chance that already the cell concentration of the first measurement lies inside the valid concentration range and no repetition of steps a ) to e ) are required .

Since the monitoring device allows high frequency measurements , the fluid system can be monitored in real time . The fluid system can be controlled by dosing additives into the fluid system wherein the monitoring device immediately allows to measure the impact of the additives on the fluid system . In particular, the additive is a biocide , fresh water or probiotics .

Depending on the measured cell concentration, the control device can automatically dose an additive into the fluid system, or an additive is added manually or by an external dosing device latest one hour, in particular latest hal f an hour, after the measurement data were available .

Other advantageous embodiments are listed in the dependent claims as well as in the description below .

Brief Description of the Drawings

The invention will be better understood and obj ects other than those set forth above will become apparent from the following detailed description thereof . Such description makes reference to the annexed drawings , wherein :

Fig . 1 shows a schematic illustration of a monitoring device and a fluid system;

Fig . 2 shows a flow chart of a method for monitoring cells , in particular bacteria, in the fluid system by the monitoring device of Fig . 1 ;

Fig . 3 shows a plot with measurements inside and outside a valid concentration range of a flow cytometer ;

Fig . 4a and 4b show plots of one single valid measurement , each . The X and Y axis represent green and red fluorescence intensity on a logarithmic scale respectively; and

Fig . 5 shows a plot of measurements over several days .

Mode for Carrying Out the Invention

Fig . 1 shows a fluid system 1 which is monitored by the monitoring device . The fluid system could be a river, an aquaculture , cooling water or a paper factory, etc . The monitoring device comprises a syringe plunger pump 2 in fluid communication with a multiport pump head 3 . The syringe of the syringe plunger pump 2 provides a mixing container for mixing di f ferent fluids . The multiport pump head connects the syringe plunger pump 2 with di f ferent inlet and outlet ports . The plunger of the syringe plunger pump 2 operates in a vertical direction 4 and moves away from the multiport pump head 3 or upwards when loading fluids or gases into the syringe and moves towards the multiport pump head 3 or downwards when emitting a fluid from the syringe .

The multiport pump head 3 is in fluid communication with the fluid system 1 via a fluid collecting connection 5 . The pump 2 pumps a fluid sample from the fluid system 1 into the syringe of the syringe plunger pump 2 via the fluid collecting connection 5 and the multiport pump head 3 . One fluid sample has an exemplary volume of 1 ml . The pump 2 , the multiport pump head 3 and the fluid collecting connection 5 form a sample collecting device as defined in the claims .

The multiport pump head 3 is connected with a diluent source 6 . The pump 2 pumps diluent from the diluent source 6 into the syringe of the syringe plunger pump 2 and mixes the diluent with the fluid sample from the fluid system 1 inside the syringe of the syringe plunger pump 2 . The pump 2 , the multiport pump head 3 and the diluent source 6 form a dilution device as defined in the claims . Alternatively, the diluent is mixed with the fluid sample inside a separate mixing container .

Furthermore , the multiport pump head 3 is connected with a dye source 7 . The pump 2 pumps the dye from the dye source 7 into the syringe of the syringe plunger pump 2 and mixes the diluted or undiluted fluid sample with the dye inside the syringe of the syringe plunger pump 2 or inside a mixing container . The pump 2 , the multiport pump head 3 and the dye source 7 form a dyeing device as defined in the claims .

After every measurement , the monitoring device has to be cleaned . The multiport pump head 3 is connected to a reagent source 8 . Additionally, the multiport pump head 3 is connected to a gas source 9 . The syringe plunger pump 2 draws a gas bubble into the syringe wherein the gas bubble helps mixing the fluid inside the syringe .

The monitoring device comprises an incubation device 10 . The syringe plunger pump 2 trans fer the sample-dye mixture from the syringe to the incubation device 10 via the multiport pump head 3 . The sample-dye mixture is incubated inside the incubation device 10 to a certain temperature , for example 37 ° C for a duration of 10 minutes . This ensures a stable and optimal dyeing of all cells . Having every mixture to be incubated on the same temperature and during the same time span allows consistent measurement results .

All , the syringe plunger pump 2 , the multiport pump head 3 , the incubation device 11 and the measurement device are arranged inside a casing 12 . Therefore , the sample collecting device , the dilution device and the dyeing device are at least partially arranged inside the casing . The diluent source 6 , the dyeing source 7 and the reagent source 8 are arranged outside the casing 12 . In an alternative embodiment they could be arranged inside the casing 12 . The casing might have a si ze of 0.5 m³. It is a small, portable casing which can easily be arranged in a factory.

Finally, after incubation, the incubated sample-dye mixture is drawn by the syringe plunger pump 2 from the incubation device 10 back into the multiport pump head 3 and is transferred to the measurement device, which is a flow cytometer 11 (e.g. Accuri C6, BD, USA; CytoFlEX, Beckman-Coulter, USA; NovoCyte, ACEA Biosciences, USA) .

A control device 13 equipped with a software and arranged inside the casing 12 controls at least the syringe plunger pump 2, the multiport pump head 3, the incubation device 10 and the flow cytometer 11. This is a complete setup for a fully automated monitoring of the fluid system 1. A more detailed description of such a system is available in WO 2018/138023.

Additionally, a dosing system 15 is available. Two different probiotics are arranged in containers 16 and 17 and can be added to the fluid system, either manually or directly controlled by the control device 13.

Fig. 2 shows a flow chart of a measurement performed by the monitoring device according to Fig. 1. The process starts with step SI cleaning the monitoring device with detergent from detergent source 8. In a second step S2, the syringe plunger pump 2 draws a fluid sample from the fluid system 1 via the fluid collecting connection into the syringe. In a third step S3, the syringe plunger pump 2 draws diluent from the diluent source 6 into the syringe. A gas bubble, in particular air, is drawn into the syringe which mixes the fluid sample and the diluent. In the fourth step S4, dye, for example a fluorescent marker, is drawn into the syringe from the dye source 7. Again, a gas bubble is drawn into the syringe which mixes the fluid sample, the diluent and the dye. A diluted sample-dye mixture is provided. In Step S5, the sample-dye mixture is transferred into the incubation device 10 and is incubated in one of the incubation cuvettes. Finally, in step S6, the incubated sample-dye mixture is transferred to the flow cytometer 11.

The cells inside the sample-dye mixture are measured by the flow cytometer 11. The control device 13 postprocesses the measurement data in step S7. A 2D scatter plot for different detection channels, for example for green and red fluorescence, is generated. The scatter plots are interpreted and particles of interest are selected. The total cell concentration, the intact cell concentration or the cell concentration of probiotics can be identified, for example. These different concentrations can by evaluated during one single measurement cycle or during separate measurement cycles. For example, a first measurement is performed to measure only the total cell concentration. The cells are dyed only by SYBR Green. This dye penetrates all cells. Subsequently, a second measurement is performed. The cells are dyed with a mixture of SYBR Green and Propidium Iodide. Propidium Iodide enters only damaged cells. The intact cell concentration is measured and the concentration of probiotics is identified. The dyes can be diluted in sterile TRIS buffer .

After having determined the measured cell concentration, the control device 13 checks in step L9 whether the measured cell concentration lies within a valid concentration range. The valid concentration range is predefined and depends on the capabilities of the flow cytometer. If the measured cell concentration lies above or below the valid concentration range, the measurement results are expected to be invalid. If the concentration is too high, it is expected that the flow cytometer could not count every single cell. If the concentration is too low, an unprecise measurement is expected.

If the measured cell concentration is outside the valid concentration range, the control device adjusts the dilution ratio, for example by a factor 10, in step CIO and repeats steps SI until S7 with the new dilution ratio. Step SI until S7 are repeated until the measured cell concentration lies inside the valid concentration range .

If the cell concentration lies inside the valid concentration range, the measured cell concentration is re-converted by the dilution ratio and the actual cell concentration of the fluid sample is determined in step Sil.

In step L12, the control device checks if the cell concentration of the fluid sample lies in an expected range. If not, an additive, in particular a biocide, fresh water or probiotics, can be added to the fluid system to stabilize the process operating in the fluid system.

A single measurement operation illustrated in Fig. 1 can be completed within a time interval between 20 and 40 minutes, depending on the number of repetitions of steps SI to S7. This allows to perform a new measurement cycle every 20 minutes or every 40 minutes, for example. A real time monitoring of the fluid system is possible.

Fig. 3 shows three subsequent measurement cycles. The three measurement cycles are separated by the vertical dashed lines. The horizontal dashed lines illustrate the upper and lower limit of the valid concentration range. The Y-Axis shows the total cell concentration of diluted sample-dye mixtures.

The first measurement cycle (the first three black dots) starts with a first measurement (first black dot) measuring a cell concentration lying clearly outside the valid concentration range. The dilution ratio is adjusted in step CIO and steps SI to S7 are repeated. The cell concentration of the second measurement is also outside the valid concentration range. The dilution ratio is adjusted again and steps SI to S7 are repeated. The cell concentration of the third measurement lies within the valid concentration range . The measurement data can be finally postprocessed in step S i l .

A second measurement cycle follows 45 minutes after the first measurement cycle . The time interval of 45 minutes between the first measurement and the second measurement is illustrated by double arrow Tl . The second measurement cycle starts with the dilution ratio of the third measurement of the first measurement cycle . Anyway, the first measurement of the second measurement cycle lies outside the valid concentration range . Steps S I to S7 are repeated with an adj usted dilution ratio . The second measurement of the second measurement cycle is valid . 15 minutes later, a third measurement cycle starts . Already the first measurement of the third measurement cycle lies inside the valid concentration range . A repetition of steps S I to S7 is not required .

Fig . 4a and 4b show exemplary scatter plots which are generated in step S7 . The plots show the measured cells of one measured fluid sample . Every black dot is one single cell detected by the flow cytometer . The X- axis represent green fluorescence intensity on a logarithmic scale and the Y-axis represents red fluorescence intensity on a logarithmic scale . In Fig . 4a, the gated area, framed by the dashes line , indicates the region where the cells are located on the plot . The black dots outside the gated area represent background particles . Counting the cells inside the gated area leads to the total cell concentration ( TCC ) . In Fig . 5a, the gated area indicates the region where intact cells are located . The number of measured cells inside the gated area leads to the intact cell concentration ( ICC ) .

In Fig . 4b, the cells inside the gated area allow the identi fication of three clusters marked by circles . The cluster bottom left illustrates cells of a first strain of probiotics which were added to the fluid system as additive . The cluster bottom right illustrates a second strain of probiotics which were added to the fluid system. The cluster top right illustrates other intact cells which are not probiotics.

Fig. 5 shows a plot of measurement data generated over five complete days. The days are marked by the time intervals dl, d2, d3, d4, and d5. Every shown time interval has a duration of 24 hours. The upper measurement data shows the total cell concentration (TCC) . The lower measurement data shows the intact cell concentration (ICC) . Every black dot represents a cell concentration determined during one single measurement cycle. A measurement cycle is performed every 15 minutes.

Every day, at 6:00 p.m., a biocide is added to the fluid system. These steps are marked by vertical dashed lines. After having added the biocide to the fluid system, the measured cell concentration immediately decreases. The variation of the cell concentration can be monitored in real-time.

Claims

1. Method for monitoring cells, in particular microbial cells, in particular bacteria, microalgae or yeast, in a fluid system (1) by a monitoring device comprising the following steps controlled, in particular automatically controlled, by a control device (13) : a) collecting a first fluid sample by a sample collecting device (2,3,5) , b) diluting the first fluid sample with a first dilution ratio by a dilution device (2,3, 6) , c) measuring a cell concentration in the first fluid sample by a measurement device (11) , in particular a flow cytometer.

2. Method according to claim 1, wherein

- the method does not comprise a step of dyeing the first fluid sample, or

- the method comprises a step of dyeing the first fluid sample by a dyeing device (2, 3, 7) , wherein the step of dyeing and the step of diluting the first fluid sample are separate steps.

3. Method according to any one of the preceding claims, wherein step b) of diluting the first fluid sample comprises at least two serial dilution steps.

4. Method according to any one of the preceding claims, wherein step b) comprises the substep where a user defines the first dilution ratio.

5. Method according to any one of the preceding claims, comprising the following steps controlled by the control device (13) : d) determining if the measured cell concentration lies within a valid concentration range by the control device, e) if the measured cell concentration lies outside the valid concentration range, the control device (13) automatically repeats steps a) to c) with a second fluid sample, wherein the second fluid sample is diluted with a second dilution ratio, which is different to the first dilution ratio.

6. Method according to claim 5, wherein steps a) to e) are automatically repeated by the control device (13) until the measured cell concentration lies inside the valid concentration range, completing a valid measurement and providing valid measurement data.

7. Method according to any one of the preceding claims, wherein a measurement with a new sample from the fluid system (1) is performed at least every 60 minutes, in particular at least every 40 minutes, in particular at least every 30 minutes.

8. Method according to any one of the preceding claims, wherein a measurement is completed and the measurement data is available latest 60 minutes after starting with step a) , in particular latest 45 minutes after starting with step a) , in particular latest 30 minutes after starting with step a) .

9. Method according to any one of the preceding claims, wherein a first valid measurement is performed for measuring a total cell concentration, and a second valid measurement is performed for measuring an intact cell concentration, in particular wherein the first and the second valid measurements are performed directly one after the other or with a delay of a maximum of 15 minutes, in particular of ten minutes.

10. Method according to any one of the preceding claims, wherein a cell concentration of probiotics is measured.

11. Method according to claim 10, wherein cell concentrations for different strains of probiotics are measured.

12. Method according to any one of the preceding claims, wherein in step e) at least the dilution device, the dyeing device and the measurement device (11) are cleaned before repeating steps a) to c) .

13. Method according to any one of the preceding claims, wherein the valid concentration range defines a range in which the measurement device can measure each individual cell.

14. Method according to any one of the preceding claims, wherein the sample-dye mixture is incubated by an incubator (10) before performing step d) .

15. Method according to any one of the preceding claims, wherein the dilution ratio can be varied within a range of at least 10, in particular of at least 10³, in particular of at least 10⁵, in particular within a range of at least 10⁷.

16. Method according to any one of the preceding claims, wherein the method with steps a) to f) is automatically controlled by the control device (13) .

17. Method according to any one of the preceding claims, wherein the sample collecting device (2,3,5) and the dilution device (2,3, 6) , and in particular the dyeing device (2,3,7) , the control device (13) and/or the measurement device (11) , are at least partially arranged in one single casing ( 12 ) , in particular wherein the casing has a si ze of less than 1 m³, in particular less than 2 m³, in particular wherein a diluent source or a dyeing source are arranged outside the casing .

18 . Method according to any one of the preceding claims , wherein the method is applied in a paper factory, in aquacultures , in cooling water systems , in bioprocessing, in particular in a probiotics production system, in a food factory, for fermentation, in hydroponic or aquaponic systems , or in a biofuel factory .

19 . Method according to any one of the preceding claims , wherein a new measurement cycle is started with the dilution ratio of the last valid measurement .

20 . Method according to any one of the preceding claims , wherein depending on the measured cell concentration,

- the control device ( 13 ) automatically doses an additive into the fluid system, or

- an additive is added manually, or

- an additive is added by an external dosing device , in particular latest one hour, in particular latest hal f an hour, after the measurement data were available .

21 . Method according to claim 20 , wherein the additive is biocide or fresh water .

22 . Method according to claim 20 , wherein the additive is probiotics .