CH670653A5 - - Google Patents

Description

DESCRIPTION

The present invention relates to an automatic device for detecting and counting coliform bacteria in an aqueous medium with a view, in particular, to bacteriological control of water.

The bacteriological control of water is a necessity for the health protection of populations, particularly with regard to:

- water intended for human consumption (drinking water) and animal water,

- water intended for direct or indirect food uses (used in the food industries),

- The waters of aquatic environments used for shellfish farming (shellfish farming) or aquaculture,

- swimming pool water,

- the waters of the natural aquatic environment devoted to leisure, swimming, etc.,

- discharges of more or less purified wastewater, especially in areas sensitive to pollution.

Control is all the more necessary as surface waters (rivers, lakes, ponds) are increasingly used for the preparation of drinking water and since these surface waters are very vulnerable to discharges contaminated bacteriologically (domestic and urban discharges, slaughterhouse rejections, etc.).

Faced with the difficulty of isolating and counting pathogenic bacteria, capable of causing human or animal diseases, we classically search for bacteria known as “test bacteria for faecal contamination”, the presence of which makes it possible to suspect that of pathogenic germs which are same origin (faeces) and generally accompany them. These test germs were also chosen on the basis of their ease of determination by the methods of conventional bacteriology.

Coliform bacteria meet the criteria for defining test germs and are conventionally used as such. In addition, certain bacteria of this type, of the species Escherichia coli, are potentially pathogenic and their identification brings an additional presumption of non-potability. Other bacteria can also be used as faecal contamination test germs 5 (faecal streptococci, among others).

The problem posed by the application of conventional bacteriological methods to the evaluation of the degree of bacterial contamination of water is the length of the laboratory determinations. This duration is in fact incompressible, owing to the fact that these methods include bacterial cultures, on appropriate nutritive media, requiring long-term incubations before reading the result.

As an indication, the conventional methods for counting coliform bacteria or Escherichia coli require at least 24 hours (method with membrane concentration) in the case of the analysis of poorly contaminated water (such as drinking water or treated water),

while the method used for contaminated water (river water type) requires two successive 48 h incubations to determine the coliform bacteria and Escherichia coli (method in liquid medium, called "most probable number").

20 This high response time constitutes a definite handicap when water quality control is carried out with a view to preventive or curative action (prohibition of the use of contaminated water, modification of the operating conditions of a treatment facility). Indeed, irreparable damage to public health 25 can be caused between the start of bacterial pollution of water and knowledge of the result of the control analysis.

Another drawback of these methods is that they are purely manual and that they cannot lend themselves to complete automation (carrying out analyzes continuously or sequentially at high speed without any human intervention).

The aim sought during the implementation of the invention is:

- to allow a very significant reduction in the response time of bacteriological control analyzes of water by identification and counting of bacteria, in particular coliforms and / or Es-

35 cherichia coli, and, if possible, reduce this duration from several days to a few hours;

- to allow complete automation, making practically continuous control possible.

At the laboratory level, the only currently known way 40 of shortening the response time of the methods of conventional bacteriology consists in carrying out a filtration of the liquid sample on a membrane whose pore size is smaller than that of bacteria (in general , pores of 0.4 micron in diameter), thus concentrating all of the bacteria present on the surface of the membrane 45 and carrying out thereon, by providing an adequate nutritive medium, a culture which, after rapid incubation, will identify the presence and count the bacteria specifically sought.

This technique has three serious drawbacks:

- the diameter of the pores of the membrane necessary for retention

50 tion of bacteria is so small that these pores can be quickly clogged by solid particles of small size, of non-bacterial nature (colloidal particles of clay, silts, hu-mics, plant or animal debris, etc.), this clogging preventing filtration from continuing and therefore making the method impractical; 55 - the number of bacteria separated by filtration and retained per unit area of the membrane may be too high, making it impossible to identify separately the bacterial colonies growing during incubation and, consequently, the enumeration of these bacteria;

60 - the preconcentration of bacteria on the membrane being irreversible, the latter cannot return to its initial state and be reused. We could design automated membrane renewal systems, for example by unwinding a strip of filtering material, but at the cost of great mechanical complexity and with a significant risk of contamination of the new membrane by that which has already been used.

The method described above, because of these drawbacks, cannot therefore be automated conveniently.

3

670,653

In order to solve the problem, new methods based on completely different principles have been developed. These are essentially instrumental methods, and in particular the electrochemical method for detecting coliform bacteria and Escherichia coli developed by Wilkins et al. ("Micro-bial detection method based on sensing molecular hydrogen", Applied Microbiology, vol. 27 , N ° 5, May 1974, pages 949-952, JR Wilkins, GE Stoner, EH Boykin; "Electrochemical method for early detection and monitoring of coliforms in water", JAWWA, May 1976, pages 257-263, JR Wilkins and E Boykin; "Use of plati-num electrodes for the electrochemical detection of bacteria", Applied and Environmental Microbiology, 1978, vol. 38, pages 683-687, Wilkins Jr.) which counts these bacteria by measuring the latency time at the end from which a culture carried out by mixing the sample studied with a suitable nutrient medium produces detectable molecular hydrogen using a platinum electrode whose potential is constantly compared with that of a reference electrode rence (eg calomel or silver chloride). We can calculate, from the knowledge of the latency time, the initial population of bacteria (bacterial count) contained in the water sample studied by assuming an exponential growth of the population, growth whose characteristics (coefficient of the law) are checked and measured by calibration with a bacterial suspension of known count.

The problem we posed is the development of a fully automatic device allowing the implementation of the electrochemical method of detection and counting of coliform bacteria and Escherichia coli contained in a water sample, described below. -before.

To this end, the subject of the invention is an automatic device for detecting and counting coliform bacteria in an aqueous medium, in which the presence of these bacteria is detected in a sample of water to which an adequate nutritive medium is added, by presence of molecular hydrogen generated by these bacteria and detected by means of a platinum electrode whose potential is constantly compared to that of a reference electrode, this device comprising at least one incubation vessel closed by a cover, provided with an external heating resistance, a combined potential measurement electrode and a thermistor for controlling and regulating the heating of the tank, while inside the tank, carried by the cover, are arranged three injection pipes and a level detector.

The nutrient medium used in the above device can be chosen from the usual culture media for bacteria based on peptone, lactose and meat extract, such as those available commercially, for example: BCP lactose broth ( Institut Pasteur), lactose broth biliated in brilliant green (Difco), Brain-Heart Infusion (Biomérieux), Lactose Broth (Difco), EC Medium (Difco), Lauryl-Tryptox Broth ("LTB") (Difco).

Indeed, after studying the influence of substances capable of modifying the metabolism of hydrogen in bacteria, we have now found that the use of a formate in the culture medium allows an unexpected increase in biological activity of culture and, as a result, better results in bacteria detection and counting devices.

The invention is set out below in detail with the aid of the appended drawings representing an embodiment, given by way of nonlimiting example, drawings in which:

- fig. 1 is a schematic view of the automatic device which is the subject of the invention;

- fig. 2 is a block diagram of the electronic circuit of an embodiment of the automatic device which is the subject of the invention.

The device is supplied with water to be controlled by a tube (not shown) connected either to a pipe for transporting this water, or to an aquatic medium. At its input, it includes a pressure regulator 1 delivering the water to be checked at a determined pressure.

The device comprises two identical incubation tanks 2 (see fig. 1) each provided with a cover 3. Inside each of the tanks 2, supported by the covers 3 which they pass through by sealed passages, are arranged :

s - a combined electrode 4 platinum / reference electrode (calomel or silver chloride reference electrode) provided with an electrolyte reservoir 5 placed in charge outside of the tanks 2;

- three injection pipes 6, 7 and 8;

- a thermistor 9 for controlling and regulating the heating of the tanks 2;

- a level 10 detector.

The lower half of each of the tanks 2 is surrounded by an electrical heating resistor 11, and the lowest part of each of the tanks 2 is provided with a drain pipe 12. The injection pipes 6, 7, 8, as well as the drainage pipe 12, are provided with solenoid valves of the pipe pinch type, respectively 13,14,15,16, allowing the flow or the stop of the flow of liquid in the corresponding tubes.

The injection pipe 6 serves to supply the tanks 2 with water 20 to be checked and it is connected to the pressure regulator 1, itself connected to the source of water to be checked; the injection tubing 7 is connected to a reservoir 17 containing a sterilizing agent, such as a concentrated solution of bleach (48 chlorometric degrees); the injection tube 8 is connected to a reservoir 18 containing an aqueous solution of a culture medium containing peptone, lactose, meat extract and mineral salts. The tanks 17 and 18 are placed under load outside the tanks 2.

Finally, the covers 3 of the tanks 2 are fitted with an overflow discharge pipe 19.

All the connecting pipes are made of flexible material preventing the attachment of bacteria, and the solenoid valves are of the pinch type of pipe to avoid any bacterial contamination of external origin.

Each of the combined electrodes 4 is connected (see FIG. 2) via an impedance adapter 20 to the input of a multiplexer 21, while each of the heating control and regulation thermistors 9 and each of the level detectors 10 are connected directly to the input of this same multiplexer 21. The output of the multiplexer 21 is connected to the input of an analog-40 as / digital 22 converter, the output of which is connected to the input of a microprocessor card 23 to which a clock 24 and coding wheels 27 are connected.

The microprocessor card 23 controls the opening and closing of the solenoid valves 13, 14, 15, 16, as well as the starting and stopping of the heating resistor 11 of the incubation tanks 2. It is also connected to a printer 25, which registers the results of the bacterial count, and to an alarm 26 provided with indicators and optical counters, alarm programmed to be triggered when the result of the count is greater than a fixed value. In addition, the microprocessor card 23 controls the different sequences in automatic operating mode.

The device can also be controlled manually using the coding wheels 27.

The nutrient medium placed in the device below contains two 55 groups of substances:

- the substrate (food for bacteria), which ensures the growth function. The presence of these substances - or analogous molecules - is necessary. These are lactose, peptone, meat extract. Lactose is the most important because it contributes

60 bue to the specificity of the measurement. The use of these substances is conventional in the media used in the laboratory, and

- substances which have the effect of accelerating the response of the bacteria detection and counting device, without it being known precisely whether they act on the growth rate of

65 bacteria, the production of hydrogen by bacteria or the response of the sensor (platinum electrode) by action on the electrochemical system. These include sodium chloride and formate.

In the case of formate, what is important is the formate anion.

670,653

4

The simultaneous presence of all these substances, in particular at the concentrations indicated below, is necessary to obtain good results:

- peptone, lactose and meat extract 10 to 20 g / 1. The effectiveness increases with the concentration until that of the mixture reaches approximately 15 to 20 g / l of the mixture. If you use more, the result is no better. If you use less, it is less good. For example, for a typical test, this response time is increased by 10% if only 10 g / 1 is used and by 20% if only 5 g / 1 is used;

- the same is true for sodium chloride, the effect of which becomes saturated above a concentration of 5 g / 1. If we only use

2 g / I, the response time increases by 10%, and it increases by 20% if not used; preferably 3 to 10 g / l will be used;

- the situation is different for formate, where there is an optimum concentration which is at a concentration of around 0.5 g / 1. The response time increases by 10% if 0.15 g / 1 or 1.25 g / 1 is used. It increases by 20% if we do not use it or if we use 2 g / 1. We can therefore consider that the optimal range is between 0.4 and 0.6 g / 1.

The concentrations found optimal in the cell during the measurement are as follows:

- peptone 7.5 g / 1

- lactose 7.5 g / 1

- meat extract 4.5 g / 1

- sodium chloride 5.0 g / 1

- sodium formate 0.5 g / 1

We recommend a concentrate of the culture medium, for example 400 g / 1, in liquid form (thanks to the high solubility of the substances used), easy to add and to dose, allowing a significant autonomy of the apparatus with a capacity of low volume reagent. By way of example, the composition of the concentrated medium can be as follows:

- lactose 150 g

- peptone 150 g

- meat extract 90 g

- sodium chloride 100 g

- sodium formate 10 g

- water 11

The addition of these constituents results in the production of 1.25 1 of solution. This concentrated solution corresponds to the proportions given above for the optimal concentrations.

The packaging of this concentrated solution in a plastic bag (blood bag type) allows sterilization of the culture medium directly in the bag, thanks to irradiation by y-rays. The sterilized medium is never in contact with the outside and cannot be contaminated bacteriologically. On the other hand, its high salinity, due to the high concentration used, would reduce the risks of alteration of the medium (fermentation and / or alteration of the results) in the event of accidental bacterial contamination of the medium.

This culture medium was compared with the medium used by Wilkins and with the base medium (lactose broth from the Pasteur Institute) under strictly identical conditions (salinity of the water studied) in the device described above. The results are as follows:

Response time for 1 coliform bacteria / 100 ml

Use of Difco LTB medium (Wilkins)

1110min * (6.30 p.m.)

Use of the base medium (Pasteur lactose broth)

1050 min (5.30 p.m.)

Use of the medium of the invention (lactose broth + NaCl + Na formate)

600 min (10 h)

* This apparently high time compared to that indicated by Wilkins in his publications can result both from the influence of the mineralization of the water studied and especially from the choice of the jump in the potential of the platinum electrode considered as an indicator of the appearance of hydrogen specific to the presence of coliform bacteria (200 mV). The time saving which results from the choice of a much lower potential jump (10 to 30 mV) is compensated by a significant loss of selectivity with respect to coliform bacteria. There is indeed a possibility of artifacts if one takes as a selection criterion a jump of too weak potential.

This culture medium therefore allows either a gain in response time of 46% compared to the medium used by Wilkins, with identical selectivity and reliability of result, or a gain in reliability of the result with identical response time. In addition, the stability and compatibility of the basic reagents has promised to develop a highly concentrated culture medium, whose low consumption (6 ml / analysis) allows a high operating autonomy for the device (2 months) without recharging. of reagents. The concentrated nutrient medium can be packaged in a sterile deformable pouch of 2.81.

The operation of the automatic device which is the subject of the invention is described below. The device is connected to the water source whose quality is to be controlled. It is assumed that a measurement sequence has just ended. The incubation tanks 2 are clean, but not sterile, and filled with water containing a small amount of bleach to prevent bacterial growth. The combined electrodes 4 are immersed to prevent their deterioration. When a new measurement cycle begins, one of the two tanks 2 is emptied of its contents, then rinsed with water. It is then sterilized. To do this, the tank 2 is completely filled with water through the tubing 6, and bleach at 48 chlorometric degrees is injected through the tubing 7, the solenoid valves, respectively 13 and 14, being open. The quantity of bleach injected is adjustable by the programmable opening time of the solenoid valve 14. The contact time to ensure correct sterilization of the tank 2 is of the order of 20 min. The tank 2 is then emptied, the solenoid valve 16 being opened, then rinsed for a long time with water, for approximately 30 min, to remove all traces of sterilizing agent and avoid interference on the measurement. After rinsing, a certain amount of solution of a culture medium is injected into the tank 2 through the injection tube 8, the amount to be injected being adjusted by a programmable opening time of the solenoid valve 15. Finally, 100 ml of the water to be checked are injected into the tank 2 through the injection pipe 6, the solenoid valve 13 being open. The quantity of water injected is determined by the level probe placed in the tank 2. The incubation phase then begins with a gradual heating of the contents of the tank 2, in order to avoid a thermal shock harmful to bacterial growth, until the temperature reaches 44 ° C. When this temperature is reached, the device performs a periodic measurement (several times per second) of the potential provided by the combined electrode 4. The values of the potential, after amplification and calculation of a floating average, are compared with programmed threshold values.

If, during the measurement cycle, the potential detected by the combined probe 4 is greater than a programmed threshold value, the microprocessor card 23 triggers the alarm 26, calculates the number of coliform bacteria present in the sample by reference to a previous calibration and displays the result with the date and time on the printer 25. The alarm threshold is adjustable by means of the encoder wheels 27. In addition, a similar measurement cycle is then automatically triggered, for confirmation, in the second tank 2 which was on standby.

The potential threshold causing the calculation sequence for the number of coliform bacteria present in the sample taken is adjustable. A low value, a few tens of millivolts, saves time on the response time, but increases the risk of error, because certain phenomena, biological, due to the presence of other bacteria, and possibly physico-chemical, are likely to derive the measured potential. So that a

5

10

15

20

25

30

35

40

45

50

55

60

65

5

670,653

such drift is not assimilated to the consequence of the production of hydrogen, it is preferable to adjust the potential threshold to a clearly higher value, of the order of 200 mV, for example.

At the end of each measurement cycle, and to avoid contamination of the downstream drainage network, the sample is decontaminated by injection of bleach. Then the tanks are emptied, rinsed and filled with water slightly added with sterilizing agent. The cells are then ready for a new measurement cycle.

The automatic measuring device which is the subject of the invention has the following advantages:

- it allows direct sampling from a water pipe;

- it allows periodic sampling and analysis to be carried out at a frequency determined both by the quality of the water controlled (high frequency in the case of highly contaminated water)

and by the number of incubation tanks involved, the device comprising at least one tank, but which may include several similar tanks, for example two in number according to the preferred embodiment; 20

- possibility of automatically monitoring the microbiological quality of water without other human intervention than periodic maintenance consisting in recharging the electrolyte reservoirs of the combined probe, sterilizing agent, solution of nutritive medium;

- possibility of varying the duration of a measurement cycle by programming the microprocessor card, the duration of a cycle possibly varying from 5 to 24 hours;

- automatic start of a confirmation measurement cycle in a second tank when bacterial contamination is detected during a measurement in the first tank;

- operating autonomy of 2 months, the electrolyte reservoirs of bleach and nutritive medium solution having a sufficient volume to cover this period without being recharged;

- triggering of alarms in the event that predefined and programmed contamination thresholds are exceeded, these alarms can either cause human intervention or automatically trigger immediate curative actions, such as an increase in the chlorinating agent used;

- great job security, the microprocessor card triggering an alarm in the event of:

- lack of water supply,

- tank temperature outside the adjustment range,

- failure to inject the nutrient medium,

- failure to inject sterilizing agent;

- operation possible with only one incubation tank in service, maintenance operations can then be carried out on the other tanks;

- a low production cost making the automatic device accessible to local authorities;

- great simplicity of implementation thanks to the automation functions.

The device according to the invention finds various applications such as:

- control of systems for the collection, treatment, storage, distribution of drinking water as well as the production and packaging chains of food liquids;

- sanitary control of water for therapeutic use;

- control of swimming pools and leisure facilities using water;

- control of the sanitary quality of bathing water (fresh and marine water);

- control and protection of fish farming, shellfish farming, aquaculture and all areas sensitive to micro-biological contamination;

- continuous monitoring of industrial waters.

30

R

2 sheets of drawings

Claims (5)

670653 1. Automatic device for detecting and counting coliform bacteria in an aqueous medium, in which the presence of these bacteria is detected in a sample of water to which an adequate nutritive medium is added, by the presence of molecular hydrogen generated by these bacteria and detected by means of a platinum electrode whose potential is constantly compared with that of a reference electrode, this device comprising at least one incubation tank (2), closed by a cover (3), provided with '' an external heating resistor (11), a combined electrode (4) for measuring potential, a thermistor (9) for controlling and regulating the heating of the tank (2), characterized in that at 'inside the tank (2), carried by the cover (3), are arranged three injection pipes (6,7, 8) and a level detector (10). 2. Device according to claim 1, characterized in that each incubation tank (2) is provided at its lowest part with a drain (12). 2 3. Device according to claims 1 and 2, characterized in that the cover (3) of each incubation tank (2) is provided with a tube (19) for overflow discharge. 4. Device according to one of claims 1 to 3, characterized in that the injection pipe (6) of the water to be checked from each tank is connected to a pressure regulator (1), itself connected by tubing to the water source to be checked. 5. Device according to any one of claims 1 to 4, characterized in that the combined electrode (4) of each tank (2) is provided with a reservoir (5) of electrolyte and that the injection pipes (7) and (8) of each tank (2) are respectively connected to a tank (17) containing a sterilizing agent and to a tank (18) containing a solution of a culture medium for bacteria, the tanks (5, 17,18) being placed under load outside the incubation tank (2).