NZ539210A - Optical micro-organism analysis using change of light to detect organisms - Google Patents

Abstract

A vial 103 is placed in an incubator, typically with a heating element. A light source 202 transmits light into the vial and a sensor 102 detects changes over time of light of at least one colour.

Description

53921 § 400081NZ_DPCS_101.doc - 1 - Optical Micro-organism Analysis Technical Field The invention generally relates to the optical analysis of bacterial growth.

More particularly the invention relates to the analysis of micro-organisms in a fluid or 5 other medium.

Background Art It is known to measure the levels of bacteria in fluids such as drinking water by placing a sample of the fluid in a test cell with a dye or indicator such as Resazurin or methylene blue with optionally a nutrient medium and incubating the sample at a set 10 temperature for a minimum time. A change in dye or indicator colour indicates the presence of bacteria as the growth reduces or otherwise reacts with the indicator chemical. It is also known to add suppressants to the sample for micro-organisms other than that being tested for.

Typically the colour change is monitored by eye and the incubation process takes from 15 14 to 48 hours. Some bacterial strains have a relatively high temperature sensitivity compared to others and the temperature may need to be maintained very close to a specific temperature in order to promote the relative growth of the species required to be detected. Thus, for instance, in some media, if it is desired to culture for the presence or absence of e.coli the required temperature of incubation may be 45°C, while if all coliform bacteria are being monitored the temperature is best set at 37°C.

The colour change is therefore a value judgement by eye, and may take considerable incubation time before it can be done.

While instrumental optical measurement of the medium colour is known the instruments for doing this are normally laboratory level instruments and are largely 25 unsuitable for use in the field and are generally not suitable for use in the field by those unskilled in the art of microbiology.

These problems increase the cost of obtaining a qualitative solution to the measurement of bacteria levels and provide no ability to realise a short term result. 400081NZ_DPCS_101.doc The present invention provides a solution to these and other problems which offers advantages over the prior art or which will at least provide the public with a useful choice.

Summary Of The Invention The invention relates to a micro-organism detection apparatus consisting of: a fluid container with at least one light path through container and contained fluid a container incubator at least partially surrounding a container receiving space a light source transmitting light into the fluid container and fixed in relation to the container incubator a light sensor fixed in relation to the container incubator and detecting light of at least one colour which has passed through the fluid from the light source, a comparator detecting changes over time in the light transmission of at least said one colour.

Preferably the sensor is an integrated circuit optical sensor.

Preferably colour from at least red, green and blue light is sensed.

Preferably the transmission change is as a result of a change in the colour of a substance in the fluid.

Preferably the colour change in transmitted light is monitored as a function of time.

Preferably the incubator has a heating element.

Preferably the heating element is controlled to maintain the incubator temperature substantially constant.

Preferably the light detected is transmitted light.

Preferably the light detected is light reflected from the light source by the contents of the container.

In a second alternative the invention relates to a method of detecting micro-organisms from a fluid colour change consisting of placing a fluid in an at least partially transparent container, 400081NZ_DPCS_101.doc maintaining the container at a selected temperature, emitting light into the container, detecting on at least one frequency band light emitted from the container, comparing changes over time in the detected light on at least one frequency band indicating when the changes are indicative of the growth of micro-organisms within the fluid.

Preferably the fluid contains a substance, the substance reacting to the growth of micro-organisms within the fluid by varying the amount of transmitted or reflected light at at least one frequency band.

Preferably the substance is resazurin Preferably the container is located in an incubator at a selected temperature and the colour change monitored over time.

Preferably the light detected is light transmitted through the fluid from the light source.

Preferably the light detected is light reflected from the light source by the contents of 15 the container.

Preferably the container is maintained at a selected temperature by locating it in an incubator, the incubator temperature being controlled.

Preferably a record of the change in transmission at different frequency bands with time in the fluid is maintained.

Preferably the indication is analysed and critical results provided to an operator.

These and other features of as well as advantages which characterise the present invention will be apparent upon reading of the following detailed description and review of the associated drawings.

Brief Description of the Drawings FIG. 1 is a general perspective view of an analytical apparatus according to the invention.

FIG. 2 is a top view of the apparatus of FIG 1. 400081NZ_DPCS_101.doc - 4 - FIG 3 is a side cross-section of the apparatus of FIG 1.

FIG 4 shows various graphs of colour transmission in a fluid medium.

Description of the Invention Referring now to FIG. 1 an optical measurement apparatus 100 is shown. The 5 apparatus includes a heating block 101, typically of aluminium, an optical sensor 102 mounted on a printed circuit board, a light source 202 (obscured) and a transparent vial 103 of fluid containing potentially measurable micro-organisms in a medium which shows a colour change on growth of the micro-organism to be detected. The heating block 101 has at least two and possibly four holes 104 which allow light from an 10 opposing light source to pass through the vial. An inserted resistive heater 105 is also provided.

The vial contains a fluid, normally water, which may be contaminated with microorganisms such as coliform bacteria, or which may contain substance which may possibly be contaminated, such as macerated shellfish. A detection agent is placed in 15 the fluid, typically a dye such as Methylene Blue or Resazurin. There may additionally be placed in the fluid agents to provide nutrients to the micro-organisms and suppressants, to suppress growth of micro-organisms which may compete with the wanted micro-organism. With the dye or indicator, as the micro-organisms grow the dye or indicator may be reduced or otherwise chemically altered in the liquid causes a 20 change in colour, for methylene blue from blue to colourless and for Resazurin from violet to pink. It is not necessary that a dye which changes colour with a reduction reaction is present, so long as there will be a colour change in the fluid as the microorganism of interest grows. This may be caused by an indicator whose structure is changed by the micro-organism to provide some optical change in the liquid, or it may 25 be caused by the growth of the micro-organism itself.

The optical sensor 102 is a colour sensor. It is preferably RGB sensitive and may also be UV sensitive. A typical sensor of this type is the TCS230, manufactured by Texas Instruments Ltd and marketed by Texas Advanced Optoelectronic Solutions Inc, which can produce an indication of the incident light level in the white, red, green and 30 blue optical bands. The sensor is also UV and infrared sensitive to some extent, though normally a UV filter may be fitted to prevent any UV light from producing an 400081NZ_DPCS_101.doc - 5 - output. The major advantage of using such a sensor is that the detection diodes are formed simultaneously on the same substrate, being differentiated only by the filter in front of the detectors and are therefore balanced.

The light source 202 is a source having output in the colour bands of interest. 5 Typically it may be a white or blue LED, and is mounted on a printed circuit board. The beam width of the LED is preferably small, between 5 and 15 degrees being desirable.

FIG 2 shows holes 104 through which the vial may be seen, LED 202 projecting into one of the holes and transmitting light to sensor 102.

FIG 3 shows a side view of the apparatus and the inserted heater 105. The temperature of the block may be sensed either by the variation in the resistance of the heater with temperature, or from a separate thermistor (not shown). Power to the heater is continuously controlled in accordance with the detected temperature to maintain the temperature of the fluid in the vial sensibly constant. The choice of temperature is 15 governed by the organism it is wished to culture, since different organisms have markedly different growth rates at slightly different temperatures. Typically the power to the heater is a pulse width modulated waveform, with the pulse width varied inversely in accordance with the temperature, and the temperature is controlled to better than 1°C.

In operation a vial 103 containing the fluid suspected of being contaminated is treated with the dye of choice and inserted in the apparatus. The output in each of the RGB bands, and possibly in white light is recorded as a starting point and changes in the relative output at each band are monitored. The transmitted light is continually monitored until a colour change becomes evident or until the required time has 25 elapsed. There are two possible scenarios.

Firstly there may be no contamination, in which case there will be little change in colour within the specified time.

Secondly there may be contamination with the suspected organism and a colour change of the expected type will occur. This may be automatically detected by a 30 supervising program which cyclically checks the detected colour and luminance and analyses it. 400081NZ_DPCS_101.doc - 6 - To attempt to provide some resolution of the type of contamination a further LED may be placed in a hole 104 with its light projected at 90 degrees to the sensor. Light from the LED is alternated with light from an LED 202 and provides a measure of reflected light from the organisms or other contents of the fluid. Again, the results obtained will 5 depend on the growth pattern of the organism and the organism per se, but a comparison of the two results will allow close identification of the organism or organisms concerned.

FIG 4 shows typical results for a series of tests showing in each the red, green and blue visible bands. In addition to this the turbidity of the fluid is indicated by measuring the 10 backscatter from a side illuminating LED in hole 104. The vertical scale in FIGs 4A to 4D is of the relative luminance of light from the container, the horizontal scale is in minutes.

FIG 4A shows the results for a test in which there are no micro-organisms present. The graph shows little change with time in either the red 401, green 402, blue 403 or 15 turbidity 404.

FIG 4B shows results for one type of coliform bacteria showing a steady conversion from red to blue at 411, 412, associated with a slight drop in green 413 and practically no change in turbidity 414.

FIG 4C shows the results for a differing coliform bacteria where a sharp rise in 20 turbidity 424 is accompanied by a sharp drop in blue 421, a rise in red 422 is immediately followed by a sharp rise in green 423 and a steady increase in turbidity 414.

FIG 4D shows yet another type of coliform bacteria in which the blue 431 degrades while red 432 shows a sharp rise followed by a fast degradation. Green transmission 25 433 shows an abrupt rise associated with the drop in red, while the turbidity 434 shows a sharp increase increase at the same time and then levels off.

This difference in light transmission with time and the micro-organism in the culture is distinctive and allows identification of the micro-organism within a relatively short time. 40008lNZ_DPCS_10l.doc - 7 - Using digital signal processing it is possible to quickly compare the growth curves found with those of one or more possible micro-organisms which could alone or in combination produce those curves.

In operation the time to normal detection of the presence of coliform bacteria by eye is 5 from 2 to 14 hours. Using an optical sensor system to merely detect the colour change in the dye the time is reduced by over 30%.

The control of the temperature, the regulation of the brilliance of the light source, and the measurement of the sensor output may all be carried out by appropriately programming a CPU, with outputs being passed to an appropriate display. The output 10 may display an indication of the number of contaminating organisms in the original sample and the type of organism. A record of the temperature and the detected light in each frequency band with time may also be recorded.

Where the environment is likely to exceed the desired temperature it is normal to use cooling to maintain the temperature constant. Since, with the knowledge of the growth 15 curves of different micro-organisms, it is possible to predict what will happen if the temperature is not maintained constant it is an option to allow the temperature of the culture medium to vary above the desired temperature. The temperature and time are tracked, the growth curves of the organisms are tracked, and from this it is possible to predict what organisms were present initially, and in what numbers.

While a cylindrical vial is shown it is possible to use a container of a different shape, so long as it is transparent along the light path between source and sensor. In particular, because the colour change being looked for does not need to be as intense as required for detection by eye it is possible to reduce the dimensions of the vial. The lower limit for the size of the vial is determined by the minimum concentration of 25 micro-organisms being sought. The sample size must be such that the contents are a statistical duplicate of a much larger sample.

While the invention is described in relation to water testing the invention is suitable for any material which may be dissolved, suspended or otherwise cultured in any fluid. Thus in the testing of crustacea a shellfish may be added whole or macerated to the 30 water with an additional growth medium and indicator for the micro-organism 400081NZ_DPCS_101.doc concemed if desired. In the testing of milk powder the powder may be dissolved in water with added growth promotant and an indicator.

The apparatus itself may be readily portable and self-contained, and this together with the comparatively rapid response allows its use in situations where a normal laboratory 5 result would too slow to be useful, as for instance in determining whether flooding has caused a pathogen problem. For use in bulk testing situations an apparatus containing multiple vials, each with a light source and detector, may be provided. The outputs are sequentially monitored by a processing and recording apparatus.

While the optical system described provides white light and is sensitive in only three 10 colour bands it is possible to substitute a system in which a variable narrow band filter is applied to either the source or the detector, allowing a continuous scan across the untra-violet, visible and infra-red spectrum.

It is to be understood that even though numerous characteristics and advantages of the various embodiments of the present invention have been set forth in the foregoing 15 description, together with details of the structure and functioning of various embodiments of the invention, this disclosure is illustrative only, and changes may be made in detail so long as the functioning of the invention is not adversely affected. For example the particular elements of the apparatus may vary dependent on the particular application for which it is used without variation in the spirit and scope of 20 the present invention.

In addition, although the preferred embodiments described herein are directed to an apparatus for use in a micro-organism in fluid system, it will be appreciated by those skilled in the art that the teachings of the present invention can be applied to other systems such as micro-organisms on an agar plate, or filter without departing from the 25 scope and spirit of the present invention. 400081NZ_DPCS_l01.doc

Claims (21)

Claims

1. A micro-organism detection apparatus consisting of: a fluid container with at least one light path through container and a contained fluid a container incubator at least partially surrounding a container receiving space a light source transmitting light into the fluid container and mounted to the container incubator a light sensor fixed in relation to the container incubator and detecting light of at least one colour which has passed through the fluid from the light source, a comparator detecting changes over time in the light transmission of at least said one colour.

2. The apparatus as claimed in claim 1 wherein the sensor is an integrated circuit optical sensor.

3. ' The apparatus as claimed in claim 2 wherein the colour from at least red, green and blue light is sensed.

4. The apparatus of claim 2 wherein at least one colour frequency band is outside the visible spectrum.

5. The apparatus as claimed in claim 1 wherein the colour change is as a result of a change in the colour of an indicator in the fluid.

6. The apparatus as claimed in claim 1 wherein the colour change in transmitted light is monitored as a function of time.

7. The apparatus as claimed in claim 1 wherein the incubator has a heating element.

8. The apparatus as claimed in claim 7 wherein the heating element is controlled to maintain the fluid temperature substantially constant.

9. The apparatus as claimed in claim 1 wherein the light detected is transmitted light. 400081NZ DPCS 101.doc - 10-

10. The apparatus as claimed in claim 1 wherein the light detected is light reflected from the light source by the contents of the container.

11. A method of detecting micro-organisms from a fluid colour change consisting of placing a fluid in an at least partially transparent container, 5 maintaining the container at a selected temperature, emitting light into the container, detecting on at least one frequency band light emitted from the container, comparing changes over time in the detected light on at least the one frequency band 10 indicating when the changes are indicative of the growth of micro-organisms within the fluid.

12. A method as claimed in claim 10 wherein the fluid contains an indicator, such that the fluid colour alters with the growth of micro-organisms within the fluid.

13. A method as claimed in claim 11 wherein the indicator is resazurin 15

14. A method as claimed in claim 11 wherein the fluid contains a nutrient medium and the colour of the medium plus micro-organism alters with growth of the micro-organism.

15. A method as claimed in claim 11 wherein at least one of the frequency bands measured is outside the spectrum of visible light. 20

16. A method as claimed in claim 11 wherein the container is located in an incubator at a selected temperature and the colour change monitored over time.

17. A method as claimed in claim 11 wherein the light detected is light transmitted through the fluid from the light source.

18. A method as claimed in claim 11 wherein the light detected is light reflected 25 from the light source by the contents of the container.

19. A method as claimed in claim 11 wherein the container is maintained at a selected temperature by locating it in an incubator, the incubator temperature being controlled. 400081NZ_DPCS_101.doc - 11 -

20.

21. 5 22. 10 15 A method as claimed in claim 11 wherein a record of the colour change with time of the fluid is maintained. A method as claimed in claim 11 wherein the indication is analysed and provided to an operator. A method of detecting micro-organisms from a fluid colour change consisting of placing a fluid in an at least partially transparent container, maintaining the container at a selected temperature, emitting light into the container, detecting on multiple frequency bands light transmitted through the container, comparing changes over time in the detected light on multiple frequency bands comparing such changes with known changes indicative of the growth of at least one known micro-organism indicating when the changes are indicative of the growth of the known microorganisms within the fluid. Rosemary Katherine Cameron Shp/pin By her Attorneys Pipers