Process and apparatus for analyzing specimens for the presence of microorganisms therein

• US 3,963,355 A
• Filed: 04/16/1974
• Issued: 06/15/1976
• Est. Priority Date: 05/22/1972
• Status: Expired due to Term

• Current Assignee: Mcdonnell Douglas Corporation (The Boeing Co.)
• Original Assignee: Mcdonnell Douglas Corporation (The Boeing Co.)
• Inventors: Jones, Paul W., Holen, James T., Meyer, Michael C., Gibson, Sandra F., Keyser, George F., Vannest, Richard D., Aldridge, Clifton Jr.
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US05/461,249
• Time in Patent Office: 791 Days
• Field of Search: 356/36, 356/73, 356/201, 356/244, 356/246, 195/103.5 R, 250/576
• US Class Current: 435/39
• CPC Class Codes: B01L 2200/028 Modular arrangements
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/875 Pseudomonas aeruginosa
• CPC Class Codes: Y10S 435/879 Salmonella
• CPC Class Codes: Y10S 435/883 Staphylococcus aureus
• CPC Class Codes: Y10S 435/885 Streptococcus
• CPC Class Codes: Y10S 435/922 Candida albicans

Abstract

Microorganisms in a specimen are detected, identified, and enumerated by introducing the specimen into a sampling cartridge and diluting the specimen with a known volume of water within the cartridge. The cartridge has a manifold and several cassettes attached to the manifold. Each cassette contains a serpentine flow channel having a series of filters therein and a detection cell located downstream from each filter. The flow channel in each cassette also contains a culture medium which is freeze dried and is highly selective in the sense that it promotes the growth of one type of microorganism, but not others. The mixture of the specimen and water flows from the manifold into the flow channel of each cassette where it rehydrates the culture medium therein and further flows through the filters. Each filter removes a known proportion of the microorganisms from the mixture of specimen, water and medium, thereby effecting a serial dilution. After the cassettes are heated to incubate the microorganisms, the detection cells are observed for growth of the microorganisms therein which is manifested in a change in the light transmitting characteristics of the mixtures within the cells. This change is detected with great precision by projecting beams of light through the detection cells and measuring the current flowing through dedicated photocells located beyond the detection cells. The amount of current flowing through each photocell is proportional to the relative optical density of the mixture within the corresponding detection cell, and this density is plotted against time to give a unique time-related signature for each microorganism. Thus, the presence and identity of microorganisms in the detection cells is determined by mere changes in the optical density of the cells, whereas the number of microorganisms may be determined by considering the number of detection cells which change.

Claims

1. A device for detecting the presence of at least one specific microorganism in a specimen, said device comprising:

receptacle means for containing a diluted specimen, a flow channel leading from the receptacle means, at least one selective culture medium in the flow channel whereby the diluted specimen will mix with the culture medium to form a diluent mixture, the culture medium being capable of sustaining a specific microorganism in the diluent mixture such that the continued existence of said living specific microorganism will change the light transmitting characteristics of the diluent mixture so as to indicate the presence of the specific microorganism in the specimen.

2. A device according to claim 1 wherein the flow channel comprises a manifold leading from the receptacle means and a plurality of branches leading from the manifold;

and wherein culture media is in the branches, the culture medium in each branch favoring a different microorganism.

3. A device according to claim 1 wherein the flow channel has a detection cell for observing the change in the light transmitting characteristics of the diluent mixture.

4. A device according to claim 3 wherein the flow channel contains a filter through which the diluted speciment flows, the filter being capable of removing a known proportion of the microorganisms from the diluted specimen passing through it.

5. A device according to claim 4 wherein the flow channel contains a plurality of filters to effect a serial dilution of the specimen.

6. A device according to claim 5 wherein a detection cell is located downstream from each filter.

7. A device according to claim 6 wherein the culture medium is disposed in discrete beds with a discrete bed being located upstream from each detection cell.

8. A device according to claim 3 wherein the flow channel comprises a manifold leading from the receptacle means and a plurality of branches leading from the manifold;

wherein the culture medium and the detection cells are in the branches, the culture medium of the individual branches being different and favoring different microorganisms; and

wherein the branches are in cassette means which are detachable for purposes of examination.

9. A device according to claim 2 wherein the receptacle means comprises an initially sealed container having a predetermined volume of liquid therein, and an initially open container for receiving the specimen and having the initially sealed container therein, the initially open container being connected with the manifold.

10. A device according to claim 9 wherein a valve is interposed between the initially open container and the manifold.

11. A device according to claim 3 and further characterized by a detector for measuring the optical density of the diluent mixture at the detection cell, said detector comprising a light source oriented to project a beam of light through the detection cell, and a photoelectric cell positioned in the path of the beam beyond the detection cell, the photoelectric cell controlling an electrical signal such that the magnitude of the signal is dependent on the intensity of the light falling upon the photoelectric cell.

12. A device according to claim 11 wherein the detector further comprises a clock, and an analogue to digital converter for converting to analogue signal derived from the photoelectric cell to a digital signal.

13. A device accordng to claim 11 wherein a plurality of filters are disposed in the flow channel to effect a serial dilution of the specimen;

wherein the flow channel has a detection cell located downstrem from each filter;

wherein the detector has a light source and photoelectric cell positioned to align optically with each detection cell; and

wherein the detector further comprises a clock and control for energizing the light sources in sequence.

14. A device according to claim 11 wherein the portion of the flow channel containing the culture medium and the detection cell is disposed within a detachable cassette;

and wherein the detector further comprises cooperating first and second members for receiving the cassette, the light source being in one of the members and the photoelectric cell being in the other member; and

heater means associated with the detector elevating the temperature of the cassette to incubate the microorganisms in the culture medium.

15. A device according to claim 1 wherein the culture medium is freeze dried.

16. A process for detecting and identifying microorganisms in a specimen, said process comprising diluting the specimen in a liquid;

mixing the liquid-specimen mixture with a selective culture medium to form a diluent mixture;

heating the diluent mixture to incubate the microorgaisms therein;

filtering the diluent mixture to remove a known proportion of microorganisms of the diluent mixture; and

thereafter examining the diluent mixture for a change in the optical characteristics thereof by measuring the relative optical density of the diluent mixture.

17. A process according to claim 16 wherein the diluent mixture is filtered a plurality of times to effect a serial dilution;

and wherein the relative optical density is measured after each filtration.

18. A process according to claim 17 wherein the relative optical density is measured by projecting a beam of light through the diluent mixture, having the light emerging from the mixture fall upon a photocell, and measuring the current flowing through the photocell.

19. A process for detecting and identifying microorganisms in a specimen, said process comprising diluting the specimen in a liquid;

evacuating air from the vicinity of a selective culture medium;

thereafter mixing the liquid-specimen mixture with the selective culture medium to form a diluent mixture; and

examining the diluent mixture for a change in the optical characteristics thereof.

20. A process according to claim 19 and further characterized by heating the diluent mixture to incubate the microorganisms therein.

21. A process according to claim 19 wherein the liquid-specimen mixture replaces the evacuated air so that the liquid-specimen mixture and culture medium are mixed in the absence of substantial amounts of air.

22. A process according to claim 19 and further characterized by incubating the diluent mixture, and wherein the diluent mixture is examined at intervals as it is incubated for detecting change in the optical characteristics thereof.

23. A process according to claim 22 and further characterized by comparing the results of the examination with known results for the selective culture medium used to culture the microorganism to which it is specific.

24. A process according to claim 22 wherein the process of examining the optical characteristics includes measuring the optical density of diluent mixture and plotting the optical density against time.

25. A process according to claim 22 wherein the step of examining the diluent mixture comprises projecting a beam of light through the diluent mixture and recording the intensity of the projected light after it passes through the diluent mixture.

26. A process according to claim 22 wherein the culture medium is dehydrated before the liquid-specimen mixture flows into it and is rehydrated by the liquid-specimen mixture.

27. A process for detecting and identifying microorganisms in a specimen, said process comprising diluting the specimen in a liquid;

causing the liquid-specimen mixture to flow into and mix with a selective culture medium without exposing the liquid-specimen mixture or the selective culture medium to the surrounding atmosphere, whereby a diluent mixture is formed; and

examining the diluent mixture for a change in the optical characteristics thereof.

28. A process according to claim 27 wherein the step of examining the diluent mixture for a change in the optical characteristics thereof comprises measuring the optical density of the diluent mixture.

29. A process according to claim 28 wherein the relative density is measured repeatedly for detecting a change in the relative optical density.

30. A process for detecting and identifying a specific microorganism in a specimen, said process comprising:

diluting the specimen in water confined within a vessel to form a water-specimen mixture;

attaching a cassette containing a flow channel to the vessel such that the flow channel communicates with the interior of the vessel, the flow channel including a detection cell which is visible from the exterior of the cassette on both sides of the cassette, the flow channel also containing a selective medium which when mixed with water will sustain the specific microorganisms and only the specific microorganism will cause a change in the light transmitting characteristics of the water-medium mixture so formed;

causing the water-specimen mixture to flow from the vessel into the flow channel within the cassette where the water-specimen mixture will mix with the selective medium in the flow channel to form a diluent mixture;

thereafter detaching the cassette from the vessel;

projecting a beam of light through the detection cell of the detached cassette and the diluent mixture therein; and

measuring the intensity of the light beyond the detection cell.

31. A process according to claim 30 wherein the water-specimen mixture is caused to flow from the vessel into the flow channel of the cassette by evacuating air from the channel and permitting the water-specimen mixture to take the place of the evacuated air.

32. A process for detecting and identifying a specific microorganism in a specimen;

said process comprising;

introducing the specimen into a vessel containing the water so that the specimen and water mix to form a water-specimen mixture;

attaching a cassette containing a flow channel to the vessel such that the flow channel communicates only with the interior of the vessel, the flow channel including a detection cell which is visible from the exterior of the cassette and when unobstructed is capable of transmitting light through the cassette, the flow channel also containing a selective culture medium which when mixed with water will sustain the specific microorganism and only the specific microorganism will cause a change in the light transmitting characteristics of the water-medium mixture;

closing the vessel so that the interior of the vessel and the flow channel are substantially isolated from the surrounding atmosphere to prevent the entry of contaminants;

causing the water-specimen mixture to flow into the flow channel and mix with the selective culture medium therein to form a diluent mixture, at least some of which locates in the detection cell of the flow channel;

thereafter projecting a beam of light through the detection cell and the diluent mixture therein; and

measuring the intensity of the beam of light beyond the detection cell.

33. A process according to claim 32 wherein the cassette is attached to the vessel by inserting a needle through a septum.

34. A process according to claim 32 wherein the water specimen mixture is caused to flow from the vessel into the flow channel of the cassette by evacuating air from the channel and permitting the water-specimen mixture to take the place of the evacuated air.

35. A process according to claim 32 wherein the cassette is detached from the vessel when the beam of light is projected through the detection cell.

36. A process according to claim 32 wherein the selective culture medium is freeze-dried and is rehydrated by the water when the water-specimen mixture is caused to flow through the flow channel.