WO1988007584A1

WO1988007584A1 - Method and apparatus for detection and quantification of bacteria

Info

Publication number: WO1988007584A1
Application number: PCT/US1988/000852
Authority: WO
Inventors: C. David Miller; Lawrence J. Loomis
Original assignee: New Horizons Diagnostics Corporation
Priority date: 1987-03-23
Filing date: 1988-03-22
Publication date: 1988-10-06

Abstract

The invention includes a photometer (10), a disposable test device (17) for use with the photometer (10), and a reagent system for monitoring the presence and amount of bacteria in a wide variety of samples. The photometer (10) provides a means for positioning (16) the disposable test device in proximity to a photodetector which measures the light emitted by a reaction specific to the bacterial adenosinetriphosphate (ATP) content of the sample. The disposable test device provides a method for pre-removal of reaction inhibiting constituents and non-bacterial ATP from the reaction mixture. The instrument includes an electrical circuit for generating a signal proportional to detected light and for generating an output signal representative of the presence and amount of bacteria in the sample being monitored.

Description

METHOD AND APPARATUS FOR DETECTION AND QUANTIFICATION OF BACTERIA

Field of the Invention The present invention relates to a method and apparatus for diagnosing the presence and determining the quantity of bacteria in a wide variety of sample specimens, particularly in biological fluids such as urine. The apparatus provides a means for elimination of interfering constituents and specific measurement of the adenosinetriphosphate (ATP) content of the bacterial cells present in the sample.

Background of the Invention This invention relates to rapid diagnosis and monitoring of bacterial levels in samples such as food, air, or water but primarily in biological specimens such as urine. Clinical methods for early detection of bacteriuria are highly desirable for screening wide patient populations for possible existence of urinary tract infections, whether or not the patients are symptomatic. Traditional procedures for assaying bacteria in urine typically require sample incubation over a twenty-four to forty-eight hour growth period with subsequent manual observation and count of the number of colonies formed. Disadvantages of this methodology are long assay times, requirements for specially trained personnel, and possible inadequate identification of certain potentially pathogenic bacterial species whose growth may not be supported by the specific media or its environment. Microscopic examinations and selected staining technologies are labor intensive, require skilled personnel, and may not provide sufficient sensitivity and specificity. Many of these disadvantages have been circumvented or eliminated by application of the highly specific reaction:

Mg⁺⁺ Adenosinetriphosphate + luciferin luciferase luciferyl adenylate + pyrophosphate

O₂

Luciferyladenylate oxyluciferyladenylate luciferase + H₂O + light

which has long been recognized as a potential method for assaying bacteria. Adenosinetriphosphate (ATP), a nucleotide present in all living matter, may be quantified, using this reaction, by photometric detection of the emitted light and, in turn, related to the quantity of bacteria present in a given sample. Early discourses on the nature of the reaction, the history of Its discovery, and its general areas of applicability are provided by E. N. Harvey (1957), A History of Luminescence: From The Earliest Times Until 1900, Amer. Phil. Soc, Philadelphia, PA. and W.D. McElroy and B. L. Strehler (1949) Arch. Biochem. Biophys. 22, 420-433. Test procedures exemplifying the use of this reaction for bacterial determinations and, specialized instrumentation for measurement of the associated light emission, are known and disclosed in U. S. Pat. Nos. 3,520,660; 3,933,592; 3,940,250; 3,971,703; 3,979,181; 4,013,418; and 4,385,113.

In those instances in which the above reaction is utilized for determinations of bacteria in non-complex sample matrices, where no interfering constituents or sources of ATP other than bacteria exist, an assay procedure might consist of initial rupture of bacterial cells by one of several means, followed by addition of excess luciferin-luciferase - magnesium ion reagent and measurement of the emitted light. In reality, however, such sample systems are practically non-existent. Urine, for example, typically contains a multitude of chemical and cellular entities, any one of which may adversely effect the determinative reaction specific to bacterial ATP. Somatic cells such as epithelials, leucocytes and erythrocytes, are invariably present in urine samples in widely varying concentrations. These cells contain ATP which, if not removed from the sample, would contribute to the signal derived from bacterial ATP, producing false positive results. Of equal or greater significance, are inhibitory constituents such as analgesics, therapeutic drugs, food derivatives, etc., which If not removed from the sample would depress the signal derived from bacterial ATP, producing false negative results. Prior art methods address the removal of somatic

ATP by addition of detergent - based reagents for selective lysis of non-bacterial cell membranes followed by an enzymatic degradation of the released ATP. An ATPase, such as apyrase, is used for this purpose but suffers the disadvantages of relative instability and requirement for extended periods of time to effect the complete hydrolysis of free ATP. Prior art methods do not address practical procedures for removal of reaction inhibitors. Most luminescence-based methodologies allow such inhibitors to remain in the sample matrix throughout the course of the reaction, relying solely upon relatively large dilution effects to reduce, to a limited extent, depression of the light output. Our observations, which are supported by extensive experimentation and data, confirm the magnitude of this inhibitory effect to be extremely large for a wide variety of constituents. As an example, urine samples from persons ingesting even less than maximum recommended dosages of aspirin often completely inhibit the signal corresponding to added bacteria in concentrations of 10⁵

CFU/ml or greater, if measures are not taken to first remove the aspirin or its metabolic by-products from the sample.

Detection of such bacterial concentrations would normally evidence the presence of a strongly positive urinary tract infection but, in this case, without removal of inhibitors, would produce a false negative result.

Additionally, previously disclosed luminiscence-based methodologies for bacterial detection have required expensive instrumentation, the use of relatively large amounts of costly luciferin-luciferase reagents, numerous labor-intensive steps for reagent preparation and conduction of the determinations (e.g., pH adjustments), and relatively extensive assay times due, in part, to the necessity for enzymatic degradation of free ATP.

It is, therefore, an object of the present invention to provide a system comprising a disposable test device and complementary photometer, and method of use, which together form a monitoring system for determining the presence and amount of bacteria in a wide variety of sample specimens, but particularly in biological fluids, such as urine.

It is another object of the invention to provide a method and apparatus for quantification of bacteria which avoids the requirements for enzymatic degradation of endogenous and/or somatic cell ATP and/or protein-bound ATP.

It is another object of the invention to avoid the inaccuracies associated with inhibitory constituents by providing a device and a method for their removal.

It is another object of the invention to provide a compact, i nexpens ive photometer wi th the requ i s ite hgi gh sensitivity for reading the emitted light signal and which is mechanically and functionally compatible with the disposable device and the conditions for the assay.

It Is another object of the invention to provide a methodology and reagent system which facilitates substantially improved accuracy and economy over prior methods and allows, for the first time, an assay for the specific determination of the total bacterial content of a sample to be conducted in less than one minute.

The foregoing and other objects, advantages and features of the invention, and the manner in which the same are accomplished will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying drawings, which illustrate preferred and exemplary embodiments and wherein:

Figure 1 is a perspective view illustrating the photometer and the method in which the disposable test device is positioned for light emission measurement.

Figure 2 is a partially opened fragmentary perspective view of the photometer of Figure 1, showing details of the slide mechanism and positioning of the optical block.

Figure 3 provides perspective views of: a) the disposable test device showing details of its construction. b) a positive pressure device with an associated cross-sectional view showing details of its construction c) a section of a support and absorption device d) the positioning of the three devices which allows them to be used in conjunction with one another

Figure 4 is a perspective view of a preferred configuration of a containment and dispensing device.

Figure 5 is a diagram of the electrical componetry of the photometer of Figure 1.

Summary of the Invention The present invention comprises a combination of a compact photometer, a compatible dual-function disposable test device with method and apparatus for removal of interfering substances and allowance of measurement of emitted light associated specifically with bacterial ATP, and a reagent addition sequence which together form a monitoring system for determination of the presence and amount of bacteria in a given sample. The combination provides means for selective lysis of somatic cells followed by elimination of the liquid phase containing endogenous and somatic cell ATP, means for freeing protein-bound or particulate-bound ATP followed by elimination of the ATP-containing liquid phase, means for lysis of bacterial cells followed by addition of luciferin - luciferase magnesium ion reagent and finally, means for generating and displaying an electrical signal proportional to the bacterial ATP content of the sample and, in turn, to the quantity of bacteria in the sample.

The photometer includes means for accommodation of the disposable test device in a manner which allows its precise and reproducible positioning with respect to the surface of the photosensor and which precludes any possible loss of the final reaction mixture during and after the measurement cycle. The photometer also includes means for integrating the signal evolving from the emitted light over a fixed period of time.

The invention also includes means for containment and dispensing of a precise quantity of luciferin-luciferase-magnesium ion reagent in a manner which eliminates reconstitution requirements, and insures long-term reagent stability.

The invention also includes a methodology using the afore-mentioned photometer, disposable device and associated apparatus and reagent system which allows diagnosis of the presence and quantity of bacteria in a sample in less than one minute. Detailed Description of the Invention

The subject invention is a system comprising an instrument, an interlocking disposable test device, associated reagents, and a method for rapid detection of the presonce and quantity of bacteria in a sample. As seen in Figure 1, the instrument is broadly designated at 10 and comprises a compact, portable photometer for reading emitted light which is eventually correlated with the amount of bacteria present in the sample.

Figure 1 shows an overall perspective view of the instrument 10, including the housing 11, the output display 12, a cycle-start switch 13, a cycle indicator light 14, a light sealing panel 15, and a slide mechanism 16 which accepts a disposable test device 17.

Figure 2 is a partially opened and expanded view of the photometer 10 and illustrates more of its particular components. These include an optical block 19 which provides a light-tight housing for an internally mounted photomultiplier tube and its associated socket and voltage divider circuit 18. When the slide mechanism 16, is completely closed, it reproducibly positions the optically clear side wall of the disposable test device 17 in close proximity to the photocathode of the photomultiplier tube. Moreover, in the closed position, the forward section 20 of the slide mechanism 16 resides within the recessed portion 22 of the panel 15 to insure a positive light seal. Precise and reproducible closure of the slide mechanism 16 is provided by a ball and detent mechanism within the optical block 19. The top surface of the slide mechanism 16 embodies a semicircular recess 21 which accepts the upper flange 22 of the disposable test device 17, thus preventing its lateral movement within the slide mechanism 16.

Included in the slide mechanism 16 is a vertical slot 24, which accepts a projected tab 25 on the sidewall of the disposable test device 17 for the purpose of alignment of the device. When captively positioned within the slide mechanism 16, the bottom surface 26 of the disposable test device 17 will be suspended in a manner such that it will remain free of contact with any adjacent surface. The purpose of such positioning is to disallow any loss of sample through the bottom surface 26 of the disposable test device 17.

Figure 3 provides a detailed perspective view of the disposable test device 17 and perspective views of a positive pressure device 27 and a support and absorption device 28. The body of the disposable test device 17 is comprised of optically clear molded plastic material, such as polystyrene, which is capable of nearly complete transmission of light within a 500-600 nm wavelength range. Fused to the lower surface of the device is a semi-permeable membrane 26 which is characterized by its strength and lack of deformation under pressure, and a pore size distribution which insures surface retention of bacterial cells, particularly urinary tract pathogens, while facilitating complete passage of any associated liquid phase during pressurization. The positive pressure device 27 is comprised of a plunger consisting of a shaft 29 and a flexible rubber cylinder 30 which is in contact with the inner surface walls of a barrel 31. The lower end of the barrel is recessed to accept the flange 23 of the disposable test device 17 and also contains an "o" ring seal 32 which, together with the recess, insures a leak-tight juncture and precise location of the positive pressure device 27 with respect to the surface of the flange 23 during pressurization. The support and absorption device 28 accepts the disposable test device 17 through the positioning hole 33 in a manner in which the membrane surface 26 remains in contact with the disposable absorbent pad 34 during pressurization.

The support and absorption device 28 which is constructed of a suitable plastic material such as polystyrene, is comprised of a linear array of positioning holes 33 allowing acceptance of a plurality of disposable test devices 17, typically six. Figure 4 shows a perspective view of a typical containment and dispensing device 35 for the luciferin-luciferase-magnesium ion reagent 36, the advantages and requirements of which will be discussed in a later section.

Figure 5 illustrates diagramatically the componentry comprising the electrical circuitry of the photometer 10. In a preferred embodiment, the power input supply consists of an external AC to 9V DC adapter 39 or, optionally, a rechargeable 9V nickel-cadmium battery pack 39, both of which connect to the instrument through a jack located on the rear panel of the housing 11. The 9V DC input is conditioned and regulated to a fixed 5V DC level by means of a voltage regulator 40 and also provides settable, regulated voltages within a range of 3 to 6 V DC by means of an adjustable voltage regulator 41. The adjustable voltages are inputted to a DC to DC converter 42 which, in turn, provides settable, high voltages ranging from essentially 400 to 1000 V DC. This high voltage is inputted to a voltage divider circuit 43 which is contained within the socket assembly 18. The current generated by the photomultiplier tube 44 is conditioned and inputted to an amplification and integration circuit 45, the output voltage of which is inputted to a digital display 46. A background suppression circuit 47 provides means for subtraction of photomultiplier tube dark current or other s ignals unre lated to the f inal l ight measurement . A timer circuit 48, which is triggered by a cycle-start switch 49, provides the required delay cycle, reset functions, integration cycle, and logic signals to the digital display 46, and also drives a dual light emitting diode 50 which provides a RED signal during the integration cycle and a GREEN signal at all other times, indicating that readings may be made. The total integrated signal corresponding to the amount of light incident upon the photocathode of the detector during the pre-selected period of integration (typically ten seconds) is displayed and held until the cycle-start switch 49 is again depressed to initiate another integration cycle.

The determinative procedure, although applicable to virtually all samples in a wide variety of matrices, is directed predominantly, in this disclosure, to the assay of urine samples for bacterial content. The procedure developed for urine assumes essentially a worst case situation due, in part, to the extreme variability and complexity of the sample constituents and the general presence of ATP-containing somatic cells and reaction-inhibiting materials. It is advocated that in those instances in which samples such as food, air, and water, and particularly biological specimens other than urine, such as cerebral spinal, fluids, saliva, and tear duct fluids are to be assayed for bacterial content, that such samples, even though the precence of interferences may not be suspected, are construed to be total unknowns and that the following procedure, without delection of any steps, is applied exactly as it is for urine samples.

Initially, a disposable test device is placed in the support and absorption device 28, in which a disposable absorbent pad 34 has been positioned. Next, a reagent which is capable of specific lysis of somatic cells, leaving bacterial cells intact, is added to the disposable test device 17. The use of a variety of non-ionic detergents, such as polyoxyethylene ethers, for this purpose has frequently been reported in the literature, but for the procedure of this disclosure, saponin, a detergent-like compound of natural origin, provides improved performance. The concentration of the saponin solution is of paramount importance in insuring complete release of ATP from somatic cells, while precluding rupture of bacterial cells.

The urine sample, after thorough mixing or vortexing, is then added using a sterile pipet tip or other sterile dispensing device. The release of ATP, as evidenced by experimental data obtained with a variety of mammalian cells, is rapid and is completed within a few seconds. The positive pressure device 27 is then used to force the liquid phase through the membrane 26, leaving on its surface the somatic cell debris, the intact bacterial cells, and any other particulate matter or large molecules present in the sample. Because of the efficiency of this procedure in implementing complete removal of the liquid phase, no additional steps are required for degrading residual ATP.

It is important to note the reasons and rationale for the use of positive rather then negative pressure in conjunction with the disposable test device 17 for the purpose of removing the liquid phase. Extensive experimental data which invariably show significantly higher and more precise values using positive as opposed to negative pressure suggest greater partial entrapment of bacterial species within the interstices of the membrane under negative pressure conditions, thereby limiting their accessibility to the bacterial releasing agent. Additionally, the absorbent pad 34, usable only with the positive pressure technique, precludes droplet formation on the underside of the membrane, avoiding potential reverse flow by capillary action.

An acidic solution, buffered at pH 3.5 to 4.0, is next added to the surface of the membrane. This serves to disassociate ATP from those complexes in which it may be bound to protein or other large molecules or particulate matter. The solubilized ATP is then removed by again using the positive pressure device 27 to force the liquid phase through the membrane 26. At this point, the bacterial cells on the surface of the membrane will be free from ATP- containing entitles and reaction inhibiting constituents, and the disposable test device 17 is transferred to the slide mechanism 16 of the photometer 10. A cationic detergent buffered at pH 7.75, which is capable of lysing all bacterial cells, inclusive of yeast, with minimum retardation of the light emitting reaction, is next added to the surface of the membrane. Cell wall rupture is again very rapid, occuring within seconds after addition of the reagent which is dispensed in a manner which insures efficient mixing.

The final reagent addition is that of a pH 7.75 luciferin-luciferase-magnesium ion mixture, and may be accomplished using one of two procedures. For a laboratory environment in which a large number of samples are expected to be assayed each day, it is convenient and economical to reconstitute the lyophilized reagent mixture with water, and to add a specified volume, of the solution to the disposable test device 17. Should considerable time elapse between series of assays, the solution should be refrigerated to preserve its activity. In those instances, such as physician office applications, in which only a single assay may be required at a given time, it will be more convenient and economical to utilize the containment and dispensing device 35 depicted in Figure 4. This provides a novel method for preserving the activity of the luciferin-luciferase-magnesium ion reagent, without requirement for refrigeration, and allows a precise quantity to be dispensed into the reaction mixture. The containment and dispensing device 35 is prepared by drawing an exact volume of a luciferin-luciferase-magnesium ion solution into a pipet tip 37 or another disposable dispensing device. The small end of the tip is temporarily sealed and the device 35 is placed in a vertical position in a lyophilizer. After completion of lyophilization, reagent addition to the disposable test device 17 may be accomplished by a series of rapid withdrawals and deliveries of the liquid in the device 17 using the containment and dispensing device 35 in conjunction with a standard micro pipettor 38. It should be noted that, irrespective of the method of delivery, the disposable test device 17, by virtue of its design and that of the slide mechanism 16, remains suspended and isolated from contact with any adjacent surfaces. This serves the dual purpose of eliminating loss of sample with attendant measurement inaccuracies, and precluding transfer of any liquid into the photomultiplier tube compartment. An additional advantage of the design is its allowance of exact and reproducible positioning of the disposable test device 17 with respect to the photocathode surface of the detector.

Immediately following addition of the final reagent, the slide mechanism 16 is closed and the cycle-start switch 13 of the photometer 10 is activated. Within one second any prior reading on the digital display 12 will be reset automatically to zero and the normally GREEN light emitting diode 14 will switch to RED, indicating that the integration cycle is in progress. After 10 seconds of integration, the light emitting diode 14 will revert to GREEN, indicating that the reading on the digital display 12 may now be recorded.

Photometer gain is set by means of the following procedure. Using results obtained for a wide variety of urine samples from a diversified patient population and corresponding 48-hour culture data for each sample, the photometer gain is so adjusted that readings of 500 to 1200 mv correlate linearly with bacterial concentrations, as determined by culture, of 1 x 10⁴ to 1 x 10⁵ CFU/ml, and readings greater than 1200 mv correlate with bacterial concentrations of 10⁵ CFU/ml or greater. Samples with readings of less than 500 mv are classified as "negative".

The example which follows is illustrative of a procedure and reagent system used for patient urine assays but applicable to virtually all other sample specimens, and the results cited are indicative of the sensitivity and specificity routinely obtainable with the method.

Example

The following procedure was used for determination of the presence and the amount of bacteria in three hundred and one clean catch or catheterized urine specimens collected from pediatric, adult, and geriatric populations. For each sample :

1. A disposable test device was placed into the support and absorption device so that the membrane contacted the disposable absorbent pad. Then, 75 ul of a 0.25% aqueous solution of saponin was dispensed into the disposable device.

2. After thorough vortexing, 75 ul of the urine sample to be assayed was pipetted, with mixing, into the saponin solution. The positive pressure device was then used to force the liquid phase through the membrane into the absorbent pad.

3. Next, 75 ul of a 0.005M citric acid / 0.002M sodium citrate solution was dispensed into the disposable test device. The positive pressure device was again used to force the liquid phase through the membrane into the absorbent pad.

4. The disposable test device was then transferred to the slide mechanism of the photometer and, 75 ul of a pH 7.75 solution containing 0.01% of mixed alkyl substituted ammonium halides was added, with mixing, to the disposable device.

5. Finally, 75 ul of a pH 7.75 luciferin-luciferase-magnesium ion solution was rapidly pipetted into the solution of Step 4 and the slide mechanism was immediately closed. The solution, which was 0.01M in magnesium sulfate, 0.0005M in ethylenediaminetetra-acetic acid and 0.00015M in luciferin, contained 1 mg/ml of firefly lantern extract.

6. The cycle-start switch of the photometer was depressed and the reading on the digital display was recorded after completion of the ten second signal integration period.

For the reference data, all specimens were cultured using a 0.001 ml calibrated loop to innoculate both a

MacConkey and a 5% sheep blood agar plate. The plates were Incubated at 35º C and examined and counted after 48 hours .

These results were compared with those us ing the method of this disclosure and are summarized in Table 1. Results of both methods corresponding to the species of bacteria determined to be present in the samples are summarized in

Table 2.

TABLE 1

Culture Results Total Method of Disclosure Positive Negative (> 500 mv) (< 500 mv)

< 10,000 CFU/ml (neg) 148 13 135 ≥ 10,000 CFU/ml (pos) 153 151 2

Sensitivity 98.7% Specificity 91.2% Predictive Positive 92.1% Predictive Negative 98.5% Overall Agreement 94.6% TABLE 2

Identification of microorganisms recovered from urine specimens containing ≥ 10,000 CFU/ml.

No. of Specimens

Yielding

Bacterial Species Positive Results (≥10,000 CFU/ml)

Culture Method of the Disclosure

Escherichia coli 42 42 Group D enterococcus 15 15 Group B Streptococcus 1 1 Proteus mirabilis 5 5 Pseudomonas aeruginosa 4 4 Yeast 3 3

Enterobacter spp. 2 2 Citrobacter spp. 2 1 Lactobacillus spp. 2 2 Other gram negative rods 19 19 Mixed Flora 57 56

The method and apparatus for detection and quantification of bacteria disclosed herein represents a significant improvement over prior art methods in terms of reduction of assay time, elimination of requirements for highly trained personnel, economy of reagents and apparatus, elimination of numerous labor-intensive assay steps and of certain reagents with limited stability, and provision of greater accuracy and precision due, primarily, to its novel procedure for pre-removal of interfering substances. It is to be recognized that the invention is not intended to be restricted to the particular instrumentation, disposables, and reagent systems which were described for illustrative purposes. For example, it is readily apparent that the single disposable device may be replaced by a matrix of such devices which, in conjunction with a compatible and expanded photometer, would provide means for a semi-automated, multi-sample assay. These and other such modifications in the method and apparatus may be considered by those skilled in the art without alteration of the spirit of the present invention.

WHAT IS CLAIMED IS:

1. The combination of a photometer, a compatible dual-function disposable test device with a method and apparatus for removal of interfering substances, and a reagent system with a method and addition sequence which together form a monitoring system for determining the presence and amount of bacteria in a given sample, the combination comprising:

a disposable test device which provides means for retention of bacteria, elimination of non-bacterial constituents, and containment of a light producing reaction mixture; and

Claims

an associated means and apparatus for removal of said non-bacterial constituents from said disposable test device; and

a photometer for receiving said disposable test device, said photometer including;

means for supporting and positioning said disposable test device within a defined optical path;

means for exclusion of ambient light;

means for generating an electrical signal proportional to the light produced by the reaction mixture within the disposable test device; and

means for generating an output signal in response to the electrical signal, said output signal being representative of the presence and amount of bacteria in the sample being monitored.

2. A monitoring system according to Claim 1 wherein said disposable test device comprises a vessel having optically clear side walls and a bottom surface consisting of a semi-permeable membrane which, under pressurization, allows passage of liquids or gases and disallows passage of bacteria.

3. A monitoring system according to Claim 1 wherein said apparatus for removal of said non-bacterial constituents from said disposable test device comprises a plunger mechanism including a means for establishing a seal between said mechanism and the top surface of said disposable test device.

4. A monitoring system according to Claim 1 wherein said apparatus for removal of said non-bacterial constituents from said disposable test device comprises a support device containing an absorbent pad which eliminates potential reverse fluid, flow through said semi-permeable membrane.

5. A monitoring system according to Claim 1 wherein said means for supporting and positioning said disposable test device within a defined optical path comprises a slide mechanism with means for locking in place and suspending said disposable device in a manner which avoids contact of its membrane surface with adjacent surfaces.

6. The apparatus of Claim 5 wherein said slide mechanism in conjunction with compatibly configured optical housing and panel assemblies eliminates intrusion of ambient light into the photodetector area, irrespective of the position of said slide mechanism.

7. A monitoring system according to Claim 1 wherein said means for generating an electrical signal comprises a photomultiplier tube including an associated voltage divider circuit and high voltage supply.

8. A monitoring system according to Claim 1 wherein said means for generating an output signal comprises: an integration circuit electrically connected to said means for generating an electrical signal; a timing circuit for controlling the duration of integration for a pre-selected time interval; and a display of the integrated output signal for producing a visible presentation representative of the presence and amount of bacteria in the sample.

9. A monitoring system according to Claim 1 wherein said means for generating an output signal comprises: a circuit for compensation for photodetector dark current and other signals not directly attributable to the light emitting reaction; and a circuit for adjusting the gain of the photometer so that the output signal for a sample of known bacterial content as determined by traditional methods may be set to a pre-selected value.

10. A monitoring procedure according to Claim 1 wherein: a) a somatic cell lysing agent is added to a disposable test device

b) the sample to be monitored is added to the solution of Step (a) with attendant lysis of non- bacterial cells and release of non-bacterial ATP

c) the liquid phase of Step (b) is forced through the semi-permeable membrane of the disposable test device into an absorbent pad by means of a positive pressure device, leaving on the surface of the membrane intact bacterial cells free from liquid phase contaminants.

d) an acidic solution buffered at pH 3.5 to 4.0 is added to the disposable test device with attendant release of ATP bound to protein or other large molecules.

e) the procedure of Step (c) is repeated leaving on the surface of the filter intact bacterial cells free from non-bacterial ATP and reaction-inhibiting constituents.

f) a bacterial cell lysing agent is added to the disposable test device with attendant release of bacterial cell ATP

g) a luciferin-luciferase-magnesium ion reagent is added to the solution of Step (f)

h) the light produced by the reaction of Step (g) is measured with the photometer which generates an integrated signal representative of the presence and amount of bacteria in the sample being monitored.

11. A monitoring procedure according to Claim 10 wherein the somatic cell lysing agent comprises a saponin solution.

12. A monitoring procedure according to Claim 10 wherein the acidic solution comprises a pH 3.5 to 4.0 citric acid- citrate buffer.

13. A monitoring procedure according to Claim 10 wherein the bacterial cell lysing agent comprises a mixture of alkyl substituted ammonium halides buffered at pH 7.75, requiring no futher readjustment of pH.

14. A monitoring procedure according to Claim 10 wherein a fixed volume of a luciferin-luciferase-magnesium ion solution, prepared by reconstitution of the lyophilized reagent, is added to the solution containing bacterial ATP.

15. A monitoring procedure according to Claim 10 wherein a fixed amount of luciferin-luciferase-magnesium ion reagent lyophilized within a pipet tip is introduced to the solution containing bacterial ATP by conducting a series of repetitive withdrawals and deliveries of the solution, using the pipet tip and a pipetting device.

16. A monitoring procedure according to Claim 10 wherein a single disposable test device provides means for retention of bacteria, acceptance of reagents, removal of inhibitory and non-bacterial ATP containing constituents, and transmission of the light emitted from the final reaction without requirement for centrifugation, sample transfer, additional reaction vessels or cuvettes or for enzymatic degradation of ATP.