Method of Detecting Living Microorganisms

Examples 1 to 11, below, demonstrate the use of visual screening to assay cultures for living organisms.

In these examples, visual screening of microorganisms is employed for determining a response to the firefly luminessence system. Each test is carried out in a dark room after a period of dark adaptation is achieved by wearing fluoroscopy goggles prior to testing the organisms. Cultures of the micro-organisms listed below are assayed. The incubation broth containing each culture is centrifuged and the supernatant is discarded. The recovered cells are resuspended in 1.0 ml. of deionized water and centrifuged again. The supernatant is discarded, the cells are resuspended in 1.0 ml. of deionized water and placed in a boiling water bath for 10 minutes. The preparation is then centrifuged and 0.1 ml. of the supernatant is added to a mixture of 1.0 ml. of 0.025 M glycylglycine buffer (pH 7.5), 0.05 ml. of 0.1 M MgSO.sub.4, and 0.05 ml. of firefly lantern extract (prepared by adding 50 mg. of dry firefly lanterns to about 4 ml. of water, centrifuging the mixture for 10 minutes at 1,700 G and recovering the supernatant). Positive responses are obtained (i.e., the emission of light was observed) with the following microorganisms:

Example Microorganism

1 Saccharomyces cerevisiae

2 Azotobacter agilis

3 Pseudomonas fluorescens

4 Bascillus cereus

5 Streptomyces bobiliae

6 Staphylococcus epidermis

7 Arthrobacter simplex

8 Bacillus subtilis

9 Xanthomonas campestris

10 Micrococcus cinnabareus

11 Xanthomas beticola

In order to observe and record small amounts of light produced by a positive response between the material to be assayed and the firefly lantern extract and to make quantitative measurements of the amount of light emitted, it is preferred to make use of instruments or an apparatus which will sense and record the intensity of the emitted light.

When instruments are used to detect and record the intensity of emitted light, the procedure consists of injecting a liquid medium containing the material to be assayed for living organisms, such as an aqueous extract of the material, into a cuvette containing the firefly lantern extract. The extract is held at pH 7.4 with potassium arsenate buffer. The light emitted as the result of the reaction between any ATP in the material to be tested and the firefly lantern extract strikes the surface of a photomultiplier tube giving rise to a current which can be measured and recorded by either an oscilloscope photograph or a linear recorder. The unit of intensity used for comparing these reactions is the millivolt. In this application, the unit of light intensity has been arbitrarily defined as being equivalent to one millivolt. Alternately, a pulse counting device with a digital or analogue read-out may be used to record the reaction.

Because the response (i.e., light emission) is almost instantaneous when a living organism is contacted with the firefly lantern extract, the extract should be positioned in front of the light detection system prior to the introduction of the material to be assayed.

There are two ways in which the bioluminescent response with ATP present in a material can be expressed. One is by measurement of the maximum intensity of the emitted light, which after reaching this maximum value, decays exponentially. With all other factors constant, the maximum intensity is directly proportional to the concentration of ATP. The alternative manner of expressing the response is by integration of the total amount of light emitted; i.e., area under the light intensity curve. This is the slower of the two methods, because of the relatively long time necessary for complete decay (up to 10 minutes). Therefore, maximum intensity has been chosen as the measure of ATP response.

The instrumentation necessary for the quantitative measurement of bioluminescence consists of a photomultiplier tube for the conversion of light energy into an electrical signal, a device for determining the magnitude of the signal, and a light tight chamber for presentation of the bioluminescent reaction to the photomultiplier tube.

In one system, part of the assembly consists of a composite sensing and reaction chamber which contains a photomultiplier tube, with appropriate circuitry, and a rotary cylinder mounted in a block of aluminum in a manner which permits removal of the reaction chamber without exposing the phototube to light. A section of the cylinder wall is cut out to accommodate a standard ten mm or five mm rectangular cuvette. Immediately above the cuvette holder is a small injection port sealed with a replaceable light-tight rubber plug. The entire unit is painted black to reduce light reflection. The photomultiplier converts the light energy into an electrical signal. An oscilloscope, which records the magnitude of the signal from the photomultiplier, is provided with a maximum sensitivity of 200 uv/cm of beam deflection which will allow an increase in system sensitivity by decreasing the bandwidth or directly reducing the noise level. There is a multiple switching arrangement at the scope input which makes it convenient to adjust the system zeros and balances. The differential input to the scope provides a means to balance the dark current output of the phototube. The response to the firefly luminescent system displayed on the oscilloscope screen is recorded with a camera which mounts directly onto the front of the oscilloscope. To observe and record the reaction, the cuvette containing the necessary reagents is positioned in the cuvette carrier without exposing the phototube. Rotation of the carrier positions the cuvette in front of the phototube. The unknown is then added through the injection port and the magnitude of the response, if any, is recorded by the camera.

A typical procedure for assaying an unknown material according to the practice of this invention utilizing electronic apparatus to detect and record the intensity of the bioluminescent reaction is described below:

One ml. of a 0.5% buffered aqueous solution of commercially available lyophilized firefly lantern extract is placed into a cuvette which is then positioned in the light detection chamber of the type previously described. The extract contains luciferase, luciferin and magnesium. Sufficient dissolved oxygen for the bioluminescent reaction is present in the solution. An aqueous suspension of material suspected to contain living organisms is subjected to ultrasonic vibration for one or more minutes. One tenth ml. of the suspension is then drawn into a hypodermic syringe and immediately injected through the light-proof seal into the cuvette. The reaction reaches maximum light intensity in less than 0.5 seconds and then decreases exponentially for several minutes. The entire procedure can be executed and the response through its maximum amplitude recorded in less than 2 minutes.

In order to make quantitative determinations of the amount of ATP present, the instrument used to measure the light response may be calibrated using known concentrations of ATP. A calibration may be plotted by injecting 1/10 ml. portions of known concentrations of ATP through the light-proof seal into the cuvette by means of a hypodermic syringe. The light response in millivolts is plotted against the ATP concentration. A straight linear function is obtained. For example, if the response from 10.sup.-.sup.1 gamma of ATP is 20,000 millivolts, that from 10.sup.-.sup.2 is 2000 millivolts, etc.

The following examples illustrate the use of an apparatus as previously described for screening the materials for the presence of living organisms:

Glycylglycine buffer (1.0 ml.) and firefly lantern extract (0.1 ml.) are placed into a rectangular, 5 mm., quartz cuvette which is then positioned in front of a phototube. A broth culture of Escherichia coli is injected into the reaction chamber through a light tight port, by using a 20 gauge needle and a 0.25 ml. syringe. The apparatus recorded a response of greater than 75 mv.

The process of Example 12 is repeated except that the cell culture is heated in a boiling water bath for 10 minutes before it is injected into the reaction chamber. The apparatus recorded a response of 700 mv.

The process of Example 12 is repeated except that before testing, the cell culture is centrifuged for about 10 minutes at 1,700 G, the supernatant is discarded, and the cells are resuspended in about 0.2 ml. of a glycylglycine buffer at pH 7.8. The buffered suspension is then injected into the reaction chamber. The apparatus recorded a response of 500 mv.

The process of Example 13 is repeated except that before testing, the cell broth culture is exposed to 2% aqueous Lysol for about 5 hours. (Lysol contains soap, orthohydroxydiphenyl, alcohol, pine oil, propylene glycol and glycerol.) The Lysol is then removed by centrifuging the cells. The cells are washed 5 more times to remove traces of Lysol, resuspended in deionized water, heated for 15 minutes in a boiling water bath, and 0.1 ml. of the sample injected into the reaction chamber. The apparatus recorded no response indicating that no living organisms are present. The effectiveness of the disinfectant is thereby established.

A soil extract is prepared by adding approximately 500 mg. of garden soil to 2 ml. of sterile, distilled water. After standing for 20 minutes at room temperature, the mixture is centrifuged for one minute at 1700 G and 0.1 ml. of the fairly clear supernatant is assayed by the method described in Procedure A, above. The apparatus recorded a response of greater than 450 mv. indicating a large microbial population in the soil extract.

One gram of dry yeast is suspended in 5 ml. of water and shaken. One tenth ml. of the supernatant is tested and a response of 4400 mv. recorded.

Twenty-two mg. of lyophilized Serratia marcescens were suspended in 0.44 ml. of deionized water. The suspension is heated in a boiling water bath for 5 minutes and 0.1 ml. is tested. The apparatus recorded a response of 210 mv.

A suspension of Bacillus subtilis spores (0.1 ml.) is incubated in yeast-dextrose broth at 37.degree.C for 11/2 hours. The sample is then tested and a response of 250 mv. is recorded.

One ml. of washed cells of Saccharomyces cerevisiae is added to 10 ml. of acetone. After standing about 1 minute, a 1 ml. aliquot of the mixture is dried and the residue is suspended in 1 ml. of deionized water. The sample (which contains about 37,000 cells) is assayed and a response of 260 mv. is recorded.

One ml. of a S. marcescens cell suspension is added to 10 ml. of a 30% aqueous solution of dimethyl sulfoxide. After standing for 5 minutes, the mixture is assayed by adding 0.1 ml. of treated cell suspension containing extract from about 10.sup.6 cells to 1.5 ml. of firefly lantern extract. A response of about 3200 mv. is recorded.

A 5 ml. suspension of S. marcescens cells is subjected to 5 minutes of ultrasonic oscillation. After cooling the tube of suspension in flowing water, the suspension is assayed by adding 0.1 ml. of sonically treated cell suspension to 1.5 ml. of firefly lantern extract. A response of 800 mv. is recorded.

To a 5 ml. suspension of S. marcescens cells there is added 0.1 ml. of perchloric acid. A sample of the mixture is assayed and a response of about 170 mv. recorded.

The overall sensitivity and perhaps reliability of the bioluminescent reaction of the material to be tested may be increased by the conversion of other nucleotide phoshates which are omnipresent in living organisms, such as adenosine diphosphate (ADP) and adenosine monophosphate (AMP), to ATP. This may be accomplished by the utilization of certain phosphorylating enzymes. One such enzyme is phosphocreatine kinase.

The following examples illustrate the use of phosphocreatine kinase to increase the sensitivity of the bioluminescent reaction:

Phosphocreatine kinase (10-30 units/mg.) is made up to a concentration of 0.4 mg./ml., in 0.05 M potassium arsenate buffer (pH 7.4) containing MgSO.sub.4 at a concentration of 10.sup.-.sup.5 M and creatine phosphate at a concentration of 0.1 mg./ml. Four ml. of a S. marcescens cell suspension are subjected to sonic disruption for 5 minutes and then boiled for 5 minutes. After cooling, an aliquot equivalent to 2 million cells is removed for assay. One tenth ml. of the phosphocreatine kinase solution is added to the remainder and allowed to stand for 5 minutes after which it is boiled for 2 minutes. At the end of 5 minutes, another aliquot equivalent to the first is removed and boiled 2 minutes for assay. The remainder is allowed to incubate for an additional 10 minutes after which another aliquot is removed and boiled 2 minutes. A fourth and final aliquot is removed 15 minutes later and boiled 2 minutes. All aliquots are assayed by adding 0.1 ml. of the aliquot to 1.5 ml. of firefly lantern extract (10 mg. extract per ml. of solution). The results are set forth in the following Table.

Although the use of this precursor conversion modification involves a longer period of time for the assay, its contribution to greater sensitivity is readily apparent.

The following examples illustrate the detection of the presence of bacterial cells and the determination of a specific bacteria by the selection of an appropriate incubation media and environment:

One ml. of water which is suspected to contain E. coli is introduced into 5 ml. of MacConkey broth and the mixture is assayed for ATP content. The mixture is then incubated for several hours, at 44.degree.C. and again assayed. The incubated sample is found to register a significantly higher bioluminescent response than the unincubated sample. The presence of E. coli in the sample is thus confirmed.

Ten ml. portions of nutrient broth containing a different one of each of the following antibiotics in concentrations of 0.5, 1, 2.5 and 5.0 ug./ml. are prepared: Penicillin G, Neomycin and Chloramphenicol. To each broth portion, there is added an equal amount of an aqueous suspension of a bacteria culture obtained from a throat swabbing. Each portion of broth is then incubated. Each broth is assayed for ATP content before and after incubation. It is found that the ATP content of the broth containing 5. 0 ug./ml. of Pencillin G has increased less than that of the broths containing the other antibiotics. It is thus established that Penicillin G in a concentration of 5.0 ug./ml. is the most effective antibiotic in treating the bacterial infection.

Two 10 ml. portions of nutrient broth are prepared, one portion containing typhoid fever antibodies and the other portion containing staphylococcusantibodies. Each broth portion is inoculated with equal aliquots of an aqueous suspension of a bacteria culture and incubated. Both before and after incubation, each culture is assayed for ATP content. It is found that the ATP content of the broth containing the staphylococcus antibodies increases much less than the ATP content of the broth containing the typhoid fever antibodies, thus indicating that the bacteria present is staphylococcus.