CA1103050A - Method for detecting toxic substances in liquid - Google Patents
Method for detecting toxic substances in liquidInfo
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- CA1103050A CA1103050A CA311,855A CA311855A CA1103050A CA 1103050 A CA1103050 A CA 1103050A CA 311855 A CA311855 A CA 311855A CA 1103050 A CA1103050 A CA 1103050A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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- Proteomics, Peptides & Aminoacids (AREA)
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- Biotechnology (AREA)
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- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Abstract One or more strains of luminous microorganisms are used as biological indicators in the detection of toxic substances or conditions in liquids. When exposed to toxic substances or conditions, the change in light output is sensed by light sensing apparatus. The amount of change in light output of the micro-organisms is related to the concentration of toxic substance to which the organisms have been exposed. The cells are grown in a variety of ways and lyophilized cells, which have a long storage life, can be reconstituted and used in detection of toxic sub-stances in accordance with the invention without further pro-pagation.
Description
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Back~round of the Invention The present invention relates to the detection of toxic substances in liquids and, more particularly, to a method for the detection of toxic substances using changes in the metabolic processes of biological organisms as the indicator.
Biological organisms provide a sensitive and relatively reliable indicator of the presence of even relatively small proportions of toxic substances in a fluid medium. With growing concern over the pollution of water and the harmful effects of toxic substances on man, the Environmental Protection Agency has been charged with the responsibility of propounding regulations and testing procedures for detecting the presence of toxic sub-stances in water. The testing protocol presently preferred by most workers in the field involves contacting biological organisms such as small fish or water fleas, with the water being tested.
The presence of toxic substances is indicated by the erratic physiological response or death of fish or water fleas and the concentration of toxic material is determined by the survival rate of the organisms. These tests are time consuming and ex-pensive. For example, when utiliæing minnows to test for toxic --~20D~135 Can. ~3~3 substances in water, the tests are run over a perio~ of 48 to 96 hours, although a longer time, on the order of one to four weeks, is required to stabilize the minnow population prior to the actual tests. In addition to long testing periods, reproducibility results can be difficult to achieve because of the uniqueness of each population used in the testing procedure.
It has been suggested that microorganisms may be used for assaying samples for the detection of various substances. For example, in U.S. patent 3,370,175, Jordan et al., the use of luminescent microorganisms for the detection of toxic substances in air is disclosed. The metabolism of the luminous micro-organisms is affected by low levels of toxicants and the in-tensity of light output is affected. By sensing changes in the light output the presence and relative concentration of a toxicant can be detected. Similar systems are disclosed in U.S.
patents 3,849,653 Sakaide et al. and 3,958,938 Doonan et al.
The systems represented by the Jordan et al. and other patents discussed above are specifically designed for the detection of toxic substances in vapor, aerosol or gaseous form and require that the microorganism cultures be in a growing state on solid substrates in order that there be maximum contact between the air being tested and the culture. Consequently, these systems are not adapted for use in a liquid environment and are not amenable for the testing of water and other liquids. It is preferred when using these systems that ambient conditions of humidity and temperature be maintained at optimum levels in order to support the continued growth of the microorganism culture. This is necessary since the same culture is re-used a number of times.
The foregoing systems are deficient either ~n that they are time consuming, difficult and expensive to run or that they are not adapted for use in the testing for toxic substances in liquid environment.
420D~135 Can.
Summary of the Invention The present invention is directed to a method for testing of liquids for the presence of toxic substances therein. The method employs a biological organism as the indicator and is particularly suited for the detection of toxic pollutants in water. Test results can be correlated with the "fish testsi', described above, which are the presently preferred biological test for the presence of toxic substances in water.
In accordance with the present invention a material suspected of containing a toxic substance is intermixed with an aqueous suspension of luminous microorganisms of a known light output.
After intermixing the material and the aqueous suspension the light output of the mixture is measured. If a substance toxic to the microorganisms is present, the light output of the mixture will deviate from the light output of the suspension before contact and the magnitude of deviation is approximately propor-tional to the concentration of toxic substance in the material being tested.
; In the practice of the invention the microorganisms are suspended in an agueous medium such as an aqueous salt solution during the testing procedure and since it is unnecessary that the culture be actually growing at the time of the test, the lumines-cent microorganisms are readily prepared for use by simply remov-ing the microorganisms from their growing culture and suspending them in the aqueous medium.
It has been found in practicing the method of the present invention that the temperature at which the microorganism suspen-sion is maintained can affect the sensitivity and light output of the microorganism. Although not critical, it is preferred that the témperature of the sample and microorganism suspension be controlled for best results.
--420D-135 Can.
In a preferred embodiment of the invention the micro-organisms used as the biological indicator are in lyophilized form prior to use. In such form, the microorganisms are readily reconstituted for substantially immediate use by the addition of distilled water to the lyophilized microorganism. The micro-organism can thus be conveniently maintained in stable condition for shipping and storage until it is needed for test purposes.
Other advantages and features of the present invention will be apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Brief Description of the Drawings Fig. 1 is a block diagram of a light output sensor employed in the present invention.
Fig. 2 shows the relationship of phenol concentration versus light output of a luminous microorganism.
Detailed Description of the Invention The invention provides a method for the detection of toxic substance or toxic condition in liquids using luminous micro-organisms as a biological indicator. The term liguid includes any life supporting liguid which typically comprises a major portion of water and a minor proportion of other constituents dissolved or suspended therein. Thus the method of the invention is used for the detection of toxic substances or toxic conditions in water supplies, run-off surface waters and the like. In addition, the method of the invention can be used to detect toxic substances or a toxic condition in body fluids and serums and other fluids which have an aqueous base and which will support life forms.
The term "condition" is used herein to mean the environment for microorganisms provided by a liquid.
A substance or condition is considered toxic if it adversely affects the metabolic processes of the biological indicators ~-~20D-135 Can.
utilized in the present invention as evidenced by a change in light output. Although a wide variety of toxic substances and conditions can be detected by the method of the present invention, certain substances are particularly well known to be toxic to life forms in relatively small concentrations. These substances may be found in streams, rivers and other water sources polluted by industrial and agricultural wastes and, accordingly, toxicity tests of water supplies are normally designed for the detection of the presence of these substances.
Thus, inorganic materials such as mercury, chromium, lead and zinc compounds, ammonia, nitrates, phosphorus compunds, sulfates and copper compounds have all been identified from various sources as present in waters polluted by industrial waste. In addition, organic compounds such as DDT, phenol, lindane, heptachlor, aldrin, toluene, 7-12-dimethylbenzanthracene, 3-methyl cholanthrene, benzedene, o-aminoazotoluene, 4-dimethyl-aminoazobenzene, pentachlorophenol, carbon tetrachloride, acetone, thimersol, sodium laurel sulfate and chlordane, are examples of various herbicides, pesticides, surfactants and other organics which can be expected to be found in waters polluted by industrial and agricultural activities.
The foregoing list is not intended to be exhaustive and only exemplifies the wide variety of substances, the presence or absence of which comprise conditions toxic to biological organisms.
Typically, most industrial wastes comprise mixtures of materials considered as pollutants, one or more of which are present in sufficient concentrations to adversely affect the metabolic processes of the biological indicator and thus give rise to toxic conditions.
The toxic substance need not be highly soluble in the liquid being tested. It will be understood, however, that in order for a substance to be toxic or to produce a toxic condition it must ~ 20D-135 Can.
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have sufficient physical or chemical properties to enable it either to be absorbed in toxic amounts or to affect the liquid in which it is contained, either physically or chemically so as to produce a toxic condition in the liquid. Substantially all toxic substances for which a test would be desired will meet one or both of these requirements.
The material being tested for toxicity need not initially be an aqueous liquid. For example, materials to be tested for toxicity may be in the form of a finely divided powder which can be subsequently solubilized or dispersed in an aqueous base or be added directly to the aqueous suspension of microorganisms.
Luminous microorganisms are broadly sensitive to toxic substances or conditions without regard to the strain or species of the microorganism. However, some microorganism strains are found to be more sensitive to a particular substance or a given condition and thus exhibit a more pronounced change in light output when in contact with such substances or condition than strains less sensitive to the same substance or condition. It is preferred, therefore, to utilize a microorganism strain which is selected based on its sensitivity when challenged by a given substance or condition. It is also within the scope of the invention to utilize a mi~ture of microorganism strains, the individual strains of which are especially sensitive to given substances or conditions. The total effect of the mixture of strains is to provide enhanced and broadened sensitivity to selected varieties of substances or conditions.
However, it is not necessary to know the precise toxic substance or condition in the material being tested. As mentioned above, luminous microorganisms are broadly sensitive to toxic substances and regardless of the strain or species, will display a change in light output in the presence of a toxic substance or condition in the test liquid. As will be explained -~2OD-135 Can.
in more detail, the change in light output of the microorganisms is a convenient indication of the concentration of toxic substance in the test li~uid.
Several strains of bacteria are known to exhibit strong luminescence in the course of their growth cycle. Particularly well known and readily available species of luminous bacteria include the Photobacterium such as P. splendidum, P. manda-pamensis, _ phosphoreum. Strains of bacteria from the genus Vibrio likewise exhibit luminescence such as, for example, V.
fischeri. as well as microorganisms from the genus Lucibacterium such as L harveyi and Achromobacter. In addition to the various bacteria, other types of microorganisms also exhibit luminosity such as, for example, certain marine dinoflagellates, which notably include Noctiluca and Gonyaulax. Certain varieties of fungi (Basidiomycetes) also exhibit luminescence including for example, Armillaria mellea, Panus stipticus, Mycena polygramma and Omphalia flavida. Luminous bacterial and fungal micro-organisms are available from the American Type Culture Collection, 12301 Parkland Drive, Rockville, Maryland, or Northern Regional Research Laboratories, U.S. Department of Agriculture, Peoria, Illinois, as well as from many commercial and university labora-tories.
Typically, the stock cultures of bacteria or fungi are grown from a preserved culture by transferring a portion of the cells from the preserved culture to a nutrient medium where they are permitted to grow and multiply. The choice of medium used for growing the culture is dependent on the type of culture being employed and the manner in which the microorganisms are to be subsequently treated in preparation of their use in the present invention.
A preferred growth medium for the more common luminescent organisms typically includes up to about 2% agar, between about --~2OD-135 Can.
~33C~
.25% to about .7% of sodium or potassium pyrophosphate, about .5%
glycerol, between 0.5% and about 1% peptones (animal and/or vegetable), up to about 1% hydrolyzed casein, and between about 0.5% to about 5% of sodium chloride and the remainder being water. The pH of the growth medium preferably ranges between about 6.5 to about 7.5, and most preferably on the order of 7.0 For marine microorganisms, sea water can be substituted for the water in the nutrient medium. Also depending on the strain of microorganism being grown, an extract such as meat extract, squid or fish extract, can be highly beneficial in the nutrient medium.
Luminous fungi are conveniently grown in an aqueous broth having about 10% bread crumbs, and artificial nutrient or cooked cherry broth. They may also be grown on a solid agar nutrient comprising, for example, 2% agar and 10% bread crumbs and water.
In one embodiment of the method of the invention, an aliquot of cells is obtained from a growing culture of luminous micro-organisms and the microorganisms are suspended in an aqueous salt solution to form a working suspension of the cells for use in a light sensing apparatus. The concentration of cells in the suspension is not critical and may vary over a broad range.
However, a sufficient quantity of cells should be present in the suspension so that the total light output of the working suspen-sion is in the detectable range on the light sensing means employed in the method. In forming a working suspension of cells from a growing culture, it is convenient to first form a stock suspension which is more highly concentrated than necessary. The working suspension is then made by diluting aliquots from the stock solution until a desired level of intensity of light output (base line light output) is obtained. With lyophils the concentration of cells is adjusted prior to lyophilization and a working sus-pension having the desired base line is prepared by simply adding distilled water to the lyophil. The base line light output is -~20D-135 Can.
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recorded for each suspension prior to introduction of the test liquid. The base line serves as a reference light output from which subsequent changes in light output are measured. It will be seen that a variation or fluctuation in base line due to causes other than the toxic substance or condition being tested for such variations is undesirable and could result in misleading or false readings unless compensated for by the light sensing means. It is not abnormal for base line light output from the working suspension to change over a period of time. The change in light output, in the absence of a toxic substance or condition, is referred to as base line drift and is typically determined using a control for each series of tests employing a particular strain of microorganism. Where test procedures permit base line drift is preferably determined for each working suspension. ~ase line drift is not uncommon in many photometric analytical systems and can be compensated for by suitable means such as electronic or manual control, or compensating instrumentation, as is well known in the art. Preferably, base line drift is maintained at minimum since a low base line drift will permit the utilization of simpler, and typically less expensive instrumentation as the light sensing means.
It has been found that the suspending medium can have a substantial effect on the amount of~rift in the base line. Good results are achieved when the suspending medium contains between about 0.5% to about 6% by weight of sodium chloride with about 2%
to about 6% sodium chloride being preferred and 3% sodium chloride highly preferred. These salt concentrations are substantially similar to the salt concentrations utilized in the nutrient medium during the growing cycle of the cells. It has also been found that good results are achieved when the aqueous suspending medium includes minor amounts of other salts such as sodium sulfate and sodium pyrophosphate and nutrients such as yeast -~2OD-135 Can.
3~
extract. Although it is not completely understood as to why inclusion of the foregoing ingredients in the suspending medium improves stability of light output and reduces base line drift in the suspension, it is believed that while the cells are not -actively growing in number, the suspending medium still should provide an environment to maintain cell metabolism at an accept-able level so that light output remains relatively stable. A
preferred suspending medium includes, in addition to sodium chloride, between 1 and about 10 micrograms/ml of yeast extract and between about 50 and 250 micrograms/ml of sodium pyrophos-phate.
Having formed the suspension of luminous microorganisms as described and having sensed and recorded the base line light output of the suspension, a sample suspected of containing a toxic substance or having a toxic condition is introduced into the container with the working suspension. The contents are agitated so as to thoroughly mix the suspension and the intro-duced material. A change in the light output is determined either by measuring the total change in light output over a given period of time or the rate of change of light output. Whether the change in light output is measured as rate of change or as the total change over a given period of time will depend on factors such as the type and concentration of the toxic substance, the sensitivity of the microorganism, the means for sensing the light output and the like, and the method employed can be readily determined by prescreening the microorganism and liquid being tested.
The presence of a toxic substance or condition is indicated by a change in the light output of the microorganism suspension over the base line output. Typically the toxic substance or condition cause a reduction in light output although depending on the strain of microorganism certain toxic substances or conditions ~2OD-135 Can.
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may cause at least a temporary increase in the light output of the microorganism suspension. The amount of light output change is related to the concentration of the toxic substance or the magnitude of the toxic condition.
Accordingly, using standard curves, the concentration of a given toxic substance can be determined, assuming that the toxic substance is identified. In addition, a correlation can be set up between the results of the present method and the results of the so-called "fish" test, the test most commonly employed at the present time in attempting to determine the presence of a toxic substance in water.
Any conventional light sensing means can be employed in the present invention to sense the light output from the aqueous suspension of microorganisms and the subsequently formed mixture of microorganisms and material being tested. As is well known in the art, there are various types of photometric and optical analytical instruments, all of which operate on the same general principles well known in the art.
In Fig. 1, a photometric system suitable for detecting the output of light from the luminescent microorganisms is shown diagrammatically and is generally indicated by reference numeral 10. In its broadest aspects, the system 10 includes a suitable cell, cuvette or container 12 in which the suspension of luminous microorganisms and the test sample are contained for light output measurement. A light detector such as a photomultiplier tube 14, or a photocell, is positioned in light impinging relationship to the container 12 for generating a low voltage current which is relative to the intensity of light impinging the detector. The output from the cell 14 is passed through a photocell amplifier 17 where the output is amplified and then passed on to a display means such as a meter 18. The choice of display means is not critical and in place of, or in addition to the meter 18, there ~20D-135 Can.
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may be included in the system a recording chart or a digital readout device of conventional design.
It should also be noted that the temperature of the working suspension will affect both the sensitivity and the actual light output of the microorganisms in the working suspension. Accord-ingly, it is preferred to run all tests with a particular strain of microorganism and a particular toxicant at the same temperature to insure reproducibility of results. Thus, with toxicants such as phenol it has been found that for a given concentration of toxicant, the actual percent decrease in light output of a working suspension of microorganism decreases with increasing temperature.
With other toxicants such as isopropyl alcohol the percent of light output decrease of the working suspension increases with increasing temperature. Accordingly, it is preferred, partic-ularly when working with new test samples, to record the light output change of the working suspension through a range of temp-eratures of between about 10C. to about 30C. The preferred test temperature will be that temperature at which the maximum change in light output is recorded. This indicates the temp-erature at which the microorganism has maximum sensitivity to the toxicant ~r mixture of toxicants in the test solution.
Where reproducibility of results or maximum sensitivity are not of primary concern, the method can be carried out with working solution at the ambient temperature within the photometer. In any case, the working suspension should be allowed to come to a stable temperature before recording light output because changes in temperature will also affect the base line light output and will result in an exaggerated base line drift. In the examples set forth below the working suspension was stabilized at the ambient temperature of the photometer before recording the light output.
--~2OD-135 Can.
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The invention is further described by the following examples which are illustrative of specific modes of practicing the inven-tion and are not intended as limiting the scope of the invention as defined by the appended claims.
EXAMPLE I
Luminescent bacterial cells for use in toxicity testing were prepared by innoculating agar media to form cultures of V.
fischeri, P. phosphoreu_, ATCC 11040; P. mandapamensis, ATCC
27561; L. harveyi, ATCC 14126 and A. fischeri, NRRL B-11177. The agar medium was made up of the following ingredients:
KH2P4 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - O.25 wt. %
NaCl - - - - - - - - - - - - - - 3.0 wt. %
Glycerol ~ - - - - - - - - 0.5 wt. %
Yeast Extract- - - - - - - - - - - 0.1 wt. %
Polypeptone- - - - - - - - - - - - 0.5 wt. %
Hydrolyzed Casein- - - - - - - - - l.O wt. %
Squid Extract- - - - - - - - - - -10.0 Vol.%
Agar - - - - - - - - - - - - - - - 1.5 wt. %
Distilled Water- - - - - - - - - -90.0 Vol.%
The squid extract was prepared by homogenizing 454 grams of whole squid with enough distilled water to make a final volume of 1 liter. The slurry was autoclaved at 121C. for 15 minutes and the resulting suspension clarified by centrifugation.
After 24 hours of growth at 22C. the cells were removed from each of the cultures and suspended in 3% aqueous solution of sodium chloride (pH 7.1) to form a stock suspension of each of the cell cultures. The stock suspension was agitated vigorously to assure a homogenous suspension.
A working suspension was prepared from each of the stock solutions by diluting 10 microliter aliquots of each stock solu-tion in 2 ml of an aqueous suspending medium. The suspending medium consisted of an aqueous sodium chloride solution (3 wt.%) ~-420D-135 Can.
which contained 135 micrograms/ml of sodium pyrophosphate and 5 micrograms/ml of commercially available yeast extract (Diffco Laboratories, Detroit, Michigan).
The base line light output of the working solutions was determined with the suspensions substantially at ambient temp-eratures in a photometer equipped with a photomultiplier tube and a chart recorder.
EXAMPLE II
Luminescent bacterial cell cultures were also grown in liquid media. Cultures of P. phosphoreum, ATCC 11041; P.
mandapamensis, ATCC 27561; L. harveyi, ATCC 14126 and A.
fischeri, NRRL B-11177 were grown as shake flask cultures. The growth medium was as used in Example I except that no agar was utilized in the culture media. The cultures were grown in S00 ml. triple baffled DeLong style flasks containing 100 ml. per flask. The cultures were grown for 18 hours at 22C. and during the growth period, the cultures were agitated at 240 rpm on a New Brunswick gyrotary shaker. The cells were harvested during the logarithmic phase of growth and separated from the growth medium by centrifugation. The resulting pellet of cells from each of the cultures was maintained at 4C. prior to formation o~ the respective stock suspension in the manner disclosed in Example I.
Each stock suspension was diluted to form a working suspension for use in the photometer in the manner of Example I
and base line drift was recorded for each of the working suspensions.
EXAMPLE III
Cultures of P. mandapamensis, ATCC 27561; L. harveyi, ATCC
14126 and A. fischeri, NRRL B-11177 were each prepared in a broth growth medium as described in Example II. The cultures were separated from the broth during the logarithmic phase of growth by centrifugation and the resulting cell pellet chilled to 4C.
-~20D-135 Can.
11~}3QS~
The chilled cell pellet was suspended in a 3% sodium chloride solution maintained at a temperature of 4C. The sodium chloride solution was added to the suspension until a homogenous cell suspension having a final optical density of 160 was prepared.
The cell suspension was mixed with an a~ueous milk solution (20 wt.% skim milk, 2 wt.% NaCl, pH 7.1) in a volume ratio of one part cell suspension to 19 parts of the milk solution. The cell-milk suspension was distributed in 1 ml. aliquots into 10 ml. vials and quick frozen at a temperature of -50C. The cells were then dried at -5C. under 55 microns vacuum for 18 hours.
The vials were then vacuum sealed.
Working suspensions of each of the lyophilized samples were prepared by the addition of 1 ml. of distilled water to the vials. The resultant suspensions exhibited good base line light output.
EXAMPLE IV
Working suspensions of the several species of luminescent bacteria grown and prepared as in Examples I, II and III were challenged with varying concentrations of substances known to be toxic to biological organisms to determine the effect on the cell light output of the condition thus created.
The following toxic substances were used to create toxic conditions: phenol; sodium laurel sulfate; a mixture of various substances referred to as mixture 401, and a wood preservative known as penta, which consisted of 3.5% pentachlorophenol, 65%
mineral spirits and 32.5% inert ingredients.
The reference mixture 401 was prepared by adding 3.82 grams NH4C1, 0.093 grams K2Cro4, 0.128 grams Na2S, .9 H2O, 0.20 grams Kaolin, 0.1 grams phenol and 1.28 ml. No. 2 fuel oil to 100 ml.
of distilled water.
The tests were conducted by adding between 10 and 100 microliters of solutions of the toxic substance to a cuvette -420D-135 Can.
3~50 containing a working suspension of cells for which the base line and base line drift had been recorded. After introducing the toxic substance, the cuvettes were inverted several times to insure mixing of the suspension and the material containing the toxic substance. The cuvettes were replaced in the photometer and the percent decrease in light was determined after a period of two minutes from the addition of the toxic substance. Control samples consisting of a working suspension of each of the cultures and 10 microliters of a 3% sodium chloride solution were run along with test samples.
The results are set forth in Tables A, B and C below. In each case, concentrations of the toxic substances used are expressed in milligrams/liter, except for mixture 401, which is expressed as volume percent of the mixture in the test solution.
Table A sets forth the results for luminescent cells grown in accordance with Example I. Tables B and C set forth results for the cells grown and prepared as in Examples II and III, respectively.
-~2OD-135 Can.
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TABLE A
Percent Decrease in Light Concentrations of ATCC ATCCATCC NRRLVibrio Toxicants Tested 2756114126 11040B-11177Fischeri 3% NaCl (control) O O O O O
Phenol 50 mg/L 20 26 9 13 10 100 mg/L 55 40 18 23 20 250 mg/L 95 91 33 51 41 Sodium Lauryl Sulfate 25 mg/L 20 19 17 67 21 50 mg/L 40 36 31 90 81 100 mg/L 94 33 12 89 83 Reference Mixture 401 .5% 9 2 3 7 6 1.0% 14 4 9 9 6
Back~round of the Invention The present invention relates to the detection of toxic substances in liquids and, more particularly, to a method for the detection of toxic substances using changes in the metabolic processes of biological organisms as the indicator.
Biological organisms provide a sensitive and relatively reliable indicator of the presence of even relatively small proportions of toxic substances in a fluid medium. With growing concern over the pollution of water and the harmful effects of toxic substances on man, the Environmental Protection Agency has been charged with the responsibility of propounding regulations and testing procedures for detecting the presence of toxic sub-stances in water. The testing protocol presently preferred by most workers in the field involves contacting biological organisms such as small fish or water fleas, with the water being tested.
The presence of toxic substances is indicated by the erratic physiological response or death of fish or water fleas and the concentration of toxic material is determined by the survival rate of the organisms. These tests are time consuming and ex-pensive. For example, when utiliæing minnows to test for toxic --~20D~135 Can. ~3~3 substances in water, the tests are run over a perio~ of 48 to 96 hours, although a longer time, on the order of one to four weeks, is required to stabilize the minnow population prior to the actual tests. In addition to long testing periods, reproducibility results can be difficult to achieve because of the uniqueness of each population used in the testing procedure.
It has been suggested that microorganisms may be used for assaying samples for the detection of various substances. For example, in U.S. patent 3,370,175, Jordan et al., the use of luminescent microorganisms for the detection of toxic substances in air is disclosed. The metabolism of the luminous micro-organisms is affected by low levels of toxicants and the in-tensity of light output is affected. By sensing changes in the light output the presence and relative concentration of a toxicant can be detected. Similar systems are disclosed in U.S.
patents 3,849,653 Sakaide et al. and 3,958,938 Doonan et al.
The systems represented by the Jordan et al. and other patents discussed above are specifically designed for the detection of toxic substances in vapor, aerosol or gaseous form and require that the microorganism cultures be in a growing state on solid substrates in order that there be maximum contact between the air being tested and the culture. Consequently, these systems are not adapted for use in a liquid environment and are not amenable for the testing of water and other liquids. It is preferred when using these systems that ambient conditions of humidity and temperature be maintained at optimum levels in order to support the continued growth of the microorganism culture. This is necessary since the same culture is re-used a number of times.
The foregoing systems are deficient either ~n that they are time consuming, difficult and expensive to run or that they are not adapted for use in the testing for toxic substances in liquid environment.
420D~135 Can.
Summary of the Invention The present invention is directed to a method for testing of liquids for the presence of toxic substances therein. The method employs a biological organism as the indicator and is particularly suited for the detection of toxic pollutants in water. Test results can be correlated with the "fish testsi', described above, which are the presently preferred biological test for the presence of toxic substances in water.
In accordance with the present invention a material suspected of containing a toxic substance is intermixed with an aqueous suspension of luminous microorganisms of a known light output.
After intermixing the material and the aqueous suspension the light output of the mixture is measured. If a substance toxic to the microorganisms is present, the light output of the mixture will deviate from the light output of the suspension before contact and the magnitude of deviation is approximately propor-tional to the concentration of toxic substance in the material being tested.
; In the practice of the invention the microorganisms are suspended in an agueous medium such as an aqueous salt solution during the testing procedure and since it is unnecessary that the culture be actually growing at the time of the test, the lumines-cent microorganisms are readily prepared for use by simply remov-ing the microorganisms from their growing culture and suspending them in the aqueous medium.
It has been found in practicing the method of the present invention that the temperature at which the microorganism suspen-sion is maintained can affect the sensitivity and light output of the microorganism. Although not critical, it is preferred that the témperature of the sample and microorganism suspension be controlled for best results.
--420D-135 Can.
In a preferred embodiment of the invention the micro-organisms used as the biological indicator are in lyophilized form prior to use. In such form, the microorganisms are readily reconstituted for substantially immediate use by the addition of distilled water to the lyophilized microorganism. The micro-organism can thus be conveniently maintained in stable condition for shipping and storage until it is needed for test purposes.
Other advantages and features of the present invention will be apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Brief Description of the Drawings Fig. 1 is a block diagram of a light output sensor employed in the present invention.
Fig. 2 shows the relationship of phenol concentration versus light output of a luminous microorganism.
Detailed Description of the Invention The invention provides a method for the detection of toxic substance or toxic condition in liquids using luminous micro-organisms as a biological indicator. The term liguid includes any life supporting liguid which typically comprises a major portion of water and a minor proportion of other constituents dissolved or suspended therein. Thus the method of the invention is used for the detection of toxic substances or toxic conditions in water supplies, run-off surface waters and the like. In addition, the method of the invention can be used to detect toxic substances or a toxic condition in body fluids and serums and other fluids which have an aqueous base and which will support life forms.
The term "condition" is used herein to mean the environment for microorganisms provided by a liquid.
A substance or condition is considered toxic if it adversely affects the metabolic processes of the biological indicators ~-~20D-135 Can.
utilized in the present invention as evidenced by a change in light output. Although a wide variety of toxic substances and conditions can be detected by the method of the present invention, certain substances are particularly well known to be toxic to life forms in relatively small concentrations. These substances may be found in streams, rivers and other water sources polluted by industrial and agricultural wastes and, accordingly, toxicity tests of water supplies are normally designed for the detection of the presence of these substances.
Thus, inorganic materials such as mercury, chromium, lead and zinc compounds, ammonia, nitrates, phosphorus compunds, sulfates and copper compounds have all been identified from various sources as present in waters polluted by industrial waste. In addition, organic compounds such as DDT, phenol, lindane, heptachlor, aldrin, toluene, 7-12-dimethylbenzanthracene, 3-methyl cholanthrene, benzedene, o-aminoazotoluene, 4-dimethyl-aminoazobenzene, pentachlorophenol, carbon tetrachloride, acetone, thimersol, sodium laurel sulfate and chlordane, are examples of various herbicides, pesticides, surfactants and other organics which can be expected to be found in waters polluted by industrial and agricultural activities.
The foregoing list is not intended to be exhaustive and only exemplifies the wide variety of substances, the presence or absence of which comprise conditions toxic to biological organisms.
Typically, most industrial wastes comprise mixtures of materials considered as pollutants, one or more of which are present in sufficient concentrations to adversely affect the metabolic processes of the biological indicator and thus give rise to toxic conditions.
The toxic substance need not be highly soluble in the liquid being tested. It will be understood, however, that in order for a substance to be toxic or to produce a toxic condition it must ~ 20D-135 Can.
3Ç~
have sufficient physical or chemical properties to enable it either to be absorbed in toxic amounts or to affect the liquid in which it is contained, either physically or chemically so as to produce a toxic condition in the liquid. Substantially all toxic substances for which a test would be desired will meet one or both of these requirements.
The material being tested for toxicity need not initially be an aqueous liquid. For example, materials to be tested for toxicity may be in the form of a finely divided powder which can be subsequently solubilized or dispersed in an aqueous base or be added directly to the aqueous suspension of microorganisms.
Luminous microorganisms are broadly sensitive to toxic substances or conditions without regard to the strain or species of the microorganism. However, some microorganism strains are found to be more sensitive to a particular substance or a given condition and thus exhibit a more pronounced change in light output when in contact with such substances or condition than strains less sensitive to the same substance or condition. It is preferred, therefore, to utilize a microorganism strain which is selected based on its sensitivity when challenged by a given substance or condition. It is also within the scope of the invention to utilize a mi~ture of microorganism strains, the individual strains of which are especially sensitive to given substances or conditions. The total effect of the mixture of strains is to provide enhanced and broadened sensitivity to selected varieties of substances or conditions.
However, it is not necessary to know the precise toxic substance or condition in the material being tested. As mentioned above, luminous microorganisms are broadly sensitive to toxic substances and regardless of the strain or species, will display a change in light output in the presence of a toxic substance or condition in the test liquid. As will be explained -~2OD-135 Can.
in more detail, the change in light output of the microorganisms is a convenient indication of the concentration of toxic substance in the test li~uid.
Several strains of bacteria are known to exhibit strong luminescence in the course of their growth cycle. Particularly well known and readily available species of luminous bacteria include the Photobacterium such as P. splendidum, P. manda-pamensis, _ phosphoreum. Strains of bacteria from the genus Vibrio likewise exhibit luminescence such as, for example, V.
fischeri. as well as microorganisms from the genus Lucibacterium such as L harveyi and Achromobacter. In addition to the various bacteria, other types of microorganisms also exhibit luminosity such as, for example, certain marine dinoflagellates, which notably include Noctiluca and Gonyaulax. Certain varieties of fungi (Basidiomycetes) also exhibit luminescence including for example, Armillaria mellea, Panus stipticus, Mycena polygramma and Omphalia flavida. Luminous bacterial and fungal micro-organisms are available from the American Type Culture Collection, 12301 Parkland Drive, Rockville, Maryland, or Northern Regional Research Laboratories, U.S. Department of Agriculture, Peoria, Illinois, as well as from many commercial and university labora-tories.
Typically, the stock cultures of bacteria or fungi are grown from a preserved culture by transferring a portion of the cells from the preserved culture to a nutrient medium where they are permitted to grow and multiply. The choice of medium used for growing the culture is dependent on the type of culture being employed and the manner in which the microorganisms are to be subsequently treated in preparation of their use in the present invention.
A preferred growth medium for the more common luminescent organisms typically includes up to about 2% agar, between about --~2OD-135 Can.
~33C~
.25% to about .7% of sodium or potassium pyrophosphate, about .5%
glycerol, between 0.5% and about 1% peptones (animal and/or vegetable), up to about 1% hydrolyzed casein, and between about 0.5% to about 5% of sodium chloride and the remainder being water. The pH of the growth medium preferably ranges between about 6.5 to about 7.5, and most preferably on the order of 7.0 For marine microorganisms, sea water can be substituted for the water in the nutrient medium. Also depending on the strain of microorganism being grown, an extract such as meat extract, squid or fish extract, can be highly beneficial in the nutrient medium.
Luminous fungi are conveniently grown in an aqueous broth having about 10% bread crumbs, and artificial nutrient or cooked cherry broth. They may also be grown on a solid agar nutrient comprising, for example, 2% agar and 10% bread crumbs and water.
In one embodiment of the method of the invention, an aliquot of cells is obtained from a growing culture of luminous micro-organisms and the microorganisms are suspended in an aqueous salt solution to form a working suspension of the cells for use in a light sensing apparatus. The concentration of cells in the suspension is not critical and may vary over a broad range.
However, a sufficient quantity of cells should be present in the suspension so that the total light output of the working suspen-sion is in the detectable range on the light sensing means employed in the method. In forming a working suspension of cells from a growing culture, it is convenient to first form a stock suspension which is more highly concentrated than necessary. The working suspension is then made by diluting aliquots from the stock solution until a desired level of intensity of light output (base line light output) is obtained. With lyophils the concentration of cells is adjusted prior to lyophilization and a working sus-pension having the desired base line is prepared by simply adding distilled water to the lyophil. The base line light output is -~20D-135 Can.
~3~
recorded for each suspension prior to introduction of the test liquid. The base line serves as a reference light output from which subsequent changes in light output are measured. It will be seen that a variation or fluctuation in base line due to causes other than the toxic substance or condition being tested for such variations is undesirable and could result in misleading or false readings unless compensated for by the light sensing means. It is not abnormal for base line light output from the working suspension to change over a period of time. The change in light output, in the absence of a toxic substance or condition, is referred to as base line drift and is typically determined using a control for each series of tests employing a particular strain of microorganism. Where test procedures permit base line drift is preferably determined for each working suspension. ~ase line drift is not uncommon in many photometric analytical systems and can be compensated for by suitable means such as electronic or manual control, or compensating instrumentation, as is well known in the art. Preferably, base line drift is maintained at minimum since a low base line drift will permit the utilization of simpler, and typically less expensive instrumentation as the light sensing means.
It has been found that the suspending medium can have a substantial effect on the amount of~rift in the base line. Good results are achieved when the suspending medium contains between about 0.5% to about 6% by weight of sodium chloride with about 2%
to about 6% sodium chloride being preferred and 3% sodium chloride highly preferred. These salt concentrations are substantially similar to the salt concentrations utilized in the nutrient medium during the growing cycle of the cells. It has also been found that good results are achieved when the aqueous suspending medium includes minor amounts of other salts such as sodium sulfate and sodium pyrophosphate and nutrients such as yeast -~2OD-135 Can.
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extract. Although it is not completely understood as to why inclusion of the foregoing ingredients in the suspending medium improves stability of light output and reduces base line drift in the suspension, it is believed that while the cells are not -actively growing in number, the suspending medium still should provide an environment to maintain cell metabolism at an accept-able level so that light output remains relatively stable. A
preferred suspending medium includes, in addition to sodium chloride, between 1 and about 10 micrograms/ml of yeast extract and between about 50 and 250 micrograms/ml of sodium pyrophos-phate.
Having formed the suspension of luminous microorganisms as described and having sensed and recorded the base line light output of the suspension, a sample suspected of containing a toxic substance or having a toxic condition is introduced into the container with the working suspension. The contents are agitated so as to thoroughly mix the suspension and the intro-duced material. A change in the light output is determined either by measuring the total change in light output over a given period of time or the rate of change of light output. Whether the change in light output is measured as rate of change or as the total change over a given period of time will depend on factors such as the type and concentration of the toxic substance, the sensitivity of the microorganism, the means for sensing the light output and the like, and the method employed can be readily determined by prescreening the microorganism and liquid being tested.
The presence of a toxic substance or condition is indicated by a change in the light output of the microorganism suspension over the base line output. Typically the toxic substance or condition cause a reduction in light output although depending on the strain of microorganism certain toxic substances or conditions ~2OD-135 Can.
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may cause at least a temporary increase in the light output of the microorganism suspension. The amount of light output change is related to the concentration of the toxic substance or the magnitude of the toxic condition.
Accordingly, using standard curves, the concentration of a given toxic substance can be determined, assuming that the toxic substance is identified. In addition, a correlation can be set up between the results of the present method and the results of the so-called "fish" test, the test most commonly employed at the present time in attempting to determine the presence of a toxic substance in water.
Any conventional light sensing means can be employed in the present invention to sense the light output from the aqueous suspension of microorganisms and the subsequently formed mixture of microorganisms and material being tested. As is well known in the art, there are various types of photometric and optical analytical instruments, all of which operate on the same general principles well known in the art.
In Fig. 1, a photometric system suitable for detecting the output of light from the luminescent microorganisms is shown diagrammatically and is generally indicated by reference numeral 10. In its broadest aspects, the system 10 includes a suitable cell, cuvette or container 12 in which the suspension of luminous microorganisms and the test sample are contained for light output measurement. A light detector such as a photomultiplier tube 14, or a photocell, is positioned in light impinging relationship to the container 12 for generating a low voltage current which is relative to the intensity of light impinging the detector. The output from the cell 14 is passed through a photocell amplifier 17 where the output is amplified and then passed on to a display means such as a meter 18. The choice of display means is not critical and in place of, or in addition to the meter 18, there ~20D-135 Can.
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may be included in the system a recording chart or a digital readout device of conventional design.
It should also be noted that the temperature of the working suspension will affect both the sensitivity and the actual light output of the microorganisms in the working suspension. Accord-ingly, it is preferred to run all tests with a particular strain of microorganism and a particular toxicant at the same temperature to insure reproducibility of results. Thus, with toxicants such as phenol it has been found that for a given concentration of toxicant, the actual percent decrease in light output of a working suspension of microorganism decreases with increasing temperature.
With other toxicants such as isopropyl alcohol the percent of light output decrease of the working suspension increases with increasing temperature. Accordingly, it is preferred, partic-ularly when working with new test samples, to record the light output change of the working suspension through a range of temp-eratures of between about 10C. to about 30C. The preferred test temperature will be that temperature at which the maximum change in light output is recorded. This indicates the temp-erature at which the microorganism has maximum sensitivity to the toxicant ~r mixture of toxicants in the test solution.
Where reproducibility of results or maximum sensitivity are not of primary concern, the method can be carried out with working solution at the ambient temperature within the photometer. In any case, the working suspension should be allowed to come to a stable temperature before recording light output because changes in temperature will also affect the base line light output and will result in an exaggerated base line drift. In the examples set forth below the working suspension was stabilized at the ambient temperature of the photometer before recording the light output.
--~2OD-135 Can.
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The invention is further described by the following examples which are illustrative of specific modes of practicing the inven-tion and are not intended as limiting the scope of the invention as defined by the appended claims.
EXAMPLE I
Luminescent bacterial cells for use in toxicity testing were prepared by innoculating agar media to form cultures of V.
fischeri, P. phosphoreu_, ATCC 11040; P. mandapamensis, ATCC
27561; L. harveyi, ATCC 14126 and A. fischeri, NRRL B-11177. The agar medium was made up of the following ingredients:
KH2P4 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - - - - O.25 wt. %
NaCl - - - - - - - - - - - - - - 3.0 wt. %
Glycerol ~ - - - - - - - - 0.5 wt. %
Yeast Extract- - - - - - - - - - - 0.1 wt. %
Polypeptone- - - - - - - - - - - - 0.5 wt. %
Hydrolyzed Casein- - - - - - - - - l.O wt. %
Squid Extract- - - - - - - - - - -10.0 Vol.%
Agar - - - - - - - - - - - - - - - 1.5 wt. %
Distilled Water- - - - - - - - - -90.0 Vol.%
The squid extract was prepared by homogenizing 454 grams of whole squid with enough distilled water to make a final volume of 1 liter. The slurry was autoclaved at 121C. for 15 minutes and the resulting suspension clarified by centrifugation.
After 24 hours of growth at 22C. the cells were removed from each of the cultures and suspended in 3% aqueous solution of sodium chloride (pH 7.1) to form a stock suspension of each of the cell cultures. The stock suspension was agitated vigorously to assure a homogenous suspension.
A working suspension was prepared from each of the stock solutions by diluting 10 microliter aliquots of each stock solu-tion in 2 ml of an aqueous suspending medium. The suspending medium consisted of an aqueous sodium chloride solution (3 wt.%) ~-420D-135 Can.
which contained 135 micrograms/ml of sodium pyrophosphate and 5 micrograms/ml of commercially available yeast extract (Diffco Laboratories, Detroit, Michigan).
The base line light output of the working solutions was determined with the suspensions substantially at ambient temp-eratures in a photometer equipped with a photomultiplier tube and a chart recorder.
EXAMPLE II
Luminescent bacterial cell cultures were also grown in liquid media. Cultures of P. phosphoreum, ATCC 11041; P.
mandapamensis, ATCC 27561; L. harveyi, ATCC 14126 and A.
fischeri, NRRL B-11177 were grown as shake flask cultures. The growth medium was as used in Example I except that no agar was utilized in the culture media. The cultures were grown in S00 ml. triple baffled DeLong style flasks containing 100 ml. per flask. The cultures were grown for 18 hours at 22C. and during the growth period, the cultures were agitated at 240 rpm on a New Brunswick gyrotary shaker. The cells were harvested during the logarithmic phase of growth and separated from the growth medium by centrifugation. The resulting pellet of cells from each of the cultures was maintained at 4C. prior to formation o~ the respective stock suspension in the manner disclosed in Example I.
Each stock suspension was diluted to form a working suspension for use in the photometer in the manner of Example I
and base line drift was recorded for each of the working suspensions.
EXAMPLE III
Cultures of P. mandapamensis, ATCC 27561; L. harveyi, ATCC
14126 and A. fischeri, NRRL B-11177 were each prepared in a broth growth medium as described in Example II. The cultures were separated from the broth during the logarithmic phase of growth by centrifugation and the resulting cell pellet chilled to 4C.
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The chilled cell pellet was suspended in a 3% sodium chloride solution maintained at a temperature of 4C. The sodium chloride solution was added to the suspension until a homogenous cell suspension having a final optical density of 160 was prepared.
The cell suspension was mixed with an a~ueous milk solution (20 wt.% skim milk, 2 wt.% NaCl, pH 7.1) in a volume ratio of one part cell suspension to 19 parts of the milk solution. The cell-milk suspension was distributed in 1 ml. aliquots into 10 ml. vials and quick frozen at a temperature of -50C. The cells were then dried at -5C. under 55 microns vacuum for 18 hours.
The vials were then vacuum sealed.
Working suspensions of each of the lyophilized samples were prepared by the addition of 1 ml. of distilled water to the vials. The resultant suspensions exhibited good base line light output.
EXAMPLE IV
Working suspensions of the several species of luminescent bacteria grown and prepared as in Examples I, II and III were challenged with varying concentrations of substances known to be toxic to biological organisms to determine the effect on the cell light output of the condition thus created.
The following toxic substances were used to create toxic conditions: phenol; sodium laurel sulfate; a mixture of various substances referred to as mixture 401, and a wood preservative known as penta, which consisted of 3.5% pentachlorophenol, 65%
mineral spirits and 32.5% inert ingredients.
The reference mixture 401 was prepared by adding 3.82 grams NH4C1, 0.093 grams K2Cro4, 0.128 grams Na2S, .9 H2O, 0.20 grams Kaolin, 0.1 grams phenol and 1.28 ml. No. 2 fuel oil to 100 ml.
of distilled water.
The tests were conducted by adding between 10 and 100 microliters of solutions of the toxic substance to a cuvette -420D-135 Can.
3~50 containing a working suspension of cells for which the base line and base line drift had been recorded. After introducing the toxic substance, the cuvettes were inverted several times to insure mixing of the suspension and the material containing the toxic substance. The cuvettes were replaced in the photometer and the percent decrease in light was determined after a period of two minutes from the addition of the toxic substance. Control samples consisting of a working suspension of each of the cultures and 10 microliters of a 3% sodium chloride solution were run along with test samples.
The results are set forth in Tables A, B and C below. In each case, concentrations of the toxic substances used are expressed in milligrams/liter, except for mixture 401, which is expressed as volume percent of the mixture in the test solution.
Table A sets forth the results for luminescent cells grown in accordance with Example I. Tables B and C set forth results for the cells grown and prepared as in Examples II and III, respectively.
-~2OD-135 Can.
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TABLE A
Percent Decrease in Light Concentrations of ATCC ATCCATCC NRRLVibrio Toxicants Tested 2756114126 11040B-11177Fischeri 3% NaCl (control) O O O O O
Phenol 50 mg/L 20 26 9 13 10 100 mg/L 55 40 18 23 20 250 mg/L 95 91 33 51 41 Sodium Lauryl Sulfate 25 mg/L 20 19 17 67 21 50 mg/L 40 36 31 90 81 100 mg/L 94 33 12 89 83 Reference Mixture 401 .5% 9 2 3 7 6 1.0% 14 4 9 9 6
2.5% 24 18 23 26 8 Penta-Chlorophenol 5 mg/L 11 71 85 71 10 mg/L 31 73 95 84 25 mg/L 61 89 99 90 ~20D-135 Can.
TABLE B
Cells Percent Decrease in Light Concentrations of ATCC ATCCNRRL Vibrio Toxicants Tested 2756114126B-11177 Flscher
TABLE B
Cells Percent Decrease in Light Concentrations of ATCC ATCCNRRL Vibrio Toxicants Tested 2756114126B-11177 Flscher
3% NaCl (control) Q 0 0 0 Phenol 50 mg/L 36 10 19 26 100 mg/L 50 29 29 37 250 mg/L 37 57 55 52 Sodium Lauryl Sulfate 25 mg/L 22 48 83 15 50 mg/L 30 45 89 28 100 mg/L 76 69 78 70 Reference Mixture 401 0.5% 7 0 12 12 1.0% 7 4 14 14 2.5% 11 10 16 15 TABLE C
Percent Decrease in Light Concentrations of ATCC ATCCNRRL Vibrio Toxicants Tested 2756114126B-11177 Fischeri 3% NaCl (control) 0 0 0 0 Phenol (PPM) 50 mg/L 7 12 25 13 100 mg/L 20 21 40 27 250 mg/L 28 35 63 50 Sodium Lauryl Sulfate 25 mg/L 12 33 33 41 50 mg/L 14 40 40 43 100 mg/L 31 56 40 43 Reference Mixture 401 0.5% 3 6 17 11 1.0% 3 10 19 17 2.5% 9 13 42 23 -~20D-135 Can.
3~0 A freeze dried sample of A. fischeri, NRRL B-11177 which had been grown and lyophilized in accordance with Example III was utilized to demonstrate the relationship between concentration of toxic substances and the decrease in light output after two minutes of exposure.
Into cuvettes containing 1 ml. of a working suspension of the reconstituted luminous microorganism ( A. fischeri) on which the base line had been previously determined, 10 microliters of the samples containing phenol in varying concentrations were introduced and the cuvettes inverted to assure good mixing.
After two minutes the reduction in light output was noted and reported as percent light decrease. The results are shown in Table D below:
TABLE D
Phenol Concentration Percent Light Decrease (PPM) After Two Minutes Exposure 1.
10.
18.
21.
25.
31.
37.
40.
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Fig. 2 is a plot of the data in Table D with concentration of phenol on the vertical log scale and the percent light decrease on the horizontal linear scale. When thus treated a direct relationship between concentration of phenol and decrease in light output is established.
In the foregoing examples the material containing toxic substances was added to the suspension of luminescent micro-organisms in the aqueous suspending medium. However, it has been found that when concentrations of toxic substance in liquid are low, good results are achieved by suspending the cells directly in the liquid containing the toxic substance to form the working suspension. In this case, sufficient sodium chloride is added to the material suspected of containing the toxic substance to form a 3% solution of the salt. The light output is measured and compared to the light output of a working suspension of the luminescent cells in suspending medium only.
From the foregoing it will be seen how the advantages of the present invention are achieved by the various embodiments and modifications described in the foregoing description and examples.
Further modifications will be apparent to those skilled in the art. Such modifications are included within the scope of this invention as defined by the following claims.
Percent Decrease in Light Concentrations of ATCC ATCCNRRL Vibrio Toxicants Tested 2756114126B-11177 Fischeri 3% NaCl (control) 0 0 0 0 Phenol (PPM) 50 mg/L 7 12 25 13 100 mg/L 20 21 40 27 250 mg/L 28 35 63 50 Sodium Lauryl Sulfate 25 mg/L 12 33 33 41 50 mg/L 14 40 40 43 100 mg/L 31 56 40 43 Reference Mixture 401 0.5% 3 6 17 11 1.0% 3 10 19 17 2.5% 9 13 42 23 -~20D-135 Can.
3~0 A freeze dried sample of A. fischeri, NRRL B-11177 which had been grown and lyophilized in accordance with Example III was utilized to demonstrate the relationship between concentration of toxic substances and the decrease in light output after two minutes of exposure.
Into cuvettes containing 1 ml. of a working suspension of the reconstituted luminous microorganism ( A. fischeri) on which the base line had been previously determined, 10 microliters of the samples containing phenol in varying concentrations were introduced and the cuvettes inverted to assure good mixing.
After two minutes the reduction in light output was noted and reported as percent light decrease. The results are shown in Table D below:
TABLE D
Phenol Concentration Percent Light Decrease (PPM) After Two Minutes Exposure 1.
10.
18.
21.
25.
31.
37.
40.
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Fig. 2 is a plot of the data in Table D with concentration of phenol on the vertical log scale and the percent light decrease on the horizontal linear scale. When thus treated a direct relationship between concentration of phenol and decrease in light output is established.
In the foregoing examples the material containing toxic substances was added to the suspension of luminescent micro-organisms in the aqueous suspending medium. However, it has been found that when concentrations of toxic substance in liquid are low, good results are achieved by suspending the cells directly in the liquid containing the toxic substance to form the working suspension. In this case, sufficient sodium chloride is added to the material suspected of containing the toxic substance to form a 3% solution of the salt. The light output is measured and compared to the light output of a working suspension of the luminescent cells in suspending medium only.
From the foregoing it will be seen how the advantages of the present invention are achieved by the various embodiments and modifications described in the foregoing description and examples.
Further modifications will be apparent to those skilled in the art. Such modifications are included within the scope of this invention as defined by the following claims.
Claims (17)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for testing a liquid for a toxic substance or condition comprising:
forming a mixture comprising a suspension of luminous microorganisms and said liquid being tested thereby to effect contact with said microorganisms by said liquid being tested;
sensing the light output of said mixture; and comparing the sensed light output with a base line light output of an aqueous suspension of said microorganisms free of said liquid being tested to determine the effect of said liquid being tested on said microorganisms.
forming a mixture comprising a suspension of luminous microorganisms and said liquid being tested thereby to effect contact with said microorganisms by said liquid being tested;
sensing the light output of said mixture; and comparing the sensed light output with a base line light output of an aqueous suspension of said microorganisms free of said liquid being tested to determine the effect of said liquid being tested on said microorganisms.
2. The method of Claim 1 further including the steps of growing a culture of luminous microorganisms;
transferring a portion of said culture to an aqueous medium thereby to form a suspension of said luminous micro-organisms therein; and sensing the base line light output from said aqueous suspension.
transferring a portion of said culture to an aqueous medium thereby to form a suspension of said luminous micro-organisms therein; and sensing the base line light output from said aqueous suspension.
3. The method of Claim 1 wherein said microorganisms are suspended in an aqueous medium comprising between about 0.5 weight percent and about 6 weight percent of a soluble alkali metal salt.
4. The method of Claim 1 wherein said luminous micro-organisms are suspended in an aqueous salt solution suspending medium comprising between about 0.5 weight percent and about 6 weight percent of sodium chloride.
5. The method of Claim 4 wherein said aqueous suspending medium comprises between about 2 weight percent and about 6 weight percent of sodium chloride.
6. The method of claim 4 wherein said aqueous suspending medium comprises about 3 weight percent sodium chloride.
7. The method of claim 4 wherein said suspending medium further comprises a minor proportion of a salt selected from the group consisting of an alkali metal pyrophosphate and sulfate.
8. The method of claim 4 wherein said suspending medium further includes yeast extract.
9. The method of claim 4 wherein said suspending medium comprises between about 50 and about 250 micrograms/ml of sodium pyrophosphate and between about 1 and about 10 micrograms/ml of yeast extract.
10. The method of claim 4 wherein said suspending medium is an aqueous solution comprising 3 weight percent sodium chloride, 135 micrograms/ml of sodium pyrophosphate and 5 micrograms/ml of yeast extract.
11. The method of claim 1 wherein said luminous micro-organisms are luminous bacteria selected from the group con-sisting of the genera Photobacterium, Vibrio, Achromobacter, Lucibacterium, and mixtures thereof.
12. The method of claim 11 wherein said luminous bacteria are selected from the group consisting of P. splendidum, P.
mandapamensis, P. phosphoreum, V. fischeri, A. fischeri, L.
harveyi and mixtures thereof.
mandapamensis, P. phosphoreum, V. fischeri, A. fischeri, L.
harveyi and mixtures thereof.
13. The method of claim 2 wherein said microorganisms are grown in a nutrient medium comprising on a weight percent basis, between about 0.5% and about 3% sodium chloride, between about 0.25% and about 0.7% of an alkali metal pyrophosphate, about 0.5%
glycerol, between about 0.5% and about 1% polypeptone, up to about 1% trypticase and up to about 2% agar.
glycerol, between about 0.5% and about 1% polypeptone, up to about 1% trypticase and up to about 2% agar.
14. The method of claim 2 wherein said nutrient medium further comprises squid extract.
15. The method of claim 2 wherein said aqueous portion of said nutrient medium comprises sea water.
16. The method of claim 1 wherein said microorganisms are in a lyophilized state and said suspension is formed by the addition of water to said lyophilized microorganism.
17. A method for the detection of a toxic substance or condition in a liquid, the method comprising the steps of:
growing a culture of luminous microorganisms selected from the group consisting of P. splendidum, P. mandapamensis, P.
phosphoreum, V. fischeri, A. fischeri, L. harveyi and lyo-philizing a portion of said microorganism culture;
forming an aqueous suspension of said lyophilized micro-organism by adding distilled water thereto to replace the liquid lost during said lyophilization step;
determining the base line light output of said suspension and thereafter contacting said suspension with a liquid suspected of containing a toxic substance or condition; and determining the light output of suspension after said contact with said liquid, whereby the change in light output is indicative of the presence of a toxic substance or condition in said liquid.
growing a culture of luminous microorganisms selected from the group consisting of P. splendidum, P. mandapamensis, P.
phosphoreum, V. fischeri, A. fischeri, L. harveyi and lyo-philizing a portion of said microorganism culture;
forming an aqueous suspension of said lyophilized micro-organism by adding distilled water thereto to replace the liquid lost during said lyophilization step;
determining the base line light output of said suspension and thereafter contacting said suspension with a liquid suspected of containing a toxic substance or condition; and determining the light output of suspension after said contact with said liquid, whereby the change in light output is indicative of the presence of a toxic substance or condition in said liquid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US83749877A | 1977-09-28 | 1977-09-28 | |
| US837,498 | 1977-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1103050A true CA1103050A (en) | 1981-06-16 |
Family
ID=25274625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA311,855A Expired CA1103050A (en) | 1977-09-28 | 1978-09-22 | Method for detecting toxic substances in liquid |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS5458492A (en) |
| BE (1) | BE870833A (en) |
| CA (1) | CA1103050A (en) |
| DE (1) | DE2841896C3 (en) |
| FR (1) | FR2404847A1 (en) |
| GB (1) | GB2005018B (en) |
| NL (1) | NL7809821A (en) |
| SE (1) | SE462166C (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1153218A (en) * | 1980-03-25 | 1983-09-06 | Malbone W. Greene | Method and apparatus for monitoring light signals from liquid medium |
| WO1985000890A1 (en) * | 1983-08-16 | 1985-02-28 | Battelle Development Corporation | Bioluminescent chemical system and method for detecting the presence of chemical agents in a medium |
| DE3833628A1 (en) * | 1988-10-03 | 1990-04-12 | Genlux Forschungsgesellschaft | METHOD FOR DETECTING AND IDENTIFYING TOXIC SUBSTANCES USING CLONED MICROORGANISMS |
| WO1990004053A1 (en) * | 1988-10-10 | 1990-04-19 | Siemens Aktiengesellschaft | Electric surface-polishing device |
| FR2680247B1 (en) * | 1991-08-07 | 1993-11-12 | Anjou Recherche Gie | AUTOMATED POLLUTION DETECTOR IN AQUEOUS MEDIA USING A MICROORGANISM TEST. |
| GB9119382D0 (en) * | 1991-09-11 | 1991-10-23 | Knight Scient Ltd | Apparatus for monitoring liquids |
| FR2686350B1 (en) * | 1992-01-17 | 1996-12-27 | Rochas | METHOD FOR EVALUATING THE SAFETY OF PRODUCTS USING BIOLUMINESCENCE MEASUREMENTS. |
| DE4230264A1 (en) * | 1992-09-10 | 1994-03-17 | Bayer Ag | Analytical method for the investigation of mixtures for toxic components |
| GB2279738A (en) * | 1993-06-18 | 1995-01-11 | Yorkshire Water Plc | Determining toxicity in fluid samples |
| DE4325482C1 (en) * | 1993-07-29 | 1994-12-15 | Lange Gmbh Dr Bruno | Medium for reactivating preserved microorganisms |
| AU7541894A (en) * | 1993-09-08 | 1995-03-27 | Merck Patent Gmbh | Cell reagent and assay |
| DE4332165A1 (en) * | 1993-09-22 | 1995-03-23 | Kolibri Umweltanalytik Und On | Method and apparatus for pollutant analysis of water samples |
| JPH09503666A (en) * | 1993-10-15 | 1997-04-15 | メルク・パテント・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツンク | Assay method |
| DE4401169C2 (en) * | 1994-01-17 | 2003-01-09 | Buehler Ag | Process for distinguishing the quality of liquids |
| DE4401868C1 (en) * | 1994-01-22 | 1995-02-16 | Lobbe Xenex Gmbh | Microbial method for detecting pollutants |
| GB2293878A (en) * | 1994-10-06 | 1996-04-10 | Stephen John Shore | Monitoring biocide content of industrial waters |
| WO1996014570A1 (en) * | 1994-11-07 | 1996-05-17 | Idexx Laboratories, Inc. | Self-contained signal generating sampling device and methods of use of same |
| GB0510709D0 (en) * | 2005-05-26 | 2005-06-29 | Cymtox Ltd | Water monitoring system |
| US7704731B2 (en) * | 2006-10-10 | 2010-04-27 | The United States Of America As Represented By The Secretary Of The Navy | System and method for quantifying toxicity in water, soil, and sediments |
| CN112198150B (en) * | 2020-07-07 | 2022-10-25 | 山东省临沂生态环境监测中心 | Method for monitoring polluted water body by bioluminescence method |
| CN113340885B (en) * | 2021-06-23 | 2022-08-09 | 同济大学 | Formaldehyde biological monitoring device and monitoring method |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3370175A (en) * | 1965-01-28 | 1968-02-20 | North American Rockwell | Toxicant detector |
| US3797999A (en) * | 1970-05-20 | 1974-03-19 | Aerojet General Co | Method and apparatus for indicating micro-organic matter by means of chemiluminescence |
| US3679312A (en) * | 1971-06-16 | 1972-07-25 | Technicon Instr | Method and apparatus for measuring bioluminescence or chemiluminescence for quantitative analysis of samples |
| US3849653A (en) * | 1973-09-27 | 1974-11-19 | Bausch & Lomb | Multichannel bioluminescent sensors |
| US3958938A (en) * | 1974-12-16 | 1976-05-25 | Bausch & Lomb Incorporated | Bioluminescent sensor system |
| AU508807B2 (en) * | 1975-10-20 | 1980-04-03 | University Of Sydney, The | Photoelectric analysis using luminous bacteria |
| GB1571466A (en) * | 1976-12-14 | 1980-07-16 | Univ California | Assay mehtod |
-
1978
- 1978-09-22 CA CA311,855A patent/CA1103050A/en not_active Expired
- 1978-09-26 DE DE2841896A patent/DE2841896C3/en not_active Expired - Lifetime
- 1978-09-27 SE SE7810154A patent/SE462166C/en not_active IP Right Cessation
- 1978-09-28 GB GB7838480A patent/GB2005018B/en not_active Expired
- 1978-09-28 JP JP11868878A patent/JPS5458492A/en active Granted
- 1978-09-28 FR FR7827822A patent/FR2404847A1/en active Granted
- 1978-09-28 BE BE190765A patent/BE870833A/en not_active IP Right Cessation
- 1978-09-28 NL NL7809821A patent/NL7809821A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| FR2404847B1 (en) | 1984-12-28 |
| SE7810154L (en) | 1979-03-29 |
| SE462166C (en) | 1997-02-02 |
| GB2005018B (en) | 1982-07-14 |
| JPS5458492A (en) | 1979-05-11 |
| DE2841896C2 (en) | 1991-10-24 |
| DE2841896C3 (en) | 1996-09-26 |
| GB2005018A (en) | 1979-04-11 |
| JPS5728272B2 (en) | 1982-06-15 |
| SE462166B (en) | 1990-05-14 |
| BE870833A (en) | 1979-01-15 |
| DE2841896A1 (en) | 1979-03-29 |
| NL7809821A (en) | 1979-03-30 |
| FR2404847A1 (en) | 1979-04-27 |
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