Detection of first generation environmental sourced microbes in an environmentally-derived sample

Abstract

The presence or absence of a predetermined target first generation environmental sourced microbe in an environmentally derived sample is determined by adding a testing medium to the sample, or vice versa. The testing medium provides a selective growth medium for the target microbe and includes a specific nutrient which only the target microbe can significantly metabolize and use for growth This specific nutrient is modified by attaching a sample-altering moiety thereto, thereby converting the nutrient to a nutrient-indicator. The sample-altering moiety is activated to alter the sample only if the specific nutrient is metabolized by the target microbe. The sample-altering moiety can be a material which changes the color of the sample (visable or non-visable) or changes an electrical characteristic of the sample, or alters some other detectable characteristic of the sample. The testing media does not have to be kept sterile, (but, obviously must be free of viable target microbes) and the test ire procedure does not have to be performed in a sterile environment. The medium also includes an accelerant which hastens the advancement of the target microbes to the log phase of growth during the testing procedure.

Description

This invention relates to the detection of microbes in an environmental sample such as water, food, or the like. More particularly, this invention relates to the detection of a target microbe through the use of a testing medium which medium contains a nutrient which can be significantly metabolized only by the target microbe during tog phase of growth in the medium, and which, once metabolized, releases a moiety which alters a characteristic of the sample. The medium is thus a "specific medium" in that it will support growth in tog phase of only the target microbes, rather than a general medium which will also support growth in log phase of microbes other than the target microbes. The medium also contains a growth accelerant for the target microbes to boost them through lag phase and into log phase during the testing procedure. The microbes which this technique can detect are first generation environmental sample-sourced organisms.

in order to detect microbial pathogens in specimens, whether of human, animal or environmental origin, the following general procedure is commonly used: the target (and other) microbes in the specimen are, in the prior art, inoculated with the specimen into a culture medium in which they are provided with all the nutrients they require for growth. The specimen may be an untreated natural sample, or it may be a sample which has been pre-treated as, for example, by membrane filtration. The culture medium has the nutrients and other selective chemicals such as antimetabolites or antibiotics, which are selectively active microbes other than the target microbes. The culture medium is a "general medium", even with the selective chemicals, in that it supports the growth of both target microbes and related microbes and thus is only partially specific to the target microbes.

The culture medium, which may be a water solution or a water gel, is sterilized to rid it of any contaminating microbes which may be present in the medium and which could, therefore, interfere with the analysis. The culture medium must be refrigerated and packaged in such a way to avoid contamination after manufacture.

After one or more of the culture media are inoculated with the specimen, the inoculated media are incubated under controlled atmospheric conditions. After incubation, the culture media are examined for growth of any microbes, if such growth is observed, a sample thereof is taken for further analysis, since the presence of the target microbe can only be established by isolating it in the pure state, not mixed with other microbes. Once isolated on subsequent culture media, the target microbes are identified by testing for a variety of physical and chemical characteristics. If the apparent target microbe growths are not isolated, false negative tests can result.

It will be readily appreciated that this most common analytical procedure is time consuming and must be carefully performed to preserve sterility.

This invention detects target microbes in a sample by using an indicator which is metabolized by the target microbe, but which cannot be substantially metabolized by any other viable microbes which may be present in the sample along with the target microbe. The invention thus uses an active selector of the target microbes. rather than the reactors used by the prior art. The indicator will change a characteristic of the sample once the nutrient is metabolized by the target microbe. The characteristic can be: color (either visible, ultra violet, or infrared); electrical conductivity; or the like. The preferred mode of performing the invention involves detecting the target microbes by use of a nutrient-indicator which, when metabolized, changes the visible or fluorescent color of an aqueous solution containing the specimen.

The nutrient-indicator actively participates in the growth of the target microbes by serving as a preferred or primary nutrient source. The target microbes can grow, metabolize and multiply into log phase, because they, and substantially only they, can use the indicator as their primary nutrient. Indicators can include chromogens attached to: salts; carbon; sulfur; aminoacids; fatty acids; peptides; or other selective primary nutrients for microbes. Because microbes other than the target microbes are prevented from growing, metabolizing, or multiplying substantially into log phase, the medium is so specific that it does not have to be sterilized before use. Competition between target microbes and other microbes in the sample for the available primary nutrient in the medium is eliminated by the subject invention. The medium can be manufactured and packaged in a powder form which is added to the sample being tested. As noted, no sterilization is necessary. The medium can be dissolved in water and the sample can be added to the solution, or: if the sample is aqueous, the medium can be added directly to the sample.

The testing medium also includes a minor amount of a growth accelerant which will boost the target microbes and all of the other viable microbes in the sample through lag phase toward log phase of growth in the testing procedure. It will be understood by those skilled in the art that when a sample, such as environmental samples, are tested in accordance with the procedure of this invention, is combined with the testing medium of this invention, all of the microbes in the sample will lapse into a lag phase of growth due to the newness of the environment they are in. In the lag phase, none of the microbes will significantly multiply and grow until they adjust to the new environment. This dormant stage, which all of the microbes, including the targets, encounter, causes the test period to be undesirably long. The growth accelerant which is incorporated into the medium of this invention is a combination of natural plant extracts, vitamins, and minerals which hasten the transition of the target microbes, and all of the other microbes in the sample, through the lag phase and into the log phase so as to lessen the time duration from the inception of the test to the alteration (or no alteration) of the sample which indicates the presence (or absence) of the target microbes in the sample. The total time lapse will be reduced by about one half by inclusion of the accelerant in the medium. The accelerant is present in a small amount so as to be dissipated by the time the microbes pass through log phase of growth.

The development of a specific color indicates the presence of the target microbes. This may occur at any time after the procedure is initiated. There is no need to isolate the target microbes from the other microbes in the sample. There is no need to perform any chemical analysis of the sample to determine whether the target microbes are present.

As used in this disclosure, the term "target microbe(s)" can refer to a single microbe; a related species of microbes; or a large genus of microbes possessing a common taxonomic characteristic. The indicator only needs to be specific to the "target microbe". For example, indicators are available for detecting a single microbe, such as Escherichia coli (E. coli), or for detecting any one of a closely related species of microbes, such Klebsilla- Enterobacter- Serratia, or any one of a large genus of microbes, such as Gram negative bacteria, for example. The chromogens used in the nutrient-indicator can produce color in the visible range; the ultraviolet range; or the infrared range. As will be appreciated from the aforesaid, the nutrient-indicator will preferably be colorless in the non-metabolized state, and will preferably release a color moiety after being metabolized by the target microbes. The color may be visible, fluorescent, machine-readable, or a combination of the aforesaid. As previously noted, using the invention, there is very little, or no, competition for food or nutrients among the microbes in the medium. Accordingly, a significant number of false-negative or false-positive tests which will occur with the procedures of the prior art are eliminated by this invention. The nutrient-indicator used will be one that the target microbes can readily assimilate and for which other microbes in the sample have little or no preference, and/or cannot significantly assimilate. Thus, only the presence of the target microbes in the specimen can result in sufficient metabolism of the nutrient-indicator to cause the color, or other characteristic change, in the sample. This is the crux of the invention.

Since the nutrient-indicator is substantially specific only to the target microbes, and is the preferred, or primary, nutrient in the medium for the target microbes, the target microbes tend to direct their metabolic pathways toward the nutrient-indicator, thus further speeding up the color change.

It is, therefore, an object of this invention to provide a procedure for detecting microbes in a specimen by metabolistically changing a detectable characteristic of the sample.

It is an additional object of this invention to provide a procedure of the character described wherein the color of the sample is changed by metabolization by a target microbe.

It is another object of this invention to provide a procedure of the character described wherein the color change is provided by metabolism by the target microbes, of a nutrient/indicator added to the sample, which nutrient includes a chromogenic moiety which is detectable only after the nutrient is metabolized.

It is a further object of this invention to provide a procedure of the character described wherein the nutrient having the chromogenic moiety can only be significantly metabolized by the target microbes.

It is another object of this invention to provide a procedure of the character described wherein the nutrient can only support growth of the target microbes in log phase, and the remaining viable microbes in the sample cannot sustain log phase of growth since they cannot metabolize the nutrient-indicator to the extent required therefor.

It is a further object of this invention to provide a procedure wherein the growth of the target microbes is accelerated into log phase to lessen the time period needed to conduct the test.

These and other objects of the invention will become more readily apparent from the following detailed description of several preferred embodiments thereof.

Three examples of the use of the invention to detect a genus and a species of gram negative microbe (Escherichia coli), a genus and species of gram positive microbe (Streptococcus faecalis), and a taxonomic class consisting of a large group containing many members (Gram negative microbes) are set forth hereinafter. When a specimen is examined for any Of these three, each is referred to as the target microbe(s).

Escherichia coli

The nutrient is a substrate of the enzyme B-glucuronidase. If one wishes to determine the presence of E. coli by a color change, the nutrient-indicator can be orthonitrophenyl-B-D-glucuronide (yellow), B-napthalamide-B-D-glucuronide (purple), alpha-napthol-B-D-glucuronide (red), or methylumbilliferyl-B-D-glucuronide (fluorescent), or the like.

The nutrient-indicator serves as the primary source of carbon. The rest of the medium is tailored so that each ingredient provides a requirement for E coli.

First, to prevent competition from microbes other than the broad catagory of Gram negative bacteria, the antibiotics vancomycin and ansiomycin are added In the percent by weight of 5%. These antibiotics may be present in the range of 1% to 10% by weight.

Second, to select E. coli from Gram negative bacteria, the following ingredients are used:

__________________________________________________________________________INGREDIENT SOURCE % BY WEIGHT RANGE % BY WEIGHT__________________________________________________________________________Nitrogen ammonium sulfate 37. 10-50Amino Acids histidine .0697 0.02-0.1 methionine .1860 0.02-0.4 tryptophan .2325 0.02-0.5Vitamins biotin .000232 0.0001-0.00 pantothenate .0093 0.001-0.03 folic acid .000232 0.0001-0.02 inositol .0186 0.01-0.02 p-aminobenzoic acid .046 0.01-.1 pyrodoxine hydrochloride .093 0.05-0.3 riboflavin .037 0.01-0.06 thiamine .037 0.01-0.06Elements ferric chloride .046 0.02-0.1 copper sulfate .00186 0.001-0.002 manganese sulfate .0037 0.002-0.007 potassium chloride .00001 0.00001-0.001 potassium iodide .0000046 0.000001-0.00001 zinc sulfate .046 0.01-0.08 boric acid .460 0.01-0.5 magnesium chloride .019 0.01-0.05Salts potassium phosphate monobasic 9.0 1-15 potassium phosphate dibasic 23.0 2-30 sodium carbonate 23.0 2-30 magnesium sulfate 4.6 1-10 sodium chloride .9 0.2-5 calcium chloride .9 0.2-5 sodium pyruvate .023 0.01-0.1Nutrient-indicator .345 0.2-2Accelerant 2.0 1.5-2.5__________________________________________________________________________

Strepococcus faecalis

Streptococcus faecalis is a microbe found to be a cause of urinary tract infection. It is the major bacterium sought out in swimming and recreational water.

The nutrient-indicator is a substrate of the enzyme L-pyronidonyl aminopeptidase. If one wishes to determine the presence of S. faecalis by a color change, the nutrient-indicator molecule can be orthonitrophenyl-B-L-pyronidonyl (yellow); B-napthalamide-B-L-pyronidonyl (purple); alpha-napthol-B-L-pyronidonyl (red); or methylumbilliferyl-B-L-pyronidonyl (fluorescent).

The nutrient-indicator serves as the essential source of carbon. The rest of the medium is tailored so that each ingredient provides a requirement for S. faecalis.

First, to prevent competition from microbes other than the broad category of Gram positive bacteria, the antibiotics colistin, naladixic acid and ansiomycin are added.

Second, to select S. faecalis from Gram positive bacteria, the same ingredient mixture specified for E. coli is used with the above-noted nutrient-indicator and antibiotics. The nutrient-indicator is present in a concentration of 0.345 percent by weight, the usable range being about 0.2 to about 2.0 percent by weight and the antibiotics are present in the concentration of 5 percent by weight, the usable range being about 1 to about 10 percent by weight.

Gram Negative Bacteria

There are two broad classes of bacteria; Gram positive and Gram negative. Gram negative bacteria are important because they contain a toxic material as part of their bodies called endotoxin. They also may contaminate pharmaceuticals and other medical preparations.

The nutrient-indicator is a substrate of the enzyme L-alanine aminopeptidase. If one wishes to determine the presence of Gram negative bacteria by a color change, the nutrient-indicator molecule can be L-alanine-B-orthonitrophenyl (yellow); B-napthalamide-B-L-alanine (purple); alpha-napthol-B-L-alanine (red); or methylumbilliferyl-B-L-alanine (fluorescent).

The nutrient-indicator serves as the essential source of carbon. The rest of the medium is tailored so that each ingredient provides a requirement for Gram negative bacteria.

First, to eliminate microbes other than the broad category of Gram negative bacteria, the antibiotics ansiomycin (eliminates yeast) and vancomycin (eliminates Gram positives) are added in amounts of 5% by weight.

The same ingredient mixture specified above is used with the nutrient-indicator being present in the amount of 0.345% by weight and in the range of about 0.2 to about 2.0% by weight, and the antibiotics may be present in the range of about 1 to about 10% by weight.

In all of the aforesaid examples, the accelerant mixture of plant extracts, vitamins and minerals which can be assimilated by all of the microbes in the sample will be included in the amount of about 2% by weight.

A sample of the specimen is added to a vessel, such as a bottle. The testing medium of this invention is added to the specimen and well mixed. If the sample is a solid, a water diluent can be used. If the target microbe or group of microbes are present, the invention will change color sometime (generally within twenty four hours) after the time of inoculation. There is no technical time or labor required after inoculation of the invention. Also, because the end-point is a defined color change, it does not require a trained individual to determine positivity.

Substrates are available to specifically detect fecal coliforms (E. coli), total coliforms, the Klebsilla-Enterobacter-Serratia group; and Streptococcus faecalis.

This invention is particularly useful in analyzing water. When water is analyzed, if necessary, sodium thiosulfate may be added to neutralize antibacterials found in water.

To analyze waste water for E. coli by the invention, the following procedure is followed:

1. A water sample is collected, using precalibrated pipettes: 1.0 milliliter; 0.1 milliliter; and 0.01 milliliter, from which amounts of the water sample are added to each of three tubes. The aforesaid medium of this invention is added in powder form (alternatively, the medium can be present in powder form in the tubes).

2. The tubes are incubated at between 20 degrees C. (70F.) to 44 degrees C. (140F.).

3. The presence of E. coli is indicated by the change in color in the tube.

4. If greater than 100 E. coli/ml are present, the 0.01 tube will be positive; if greater than 10 E. coli/ml are present, the 0.1 ml tube will be positive; if less than one E. coli/ml is present, only the 1 ml tube will be positive.

A positive test can occur anytime from shortly after inoculation with a heavily inoculated sample, to 20 hours, if there is only one target bacterium initially present in the sample in the container. Whatever the time period needed to produce the color change, this time period will be substantially reduced by the accelerant which will boost the microbes into log phase growth. The only technical manipulation is the addition of the water to the tubes by the pre-calibrated pipettes.

The same medium described above was used to analyze water in the presence or absence (P-A) test for E. coli.

1. A 100 ml sample of water was added to a vessel containing the aforesaid medium of this invention.

2. If the reaction mixture changes color, E. coli is present and the test is positive.

3. Confirmatory or other tests are not necessary.

The procedure of this invention was tested with several B-glucuronidase and B-galactopyranoside substrates in the field. A comparison of the procedure of this invention in a P-A test format was made with the conventional membrane filtration technique, and was analyzed according to the EPA protocol for the certification of new devices. The procedure of this invention is specific and requires no confirmatory tests. The test was conducted for two target microbes; E. coli; and total coliforms. The base formula was made as described above; only the hydrolyzable substrate was changed for the detection of the particular target microbes.

In general, with respect to this invention, after the specific medium and the sample are combined, during the lag phase while the microbes are adjusting to the presence of the medium, no substantial microbial metabolism will occur with either the target or non-target microbes. At the beginning of the log phase, all of the microbes will begin to metabolize the vitamin and mineral, and the accelerent components of the medium, but only the target microbes will also significantly metabolize the specific nutrient-indicator component of the medium. This specific nutrient-indicator is the only carbon source ingredient in the medium which will allow substantial growth, ie, growth which will allow microbial reproduction at logarithmic rates (log phase), of any microbes in the sample. Thus the medium will only support continued reproductive growth of the target microbes. For this reason the population of non-target microbes in the sample will increase much less than the population of the target organism. By the time that the log phase has progressed to the equillibrium phase, the population growth rate of the target microbes will be at least ten times that of any other microbes in the sample due to the selective reproductive growth of the target microbes. Commonly, in the log phase, the target microbe population will be ten thousand times or more, than that of any other microbes in the sample.

It will be readily appreciated that the specific medium of this invention can be produced in powder form and packaged in ready-to-use quantities specific to a variety of target microbes. The medium, as produced, can include antibiotic components, if desired.

Since many changes and variations of the disclosed embodiments of this invention may be used without departing from the inventive concept, it is not intended to limit the invention otherwise than as required by the appended claims.

Claims

1. A target microbe-specific medium for detecting the presence or absence of a target microbe in a liquified environmental or biological sample, said medium comprising:

a) an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and log phase reproduction of said target microbe in the presence of a nutrient-indicator and to aid the target microbe through lag phase and into log phase of growth in the sample;

b) an effective amount of at least one antibiotic which inhibits growth of a potential non-target microbe in said medium; and

c) an effective amount of a nutrient-indicator which is provided in an amount sufficient to support log phase growth of said target microbe until a detectable characteristic signal is produced in the medium during said log phase growth; said nutrient-indicator in the presence of said antibiotic being incapable of supporting continued logarithmic growth of any viable non-target microbes in the sample to produce a detectable characteristic signal; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to confirm the presence or absence of the target microbe in the sample;

wherein said medium lacks a gelling agent so that when said medium is mixed with said liquified sample a liquid is formed, and wherein said ingredients in (a) and (b), and said nutrient-indicator are chosen such that growth of non-target microbes do not interfere with growth of said target microbe wherein said medium allows growth of Escherichia coli and is specific for Escherichia coli.

2. A target microbe-specific medium for detecting the presence or absence of a target microbe in a liquified environmental or biological sample, said medium comprising:

a) an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and log phase reproduction of said target microbe in the presence of a nutrient-indicator and to aid the target microbe through lag phase and into log phase of growth in the sample;

b) an effective amount of at least one antibiotic which inhibits growth of a potential non-target microbe in said medium; and

c) an effective amount of a nutrient-indicator which is provided in an amount sufficient to support log phase growth of said target microbe until a detectable characteristic signal is produced in the medium during said log phase growth; said nutrient-indicator in the presence of said antibiotic being incapable of supporting continued logarithmic growth of any viable non-target microbes in the sample to produce a detectable characteristic signal; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to confirm the presence or absence of the target microbe in the sample;

wherein said medium lacks a gelling agent so that when said medium is mixed with said liquified sample a liquid is formed, and wherein said ingredients in (a) and (b), and said nutrient-indicator are chosen such that growth of non-target microbes do not interfere with growth of said target microbe wherein said medium allows growth of one or more coliform bacteria and no other bacteria and is thereby specific for coliform bacteria.

3. The medium of claims 1 or 2 wherein said ingredients include: a source of salts present in an amount of about 62 percent by weight; and a source of nitrogen present in an amount of at least 10 percent by weight.

4. The medium of claims 1 or 2 wherein said ingredients are present in an amount in the range of at least about 1.5 to about 2.5 percent

5. The medium of claim 4 wherein said nutrient-indicator is present in an amount in the range of about 0.2 to about 2.0 percent by weight.

6. The medium of claims 1 or 2, wherein said nutrient-indicator comprises a chromogen which, when released by metabolization of the nutrient-indicator, will alter the color of the sample.

7. The medium of claim 6 wherein said chromogen is a glucuronide selected from the group consisting of orthonitrophenyl-B-D-glucuronide, B-naphthalamide-B-D-glucuronide, alpha-naphthol-B-D-glucuronide, and methylumbilliferyl-B-D-glucuronide.

8. The medium of claim 6 wherein said chromogen is selected from orthonitrophenyl-B-L-pyronidonyl .beta.-naphthalamide-B-L-pyronidonyl, alpha-naphthol-B-L-pyronidonyl, and methylumbilliferyl-B-L-pyronidonyl.

9. The medium of claim 6 wherein said chromogen is selected from the group consisting of L-alanine-B-orthonitrophenyl, B-naphthalamide-B-L-alanine, alphanaphthol-B-L-alanine, and methylumbilliferyl-B-L-alanine.

10. A method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample, said method comprising the steps of:

a) mixing the liquid sample with a medium which includes an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and log phase reproduction of said target microbe in the presence of a nutrient-indicator and to aid the target microbe through lag phase and into log phase of growth in the sample; an effective amount of at least one antibiotic which inhibits growth of a potential non-target microbe in said medium; and an effective amount of a nutrient-indicator which is provided in an amount sufficient to support log phase growth of said target microbe until a detectable characteristic signal is produced in the medium during said log phase growth; said nutrient-indicator in the presence of said antibiotic being incapable of supporting continued logarithmic growth of any viable non-target microbes in the sample to produce a detectable characteristic signal; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to confirm the presence or absence of the target microbe in the sample; wherein said medium lacks a gelling agent so that when said medium is mixed with said liquified sample a liquid is formed, and wherein said ingredients, said antibiotic, and said nutrient-indicator are chosen such that growth of non-target microbes do not interfere with growth of said target microbe; wherein said medium allows growth of Escherichia coli and is specific for Escherichia coli,

and (b) thereafter evaluating the sample to determine whether said detectable characteristic has been altered to allow detection of the presence or absence of the target microbe.

11. A method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample, said method comprising the steps of:

a) mixing the liquid sample with a medium which includes an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and log phase reproduction of said target microbe in the presence of a nutrient-indicator and to aid the target microbe through lag phase and into log phase of growth in the sample; an effective amount of at least one antibiotic which inhibits growth of a potential non-target microbe in said medium; and an effective amount of a nutrient-indicator which is provided in an amount sufficient to support log phase growth of said target microbe until a detectable characteristic signal is produced in the medium during said log phase growth; said nutrient-indicator in the presence of said antibiotic being incapable of supporting continued logarithmic growth of any viable non-target microbes in the sample to produce a detectable characteristic signer; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to confirm the presence or absence of the target microbe in the sample; wherein said medium lacks a gelling agent so that when said medium is mixed with said liquified sample a liquid is formed, and wherein said ingredients, said antibiotic, and said nutrient-indicator are chosen such that growth of non-target microbes do not interfere with growth of said target microbe; wherein said medium allows growth of one or more coliform bacteria and no other bacterium, and thereby is specific coliform bacteria,

and (b) thereafter evaluating the sample to determine whether said detectable characteristic has been altered to allow detection of the presence or absence of the target microbe.

12. The method of claims 10 or 11 wherein said medium includes: a source of salts present in an amount of about 62 percent by weight; and a source of nitrogen present in an amount of at least 10 percent by weight, both of which ensure viability of the target microbes.

13. The method of claim 12 wherein said ingredients are present in an amount in the range of at least about 1.5 to about 2.5 percent b weight.

14. The method of claim 13 wherein said nutrient-indicator is present in an amount in the range of about 0.2 to about 2.0 percent by weight.

15. The method of claims 10 or 11 wherein said medium is in a solid form.

16. The method of claims 10 or 11 wherein said medium is in a powder form.

17. The method of claims 10 or 11 wherein said method further comprising the steps of:

a) obtaining at least one known volume of the liquid sample;

b) forming a specimen sample and medium mixture by adding to the specimen sample a predetermined amount of a solid form of said medium which is soluble in the specimen sample; and

(c) monitoring the specimen sample and medium mixture for at least about twenty hours or until said detectable characteristic has been altered to determine the presence or absence of the target microbe.

18. The method of claim 17, wherein said medium is added to a 1.0 ml sample of the specimen, to a 0.1 ml sample of the specimen, and a 0.01 ml sample of the specimen and each specimen sample and medium mixture is monitored for detectable characteristic alteration to detect the presence of said target microbe in the specimen.

19. The method of claim 18 further comprising the step of incubating the specimen sample and medium mixture at a temperature in the range of about 20.degree. C. to about 44.degree. C. during monitoring thereof.