Note: Descriptions are shown in the official language in which they were submitted.
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PRECIPITATE TEST FOR MICROORGAI'~ISMS
The present invention relates to an improved method for assaying
for a particular microorganism or group of microorganisms wherein the
particular
microorganism or group of microorganisms are characterized by the presence of
a
particular enzyme. The assay uses a dye-forming substrate within a polymer
matrix or on the surface of a solid support. The dye precipitates when cleaved
by
the enzyme. The present invention further comprises an enzyme indicator device
comprising a dye-forming substrate, polymer system, and a solid support.
Background of the Invention
Human gastrointestinal disease can be caused by a variety of
microorganisms. Common vehicles for infection are contaminated foods and
water. To reduce the incidence of such disease, foods and water destined for
human consumption are routinely tested for their sanitary quality. Instead of
testing for a multitude of different enteric pathogens, laboratories test for
the
presence of indicator organisms. Coliform bacteria are typically used as the
primary indicators of sanitary quality because they are commonly associated
with
the gastrointestinal tracts of warm-blooded animals. The presence of coliform
bacteria, especially Escherichia ~, in high numbers in foods or water suggests
that there may be fecal contamination, and contraindicates human consumption.
Coliform bacteria are distinguished from other organisms and from
their close relatives in the family Enterobacteriaceae by their ability to
ferment
lactose to acidic and gaseous (C02 and H2) end products. Certain non-coliform
bacteria may ferment lactose to the same end products, but the growth of these
organisms in the coliform assay is usually minimized or prevented by the
selective
properties of the bacteriological media used for testing.
Typically, the presence of coliform bacteria in a sample is
determined by adding the material to a liquid bacteriological growth medium
and
incubating the mixture at a temperature which is conducive to bacterial
growth.
Incubation of the mixture of sample and growth medium is important because the
assay must detect low levels of coiiform bacterial contamination in the
sample.
Incubation of the mixture results in the multiplication of coliforms to a
level of
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approximately 106 to 108 cells per milliliter of culture, wherein their
presence may
be detected by any of a number of techniques. The variety of different liquid
bacteriological growth media which have been used for coliform detection share
two common properties: they contain the disaccharide lactose and they also
contain chemical agents which selectively inhibit the growth of non-coliform
microorganisms. Selection is important because the sample invariably contains
a
variety of microorganisms, and the success of the assay depends on the
coliforms
not being overgrown by the non-coliforms.
Coliform assays can be performed using either solid or liquid media.
Assays which employ a solid medium allow a viable cell count. Sample is added
to
the solid medium and discrete colonies of coliforms are enumerated.
Alternatively, samples may be added to liquid media. Coliforms are detected
through the formation of characteristic metabolic end products. The liquid
medium format may be qualitative or quantitative. The liquid medium format is
preferred for samples containing fewer coliforms (e.g., less than 10 organisms
per
milliliter), or samples containing particulate material (e.g., food or dairy
samples)
which obscures colony visualization.
Most coliform assays in a broth or agar take place in two discrete
stages: presumptive and ~nfirmed. First, a presumptive assay provides an
indication of possible coliform presence. In the confirmed stage,
presumptively
po~dve cultures or typical colonies are subcultured into a second, more
selective
medium. In principle, the confirmed medium eliminates false positive results.
Together, the two stages of the assay require 48 to 96 hours for completion.
Therefore, there is a need in the art to provide accurate results in a more
timely
manner.
Generally, detection is based upon the end products of metabolic
pathways of coliform bacteria. For example, acid production by coliform
bacteria
is generally detected through the incorporation of pH indicators in the
medium.
Acid formation can be detected by a change in color from purple to yellow of
the
indicator bromocresol purple in a liquid medium such as Clarke's medium. Acid
production is detected by the formation of colonies which are dark-centered
due
to the reaction between the acid and the indicator neutral red in a solid
medium
such as violet red bile agar (VRBA) and MacConkey agar.
Further, gaseous end products are usually detected by the presence
of gas bubbles in a liquid. Gas bubbles may be entrapped in a smaller,
inverted
test tube or an inverted vial within the culture tube or in a special portion
of the
culture device. For example, the BioControl Coh'Trak'" product entraps gas
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bubbles in a dome associated with the top portion of the device. Petrifilm~"
(3M),
with a solid growth medium, entraps gas bubbles in close proximity to a
bacterial
color. Alternatively, gaseous end products may be detected by nonvisual means
such as by electrochemical detection of hydrogen, by radiometric detection of
14C02 released from the fermentation of radiolabelled lactose, or by
impedimetric or gas chromatographic detection of organic compounds produced
during fermentation.
An alternative approach to coliform detection is based upon the
detection of coliform-associated enzyme activity rather than metabolic end
products. Generally, an enzyme assay approach can yield quantitative estimates
for coliform bacteria comparable to confirmatory results using lengthy culture
testing procedures. The enzyme beta-galactosidase is a bacterial enzyme used
for
the fermentation of lactose. Beta-galactosidase hydrolyzes lactose to its
component sugars glucose and galactose. Coliform bacteria typically express
this
enzyme. For example, Warren et al., Cpl. Emriron. Microbiol. x:136-41, 1978,
refers to a coliform ttsting method which uses the chromogenic beta-
galactosidase
substrate, o-nitrophenyl-beta-D-galactoside (ONPG), for quantitating fecal
coliforms in water. In Warren et al., the time of appearance of the yellow
reaction
product o-nitrophenol (ONP) is inversely related to the initial concentration
of
coliforms in the test sample.
The product Colilert'" (Access Medical) uses the ONPG enzyme
~dicator for detecting coliforms in drinlang water. The water sample is used
to
solubilize a basal growth medium containing ONPG. Beta-galactosidase from the
bacteria growing in the culture hydrolyzes the ONPG to produce galactose,
which
serves as the sole carbon and energy source for growth. ONP formation
indicates
enzyme activity. After 24 hours of incubation, the presence of a yellow color
throughout the liquid culture of 10 mil>ivters signifies that coliform
bacteria were
present in the water sample.
The fluorogenic beta-galactosidase substrate fluorescein-beta-D
galactoside is another indicator of activity. Cundell et al., Proc. Water
Reuse
x:1895-99, 1979, refers to an assay incubating coliform bacteria in a medium
containing the fluorescein-beta-D-galactoside. Coliform bacteria are
determined
quantitatively by flow cytometry. The fluorescein moiety of the substrate
becomes
concentrated within the cell after cleavage by beta-galactosidase and imparts
fluorescence to the cells under ultraviolet illumination. U.S. Patent No,
4,242,44?
refers to a fluorescent assay for enumerating coliforms using a fluorogenic
substrate. Specifically, U.S. Patent No. 4,242,447 refers to a process
emulsifying a
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water sample and a fluorogenic substrate with an oil to form oil droplets
containing coliform bacteria. Beta-galactosidase activity cleaves the
fluorogenic
substrate and forms a fluorescent oil droplet which is counted in a
fluorescence
detector. Cundell et al., I?ev. Ind~jcrobiol. x:571-77, 1979, refers to a
similar
technique that yields oil-encapsulated cells on a microscope slide.
Fluorescent
droplets are counted under a fluoresence microscope. The Cundell method has
been used with sewage samples. The sensitivity of the Cundell method is 105
cells
per milliliter.
The bacterium, ~, sue, is a coliform which may be assayed separate
and apart from the coliform bacteria. ~, ~ presence is considered to be a more
reliable indicator of fecal contamination. Additionally, certain strains of ~,
Vii.'
are pathogenic for humans and animals. The standard ~ ~ detection method
subcultures positive presumptive cultures into EC broth and incubates at 45.5'
C.
Gas-positive EC cultures are then streaked onto a differential agar medium.
The
isolates are identified by biochemical characterization.
_ ~ S~li is a relatively unique organism by possessing the enzyme
,_ beta-glucuronidase. Consequently, the fluorogenic substrate
4-methylumbelliferyl-~-D-glucuronide (MUG) is used to detect E< ~ in food
and water samples. The ~ ~ detection procedure consists of inoculating
cultures and incubating by standard methods in the presence of MUG.
Fluorescence which develops during 24 hours incubation indicates the presence
of
~ ~ because beta-glucuronidase from $,, ~ cleaves MUG to a fluorescent
product. Thus, the use of MUG-containing media can shorten the detection time
. , for ~ ~. Unfortunately, MUG is an expensive reagent and large sample
sizes,
such as water samples, require large quantities of MUG and make the test
prohibitively expensive. Currently, the recommended concentration of MUG is
50-100 micrograms per milliliter of final culture. Typical volumes for food
and
water tests are 90 and 100 milliliters respectively. Thus, the cost of the MUG
reagent makes it unattractive for food testing and for use in the PA type of
water
testing.
One can assay for beta-galactosidase and beta-glucuronidase
activities as a means for determining the presence of coliforms and ~ ~,,
respectively. Enzyme detection assays offer advantages over the detection of
fermentation end products. First, the enzyme assay approach is more sensitive.
One enzyme can cleave many substrate molecules. Each molecule of substrate
cleaved by an enzyme yields a fluorescent or colored reporter product.
Therefore,
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the signal is amplified by an enzyme. With certain substrates, as few as 104
metabolically active cells can yield a signal within 24 hours.
By contrast, it takes approximately 107 to 10$ metabolically active
cells to produce a visible gas bubble within 24 hours, bemuse beta-
galactosidase
cleavage of lactose does not result in stoichiometric amounts of acid and gas
end
products due to physiological regulation of metabolism. The majority of the
carbon in the lactose molecule is incorporated into cellular biomass and is
not
used to regenerate oxidized NAD during fermentation. The bacterial
fermentation enzymes responsible for acid and gas production are at the end of
the fermentation pathway and are tightly regulated. For instance, pyruvate-
formate lyase is inactive under aerobic conditions and is activated only when
the
culture becomes anaerobic. Formate dehydrogenase, a key enzyme in gas
production, is not synthesized in the presence of oxygen or when the culture
pH is
6 or greater. Hydrogenase activity and synthesis are negatively regulated by
alternative oxidants such as oxygen, nitrate and nitrite. Accordingly, it is
possible
that growth conditions of anaerobiosis, acidic pH and the absence of external
oxidants are either not achieved or are achieved only slowly . Moreover, much
of
the lactose in the culture will have been cleaved by the time the acid- and
gas-
producing enzymes become active. Therefore, enzyme assays for beta-
galactosidase circumvent the fermentation pathway regulation problems and
yield
a reporter group with every substrate cleavage event.
The presence of anaerogenic strains of genera which belong to the
coliform group further complicates gas detection assay procedures. These
strains
possess beta-galactosidase activity, but do not produce gas from lactose.
Thus,
only an assay for beta-galactosidase activity would detect the anaerogenic
strains
of the coliform group.
It is possible to detect coliform-associated enzymes within 24 hours
of incubation. Beta-galactosidase and beta-glucuronidase a$say results
correlate
with the confirmed presence of coliforms and ~ ~l'. When aliquots from 24 hour
enzyme assay-positive coliform cultures are streaked onto a differential
medium,
such as eosin methylene blue agar, and the agar incubated, typical coliform
and
~, ~ colonies are almost always recovered. Members of the coliform genera
are the taxonomically identified colonies from beta-galactosidase-positive
cultures.
~, ~ is the taxanomicaily identified organism from beta-glucuronidase-positive
cultures. These observations indicate that the first incubation (presumptive)
stage
of the standard coliform assay can be shortened from 48 to 24 hours, and that
the
second (confirmed) incubation stage is unnecessary. Consequently, enzyme
assays
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for coliforms and ~,, ~ can reduce the time required to produce a con~rmatory
result by as much as 72 hours, and simultaneously, provide significantly
better
sensitivity. However, the cost is more expensive due to the reagents,
especially for
large sample sizes.
Food and dairy samples present problems detecting coliforms.
There is a large diversity of foods and the physical/chemical nature of some
makes the detection of either acidic or gaseous end products or certain types
of
enzyme assays difficult. For example, gas bubbles may be difficult to detect
in a
broth due to the turbidity from a food sample such as nonfat dried milk. The
color associated with a food product such as dried cheese, chili powder or
green or
yellow vegetables can obscure the color of a pH indicator or a colored
reporter,
_ such as ONP, in a beta-galactosidase assay using ONPG as a substrate.
Further, a
food may be inherently acidic, or it may internally trap gas bubbles during
sample
preparation. Finally, food chemistry may interfere with an assay. Foods add
additional nutrients to a culture broth and these nutrients may be mexabolized
by
non-coliform bacteria to produce gaseous or acidic end products leading to a
false-positive result. For example, cake mikes or other sweetened products
contain significant amounts of disaccharides and monosaccharides, which can be
converted to acid and/or gas by non-coliforms.
It is important to choose an appropriate substrate for beta-
galactosidase and beta-giucuronidase assays for coliform bacterial and ~
detection. For example, beta-galactosidase cleaves the substrate ONPG to a
yellow product, ONP. However, certain colors inherent in or added to food
products may obscure the yellow color of the ONP reporter product. Food
substances which can obscure the yellow color include hemoglobin in red meats,
chlorophyll in leafy greens and vegetables, beta-carotene and other yellow and
orange pigments in fruits and vegetables, natural pigments in spices, and
artificial
and natural colorants. Turbid water and water with a high hunuc content can
also
obscure weakly yellow positive ONP reactions. Moreover, standard
bacteriological growth media ingredients, such as protein or yeast
hydrolysates,
impart a yellow or golden color to the media and ran obscure the presence of
ONP. For example, the standard method coliform medium, lauryl sulfate tryptose
(hSZ~ broth, has a golden hue. The result of performing an ONPG-type enzyme
assay in LS'T broth and other supplemented media will lead to a significant
number of false-negative results.
An additional problem associated with the use of ONPG is the
hazardous nature of the ONP cleavage product. ONP is a volatile compound and
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a known respiratory eye and skin irritant. The presence of ONP makes
activities
associated with handling of cultures and washing of glassware hazardous to lab
workers. There are also toxic waste disposal problems when using ONP.
Similarly, beta-giucuronidase assays use MUG, which generates a
fluorescent signal. Pigments associated with foods have been observed to
quench
the fluorescent signal. For example, the pigment in chili powder obscures the
fluorescence reaction of the reporter product. A further problem with MUG is
that the pH range for maximal fluorescence is outside of the pH range produced
during lactose fermentation. MUG is maximally fluorescent above pH 10. Yet,
acids produced from lactose fermentation typically lower the coliform culture
medium to pH 5 to 5, obscuring fluorescence reactions. Finally, as noted
above,
beta-glucuronidase assays which employ chromogenic substrates may be subject
to
interference from some types of samples. Therefore, current enzyme assays for
beta-glucuronidase have pH and interference problems from the food samples,
and are inappropriate for use in certain types of foods.
Fluorogenic substrates, such as MUG have been used in agar to
detect ~ ~ in food samples. The fluorescent cleavage product,
4-methylumbelliferone, is soluble and will diffuse from ~, ~ji colonies in
agar (a
solid medium) to add to the difficulties associated with the determination of
which
colonies contain the enzyme beta-giucuronidase and which do not.
An additional complication is that the final volume of culture broth
for different types of samples varies from 1 ml to 1 liter. For each test, the
molarity of the chromogenic or fluorogenic substrate within the culture medium
would need to be constant. This would require consumption of large amounts of
expensive test reagents. It would be advantageous to a food processor if all
coliform testing could be done in a single assay format which features easily
read
results across all test samples, regardless of size, and which economizes on
test
reagents.
A typical food processing company or water treatment facility tests
incoming and outgoing foods, process water, waste water or environmental
samples. Therefore, an ONPG- or MUG-based assay may only be appropriate for
certain types of samples. Thus, there is a need in the art for assays which
are
functional for a variety of samples, such as foods, turbid and/or colored
water and
em~ironmental samples and which eliminate the difficulties associated with
correlating different test methodologies.
The indigogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-
galactoside (X-Gal) has been used in solid media for the detection of ~ ~i_
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colonies which express beta-galactosidase activity. Beta-galactoside cleaves X-
Gal
to galactose and the indolyl derivative. The indolyl derivative dimerizes to
form a
substituted indigo, which has an intense blue color.
Frampton et al., J. Food Prot. x:402-04, 1988, refers to a peptone
s tergitol agar supplemented with 5-bromo-4-chloro-3-indolyl-S-D-glucuronide
(X
Gluc) to differentiate E, ~ from other bacterial colonies in artificially
inoculated
raw minced chicken. Ixy et al., Ann. Meet. Am. Soc. Microbiol.. Abstract
Q35:288, 1988, refers to a method to enumerate ~, ~, in water and sewage using
a membrane filtration technique with a glycerol medium and 3-indolyl-beta-D
glucuronide subsuate. ~, ~ colonies appeared blue in the semisolid medium.
Watkins et al., ~~ Environ. Microbiol. x:1874-75, 1988, refers to a pour plate
method using X-Gluc for detecting ~ ~ in shellfish and waste water.
A product Pevifilm'" (3M) uses a solid medium and X Gluc for
detecting ~, Eli. E" ~ colonies appear blue.
Because of the high cost of the indogcnic compounds X-Gal and X
Gluc, for the detection of beta-galactosidase and beta-glucuronidase activity
have
_ not found widespread use or have been used with relatively small culture
volumes.
Therefore, there is a need in the art for a beta-galactosidase test system
that can
economically use small quantities of X-Gal and X-Gluc for food and water
testing,
because the intense blue color will solve many of the problems associated with
the
yellow color of ONP and other colored reporters.
In summary, there is a need in the art, therefore, for a coliform
detectioa assay which offers the improved sensitivity and speed associated
with
enzyme assays, the capacity to detect anaerogenic coliforms, successful
applications to food, water and environmental samples, safety of handling and
disposal, and economy in the consumption of expensive test reagents.
Summary of the Invention
The present invention comprises a method for assaying an enzyme
associated with a particular microorganism or a group of microorganisms and an
enzyme indicator device that is useful for assaying for a particular
microorganism
and/or group of microorganisms. In one aspect, the method ~mpris~s adding
sample and an enzyme indicator device to growth medium, incubating at a
permissive temperature and reading the results on the enzyme indicator device.
Preferably, the growth medium is a liquid. Another method comprises adding a
sample to a growth medium; incubating the sample and growth medium at a
permissive temperature for the growth of microorganisms until there is
evidence
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of microorganism growth within the growth medium; adding an enzyme indicator
device to the growth medium, wherein the enzyme indicator device comprises a
dye-forming and/or fluorogenic-generating substrate in a polymer system on a
solid support, or a dye-forming or fluorogenic-generating substrate dried on a
solid support, and wherein the dye-forming substrate is cleaved by the enzyme
to
form a colored precipitate or fluorescent reaction; incubating the sample,
growth
medium, and enzyme indicator device at a permissive temperature and in an
oxidizing environment from about one hour to about forty-eight hours; and
visually detecting the presence of the particular microorganism or group of
microorganisms by a color change or fluorescent reaction on the enzyme
indicator
device. Preferably, an oxidizing agent is added to the growth medium along
with
the enzyme indicator device. The enzyme indicator device preferably comprises
a
- dye-forming and/or fluorogenic-generating substrate in a polymer system.
Alternatively, the enzyme indicator device comprises a dye-forming and/or
fluorogenic-generating substrate adhering to the surface of a solid support.
More
particularly, the enzyme indicator device further comprises a solid support
member, wherein the dye-forming and/or fluorogenic-generating substrate in the
polymer system is layered onto a surface of the solid support system. It is
preferred that the solid support floats on the surface of a liquid so as to
have an
oxidizing environment from air without the need for additional oxidizing
agents.
Within another aspect of the present invention, a method for
assaying for an enzyme associated with a particular microorganism or a group
of
microorganisms is provided, comprising adding a sample to a growth medium;
adding a dye-forming substrate in a polymer system, wherein the dye-forming
substrate forms a precipitate in association with the polymer, when cleaved by
the
enzyme; contacting the dye-forming substrate in the polymer with the sample
and
the growth medium; incubating the sample, growth medium and dye-forming
substrate in polymer at a permissive temperature for the growth of particular
microorganism or group of microorganisms; and detecting the presence of the
particular microorganism or group of microorganisms by a colored precipitate
in
association with the polymer.
The dye-forming substrate forms an insoluble precipitate upon
enzymatic cleavage. Preferably, the dye-forming substrate is a substituted
indigo
derivative of the formula:
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R1 O-sugar
H
wherein each R substituent is a halo group or hydrogen. The halo group is a
chloro, bromo, or iodo group. When Rl, R2 and R3 are H, the precipitate dye
formed is indigo.
It is preferred that the dye-forming substrate is insoluble in aqueous
systems so as to prevent the dye-forming substrate from leaching out of the
polymer or from coming off the surface of the solid support when exposed to
the
growth medium. In this way, it is possible to conserve costs by reducing the
amount of dye-forming substrate needed for each test. Thus, the amount of dye
fonming substrate used is independent of the test size or the sample size.
Moreover, the analysis of particular microorganisms or group of microorganisms
~by enzyme assays provides a more rapid and more sensitive assay system than
comrentional growth and pure culture detection methods.
The advantages of the im~entive method and the use of the imrentive
enzyme indicator device allow the dye-forming substrate to be deployed on a
ZS , defined reaction surface rather than dispersed throughout a solution. The
reaction surface is the surface of a solid support member or a polymer matrix.
'This results in an assay format which is independent of culture volume, thus
leading to an economy of reagent consumption. Second, the insoluble indigo dye-
forming substrates from the indigogenic substrates range in color from blue to
purple, colors which are not masked or quenched by pigments or turbidity
associated with foods or certain types of water. Thus, the indigogenic
substrates
are amenable to detection of coliform bacteria in both food and water samples.
Third, the indigogenic substrates offer the speed and sensitivity associated
with
enzyme assays. Fourth, the assay for beta-galactosidase eliminates the
problems
associated with enzyme-regulation in the lower portion of the lactose
fermentation
pathway. Fifth, an enzyme assay ensures that signal is being generated in the
$rst
step of the pathway every time the enzyme performs a hydrolysis of the dye-
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forming substrate, rather than at the end of the pathway where only a portion
of
the cleaved galactoside ends up.
Within another aspect of the present im~ention, the method for
assaying for an enzyme associated with a particular microorganism and a group
of
microorganisms comprises (a) adding a sample to a liquid growth medium;
(b) adding a dry-forming or fluorogenic-generating substrate on a de5ned
reaction
surface wherein the substrate forms a precipitate or a fluorescent by-product
when
cleaved by the enzyme; (c) incubating the sample, growth medium and substrate
at
a permissive temperature for the growth of the particular microorganism and
group of microorganisms; and (d) detecting the presenec of the particular
microorganism and group of microorganisms by ,. a colored precipitate or
fluorescence on the defined reaction surface or in the surrounding medium.
Within a preferred embodiment, the fluorogenic-generating substrate is 4-
methylumbelliferyl-~-D-glucuronide.
brief DescriRtion of the Drawing
The Figure shows the reaction pathway for
bromochloroindolylgalactoside (X-Gal) hydrolysis to form bromochloroindigo.
Bromochloroindolylgalactoside is first cleaved by beta-galactosidase from the
particular microorganism or group of microorganisms to remove the galactose
group and form bromochloroindoxyl, which then dimerizes by an o~ddation
reaction to the highly colored blue-green precipitate, bromochloroindigo.
Oxidation can be achieved either by an oxidizing agent in solution or by
exposure
of bromochloroindoxyl to air.
detailed Description of the Invention
The present im~ention is a method for assaying an enzyme
associated with a particular microorganism and/or a group of microorganisms.
Preferably, the particular microorganism is ~ ~ and the group of
microorganisms is the coliform bacteria. The enzyme beta-galactosidase is
characteristic of the coliform bacteria and beta-glucuronidase activity is
characteristic of ~ ~. The dye-forming substrates preferably are mixed in a
polymer system and form an insoluble precipitate upon enzymatic cleavage.
Alternatively, the dye-forming substrates may adhere to a solid support member
which also is the reaction surface for forming the insoluble precipitate.
Preferably, the dye-forming substrate is insoluble in an aqueous liquid, such
as a
growth medium so as to avoid dispersion of the dye-forming substrate
throughout
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the growth medium and instead, have the dye-forming substrate concentrated
within the polymer system or on the surface of the solid support member.
The reaction of the dye-forming substrate to form a highly colored
precipitate requires the presence of the specific enzyme to cleave off the
sugar
group and an wddizing emrironment to form the highly colored diner precipitate
from the cleaved dye-forming substrate. The oxidizing environment can be
created by having the dye-forming substrate in the polymer exposed to air or
by
the addition of an oxidizing agent to the growth medium. Examples of oxidizing
agents include hydrogen peroxide, N03-, Fe3+, Ag+, pyrogallol, G~+ and
others. Most preferably, the oxidizing agent will have a redox potential
greater
than 0.10 and be used at a concentration that avoids toxicity to the growing
microorganisms.
The dye-forming substrate is preferably a substituted indigo
derivative of the formula:
R1 O-sugar
R2
_ R3
N
H
wherein each R substituent is a halo group or hydrogen. The sugar group is air
monosaccharide, derivative thereof or an aldehydo acid. Preferably, the sugar
group is a galactoside or a glucoside and the aldehydo acid is glucuronic
acid.
Most preferably, the sugar or aldehydo acid is attached to oxygen by a beta
linkage. Examples of the substituted indigo dyes include 3-indolyl-, 5-bromo-4-
chloro-3-indolyl-, 5-bromo-3-indolyl-, 5-bromo-6-chloro-3-indolyl, and 5-iodo-
3-
indolyl- derivatives in beta-D linkages with galactose and glucuronic acid.
The
substrate is colorless, however when hydrolyzed by the appropriate enzyme, the
indoxyl or substituted indoxyl portion of the substrate oxidizes to form an
intensely
colored, insoluble indigo precipitate or indigo derivative as shown, for
example, in
the Figure. The dimerizing reaction requires an oxidizing environment Further,
non-indigogenic beta-sugar substrates which form colored insoluble products
after
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enzymatic cleavage could be used for the inventive method and the inventive
enzyme indicator device.
In another embodiment, a culture of a particular microorganism or
a group of microorganisms can grow in a growth medium or have yielded a
S presumptive positive result for a particular microorganism or a group of
microorganisms. The inventive enzyme indicator device for assaying for a
particular enzyme assoaated with a particular microorganism or group of
microorganisms can be added to the growth medium after growth of the sample in
the growth medium. For example, a dye-forming substrate can be mixed with a
polymer system and then layered on top of a solid support. Alternatively, the
dye-
forming substrate can be adhered onto the surface of a solid support member.
It
is preferred that the solid support be a floatable plastic means to remain on
the
surfa~,~e of a liquid growth medium. Alternatively, a dye-forming substrate
can be
mixed in with a polymer material, such as latex, to form a matrix containing
the
dye-forming substrate throughout. The latex matrix with dye-forming substrate
can be dropped into a liquid growth medium or suspended on the surface of the
liquid growth medium.
If the dye-forming substrate is not in contact with air, then a
developer solution should be added to the growth medium concurrently with the
dye-forming substrate or shortly thereafter. The developer solution comprises
an
oxidizing agent, such as hydrogen peroxide, Ag+, Hg+ +, I3 , I04-, persulfate,
Pd+ +, nitrobenzenes, azobenzenes, quinones, 2-chloro-2-nitro-propane,
K3Fe(CN)6, indolones, and p-hydroxymercaribenzoate. The developer solution
preferably further comprises a precipitation enhancer and a cell
permeabilizer.
The cell permeabilizer facilitates entrance of an indigogenic substrate into
the cell
to result in a faster turnover of the substrate, and a faster exiting of the
indoxyl or
indigo from the bacterial cell. Examples of cell permeabdizers include,
toluene,
anionic and nonionic detergents, and cetyltrimethylammonium bromide. 'The
precipitation enhancer helps to prevent the precipitate from migrating from
the
solid support and/or the polymer matrix. An example of a precipitation
enhancer
is an alkaline detergent mixture, such as sodium dodecylsulfate at a 0.1% to
1.0%
concentration and a pH of 8.0 to 10Ø The precipitation enhancer is
preferably
used in a growth medium which shows turbidity or potential quenching of the
color reaction.
The enzyme indicator device may be made by dissolving a dye-
forming substrate, such as X-Gal, in a solvent such as dimethylformamide
(DMF),
methyl cellosolve (2-methoxyethanol), or DMSO and mixing with a liquid polymer
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system, such as latex. Other adhesive-type polymers that can be dried into a
solid
or semisolid state include, for example, ethylene vi~rl acetate (EVA),
polyvinyl
alcohol (PVA), and polyvinyl pyrrolidone (PVP). EVA is a polymer soluble in
methylene chloride. _ Additionally, polymer systems that can be liquefied in
solvents, such as methylene chloride are useful to liquefy the adhesive-type
polymer for mixing with the dye-forming substrate. The mixture of dye-forming
substrate and polymer is dried to form a solid or pliable solid. Preferably,
the
fiquid dye-forming substrate and polymer, in a more viscous state, is layered
onto
the surface of a solid support. Most preferabl.X-asolid support is a porous
plastic
material that can float on the surface of a liquid.
Alternatively, the dye-forming substrate in solvent can be added
directly to the surface of a solid support member and allowed to dry (solvent
evaporated). Additionally, the dye-forming substrate can be precipitated out
of
the solvent, such as DMF, by adding a reagent, such as methylene chloride, in
which the dye-forming substrate is insoluble. The precipitate is then layered
directly onto a solid support member.
The concentration of dye-forming substrate is sufficient to produce
a visible response given the surface area and volume of the reaction surface.
For
example, the concentration X-Gal on a circular solid support member of
polyethylene with a 70 micron pore size of dimensions of one-half inch
diameter
and one-sixteenth thickness ranges from about 50 ~tg to about 1 mg.
The solid support may float on the surface of a liquid, may reside
below the surface of the liquid, or be mechanically placed near the surface of
a
~~_ liquid growth medium by physically attaching it to the culture container
or culture
, container closure. Placing the solid support at or near the liquid-air
interface
offers close proximity to oxygen in the air to facilitate the oxidation of
indoxyl to
indigo, thereby improving the speed and sensitivity of the assay. Further, if
the
solid support and ultimate color localization is located at the top of the
culture
medium, the reaction is not obscured by the culture broth and it becomes
easier to
read. If the solid support resides below the surface of the liquid, it is
preferred
that a developer solution be added to facilitate oxidation of the dye-forming
substrate.
When testing for coliform bacteria, common components in the
medium, such as in LST broth, can obscure the color reaction from a soluble
substrate such as ONPG. Thus, ONPG can only be tested in clear media so that
weak reactions can be read. Here, the incorporation of the dye-forming
substrate
onto a solid support of literally any size can conserve reagent use
independent of
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sample size. The same solid support and surface area of dye-forming substrate
associated with a polymer system can be used for any volume of culture,
because
the substrate reaction with the bacterial enzyme tames place within a defined
surface area rather than throughout the entire volume of the culture. The blue
color which is generated is not obscured by ordinary bacteriological media
components and therefore an assay with the dye-forming substrate in a polymer
system can be performed in a complex medium such as LST broth. The insoluble
blue precipitate that is formed from hydrolysis of the dye-forming substrate
in the
presence of an oxidizing environment remains at the surface of the solid
support,
thus concentrating the reaction product into a comparatively small, more
readily-observed space. Irocalization of the precipitate makes the test easier
to
read and increases its sensitivity. The localization of the enzyme assay to a
solid
support also results in an economy of reagent use.
In another aspect, the inventive method and enzyme indicator
device can be used in a quantitative assay to estimate the number of coliform
bacteria in a food or water sample using the most probable number (MPN)
method. An MPN coliform assay is performed by incorporating a dye-forming
substrate in a polymer system, such as latex into a standard coliform medium,
such
- _ as LST broth. Preparation of the sample is done by standard techniques.
After
. overnight incubation, the tubes are read for color production in the polymer
system and the positive tubes are used to calculate a ~nfirmed MPN per gram or
MPN per milliliter value, according to standard MPN techniques. See
o~nendium of Methods for the Microbioioaical Examination of Foods (2nd Ed.,
American Public Health Association, 1984), Official Methodls o~~ (14th
Ed., Assoaation of O~cial Analytical Chemists, 1984), and Standard Methods for
lhP Fx~minatir~n of Water and Wastewater (17th Ed., American Public Health
- Association, 1981).
The inventive method and enzyme indicator device are used, for
example, for qualitative testing for coliform bacteria. An indigogenic
galactoside
substrate is incorporated into the medium used for the PA test for coliforms
in
. . , drinking water. The water sample is added to a medium containing the dye
.- forming substrate in a polymer system, preferably layered onto a floatable
plastic
solid support. Precipitated indigo, on the solid support member, within the
culture vessel after a suitable incubation period signifies the presence of
coliforms
, in the sample. This procedure is also used for qualitative testing of
environmental
samples for the presence of coliforms. For example, a surface or drain could
be
assayed for the effectiveness of sanitation by swabbing with a cotton-tipped
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applicator and then placing the applicator in a coliform medium containing the
indigogenic galactoside dye-forming substrate incorporated into a polymer
system,
preferably on a solid support member. A precipitated indigo color within the
. culture vessel, or more specifically on the surface of the solid support
after a
suitable incubation period, such as 1 to 24 hours at a permissive temperature
for
bacterial growth would signify the presence of coliforms in the sample.
In addition to the use of a dye-forming substrate as described above,
the present imrention provides for the simultaneous confirmatory determination
of
both ~, X11 and coliform bacteria without the need for additional culture
transfers, reagent preparation or diagnostic manipulating. More specifically,
a
disc as described above containing dye-forming and fluorogenic~enerating
substrates is added to a container of microbiological culture media to be
tested for
the presence of ~, ~ and/or the coliform group of bacteria. The container with
the medium and reaction disc is incubated at a permissive temperature for
growth
of the microorganisms of interest. Following an incubation step, the container
is
observed for the presence of a distinguishing color on the reaction disc or in
the
surrounding medium. The container is then exposed to long wave ultraviolet
light
(366 nm) and observed for fluorescence in the surrounding medium. The
observance of color on the disc indicates a reaction for the coliform
_. 20 group of organisms. The presence of fluorexence indicates a reaction
for the presence of the specific microorganism, E, ~.
The following examples are offered by way of illustration and not by
way of limitation.
This example illustrates a method for detecting coliform bacteria in
drinking water by the PA (presence-absence) technique, as described in
Standard
Methods for the Examination of Water and Wastewater, (17th Ed., American
Public Health Association, 1981). Briefly,100 ml of water are aseptically
added to
50 ml of triple strength Clark's medium in a 250 ml bottle. Clark's medium had
been modified to exclude a pH indicator. After inoculation, a plastic support
containing an indigogenic galactoside substrate in a latex polymer system was
added to the culture vessel so that it floated at the liquid surface. The
indigogenic
galactoside substrate in a latex polymer system on the plastic support was
made as
described herein. The mixture was incubated at 35' C and the plastic support
was
inspected for indigo formation at 24 hours. The indigo-positive reaction
signified
the confirmed presence of conforms in the water sample.
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This example illustrates a method for detecting coliform bacteria
and ~ X11, in an environmental sample using the inventive method. The
S environmental sample was taken in an effort to assess the effectiveness of
sanitation or as part of a routine environmental screening program. A
cotton-tipped applicator was wetted with sterile diluent and used to swab a
surface. The swab was placed in a bacteriological lauryl sulfate tryptose
broth
growth medium. A buoyant circular plastic support layered with an indigogenic
galactoside substrate in a polymer system on one-half of the circle and an
indigogenic glucuronide substrate on the other half of the circle was added
after
inoculation. The mixture was incubated for 35' C for 24 hours. An
indigo-positive reaction on one half of the plastic support signified the
presence
of coliform bacteria in the environmental sample. An indigo-positive reaction
on
both halves of the plastic support signified the presence of ~, ~ in the
sample.
This example illustrates a method for estimating the level of
coliforms and ~, ~ in a food or water sample by the Most Probable Number
(MPN) analysis using the imrentive method. The MPN technique is described in
Compendium of Methods for the Microbiological Examination of Foodc (2nd Ed.,
American Public Health Association, 1984), Official Methods of A_nalysic (14th
Ed, Association of Official Analytical Chemists, 1984), and Standard Methods
for
Examination ~~f Water and Wastewater (17th Ed., American Public Health
Association, 1981).
Water samples ( 10 ml) were aseptically added to 10 ml of double-
strength lauryl sulfate tryptose (LST) broth. A buoyant circular plastic solid
support containing an indigogenic galactoside substrate in a polymer system on
one-half of the circular plastic and an indigogenic glucuronide substrate in a
polymer system on the other half was added to each test tube after
inoculation.
The mixture was incubated at 35' C for 24 hours. An indigo-positive reaction
on
one half of the plastic support signifies the confirmed presence of coliform
bacteria in the water sample. An indigo-positive reaction on both halves of
the
plastic support confirms the presence of ~ ~ in the sample. The number of
indigo-positive cultures was used to estimate the number of coliform bacteria
and
~ ~ present within the original water sample through the use of a standard
statistical probability table (MPN Table).
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The food samples were diluted in serial 10-fold fashion prior to the
MPN assay. One part (by mass) of solid food sample was added to 9 parts (by
volume) of sterile diluent ButterFeld's buffered phosphate buffer. The mixture
was blended at high speed for two minutes. Two additional 10-fold dilutions
were
made from the 1:10 diluted food sample. One ml of each 10-fold dilution was
added to each of three 10 ml test tubes of LST broth. A buoyant circular
plastic
support containing an indigogenic galactoside substrate in a polymer system on
one half and an indigogenic glucuronide substrate in a polymer system on the
other half was added to each test tube after inoculation. The mixture was
incubated at 35'C for 24 hours. An indigo-positive reaction on one half of the
plastic support confirmed the presence of coliform bacteria in the sample
dilution.
An indigo-positive reaction on both halves of the plastic support confirmed
the
presence of ~, ~ in the sample dilution The number of indigo-positive cultures
was used to estimate the number of coliform bacteria and ~, ~ present within
the original food sample through the use of standard statistical probability
tables
(MPN Table).
This example illustrates a method for the confirmation of
presumptively positive coliform cultures for total coliforms and for ~, ~j~,
using an
indigogenic substrate mixture. Cultures which were presumptively gas-positive
for
coliforms were confirmed for coliform and jor ~, ~ presence according to the
_. following procedure. A buoyant circular solid support containing an
indigogenic
4 ~ galactoside substrate on one half and an indigogenic glucuronide substrate
on the
other half was added to the culture. An oxidizing agent (hydrogen peroxide at
10% concentration) was added to the culture. The presumptive portion of the
coliform MPN analysis was performed in LST broth on a food or water sample
using the procedure described in Example 3. Cultures which were gas-positive
after 24 to 48 hours of incubation were confirmed with the buoyant, circular
solid
support described herein along with the addition of a developer comprising the
oxidizing agent. The culture was incubated at 35'C for approximately one hour
and the plastic support was read for indigo formation. Cultures which were
indigo-positive on one half of the circular solid support were considered to
be
confirmed for the presence of coliform bacteria. Cultures which were indigo-
positive on both halves of the circular solid support were confirmed for the
presence of ~ ~. Negative cultures were reincubated for an additional hour
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and reread. Cultures which were still negative for indigo at two hours
remained
unconfirmed for the presence of coliforms or ~ ~.
This example illustrates a method for detecting coliform bacteria in
drinlang water by the PA (presence-absence) technique, as described in ~
Methods for the Examination of Water and Wastewater. (17th Ed., American
Public Health Association,1981). Briefly,100 ml of water are aseptically added
to
SO ml of triple strength Clark's medium in a 250 ml bottle. Clark's medium had
been modified to exclude a pH indicator. After inoculation, a plastic support
containing an indigogenic galactoside substrate and a fluorogenic-generating
. substrate in a latex polymer system was added to the culture vessel so that
it
floated at the liquid surface. The indigogenic galactoside substrate in a
latex
polymer system on the plastic support was made as described herein. The
mixture
was incubated at 35~C and the plastic support was inspected for indigo
formation
at 24 hours. The indigo-positive reaction signified the confirmed presence of
coliforms in the water sample.
This example illustrates a method for detecting coliform bacteria
and ~" ~, in an environmental sample using the inventive method. The
em~ironmental sample was taken in an effort to assess the effectiveness of
sanitation or as part of a routine emrironmental screening program. A
cotton-tipped applicator was wetted with sterile diluent and used to swab a
surface. The swab was placed in a bacteriological lauryl sulfate tryptose
broth
growth medium A buoyant circular plastic support layered with an indigogenic
galactoside substrate in a polymer system and a fiuorogenic glucuronide
substrate
was added after inoculation. The mixture was incubated for 35~C for 24 hours.
An indigo-positive reaction on the plastic support signified the presence of
coliform bacteria in the emiironmental sample. A fluorescent-positive reaction
in t>;e container when received under long wave ultraviolet light (366 nm) of
the
plastic support signified the presence of ~ ~ in the sample.
Although the foregoing irnention has been described, in part, by
way of illustration and example for the purposes of clarity and understanding,
it
will be apparent that certain changes or modifications may be practicxd
without
deviating from the spirit and scope of the irn~ention.
