Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-07-20
2002-02-26
Woodward, Michael P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S038000, C435S007320, C435S014000
Reexamination Certificate
active
06350588
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a test medium and method for the detection, quantification, identification and/or differentiation of biological materials in a sample which may contain a plurality of different biological materials.
Bacteria are the causative factor in many diseases of humans, higher animals and plants, and are commonly transmitted by carriers such as water, beverages, food and other organisms. The testing of these potential carriers of bacteria is of critical importance and generally relies on “indicator organisms.” Borrego et al., Microbiol. Sem. 13:413-426, (1998). For example,
Escherichia coli
(
E. coli
) is a gram negative member of the family Enterobacteriaceae which is part of the normal intestinal flora of warm blooded animals, and its presence indicates fecal contamination (e.g., raw sewage). Even though most strains of
E. coli
are not the actual cause of disease, their presence is a strong indication of the possible presence of pathogens associated with intestinal disease, such as cholera, dysentery, and hepatitis, among others. Consequently,
E. coli
has become a prime indicator organism for fecal contamination, and as a result, any method which differentiates and identifies
E. coli
from other bacteria is very useful.
Others members of the family Enterobacteriaceae, commonly referred to as “general coliforms,” especially the genera Citrobacter, Enterobacter and Klebsiella, are also considered to be significant indicator organisms for the quality of water, beverages and foods. Therefore, tests to identify and differentiate general coliforms from
E. coli
are also very useful. Also, various species of the genus Aeromonas have been shown to not only be potential pathogens, but to have a correlation to other indicator organisms (Pettibone et al.,
J. App. Microbiol.
85:723-730 (1998)). Current test methods to identify, separate and enumerate Aeromonas spp. from the very similar Enterobacteriaceae have been lacking and most of the current methods utilizing enzyme substrates do not separate Aeromonas spp. from Enterobacteriaceae due to their almost identical biochemical profiles. Any method that depends upon the identification of general coliforms by means of a &bgr;-galactosidase substrate either does not differentiate Aeromonas spp. from general coliforms or eliminates Aeromonas from the sample by the use of specific inhibitors (antibiotic such as cefsulodin). Brenner et al.,
Appl. Envir. Microbio.
59:3534-44 (1993). They do not differentiate, identify and enumerate Aeromonas along with
E. coli
and general coliforms. Landre et al.,
Letters Appl. Microbiol.
26:352-354(1998). Improved test methods to effectively identify, separate and enumerate such bacterial types are needed, and there is a continuing search for faster, more accurate, easier to use and more versatile test methods and apparatus in this area.
Numerous test methods have been utilized to determine, identify and enumerate one or more indicator organisms. Some of these test methods only indicate the presence or absence of the microorganism, while others also attempt to quantify one or more of the particular organisms in the test sample. For example, a qualitative test referred to as the Presence/Absence (or P/A) test, may be utilized to determine the presence or absence of coliforms and
E. coli
in a test sample. A test medium including the &bgr;-galactosidase substrate O-nitrophenyl-&bgr;-D-galactopyranoside (ONPG), and the &bgr;-glucuronidase substrate 4-methyl-umbrelliferyl-&bgr;-D-glucuronide (MUG), is inoculated with the test sample. To differentiate the general coliforms from
E. coli,
this test relies on the fact that generally all coliforms produce &bgr;-galactosidase, whereas only
E. coli
also produces &bgr;-glucuronidase in addition to &bgr;-galactosidase. If any coliforms are present (including
E. coli
), the broth medium turns a yellow color due to the activity of the galactosidase enzyme on the ONPG material, causing the release of a diffusible yellow pigment. If
E. coli
is present, the broth medium will demonstrate a blue fluorescence when irradiated with ultraviolet rays, due to the breakdown of the MUG reagent with the release of the fluorogenic dye caused by the production of the glucuronidase enzyme. These reactions are very specific, and allow the presence of both coliforms in general, as well as
E. coli
to be identified in a single sample. A disadvantage of this test is that it is not directly quantitative for either bacterial type, since both reagents produce diffusible pigments. A second disadvantage is that there may a false positive coliform reaction if Aeromonas spp. are present in the test sample. This has been shown to be possible even when there are inhibitors present to supposedly prevent this from occurring (Landre et al.,
Letters Appl. Microbiol.
26:352-354 (1998)). The test also requires specific equipment for producing the ultraviolet rays. Further, this test may only be used to detect coliforms and
E. coli.
Other important microorganisms, such as the strain
E. coli
0157 which is glucuronidase negative, are not detected, nor are other non-galactosidase-glucuronidase producing microorganisms.
The Violet Red Bile Agar (VRBA) method has been used to determine the quantity of both coliform and
E. coli
in a test sample. The test medium used in this method includes bile salts (to inhibit non-coliforms), lactose and the pH indicator neutral red. As coliforms (including
E. coli
) grow in the medium, the lactose is fermented with acid production, and the neutral red in the area of the bacterial colony becomes a brick red color. The results of this test are not always easy to interpret, and in order to determine the presence of
E coli,
confirming follow-up tests, such as brilliant green lactose broth fermentation, growth in EC broth at 44.5° C. and streaking on Eosin Methylene Blue Agar (EMBA), must be performed.
The Membrane Filter (MF) method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. This method is used most often when bacterial populations are very small, and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated, and the bacterial colonies growing on the membrane filter surface are counted and evaluated. This method is widely used and provides good results when combined with proper reagents and media A disadvantage of this method is that it is expensive and time-consuming. It also does not work well with solid samples, or samples with high particulate counts. The MF method can be used in conjunction with the inventive method described in this application.
The m-Endo method is also used to determine the quantity of
E. coli
and general coliforms and is an official USEPA approved method for testing water quality. The medium is commonly used with a membrane filter and
E. coli
and general coliform colony forming units (CFU) grow as dark colonies with a golden green metallic sheen. Due to a proven high rate of false positive error, typical colonies must be confirmed by additional testing.
Standard Methods for the Examination of water and Wastewater,
20
th
Edition, 9-10 &9-60 (1998).
Other tests, such as the Most Probable Number (MPN), utilize lactose containing broths (LST, BGLB, EC) to estimate numbers of general coliforms and
E. coli,
but have also been shown to have high rates or error as well as being cumbersome and slow to produce results. Evans et al.,
Appl. Envir. Microbiol
41:130-138 (1981).
The reagent 5-bromo-4-chloro-3-indolyl-&bgr;-D-galactopyranoside (X-gal) is a known test compound for identifying coliforms. When acted on by the &bgr;-galactosidase enzyme produced by coliforms, X-gal forms an insoluble indigo blue precipitate. X-gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. X-gal has the advantage over the compound ONPG,
Roth Geoffrey N.
Roth Jonathan N.
Baker & Daniels
Micrology Laboratories, LLC
Woodward Michael P.
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