Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-01-18
2001-12-04
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287900, C435S303300, C435S305300
Reexamination Certificate
active
06326190
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and apparatus for the analysis of biofilms, and sensitivity of biofilms to anti-bacterial reagents.
BACKGROUND OF THE INVENTION
Extensive study into the growth properties of bacteria in recent years has shown that they form complex layers that adhere to surfaces. These complex forms of bacteria are known as biofilms, or sessile bacteria. Biofilms may cause problems in a variety of areas including the bodies of humans and animals, food processing, health care facilities, metal-working shops, dairy farms and other industries.
It is now widely known that bacteria in the form of biofilms are more resistant to antibacterial reagents than planktonic bacteria. Yet testing for the presence of bacteria and the testing of the efficacy of antibiotics against bacteria has traditionally involved testing for planktonic bacteria. Thus, bacterial inhibitory concentration of the antibacterial reagent may be underestimated, with the result that the wrong antibacterial reagent or wrong amount of antibacterial reagent may be used for the treatment of the bacteria.
One type of device for monitoring biofilm buildup is described in the Canadian Journal of Microbiology (1981), volume 27, pages 910 to 917, in which McCoy et al describe the use of a so-called Robbins device which comprises a tube through which water in a recycling circuit can flow. The tube has a plurality of ports in its walls, each being provided with a stud having a biofoulable surface and being capable of being retained in the port in fixed relationship with respect to the tube so that the biofoulable surface forms part of the internal surface of the tube. The studs may be removed from the ports after a desired time interval and the test surfaces by microscopy of the surfaces analyzed for the growth of microorganisms or by removal of the microorganisms from the surfaces and subsequent estimation of the degree of growth. The number of microorganisms can be estimated for instance by physical or chemical means, e.g. by detection of bacterial ATP or by further culturing the microorganisms and analyzing the products.
In another device described in U.S. Pat. No. 5,349,874, biofilm growth in a water carrying conduit is determined by providing plural removable studs in the conduit or in a second conduit parallel to the first. The studs may be removed for analysis of biofilm on the studs. Such devices as the Robbins device, or others using removable studs in a single conduit, result in rather lengthy processing times, and do not provide rapid response times for the testing of several different antibacterial reagents.
In a still further device, described in “Simple Method for Measuring the Antibiotic Concentration Required to Kill Adherent Bacteria”, Miyake et al, Chemotherapy 1992; 38, 286-290
, staphylococcus aureus
cells adhered to the bottom of a 96 well plastic tissue culture plate were treated with serially diluted antibiotic solutions, and viability of the cells was judged by their growth after a further 24 hours incubation. This method has the disadvantage of inconsistent colonization of sessile bacteria and settling of planktonic bacteria.
The device described in this patent document allows for an efficient and automated biofilm killing assay that has particular use with the 96 well platform common to many diagnostic assay systems.
SUMMARY OF THE INVENTION
There is therefore provided in accordance with one aspect of the invention, a method of analyzing characteristics of a biofilm, in which bacteria are incubated to form a biofilm on plural biofilm adherent sites by providing a flow of liquid growth medium across the plural biofilm adherent sites, the direction of the flow of liquid being repeatedly changed, and an assay made of the resulting biofilm.
In a further aspect of the invention, bacteria are incubated to form a biofilm on plural biofilm adherent sites arranged in plural rows, with plural biofilm adherent sites in each row, while providing a flow of liquid growth medium across the plural biofilm adherent sites, and an assay made of the resulting biofilm.
In a preferred method of the invention, the characteristic of the biofilm is the sensitivity of the biofilm to antibacterial reagent and the method further includes, before assaying the biofilm, treating the biofilm adherent sites with antibacterial reagent.
In a further aspect of the invention, there is also included the step of, after treating the biofilm adherent sites with antibacterial reagent, dislodging the biofilm from the biofilm adherent sites and further incubating the biofilm. Dislodging the biofilm from the biofilm adherent sites may include dislodging the biofilm from each biofilm adherent site into a separate well of a microtiter plate.
When the biofilm adherent sites are formed in rows, treating the biofilm adherent sites with an antibacterial reagent may include treating each row of biofilm adherent sites with a different antibacterial reagent, and treating each of the biofilm adherent sites in a row with a different concentration of antibacterial reagent.
Preferably, the flow direction of the liquid growth medium is repeatedly reversed. In this aspect of the invention, the liquid growth medium may flow in channels of a vessel, and the direction of flow of the liquid growth medium is reversed by rocking of the vessel.
In a further aspect of the invention, the biofilm adherent sites are projections from a lid and incubating the biofilm includes suspending the projections in liquid growth medium in the channels while rocking the vessel so as to provide shear forces on the biofilm during growth of the biofilm.
There is further provided, in accordance with an aspect of the invention, apparatus for analyzing biofilms, the apparatus comprising:
a vessel including at least one channel for flow of liquid growth medium;
plural biofilm adherent sites arranged in at least one row and having a support for supporting the plural biofilm adherent sites within the channel; and
means to flow liquid growth medium along each channel in different directions across the plural biofilm adherent sites.
In accordance with a still further aspect of the invention, there is provided apparatus for analyzing biofilms, the apparatus comprising:
a vessel including plural channels for flow of liquid growth medium;
plural biofilm adherent sites arranged in plural rows and having a support for supporting the plural biofilm adherent sites within the channels; and
means to flow liquid growth medium along each channel across the plural biofilm adherent sites.
Preferably, the plural biofilm adherent sites are formed in plural rows, with plural sites in each row; and
the vessel includes plural channels, with one channel for each row of plural biofilm adherent sites.
In a further aspect of the invention, the support for the plural biofilm adherent sites forms a lid for the vessel.
In a still further aspect of the invention, the means to flow liquid growth medium across the plural biofilm adherent sites is a tilt table.
These and other aspects of the invention will be made apparent in the description and claims that follow.
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Gjaltema, A., et al., “Heterogeneity of Biofilms in Rotating Annular Reactors: Occurrence, Structure, and Consequences”,Biotech. Bioeng., 44:194-204 (1994).
Zimmerli, W., et
Buret Andre Gerald
Ceri Howard
Morck Douglas Walter
Olson Merle Edwin
Read Ronald Rae
Beisner William H.
Burns Doane , Swecker, Mathis LLP
University Technologies International Inc.
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