US-20260125733-A1 - SEALING MICROLID FOR TESTING VOLATILE AGENTS

Abstract

A sealing microlid (novel microplate sealing lid) designed to evaluate the biological effects of volatile agents in vapour phase in combination with a microplate is disclosed. The sealing microlid includes a plate having a surface, a plurality of flanges extending from the surface, and a protruding opening tab. The flanges of the sealing microlid serve as reservoirs for growth medium containing organisms or cells, which will grow on it, and, at the same time, generate an airtight condition with the well of the microplate necessary for leakage-prevention of the volatile agents tested. The apparatus of the present invention allows quantitative, rapid, simple, labour-and cost-effective susceptibility testing of organism or cell cultures to volatile agents in vapour phase.

Inventors

• Ladislav KOKOSKA
• Marketa HOUDKOVA

Assignees

• CESKA ZEMEDELSKA UNIVERZITA V PRAZE

Dates

Publication Date

20260507

Application Date

20221006

Claims (2)

1. 1 - 4 . (canceled)
2. 5 . A method of susceptibility testing of organisms or cell cultures, comprising: providing a microplate having a plurality of wells, the plurality of wells arranged in plural rows and plural columns; providing a sealing microlid for the microplate, the sealing microlid comprising a plate having a substantially planar base structure and a plurality of flanges extending from a lower surface of the plate, the plurality of flanges arranged in plural rows and plural columns so that each of the plurality of flanges corresponds to one of the plurality of wells, each of the plurality of flanges adapted to air-tight seal and tightly fit into the corresponding well on the microplate; filling each of the plurality of wells with a growth medium containing organisms or cell cultures; adding a volatile agent to each of the plurality of flanges; positioning the sealing microlid over the microplate so that each of the plurality of flanges seals the corresponding well on the microplate; and after an incubation period, assessing an effect of the volatile agent on growth of the organisms or cell cultures in each of the plurality of wells.

Description

A microplate (microtiter plate), a flat plate with multiple wells used as small test tubes, is common labware used for numerous assays in genetics, chemistry, microbiology, and pharmacy. The most common well densities are 6, 12, 24, 48, 96, 384 or 1536 wells per plate. Each well of the microplate typically holds somewhere between tens of nanolitres to several millilitres of liquid. The wells are available in different shapes of bottom such as flat, U- and V-shaped and bottom with minimal rounded edges. Microplates are, in general, designed to be disposable and are manufactured from a variety of materials such as cyclic olefin copolymer, polypropylene, and polystyrene. They are typically produced in opaque white, opaque black, or translucent colours. There are also microplates constructed from solid pieces of glass and quartz for special applications. The standardization of the microwell plates is conducted by the Society for Biomolecular Sciences with the American National Standards Institute and Society for Laboratory Automation and Screening. Plate lids and seals are options for the protection of the well's contents from leakage, contamination, and evaporation during assay processing, incubation, or storage. They are available for a variety of plates, and in a variety of materials, many with specific applications, including storage, biological assays, microscopy, and cell culture (Markossian et al. 2004, Assay Guidance Manual, Eli Lilly & Company and NCATS, Bethesda). The minimum biofilm eradication concentration assay using a microplate/peg lid system for testing the susceptibility of biofilms to antimicrobial agents is an example of a patented (patents U.S. Pat. Nos. 6,051,423, 6,326,190, 6,410,256, 6,596,505 and 6,599,714) and commercially available product (Innovotech, Edmonton, Canada) based on a modified microplate lid. Volatile agents, being compounds of low molecular weight and high vapour pressure at ambient temperature, are present in various matrices, including plant materials, from which they can be obtained, for example, in the form of essential oils. They are of great potential for the development of novel medicinal, pharmaceutical, food and agricultural products and technologies, such as inhalation therapy, active food packaging, and controlled-atmosphere storage. However, the industrial applications based on the most typical physico-chemical feature of these agents, which is volatility, have not been fully developed yet. One of the main reasons for this situation is the lack of efficient quantitative methods suitable for the high-throughput screening of the biological activity of the volatile agents. A disc volatilization method based on the evaporation of volatile agents from a solid matrix (e.g. paper disc) is the most commonly used assay; however, it is time- and labour-consuming and allows qualitative evaluation only. With the exception of several experimental apparatuses that are not commercially available, there is no special labware designed for testing the biological activity of volatile agents in vapour phase. Therefore, there remains a need for a novel device and method that will overcome these disadvantages (Houdkova & Kokoska, 2020, Planta Med. 86,822). Recently, our team designed a high-throughput screening assay performed with microplates covered by lids with flanges designed to reduce evaporation. This broth microdilution volatilization method is suitable for the simple and rapid simultaneous determination of the antibacterial potential of volatile agents in liquid and vapour phases at different concentrations. It allows for the cost-and labour-effective high-throughput screening of volatile agents using commercially available microplates. However, since the assay described above is performed using serially produced microplates that are not designed for this purpose, the method suffers from several weaknesses. For example, clamps are necessary for fastening the plate and lid together and only a limited volume of agar can be applied on the lid, which could affect bacterial growth (Houdkova et al. 2017, Fitoterapia 118,56). With the aim of overcoming the problems related to the assessment of the biological activity of volatile agents inherent in the prior art solutions, the present invention provides an apparatus for testing the effects of volatile agents on the growth of organisms or cells in vapour phase. The present invention utilizes a sealing microlid (novel microplate sealing lid) with flanges designed to carry growth medium for the cultivation of organisms or cells, which is placed onto the top of the standard microplate. The flanges of the sealing microlid serve two important functions. The first function is as a reservoir for the growth medium (e.g., agar) containing cells or (micro)organisms such as bacteria, yeast, and fungi, which will grow on it. The second function of the flange is to generate an airtight condition with well of the microplate, necessary for leaka