Microbiological culture bottle, and method of making and using same

IS 2 199 239 T3

DESCRIPTION

Microbial culture flask and method for making and using this container.

The present invention generally relates to a method of monitoring the presence of aerobic microorganisms in a fluid sample.

The culture of body fluids such as blood, sputum, and urine is commonly used in medicine to establish the presence or absence of microorganisms.

Typically, the body fluid sample to be studied is obtained from the patient. The sample is then analyzed for the presence or absence of microorganisms. Various methods are commonly used to determine the presence or absence of microorganisms. The most common technique involves preparing a culture by inoculating a sample of the body fluid into a growth medium and incubating said culture. After sufficient incubation, visual inspection is performed by a technician to observe and evaluate the presence or absence of bacterial growth.

It is standard practice in microbiology to detect the presence and evaluate the number of microorganisms in samples. The medical samples under study include body fluids such as blood, cerebrospinal fluid, and urine. Industrial samples include pharmaceuticals, food, and other samples that must be tested for the presence or levels of microorganisms. All these samples are cultured by placing them in a container with a sterile growth medium. The growth medium contains the appropriate nutrients to support the growth of the target microorganisms.

The microbial presence is detected through changes in the liquid medium or in the atmosphere that covers the specimen after a period of time. For example, in US Patent No. 4,812,656 to Ahnell et al. A medium with a carbon 13-labeled substrate is used. After subjecting the sample to conditions conducive to microbial growth, the ratio of carbon-13 to carbon-12 in the gaseous atmosphere is determined. In United States Patent No.^° 5,232,839 to Eden et al., Assigned to the assignee of the present invention discloses a method for the timely detection of microbiological growth in a sealed container by monitoring oxygen consumption in the headspace or the production of CO2 or any other gas. as an indication of microbial metabolism. In United States Patent No.^° 5,217,876 describes a CO2 sensor present at the bottom of a vial, which detects the presence of microorganisms by detecting changes in the pH of the specimen or by the production of CO2. In United States Patent No.^° 5,047,331 to Swaine et al. A blood culture bottle is developed with a sterile container and the nutrient growth medium in which the increase in pressure in the headspace is monitored.

Other known methods of measuring microbial contamination of samples include measuring the smallest changes in temperature, pH, turbidity, color, bioluminescence, and impedance. All of these methods determine microbial contamination by determining the final microbial products or metabolites.

For diagnostic purposes, it is an advantage to determine as quickly as possible whether or not microorganisms are present in the clinical specimen. Diagnosis and initiation of effective drug therapy are greatly improved by rapid evaluation of the presence or absence of microorganisms in a clinical sample. Therefore, if the growth of the microorganism is optimized, the diagnostic process is accelerated. To achieve optimal growth rates of aerobic microorganisms, the concentration of dissolved oxygen in the culture can be increased. In other words, preventing the culture medium from becoming anaerobic enhances the growth of aerobic microbes.

Oxygen has low solubility in water and poor diffusion through the air-water interface limits achieving an achievable oxygen concentration in the culture medium. Agitation, shaking, or passage of air bubbles through a porous stem can be used to increase the dissolved oxygen content of the culture. The agitation, shaking or passage of air bubbles through the culture increases the amount of oxygen in the growth medium and, therefore, increases the oxygenation of the aerobic bacteria improving their metabolism and growth while preventing the culture medium to become anaerobic. To achieve better oxygen concentrations in the growth medium it is necessary to shake the flasks during growth. (US Patent No.^° 5,047,331 and US Patent No.^° 5,217,876). However, shaking or shaking a culture requires the aid of more complex and expensive apparatus, giving the possibility of breakage or contamination of the culture flask or tube, and can cause splashing of the culture. Furthermore, the stirring apparatus is usually expensive and prone to mechanical failure or difficulties.

Yet another method for detecting the presence of oxygen-consuming bacteria is disclosed in US Patent No.^° 4,152,213 to Anhell. In US Patent No.^° 4,152,213 discloses the introduction of a sample of material to be detected in a container, the sealing of the container that has the sample inside, the agitation of the sample to increase the microbial exposure to the oxygenated medium and the monitoring of the production of the vacuum (which indicates the presence of bacteria, since the oxygen present in the container is used by them, if they are present). However, such a method has a disadvantage, since the agitation is usually carried out using expensive apparatus that are usually prone to failure.

Therefore, it would be an advantage to provide means to increase the oxygenation of any bacteria present by increasing the amount of oxygen available to the microorganism in the medium, thereby increasing the oxygenation of the aerobic bacteria and enhancing their metabolism and growth rate without the need for shake, shake, or blow air bubbles through the medium.

In patent FR 403203A an apparatus arranged to cultivate, develop, preserve, package and dispatch specific known aerobic or anaerobic microorganisms is disclosed. However, patent FR 403203A does not disclose a method for detecting the aerobic growth of microorganisms within a receptacle.

Therefore, according to the present invention,

ES 2 199 239 T3 provides a method for detecting the aerobic growth of microorganisms, the method of which consists of:

providing a sterilizable sample container (10), having a headspace (16);

providing a non-toxic, porous, and hydratable insert (22) within the container;

providing a microbial growth medium (24) within said container such that said insert (22) is hydrated within;

sealing said container (10) with said insert (22), hydrated by said growth medium (24), together with an oxygen atmosphere in said container (10);

aseptic introduction of a fluid sample into said container; connection of the container to a head space pressure sensing device;

and monitoring of pressure changes in the headspace by which indications of microbial metabolism can be obtained within said sealed container (10) as an indicator of the presence of microorganisms in said sample, in which the container and insert are maintained in a substantially static state while monitoring the pressure changes in the headspace.

It is preferred that the package and the contents of the package are sterilized prior to aseptic introduction of the fluid sample into the package.

Typically, the insert is made of sponge, cotton, fiberglass, glass beads, plastics, resinous material, sponge beads, or a foam material.

Other advantages of the present invention will be readily appreciated as they become better understood by referring to the following detailed description when considered in connection with the accompanying drawings, in which:

Figure 1 is a perspective view of the microbiological culture flask used in the method of detecting the aerobic growth of organisms according to the present invention;

Figure 2a is a graphic illustration of a pressure change in a sample with M. tuberculosis in a 20% oxygen atmosphere without the sponge insert;

Figure 2b is a graphical illustration of the pressure change in a sample with M. tuberculosis in a 20% oxygen atmosphere with the sponge insert;

Figure 3a is a graphical illustration of the pressure change in a sample with M. tuberculosis in a 40% oxygen atmosphere without the sponge insert; and Figure 3b is a graphic illustration of the pressure change in a sample with M. tuberculosis in a 40% oxygen atmosphere with the sponge insert;

Figure 4a is a graphical illustration of pressure change in a sample with C. neoformans in a 20% oxygen atmosphere without the sponge insert;

Figure 4b is a graphical illustration of the pressure change in a sample with C. neoformans in a 20% oxygen atmosphere with the sponge insert;

Figure 5a is a graphical illustration of pressure change in a sample with C. neoformans in a 40% oxygen atmosphere without the sponge insert; and Figure 5b is a graphic illustration of the pressure change in a sample with C. neoformans in a 40% oxygen atmosphere with the sponge insert.

Figure 1 shows a container 10 for use in the method of detecting aerobic microorganisms according to the present invention such as Mycobacterium tuberculosis, Mycobacterium avium, and fungi or other microorganisms capable of growing within an oxygenated environment. The container or vial 10 consists of a bottle having an internal chamber 12 having a bottom surface 14, a headspace 16, a cap 18 with an elastic rubber stopper 20, and a non-toxic insert 22 hydrated with promotion medium. of bacterial growth 24 arranged inside the internal chamber 12 for a better dispersion of the microorganisms and to increase the microbial exposure to the oxygenated medium 24 and to improve the microbial metabolism. Furthermore, the container has a neck portion 26 and a hump portion 28.

Container 10 can be made of any suitable material such as glass or plastic. Suitable plastics include polystyrenes, polypropylenes, and polycarbonates. Obviously, any suitable material must be non-toxic to microorganisms and must be capable of being sterilized by suitable means such as autoclaving or irradiation. Preferably, container 10 will be made of a transparent material to facilitate not only the visual detection of microorganisms but will also allow the technician or user to visually confirm, before introducing a sample, such as a body fluid, that container 10 is free of contamination.

The non-toxic insert 22 is disposed within the internal chamber 12 of the container 10. In the preferred embodiment, the insert is made of a highly porous material that greatly increases the surface area of ​​microbial exposure to the oxygenated culture medium 24. The increase Microbial exposure to the oxygenated culture medium is a fundamental characteristic of the non-toxic insert 22. By increasing exposure to the oxygenated medium in this way, agitation of the container is not necessary. In other words, insert 22 provides sufficient oxygenation to growth medium 24 to promote and maintain microbial growth without the need for other supplemental oxygenation methods.

In the preferred embodiment, the non-toxic insert 22 has been made with a sponge. A sponge is an ideal medium for insert 22 because its high porosity provides increased oxygenation of the growth medium. The large surface area provided by the porosity of the sponge allows the best oxygen exchange between the air and the growth medium 24. Other materials for the insert include cotton; fiberglass; glass beads, plastic (resinous material) and Porex porous plastic and sponge beads^TM (made of polyethylene, polypropylene, polyvinylidene fluoride, ethylene vinyl acetate, styrene acrylonitrile, etc.). It should be noted that whatever material is selected to serve as insert 22, the material must be non-toxic to microorganisms, that is, the material must be essentially inert and not affect microbial growth.

When hydrated with sufficient culture medium 24, the non-toxic insert 22 occupies approximately 25-80% of the volume of the inner chamber.

IS 2 199 239 T3

12. By occupying a volume in this volume range within inner chamber 12, growth conditions within container 10 are optimized. In other words, the relationship between culture medium 24, surface area, and oxygen is optimal when the insert hydrated 22 occupies a volume of container 10 within the range set forth above and, therefore, the metabolism of microorganisms is increased. As certain aerobic microorganisms grow better in suspension at the liquid-air interface, where O2 is more available, insert 22 greatly improves the oxygenation of the microbial culture medium and, consequently, the oxygenation of the aerobic microorganisms. Another means of increasing oxygen availability is by increasing the oxygen concentration in the headspace.

In essence, insert 22 establishes an atmosphere with conditions similar to those found in the lungs. Establishing an "artificial lung" atmosphere allows the in vitro growth of microorganisms, such as M. tuberculosis and M. avium, which were previously difficult to culture in vitro. This effect is also seen with microorganisms that need oxygen, such as fungi. This microenvironment exposes the microorganisms to a highly oxygenated culture medium 24 that encourages and supports microbial growth.

The microbial growth medium 24 comprises all the nutrients required for the growth of the target microorganism. For example, microbiological growth medium such as the material marketed under the brand name Middlebrook 7H9 is used for the growth of Mycobacterium sp. Those skilled in the art understand that the microbiological growth medium 24 is chosen according to the particular microorganism to be selected. In other words, the particular microbial growth medium 24 is selected on the basis of biochemical or nutritional requirements of the microorganism to be cultured.

In addition to the liquid culture medium, the microbial growth medium 24 can include other selective additives or differential additives such as antibiotics. These additional additives can be used to screen for the presence or differentiate particular microorganisms based on specific and unique characteristics, ie antibiotic resistance or sensitivity or growth requirements.

The non-expanded non-toxic insert 22 is preferably a dehydrated or compressed sponge material. Furthermore, the non-toxic insert 22 may be a non-foaming or unexpanded material such as polyurethane, which is inserted into the container 10. Once inside the container 10, the non-expanded non-toxic insert 22 is expanded by means known in the art. the formation of foams. Glass or plastic (resin) beads, as well as sponge beads, can also be added to the containers. All insert materials serve the purpose of increasing the interface of the medium with oxygen, thus making oxygen more available to microorganisms.

When foam is used for the insert, expanding the non-expanded non-toxic insert 22 into container 10 includes the step of rehydrating the sponge material with microbial culture medium 24 such as Middlebrook 7H9 medium or other suitable culture medium. Therefore, upon expansion, the entire insert hydrates with the medium thus providing a homogeneous growth promoting medium throughout the material.

Foaming material can be filled into a bottle followed by the addition of the medium. It is critical that the material used for insert 22 is not toxic to microorganisms as described above.

In the method according to the invention the non-toxic insert 22 saturated with the microbiological growth medium 24. The insert 22 located within a sealed sample container 10 is inoculated with a sample, such as a body fluid, into which is you are going to analyze the presence or absence of microorganisms. The sealed sample container 10 with the inoculated insert 22 is monitored for signs of microbial metabolism.

The sealed sample container 10 with insert 22 saturated with microbiological growth medium can be provided in a sterile ready-to-use form. Furthermore, the sealed sample container 10 with insert 22 can be obtained in a form in which a sealed sterile container 10 having a dehydrated insert 22 is provided and the user aseptically adds his or her own specific or preferred microbiological growth medium 24 into the sealed container 10 through the rubber stopper 20.

Inoculation of insert 22 into container 10 is generally accomplished by injecting a sample, such as body fluid via a sterile syringe and needle. The needle is inserted by piercing the elastic rubber stopper 20 and the contents of the syringe are injected into the porous insert 22.

The inoculated container 10 is then monitored for evidence of microbial metabolism, namely, a pressure change in the headspace of container 10 as a function of the rate of pressure changes in the headspace.

It should be noted that the present invention is not limited to the detection of microorganisms in body fluid. Various types of samples can be inoculated into container 10, such as food products or other industrial samples tested by procedures well known in the art.

The following examples illustrate the preparation, use, and utility of the present invention.

Examples

Example 1

Materials and methods

Containers of sponge material hydrated with a quantity of Middlebrook 7H9 broth sufficient to completely moisten the sponge (approximately 30 ml) were sterilized by autoclaving. The sponge material occupied approximately 80% of the volume of the package. Samples with 2.0 x 10² CFU / ml (colony forming units / milliliter) of Mycobacterium tuberculosis H37RV were inoculated into the containers. An ESP connector (Difco Laboratories, Inc.) was fitted into the inoculated containers and connected with an ESP machine (Headspace Pressure Detection Device, Difco Laboratories, Inc.) and incubated statically at 35 ° C. . The initial amount of oxygen in the headspace was 20%. An experimental control was analyzed together with the experimental container and it was varied in that it did not contain the sponge material.

IS 2 199 239 T3

Results

With respect to Figures 2a and 2b, after two hundred and ten (210) hours of monitoring the pressure change in the headspace, the experimental package with the sponge material insert (see Figure 2b) showed a signal much better and faster indicating the presence of a microorganism than in the control container (see Figure 2a). The experimental container showed a more defined signal-to-noise ratio than the control container, that is, the point at which detection was possible was much more defined in the experimental container than in the control container. This indicates that even in the absence of agitation, the exposure of the microorganisms to the oxygenated medium is improved by using the non-toxic insert.

Example 2

Materials and methods

Containers of sponge material hydrated with a quantity of Middlebrook 7H9 broth sufficient to completely moisten a sponge (approximately 30 ml) were sterilized by autoclaving. The sponge material occupied approximately 80% of the volume of the package. Samples with 2.0 x 10² CFU / ml (colony forming units / milliliter) of Mycobacterium tuberculosis H37RV were inoculated into the containers. An ESP connector (Difco Laboratories, Inc.) was fitted into the inoculated containers and connected with an ESP machine (Headspace Pressure Detection Device, Difco Laboratories, Inc.) and incubated statically at 35 ° C. . The initial amount of oxygen in the headspace was 40%. An experimental control was analyzed together with the experimental container and it was varied in that it did not contain the sponge material.

Results

With respect to Figures 3a and 3b, after two hundred thirty (230) hours of monitoring the change in pressure in the headspace, the experimental package with the sponge material insert (see Figure 3b) showed a signal much better and faster indicating the presence of a microorganism than in the control container (see Figure 3a). The experimental package showed a more defined signal-to-noise ratio than the control package, that is, the point at which detection was possible was much more defined in the experimental package. These results also indicate that growth at a higher oxygen concentration gives more rapid and differentiated results, that is, a more differentiated signal-to-noise ratio that indicates detection of the presence of microorganisms and also indicates improved microbial metabolism. Example 3

Materials and methods

Containers with sponge material hydrated with sufficient ESP medium to completely wet the sponge (approximately 30 ml) were sterilized by autoclaving. The sponge material occupied approximately 80% of the volume of the package. The samples with 0.6 CFU / ml (colony forming units / milliliter) of Cryptococcus neoformans ATCC 14116 were inoculated into the containers. An ESP connector (Difco Laboratories, Inc.) was fitted into the inoculated containers and they were connected to an ESP machine (Headspace Pressure Detection Device, Difco Laboratories, Inc.) and statically incubated at 35^°C. The initial amount of oxygen in the bottle in the headspace was 20%. An experimental control was tested in conjunction with the experimental container and varied in that it did not contain the sponge material.

Results

With reference to Figures 4a and 4b, after fifty-four (54) hours of monitoring the change in pressure in the headspace, the experimental package with the sponge material insert (see Figure 4b) showed a signal much better and faster, indicating the presence of a microorganism, than the control container (Figure 4a). The experimental container showed a more defined signal-to-noise ratio than the control container, that is, the point at which detection was possible was much more defined in the experimental container than in the control container. This indicates that even in the absence of agitation, the exposure of the microorganisms to the oxygenated medium is improved by using the non-toxic insert. Example 4

Materials and methods

Containers with sponge material hydrated with an amount of ESP aerobic medium sufficient to completely moisten the sponge (approximately 30 ml) were sterilized by autoclaving. The sponge material occupied approximately 80% of the volume of the package. The samples with 0.6 CFU / ml (colony forming units / milliliter) of Cryptococcus neoformans ATCC 14116 were inoculated into the containers. An ESP connector (Difco Laboratories, Inc.) was fitted into the inoculated containers and they were connected to an ESP machine (Headspace Pressure Detection Device, Difco Laboratories, Inc.) and incubated without shaking at 35^°C. The initial amount of oxygen in the headspace was 40%. An experimental control was tested in conjunction with the experimental container and varied in that it did not contain the sponge material.

Results

With respect to Figures 5a and 5b, after fifty-two (52) hours of monitoring the change in pressure in the headspace, the experimental package with the sponge insert material (see Figure 5b) showed a signal much better and faster, indicating the presence of a microorganism, than the control container (see Figure 5a). The experimental package showed a more defined signal-to-noise ratio than the control package, that is, the point at which detection was possible was much more defined in the experimental package. These results also indicate that growth at higher oxygen concentrations gives faster and more differentiated results, that is, a more defined signal-to-noise ratio that indicates detection of the presence of microorganisms and also indicates improved microbial metabolism.

The invention has been described in an illustrative manner, and it should be understood that the terminology used is intended to be descriptive in nature and does not limit its scope.

Obviously, many modifications and variations of the present invention are possible in view of the above teachings. Therefore, it must be en5

It should be noted that within the scope of the appended claims, the reference numerals of which are merely conventional and are not limiting in any way, the invention may be practiced in other ways than those specifically described.