PROCEDURE AND GAS GENERATING UNIT FOR USE IN MICROBIOLOGICAL CULTIVATION

Abstract: A procedure, a gas generation unit, a reagent combination and an apparatus applicable in microbiological cultivation by the aid of which it is possible in an enclosed vessel to create an essentially accurate desired atmosphere. The aims of the invention are attained by the expedient that the culture units in the culture vessel are provided with requisite indicators and with potential catalysts and into the culture vessel are dispensed in accordance with the invention with the aid of tables the gas generation units, and the culture vessel is tightly closed. Hereafter into the vessel is added the quantity of liquid needed for initiation of gas generation, for instance the equivalent quantity of water. As taught by the invention, gas generating units may be used of which the gas generation quantity is given e.g. with the aid of tables and which have been hermetically packaged. The apparatus of the invention is provided with a hermetically closable cock, valve or equivalent to enable the requisite liquid to be introduced into the vessel, whereby the gas that is generated will remain in the culture vessel and the desired atmosphere is obtained with an accuracy even higher than 1%.

Description: PROCEDUREANDGASGENERATINGUNITFORUSEINMICROBIOLOGICALCULTIVATION. The object of the present invention is to provide a procedure and apparatus for enabling a suitable gas atmosphere to be created, especially in microbiological cultivation. Such apparatus is known in prior art by which it is possible to obtain an anaerobic environment suitable for cultivating microorganisms, for instance bacteria. An apparatus of this kind is disclosed e.g. in U.S. Patent No. 3,246,959 (Ci 23-232) and in the British Patent No. 1,544,995. Both references disclose an apparatus by which it is possible to produce in an enclosed vessel an anaerobic environment for bacterial culture. The apparatus disclosed by both references cited is largely similar in principle: they are based on the same basic chemistry, the two types of apparatus are greatly similar in appearance, and the idea of operation is quite nearly of the same type in both: gas generation tablets or equivalent have been packed in a tight bag. This bag >nay be orovided with a porous partition, as in the said U.S. Patent, or the gas generation tablets may be packed in a bag made of porous material and enclosed in a tight bag, as in the British Patent. In commercial products based on said patents there has been packed usually in an impervious foil bag, one tablet of sodium borohydride, which on adding water produces hydrogen, and two tablets of a mixture of sodium carbonate and citric acid, from which water addition elicits carbon dioxide. Whan using such a bag, its corner is cut open, water is poured into the bag and the water ' will gradually, within the bag, be absorbed by said tablets. In an enclosed vessel the oxygen is used up as hydrogen and carbon dioxide are formed. As indicator to ascertain the presence of a proper atmosphere is used methylena blue or a biological indicator, in other words a ' bacterium which grows or fails to grow in the atmosphere in question. ^Sl E OMPI The formation of gas starts as soon as the liquid comes into contact with the gas generation tablet, and this usually happens immediately after liquid addition has been commenced and, generally, before the apparatus has been closed; therefore In order that anaerobic conditions might be attained with full assurance, the tablets have been so dimensioned that gases which are produced are generated in excess. It is known through the German publicizing print No 2 513 282 to use for gas generation, tablets or equivalent which have been coated with an insulating layer or layers resisting the liquids a short time and which for instance during incubation permit the substance therewithin to be activated. It is reported in the said print that with the aid of such tablets an accuracy of 5 to 15% has been achieved with regard to CO gas. 2 From experiments a very great number of substances is known from which it is possible to generate e.g. gases needed in microbiological procedure, such as hydrogen and carbon dioxide for instance. Various tablets have also been prepared which are suitable for generating hydrogen, which as it reacts with oxygen produces water. As a rule, the tablets have been so made that also a shielding gas is formed, usually carbon dioxide CO • Generally speaking there are several different practical methods by which an anaerobi ' c volume can be obtained. In practice nowadays both in scientific research and for instance in exa. ination of foodstuffs such gas mixtures are Increasingly needed which have a given, essentially accurate oxygen content (in other woris, the oxygen content is within given permissible percent limits). Likewise, in various investigations nowadays an increasing need exists for gas mixtures in which the content of a given gas is pre-determinable with essential accuracy. For instance in studies of the intestinal bacterial flora, one should be able to create a milieu equivalent to that in the human body. Until now, for instance when a culture environment has been needed with for instance its oxygen content being of a given kind with essentially high accuracy, one has been compelled to use gas mixtures, the consequence being that the procedures take time and require suitable special apparatus and, frequently, highly trained qualified personnel. This often makes the research costly, and the attempts of culture often tend to fail e.g. in conventional microbiological research laboratories. The present invention aims to eliminate the drawbacks mentioned. The procedure of the invention is characterized by the features stated in the characteristic features part of claim 1. The features characteristic of the gas generation unit according to the invention are stated in the characteristic features part of claim 4. The features characteristic of the reagent combination of the invention are readable in the characteristic features part of claim 6. The culture vessel employed in the procedure of the invention is characterized by the features stated in the characteristic features part of ciai * a 7. The procedure of the invention is characterized in that with its aid is obtained a culture environment for microbiological purposes which is essenti ally accurate as regards the gas atmosphere. This object of the invention is attained by using a culture vessel according to the invention, and suitable prepared gas generation units, which one understands by the aid of data from the tables or equivalent used in the procedure to apply in an appropriate manner, in each case the required number of required units, this number depending on the enclosed volume available, on the number of culture units and on which organism is being cultivated. The invention is described more closely in the following, referring to the figures of the drawings attached and to the embodiment examples. - Fig. 1 presents in elevation l view, and cut open, a culture vessel in which has been placed a culture rack and therein the culture units, for instance Petri dishes, and in the topmost of these, the gas generation units; - Fig. 2 displays the elevational view of the enclosed culture vessel, cut open; — Fig. 3 presents the dosage table for the gas generation units. A culture vessel I of standard capacity is selected which Is provided with sealing elements or equivalent 11 and can be tightly closed with a preferably transparent cover 10, and in which can be placed racks 2 or equivalent for the culture units 3, such as Petri dishes, test tubes or equivalent. Of the culture units 3 on the culture rack 2, one unit 4 may be reserved for gas generation purposes, or the gas generation units may be added on the substrate on a base on the bottom of the culture vessel 1. In this unit 4, or on the base on the bottom of the culture vessel 1 are added the gas generation units, comprising the gas generation reagent in powder form, as tablets, pressed pellets, capsules or equivalent with their base, if any, which base may be coated with a suitable material if necessary. If a catalyst 12 Is required in the gas generation process, the catalyst may be placed in a suitable location, for instance mounted in the cover 10 of the culture vessel 1. It is possible for ascertaining the presence of aproper atmosphere, to provide the culture vessel 1 with a suitable indicator 13, such as a strip of indicator paper or equivalent. It is for instance possible as indicator 13 to use a chemical Indicator, a suitable pure culture, or a combination of the preceding. It is possible to draw up for each microorganism culture a dosage table 14, or equivalent, the data therein contained enabling one to find out directly the quantity of gas generation reagents required for each culture. In order to achieve transport, storage and easy dispensing, the afore-mentioned gas reagent units may be packaged in suitable packages, for instance in press-out packages. In the above-mentioned tightly closable culture vessel 1 of standard size are placed the culture unit racks 2, with duly prepared cultures, nutrients etc. To one culture unit 3 - in case a culture vessel 1 of the kind depicted in Fig. 1 or 2 is used, comprising Petri dishes 3 or equivalent stacked in a rack 2 - and pref rentially to the topmost of them, 4, or on a base on the bottom of the culture vessel 1, is added the quantity of gas generating tablets, pellets, capsules or equivalent needed for gas generation and proper for producing the atmosphere required in the cultivation process. It Is possible to look up in the dosage table 14, for instance how many gas generation tablets should be added into the culture vessel 1 if the full number of culture units 3 has been put in (e.g. 24 units) and if an anaerobic gas atmosphere is desired (e.g. 2 units producing hydrogen and one unit producing carbon dioxide). Likewise, it can immediately be seen in the dosage table 14 how many gas units are required if the full number of culture units has not been entered, or if one desires, instead of an anaerobic space, a certain type_of gas environment with regard to oxygen percentage. For example, when a given microbe is being cultivated which requires a certain kind of atmosphere as regards oxygen and carbon dioxide, one may directly read from Table 14 the number of gas units required, for instance of H tablets producing carbon dioxide 2 and hydrogen, if the full number of culture units has been inserted (24 in the example) or if the number of culture units is not complete. In such case, the oxygen cortent (in per cent) within the vessel may be controlled within the limits of 21-0%, ' i.e. , from the oxygen content of atmospheric air, 21%, down to anaerobic conditions. The volumetric capacity of the vessel with standard dimensions is easy to calculate by subtracting from the capacity of the closed space the volume of the contents, which is simply the number of culture substrates times the volume of one substrate. The culture vessel is tightly closed after the gas units 9 have been added. The liquid required for gas generation is inserted into the tightly closed vessel e.g. through a suitable valve 5, which may be any kind of gas-tight check valve known in itself In the art and which may have been fitted to the vessel or to its cover, and the valve is at once tightly closed and the liquid wets the gas generation tablets and the gas generation starts. The procedure of adding the liquid into the closed vessel through a valve or equivalent which is immediately closed to be hermetically sealed enables all the gas that is being formed to be retained in the enclosed space. According to theoretical calculations, to eat up the oxygen from one litre the quantities of sodium borohydride given in the following Table are required:- TABLE I aten up 0 0 93 11 8 1 102 12 17 2 110 13 " 25 3 119 14 34 4 127 15 42 5 136 16 51 6 144 • 17 59 7 153 18 63 3 161 19 76 9 159 20 35 10 173 21 On the basis of these calculations, pellets ware prepared, each containing 135 g N ' aB.4 , which thus in theorv should suffice to eat 4 up the oxygen from -a volume of 1 litre, and the following experiments were carried out:- Experlments with anaerobes: Using gas generation " units as taught by the invention, experiments on growing anaerobic bacteria have been carried out. For producing hydrogen gas, sodium borohydride pellets according to the invention were used, " having the average weight of about 260 mg. whereof 185 mg consisted of the substance in question. For producing carbon dioxide, soda tablet; ware used, containing 468 ag of Na bicarbonate per tablet. Conventional cultivation jars were used for culture vessels. For catalysts were used palladium-coated aluminium granules. The •*^fRE catalysts were hot-regenerated every time before use. All gas generator tablets were dispensed into the bottom part of an empty bacterial dish of plastic. The requisite water -was added. The tests were performed with anaerobic test strains:— 1. Bacterioides fragilis No. 19500/81 2. Fusobacter necrophoru ATCC 25286 3. Fusobacter nucleatum ATCC 10953 4. Fusobacter variu ATCC 8501 5. Clostridium novyi ATCC 19402 6. Clostridium tetanl EK 574 II 7. Peptococcus 2943 The aerobic control strain was Pseudo onas aeruginosa. As chemical control for anaerobicity was used an Oxoidin reazurine or BLL-methylene blue strip. The quantity of carbon dioxide was maintained at the same level (about 3%) throughout the set of test series. Scrutiny of the culture results obtained revealed that by calculation 1.06 tablets of sodium borohydride were needed per one litre of capacity of the anaerobic jar, corresponding to 196 mg of the substance. It was found that the use of the borohydride pellets was easy and that the required anaerobic conditions could be easily generated with them. The anaerobic state could be ascertained in the manner known in the art, with the aid of indications, and it was found that it is indicated, as a rule, to apply a slight over-dosage compared with the theoretical values so that not only an aerobic state might be achieved but that this state might be achieved within a given time. Aerobic microbes The invention has a particularly essential significance in the research concerning those microbes which, in order to grow, require an essentially accurate gas atmosphere. With the aid of the invention excellent results have been obtained in studies on intestinal bacteria (cambylo-bacteria), of which the study has been difficult heretofore and rεpeatable study, almost impossible. The growth experiments with Carabylo-bacteria were carried out using dosable gas generation units according to the invention and using for hermetically enclosed culture vessel, a culture jar provided with a valve, by which valve the liquid could be dispensed into the enclosed volume. The cultivation units, catalysts and indicators were introduced into the culture vessel in conventional manner, the gas generation units of the invention were dispensed, the vessel was closed to be air-tight, and the liquid was added with a syringe through the valve. Reference tests ware carried out in a manner known in the art, using a ready-made mixture gas from a gas flask, this gas mixture containing 5% oxygen, 10% carbon dioxide and 35% nitrogen. The experiments were carried out as follows. Nutrient substrate Miller-Hinton + Iso Vitalez agar Growing time 48-72 hours Temperature for various microbes: o C fetus ss. intestinalis +37 C o fetus -- +37 C o jejuni +43 C Comparison between the cultures performed as taught by the invention and those according to prior art yielded the following results in a total of 37 trials:- better growth (difference 0.2 g or more) with tablets than with gas mixture In 13 trials equal growth in 13 trials - gas mixture superior in 1 trial In general, equal or better results were obtained with 2-3 H tablets and 4 CO tablets as/than with gas mixture when cultivating 2 C. jejuni strains. The cultivation modes were compared with each other during three weeks In routine fecal cultures. The number of positive specimens obtained totalled 20, whereof one failed to grow in the gas mixture; otherwise the " results were equal regarding the number of findings. Some of the strains failed to grow in larger colonies in some gas environment, others again in other environments, but no regular differences were noted in the growth of Cambylo-bacteria. The nutrient medium employed in the routine cultures was blood a ar with five different antibiotics to achieve selectivity. In addition, the clearly evident circumstance was noted that when using tablets, interfering growth of extraneous bacterial flora would occur in lesser degree than in the gas mixture. The experiments that were carried out have demonstrated that with the aid of the invention a procedure well applicable in microbe cultivation is obtainable wherein one is enabled, by using constant dosaga of gas generation units, to create in an enclosed space an essentially accurate gas atmosphere.. With the aid of the invention a wetting system is achieved which is such that all _thε gas that is produced remains within the culture vessel. The gas generation reagents may be condensed in dosage units, tablets, pressed pellets, capsules or equivalent also in such manner that the surface coating protecting them allows absorption of the liquid through the surface layer into contact with the gas generation reagent not before the passage of a given, pre-determiτied time. It is also possible to pre ~ pare capsules or tablets or equivalent which have been shaped to be "stratified" so that the liquid will penetrate by absorption to the generation reagent through the first protective layer, producing a first atmosphere which is required e.g. for growing a certain microorganism, and after a given time the liquid penetrates through a second protective* layer, thus causing a suitable change of the atmosphere so that an atmosphere is obtained which either terminates the growth of the afore-raentionad organism or, for instance, produces an atmosphere which Is proper for the growth of another given organism. It is possible by applying the procedure of the invention, for instance, to simulate the atmosphere within the digestive system and to study for instance the microbial flora of the digestive system. The invention may also be applied in foodstuff studies in studying the keeping quality of foodstuffs. It is essential that it is possible with the aid of the present invention to obtain a method by which it is possible repeatably to produce gas contents and/or gas mixtures which are substantially conformable as to their content, because the equivalent liquid addition required for generation of water or for other gas generation is made to the gas generation unit already placed in an enclosed space, with suitably high speed through a closable cock or equivalent. It can be experimentally demonstrated that inside the culture vessel for instance the oxygen content can be regulated with an accuracy of about +1%. The same also applies for instance to carbon dioxide contents, or to methane or any other desired gas. It Is possible by the procedure of the invention to obtain for ■ microbiological cultures, in each instance, the desired gas environment in usual conditions, under normal pressure, without any special equipment, without specially trained personnel. In the pocedure of the invention it is possible to utilize normal apparatus employed in microbiological culture operations, such as Petri dishes, flasks, culture racks, etc.; naturally it is feasible in appropriate conditions to evacuate the culture vessel before -*_ adding the liquid required for gas generation. The invention may also be usefully applied in transports which require a given kind of gas environment. The invention Is particularly well applicable In field conditions, where inconvenience, high expenses and loss of time would be incurred in transporting gas flasks and equivalent equipment to the site and in procuring operating personnel. The procedure in question may also be applied when examining extensive series of samples. The procedure is also applicable in automated microbiological analyzers, and in such case the adding of gas generation units, as well as for instance the monitoring of the indicator revealing the presence of a proper atmosphere, can be automated. In the drawings only one example has been depicted of the vessel which can be used as taught by the invention. However, the invention is not meant to be confined to this example: the invention may be altered and modified within the scope of the claims, in accordance with existing conditions.

Claims: Claims . 1. Procedure usable in microbiological cultivation^ for gas generation, in particular whan a given type of gas atmosphere Is required, gas generation units (6) being used to produce said gas atmosphere, characterized in that in a closable vessel (1) are introduced the culture units (3), and the desired number of gas generation units (6) is dispensed, and in case the reaction is catalytic the vessel (1) is also charged with the requisite catalysts (12), whereafter the vessel (1) is closed, after closing the vessel the. gas generation is brought about by addition from the outside of a liquid, water for instance, whereby the gas that is generated all remains in the culture vessel, to create a pre-determined and essentially accurate gas atmosphere. 2. Procedure according to claim 1, characterized in that the culture vessel (1) is also provided with a suitable indicator (13) for ascertaining the presence of a proper atmosphere, which indicator (13) may be chemical or, for instance, a suitable pure culture. 3. Procedure according to claim I or 2, characterized in that in order to obtain a given gas atmosphere in the vessel (1) is/are placed a gas generation unit or units (6) with different gas-generating parts arranged in layers. 4. Gas generation unit (6) for use in a procedure.according to claim 1, 2 or 3, characterized in that it is a tablet (9) or equivalent, of which the " as generation quantity is given e.g. with the aid of tables (12) and which have for storage been hermetically packaged, and with the aid of which an essentially accurate atmosphere is obtained in an enclosed space. 5. Gas generation unit according to claim 4 or 5, characterized in that with Its aid can be obtained in an enclosed space a gas atmosphere which is essentially accurate regarding oxygen and/or ' - OM?I carbon dioxide. 6. Combination of reagents for use in a procedure according to claim 1, characterized in that it comprises a gas generation unit or gas generation units according to claim 4 and a table for dosage of the units. 7. Apparatus, culture vessel or equivalent for use in a procedure according to claim 1, characterized in that it has been provided with a gas-tight cock, valve (5) or equivalent for adding liquid.

Inventors: MATIKAINEN TAPIO, MATIKAINEN, TAPIO
Applicants: FENNOPORT KY
Application year: 1982
Main classification: C12M1
IPC codes: C12M1