JPH0461635B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing anaerobic bacteria. More details: dithionite, sodium bicarbonate,
An oxygen scavenger containing sodium carbonate and water and a medium inoculated with anaerobic bacteria are placed in a sealed container, and cultured within 5 hours at an oxygen concentration of 0.1% or less and a carbon dioxide concentration of 5 to 20%. The present invention relates to a method for culturing anaerobic bacteria, which is characterized by the following. Bacteria are classified into (1) obligate aerobes, (2) facultative anaerobes, and (3) obligate anaerobes based on their oxygen requirements. Among these obligate anaerobes, Clostridium botulinum , Clostridium perfringens , and other food-poisoning bacteria, and in recent years, anaerobic bacterial culture methods have been attracting attention as part of research methods from the standpoint of prevention or as pathogen testing methods. However, culturing of anaerobic bacteria has conventionally required special instruments and devices, and operation has required considerable skill. Conventional methods include (1) multilayer method, (2) multilayer solid medium culture method, (3) air displacement method, (4) chemical oxygen absorption method, (5)
There are methods such as adding a reducing agent etc. to the culture medium and (6) biological methods, but all of these methods require complicated operations to create an anaerobic atmosphere. Of (4), the most commonly used method is the gas pack method.The principle is to generate hydrogen gas and carbon dioxide gas by a chemical method, and to react the hydrogen with oxygen in a container using a palladium metal catalyst. This creates an anaerobic state. However, this method involves the generation of flammable hydrogen gas, and even after oxygen removal, 10% of hydrogen gas remains, which is dangerous.
Disadvantages include the catalyst temperature reaching a high temperature of 100°C or higher, the need to regenerate the catalyst, and poor economic efficiency. A possible solution to these drawbacks is to use an oxygen scavenger to remove oxygen. However, many anaerobic bacteria require carbon dioxide gas for their growth, and simply using an oxygen scavenger is not sufficient as a method for culturing anaerobic bacteria. The present invention overcomes these drawbacks.
That is, the present invention contains dithionite, sodium bicarbonate, sodium carbonate and water,
An oxygen scavenger with a weight ratio of (sodium hydrogen carbonate)/(sodium hydrogen carbonate + sodium carbonate) of 0.4 to 0.9 and a medium inoculated with anaerobic bacteria are sealed in a sealed container, and the oxygen concentration is reduced within 5 hours. This is a method for culturing anaerobic bacteria, characterized by culturing at a carbon dioxide gas concentration of 5 to 20% with a carbon dioxide gas concentration of 0.1% or less. According to the present invention, there is no need for a catalyst or special equipment or equipment, and the above-mentioned dithionite-based oxygen scavenger is stored in a sealed container, for example, with an oxygen permeability of 100 c.c./m ² / 24 hr / atm.
It is only necessary to seal the bag together with a medium inoculated with anaerobic bacteria in a bag made of the following packaging material, and it is extremely easy to create an anaerobic atmosphere in which carbon dioxide gas coexists. It is also possible to use an oxygen detection agent to easily check whether an anaerobic atmosphere has been reached. In the present invention, sodium dithionite (Na ₂ S ₂ O ₄ ) and zinc dithionite (ZnS ₂ O ₄ ) are generally used as the dithionite salt, and sodium dithionite is preferably used. It will be done. The oxygen scavenger in the present invention consists of 10 to 400 parts of NaHCO ₃ , 1 to 950 parts of Na ₂ CO ₃ and 5 to 40 parts of water per 100 parts of Na ₂ S ₂ O ₄ , and (NaHCO ₃ )/
The weight ratio of (NaHCO ₃ +Na ₂ CO ₃ ) is 0.3 to 0.9. More preferably for 100 parts of Na ₂ S ₂ O ₄
50-200 parts of _NaHCO3 , 15-200 parts _{of Na2CO3} and water
Consisting of 10 to 20 parts, and (NaHCO ₃ )/
The weight ratio of (NaHCO ₃ +Na ₂ CO ₃ ) is 0.4 to 0.9. The water contained in the oxygen scavenger may be pure water or various aqueous solutions such as an aqueous solution of salts or an aqueous solution of an organic substance. These waters or aqueous solutions are Na ₂ S ₂ O ₄ and
Although a method of directly adding it to the mixture of NaHCO ₃ and Na ₂ CO ₃ can be used, it is preferable to add it in the form of impregnating it into granular material, taking into account the difficulty in handling the bulk powder and the suitability of storage. The particulate material used in this case is a material that is insoluble in water or an aqueous solution, and includes, for example, diatomaceous earth, perlite, zeolite, activated alumina, silica gel, activated carbon, activated clay, and other inorganic particulate materials. The particle size of these particulate materials is generally 0.1 to 5 mm, preferably
It is 0.5-3.5mm. In addition, the packaging material used to wrap these oxygen absorbers may be any air-permeable packaging material, but in consideration of the deterioration in performance during the filling process and handling of the oxygen absorber, the Gurley air permeability is 10 to 50,000 sec/min. 100 c.c.·inch ² , preferably 50 to 10000 sec/100 c.c.·inch ² . An appropriate carbon dioxide atmosphere is required for culturing anaerobic bacteria, and its concentration is usually 5 to 20%. The present invention utilizes this carbon dioxide gas atmosphere (NaHCO ₃ )/
It is controlled by changing the weight ratio of (NaHCO ₃ + Na ₂ CO ₃ ), and this weight ratio
If it is less than 0.3, the amount of carbon dioxide generated is less than 5%, and if it is more than 0.9, it will be more than 20%.
A range of 0.3 to 0.9 is preferred. The sealed container used in the present invention has an oxygen permeability of 500
cc/ ^m2・24hr・atm or less, preferably 100c.c./
It can be used regardless of its form, as long as it is made of a material that is less than ^m2 /24hr/atm and can be completely sealed. For example, the simplest container used in the present invention is a bag made of various vinylidene chloride coated films (KOP (product name), KON, KPET, etc.), vinylon, etc., and contains a medium inoculated with anaerobic bacteria. It may be sealed together with an oxygen scavenger consisting of dithionite and additives and heat-sheeted. By using such a transparent film, it is possible to observe the growth status of anaerobic bacteria during culturing, which is convenient. In order to grow the anaerobic bacteria sufficiently, the growth time is as short as possible (preferably within 5 hours, more preferably 3 hours or less).
(within 5 hours) and creating an anaerobic atmosphere (within 5 hours).
It is necessary to have carbon dioxide (20%) present, and this condition can be easily satisfied by using the dithionite-based oxygen scavenger of the present invention. Example 1 Anaerobic bacterium Bacteroides fragilis GAM broth
Pour 0.1 ml of the diluted 24-hour anaerobic culture solution onto GAM agar medium, add 1.2 g of Na ₂ S ₂ O ₄ ,
NaHCO ₃ 1.7g, Na ₂ CO ₃ 0.7g, water 0.2g,
CaCl ₂ 0.02g, granulated coal 0.4g [The weight ratio of (NaHCO ₃ )/(NaHCO ₃ +Na ₂ CO ₃ ) in this composition is
0.7] in a transparent bag made of a two-layer film of stretched polypropylene/polyethylene coated with vinyl chloride, and then heat-sealed and sealed. After culturing this at 37°C for 48 hours, the number of colonies was measured. In addition, as a comparative example, the same sample was tested in the atmosphere (Comparative Example 1).
and an iron-based oxygen absorber (Comparative Example 2, equivalent to 12 g of iron powder) were sealed in the above bag and cultured under anaerobic conditions without carbon dioxide gas, and then the number of colonies was measured. The results are shown in Table 1.

[Table] Example 2 To compare the weight ratio of (NaHCO ₃ )/(NaHCO ₃ +Na ₂ CO ₃ ) and the amount of CO ₂ generated, 1.2 g of hydrosulfite (sodium dithionite), 0.2 g of water,
Keeping CaCl ₂ 0.02g constant, NaHCO ₃ and Na ₂ CO ₃
Mixtures with different ratios of were prepared and each bag was packed in the same manner as in Example 1. After 5 hours at 37°C, the gas concentration in the bag was measured using a gas chromatograph. The results are shown in Table 2.

[Table] Example 3 A premix of 2 parts of an aqueous solution containing 12 parts of Na ₂ S ₂ O ₄ , 17 parts of NaHCO _{3 ,} 7 parts of Na ₂ CO 3 and 0.2 parts of CaCl ₂ was packaged in 3.82 g portions using a packer. , 12 parts of Na ₂ S ₂ O ₄ , 17 parts of NaHCO ₃ , 7 parts of Na ₂ CO ₃ mixed in advance, and a mixture in which 4 parts of granular zeolite was impregnated with 2 parts of an aqueous solution containing 0.2 parts of CaCl ₂ in advance. , packaged in a packer for mixing two types so that the weight after mixing would be 4.22 g. When packaging the above-mentioned oxygen absorber, the bulk powder had poor fluidity, and the packaging process took about four times as long as when packaging the oxygen absorber. The packaging material used had a Gurley air permeability of 200 sec/100 c.c.·inch ² . Regarding the above oxygen scavengers and anaerobic bacteria
The number of colonies was measured in the same manner as in Example 1, except that Clostridium batulium was used. Further, as Comparative Example 3, the same sample as in Example 3 was cultured in the air, and then the number of colonies was measured. The results of Example 3 and Comparative Example 3 are shown in Table 3.

[Table] Example 4 Same anaerobic bacterium as Example 1 Bacteroides fragilis
After culturing at 37°C for 48 hours in the same manner as in Example 1, the colony diameter was measured. In addition, as Comparative Example 4, carbon dioxide concentration 3 to 4%
Culture was carried out in the same manner as in Example 1 using an ascorbic acid-based oxygen scavenger adjusted to give the following conditions, and then the colony system was measured. Table 4 shows the ratio of colony diameters obtained in Example 4 to the colony system of Comparative Example 4.

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Claims (1)

[Claims] 1 Oxygen scavenger containing dithionite, sodium bicarbonate, sodium carbonate and water, with a weight ratio of (sodium bicarbonate)/(sodium bicarbonate + sodium carbonate) from 0.3 to 0.9, and anaerobic bacteria. Seal the inoculated medium in a sealed container, and
A method for culturing anaerobic bacteria, which comprises culturing at an oxygen concentration of 0.1% or less and a carbon dioxide concentration of 5 to 20% within an hour.