JPS5823074B2 - Anaerobic culture method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing microorganisms anaerobically without performing troublesome operations such as depressurization and gas replacement.

For anaerobic cultivation, oxygen must be removed from the culture medium, and for this purpose a free surface is required from which the oxygen can escape, and the gas phase in contact with the free surface is blown away and an inert gas stream is created. or had to be replaced with an inert gas after being evacuated.

The former required full-scale equipment and large amounts of carbon dioxide, while the latter required extremely complicated operations.

On the other hand, polyethylene and polypropylene membranes do not allow water to pass through, but allow oxygen to pass through, so if you put water in a bag made of these highly breathable polymer membranes, the water will be kept aerobic, allowing goldfish, etc. is known to not cause suffocation.

Therefore, we reversed this and sealed a culture solution inoculated with anaerobic bacteria in a small bag made of a highly breathable polymer membrane.
When the anaerobic bacteria were cultured by immersing them in an oxygen-absorbing solution, they grew well.

Polymer membranes include polyethylene, polypropylene,
Polybutadiene, Teflon, silicone rubber, etc. can be used, but when considering price, strength, transparency, heat resistance, heat adhesiveness, etc., polypropylene is most convenient, followed by polyethylene.

As a liquid with high oxygen absorption, sodium sulfite is ranked 3rd in terms of price, not being highly toxic or corrosive, and transparency.
% aqueous solution is reasonable.

This method does not require a full-fledged carbon dioxide injection device, a pressure-resistant vacuum container, a vacuum pump, or an inert gas, and instead just fills the bottle with an aqueous sodium sulfite solution, caps it, and seals the bottle with a polypropylene membrane. All you need is a simple heating device.

It is also possible to culture by putting 100 or more polypropylene bags containing 1 ml or less culture solution into a bottle containing 500 rfL, and if you need to test it midway, use tweezers. Just take out one bag and put the lid back on.

Also, when used as a culture medium, it would be a problem if it spewed out when a vacuum was applied.

This method is particularly advantageous when using human waste and the like.

The above is a case where the culture medium is sealed in a container made of a material that is highly permeable to oxygen, but conversely, the culture medium is placed in a normal closed container such as a syringe, test tube, or bottle, and then placed inside it. Alternatively, a method of culturing by introducing an oxygen-absorbing substance sealed in a container with high oxygen permeability is also possible.

In this case as well, as a substance with high oxygen permeability,
Polypropylene membranes, polyethylene membranes, and other oxygen-absorbing substances such as sodium sulfite aqueous solution can be used, but since there is a culture medium on the outside, opaque substances,
The use of corrosive substances is also possible.

For example, a small ampoule is placed in a bag made of an oxygen-permeable material, and two or more substances, which are stable as they are, but have increased oxygen absorption properties when combined, are placed inside and outside the ampoule, and then the bag is made. The ampoule can be sealed and used by applying force from outside the bag to break the ampoule if necessary.

Such substances include ferrous sulfate or manganous sulfate or pyrogallol and aqueous sodium hydroxide;
These include aluminum and mercuric chloride.

Oxygen-permeable bags containing oxygen-absorbing materials can also be sterilized in an autoclave.

By the method of the present invention, obligate anaerobic bacteria and obligate anaerobic protozoa can be easily cultured, and facultative anaerobic microorganisms can be anaerobically cultured.

Examples will be described below.

Example 1 Diffco's cooked meat medium was dispensed and sterilized into nine test tubes fitted with cotton plugs, and the following bacteria were added to each test tube in an amount of 1 ml of medium.
Inoculation was carried out at a ratio of 1 o to the 5th power per 2.

Clostridium butyricum (Clostridium)
mbutyricum I AM 19001),
Propionibacterium freudenreichii (Pro
pionibacterium freudenrei
chii I AM1725), Eubacterium aerofaciens
ATCC25986), Bifidobacterium adolesc
Entis ATCC15705), Peptostreptococcus m1cro
s RIMDl, 636001), Fusobacterium barium (Fusobacterium barium)
umvarium ATCC8501), Bacteroides fragilis
fragilis RIMD 0230001), Escherichia coli (Escherichia coli)
iIAM1264), Micrococcus luteus
uteus IAMI 1008).

Meanwhile, a 200 ml Erlenmeyer flask was filled with a 3% aqueous solution of sodium sulfite and fitted with a rubber stopper.

Using an electric heating type polymer film sealing machine, commercially available polyethylene bags and polypropylene bags are processed to a width of 2 crrt.
, I made an envelope-shaped pouch with a length of 10 crIL, sterilized it by irradiating it under a 20 watt cocoon-killing lamp at 10 crIL for 30 minutes, and put in each glass beads with a diameter of 3 mm that had been dry heat sterilized. I used it as a weight.

Using a sterile pipette, transfer the culture medium inoculated with the bacteria into a polyethylene bag and a polypropylene bag. Pour in the culture medium, remove any bubbles, and melt-seal the bag approximately 2 cm from the bottom edge of the bag. Cut off the excess above the sealing point with scissors, and seal the part containing the culture medium with 3.5 cm of sodium sulfite. % aqueous solution, and kept at 37°C for 4 days with a rubber stopper.

The portion left in the test tube was also kept at 37°C for 4 days to serve as a control.

Table 1 shows the results of observing the growth state of the bacteria after 4 days.

Namely, spore-bearing rods (Clostridium) and non-spore-bearing rods (Gropionibacterium) with positive Durham staining.

A wide variety of obligately anaerobic bacteria, including branched bacteria (Bifidobacterium), cocci (Peptostreptococcus), and Durham stain-negative rods (Fucibacterium and Bacteroides), grow in test tubes. However, with the method of the present invention, it grew well.

Escherichia, a facultative anaerobe, grew both in the method of the present invention and in the control, and Micrococcus, an aerobic bacterium, grew well in the control (but not in the method of the present invention). According to the method of the present invention, it has been shown that anaerobic conditions are maintained.

Example 2 A broken test tube was heated with a burner and stretched to make a glass capillary tube with an inner diameter of 2 mm, leaving about 1 crfL.
(Stretch both ends into thin hair-like shapes and hydroxylate) IJ
Fill it with a 26% aqueous solution of umum, and seal both ends with a small flame.

This is inserted into a rubber tube with an inner diameter of 2m7IL and a length of 1.2C1rL to protect it.

Width 1.2crrL, length 4C made of polypropylene membrane
A glass ampoule containing a sodium hydroxide solution protected by a rubber tube is pushed into the bottom of the rIL envelope-shaped bag, and 15.1 to 15.1 of manganese sulfate monohydrate is added to the bottom, followed by approximately 0.1 N of 0.1 N dilute sulfuric acid. Add 2 ml of the bag, and while removing bubbles, seal the bag approximately 2 cm from the bottom using an electric polymer film sealer, and cut off the top of the bag.

Put this into a 100ml Erlenmeyer flask that has been sterilized by dry heat with a cotton stopper, add about 50rrL of water, and sterilize by heating in an autoclave.

When this is placed in water and pressure is applied from the outside to break the ampoule, manganese sulfate and sodium hydroxide react in the polypropylene bag to form a precipitate of manganese hydroxide, which changes to manganese hydroxide. It has the ability to completely absorb dissolved oxygen in about 160 ml of room-temperature water into which air has been blown.

Place the above ampoule and a polypropylene bag filled with manganous sulfate solution into the cylinder of a large 1001fL syringe, forcefully press the inner tube to break the ampoule, and immediately suck in 20ml of culture solution with the following composition. Point the tip of the syringe upwards to force out the foam.

The numbers are ~ per 100 ml of culture solution.

Glucose 70. Maltose 70. soluble starch 70,
Hefton 70, yeast extract 70, ammonium sulfate 70
, L cistine 70, calcium chloride 10, magnesium sulfate 10, potassium phosphate 50, dipotassium phosphate 50, sodium chloride 100, sodium bicarbonate 500° Next, the freshly removed rumen fluid s, 6 m
Inhale i, stick the tip of the syringe needle into the rubber ring, and press 3.
The cells were cultured at 7°C, and each day a drop of liquid was squeezed out and examined under a microscope to determine whether motile microorganisms were present.

As a control, a similar product except that the polypropylene bag was not placed was placed.

The results were as shown in Table 2.

That is, according to the method of the present invention, the inside of the syringe is maintained in a strongly anaerobic state from the beginning, and as a result, the strongly anaerobic microorganisms in the rumen continue to live, whereas the method of the present invention is not applied. It is clear that only facultatively anaerobic or relatively oxygen-tolerant microorganisms can survive.

Example 3 Instead of the 3% aqueous sodium sulfite solution in Example 1, Escherichia coli was added to an aqueous solution consisting of 0.5% yeast extract, 0.5% peptone, and 1% glucose.
richia coli I AM 1264)
The same results as in Example 1 were obtained by inoculating and culturing at 37°C for 12 hours without sterilization.

This is thought to be because E. coli consumed the dissolved enzyme well.

Example 4 In Example 2, when 30% sodium chloride and 60 ppm of cobalt chloride were added to a 3% aqueous solution of sodium ammonium, the survival period of protozoa was extended to 5 days.

This is due to the addition of these two substances, resulting in sulfite) IJ
This is thought to be because the carbon dioxide absorption of the aqueous solution decreased and the oxygen absorption increased.

Example 5 A polypropylene bag with a width of 2 CrrL and a length of 18 cfIL was sterilized by heating it at 120° C. for 20 minutes using an autoclave.

Prepare the following four types of culture media in a flask that has been separately detensified by dry heat, and sterilize it by heating at 120°C for 20 minutes using an autoclave.
This was dispensed into the aforementioned polypropylene bags in an amount of 10 rfl.

The culture medium is as follows.

Medium 1: Yeast extract 0.5%, peptone 1%, salt 0.
2%, agar 0.1%.

Medium 2: Add 1% glucose to medium 1.

Medium 3: Sodium nitrate 1500ppm! Potassium chloride 39 PI) rrL, magnesium sulfate 75p
pm, sodium carbonate 20 pI) m, calcium chloride 27 pprrL, sodium silicate 58 ppm, ethylenediaminetetraacetic acid disodium lppm, iron citrate 6
ppm, oxalic acid 2.86 ppm, manganese chloride 1.
81 ppm, zinc sulfate 0.08 ppm, sodium molybdate 0.021 ppm, Vitamin"B120
．． 001 pprll, vitamin B O, Ol pp
m, Via-on 0.001 ppm, agar 0.1%.

Medium 4: Medium 3 with yeast extract 0.1% and glucose 0.1
Add %.

Medium 1 contains Escherichia coli (E 5cherich
iacoli IAM1264), Salmonella typhimu
rium NI AH1231), Pseudomonas aerugi
nosa I AM 1007), Corynebacterium citreum
I AM 1614) and Staphylococcus
aureus (5taphylococcus aur)
eus I AM 1098).

Medium 2 contains Saccharomyces cerevisiae (Saccharomyces cerevisiae).
aromyces cerevisiae I,
AM4125), Rhizopus nigricans I AM
6021), Aspergillus niger #
gillus nigerIAM2020), Lactobacillus plantarum (Lactobacillus
plantarum I AM 1041) and Leuconostoc mesenteroides (Leuconos
toc mesenteroides I AM
1046) was inoculated.

Medium 3 contains Chlorella vulgaris
vulgaris RI FY 5464),
Nitzschia palea
RIFY5800), Anabaena cylindrica RI
FY 7100) and Ringvia Contorta (L
yngbyacontorta RI FY 730
6) was inoculated.

Medium 4 contains Scenedesmus quadricauda (Scene
desmus quadricauda RI F
Y 5041 ) and Chlorella pyrenoidosa (Ch
lorella pyrenoidosa RI FY
546.3) was inoculated.

Mix both well and temporarily seal the opening of the bag using an electric polymer melt-sealing machine.
were precultured at 30°C for 3 days in the dark, and medium 3 and medium 4 were precultured at 25°C for 7 days while illuminated at 5000 lux.

After inoculating Lactobacillus plantarum and Leuconostoc mesenteroides for 2 days,
The mouth of the bag was cut open and 0.51 portions of dry heat sterilized precipitated calcium carbonate was added, pinched from the outside of the bag, and mixed well.

After the preculture was completed, the mixture was melt-sealed using an electric polymer melt-sealing machine to form a series of 12 rectangles each having a width of 2 cm and a length of 1 crIl.

Make all of the above in two series, and the ■ series is 1 at 170℃.
After heating for an hour to drive out dissolved oxygen, the 500
The cells were immersed in ml of liquid paraffin, sealed tightly, and cultured at 10°C.

Another series is 500ml of 3% aqueous solution of sodium sulfite.
Then, 37ml of anion exchange resin IRA400 saturated with sulfite ions and 40 g of calcium sulfite powder was immersed in the solution, the mixture was tightly stoppered, and cultured at 10°C.

To saturate the anion exchange resin IRA400 with sulfite, add IRA400 to approximately 10 Qd of boiled and cooled distilled water.
Submerge and add sodium sulfite 151 (shake, discard the supernatant after 10 minutes, wash with boiled and cooled water, and use immediately.

As a control, an agar slant medium containing 2% agar added to a medium having the same composition as in this experiment was used.

Among the above, in this experiment, one section was cut out with scissors every three months, and for the control, one platinum loop was taken, inoculated onto the same agar slant medium as the control, and cultured under the same conditions as the preculture. I investigated whether it would grow or not.

For items soaked in sodium sulfite solution, place the cut polypropylene bag in a 1N aqueous sodium hydroxide solution for 30 minutes to sterilize the outside, then soak it in 0.1N diluted sulfuric acid to neutralize it, and then sterilize it. Wash thoroughly with water, cut the ends with flame sterilized scissors, and squeeze out the insides.

Those soaked in liquid paraffin were treated in the same way as the examples soaked in aqueous sodium chloride solution after washing the outside of the bag with ethyl ether.

The test results are shown in Table 3.

The numbers in the table indicate the number of months the creature lived.

It is thought that those that survived for 24 months will survive even longer, but this has not been tested.

None of the microorganisms used in this example are obligate anaerobic organisms, and rather require oxygen for active growth. live longer.

Conventionally, the best method was to pre-culture on an agar slant, then layer liquid paraffin heated to drive out dissolved oxygen, and then apply a butyl rubber stopper on top, but this method takes up space and requires a large number of layers. The test tube is smeared with liquid paraffin, and it is not suitable for algae that dislike contact with oil, and due to its troublesome nature, it was not widely used.

If the anaerobic culture method of the present invention is applied, it does not take up much space, is extremely simple, and is safe because microorganisms do not come into direct contact with the oxygen-absorbing liquid. It is possible to preserve and culture blue-green algae, eukaryotic algae, and a wide range of microorganisms for a long time.

In addition, the method using an aqueous sodium sulfite solution has better preservation of microorganisms than the liquid paraffin method.
Because the liquid is water-soluble, it is difficult to handle (you can also enclose it in a laminated film that is impervious to gas and water while wet with the sodium sulfite aqueous solution, then put it in an envelope and mail it. can.

When taking out a sample, each time the container lid is opened, the sample is exposed to air, and the sulfite ions are gradually oxidized to sulfate ions, which have no ability to absorb oxygen.

However, sulfate ions have a greater ability to bind to resin and calcium than sulfite ions, so the sulfite ions that were bound to resin and calcium are replaced and come out, keeping the dissolved sulfite constant. Long-term anaerobic retention is possible by using a combination of IJium (sulfite) and ion exchange resins or insoluble metal salts bound to sulfite as an external solution.

Microorganisms are widely used in the production of industrially useful substances and in the assay of physiologically active substances, and the development of simple long-term storage methods for them is extremely useful for industry.

Claims (1)

[Scope of Claims] 1. A thin film made of a substance with high permeability to oxygen and low permeability to water is laid down, and a culture medium is present on one side and a substance with high oxygen absorption is present on the other side. An anaerobic culture method characterized by: 2. The anaerobic culture method according to claim 1, wherein a polymer material such as polyethylene, polypropylene, polybutadiene, Teflon, silicone rubber, etc. is used as the thin film. 3. Claims characterized in that a reducing substance such as a sodium sulfite aqueous solution, an alkaline pyrogallol aqueous solution, a manganese hydroxide suspension, a ferrous hydroxide suspension is used as a substance with high oxygen absorption properties. The anaerobic culture method described in paragraph 1. 4. The anaerobic culture method according to claim 1, characterized in that a culture medium is placed in a container made entirely or partly of a thin film that is highly permeable to oxygen, and a substance that is highly oxygen-absorbing is present on the outside of the container. . 5. The anaerobic culture method according to claim 1, characterized in that a substance with high oxygen absorbing properties is placed in a container that is entirely or partially made of a thin film with high permeability to oxygen, and a culture medium is present outside the container. .