Experiments on the cultivation of anaerobic microorganisms

Summary

Anaerobic microorganisms are widely distributed and diverse in nature, and their role is increasingly being emphasized. The technical key to cultivating anaerobic microorganisms is to keep the microorganisms in an environment where oxygen is removed or the redox potential is low.

Operation method

Anaerobic microbial culture methods

Principle

Focal gallic acid and alkaline solution, the formation of alkaline gallates, in the process of this reaction can absorb oxygen and create an anaerobic environment; beef scraps contain both unsaturated fatty acids can absorb oxygen, but also contains glutathione (glutathione) can form a negative redox potential difference; anaerobic tanks is to use a certain method of removing the oxygen in it, for example, will be made of magnesium and zinc oxide to produce hydrogen bags, put into the tank and add water to react to produce hydrogen. Tank reaction with water to produce hydrogen, palladium or platinum is a catalyst, at room temperature catalyzed hydrogen and oxidation synthesis of water, then you can remove the oxygen in the sealed anaerobic tank.

Materials and Instruments

Clostridium pasteurianum, Pseudomonas fluorescens.
Pyrogallic gallic acid Cotton NaOH Paraffin Wax Vaseline Beef Protein Chen Glucose NaCl Peptone Agar Media for Meat Paste
Anaerobic tanks Catalyst bags Gas generation bags Indicator bags Test tubes Glass plates Dropper tubes Flasks Knives

Move

1. Pyrogallic gallic acid method
(1) large tube set small tube method in a large test tube into a small amount of cotton and pyrogallic gallic acid, pyrogallic gallic acid dosage according to it in excess of lye can absorb 100 ml of oxygen in the air per gram to estimate the amount of this experiment is about 0.5 g. Inoculation of Clostridium perfringens in a small test tube on the slant of the bolognese peptone agar, and quickly drop 10% NaOH in a large test tube, so that the pyrogallic gallic acid wetting and immediately put into the small test tube slant (small test tube mouth upwards), plugged with a rubber stopper or screw cap and set at 30 ℃ culture. Put into the small test tube slant that has been inoculated with bacteria by removing the cotton plug (the mouth of the small test tube is facing up), plug the rubber plug or screw on the screw cap, and incubate at 30℃.
(2) Petri dish method: Take a glass plate or a petri dish cover, spread a thin layer of sterilized cotton wool, and put 1 g of pyrogallic gallic acid on it. Pour the plate with meat paste protein agar medium, after solidification and drying a little, half of the plate line inoculation of Clostridium pasteurianum, the lower half of the line inoculation of Pseudomonas fluorescens, and mark the bottom of the dish with a marker pen to mark. Add about 2 ml of 10% NaOH solution on the pyrogallic gallic acid, do not make the solution overflow the cotton, immediately inoculated plate covered with a glass plate or Petri dish cover, must cover all the skimmed cotton, pyrogallic gallic acid reaction should not be in contact with the surface of the culture medium, with dissolved paraffin petroleum jelly solution (i.e., vaspar) to seal the bottom of the Petri dish in contact with the glass plate or Petri dish cover. Place in a 30°C incubator for incubation.
2. Blister medium method
(1) Take 500 g of beef with the sinew and fat removed, cut it into small cubes, put it into 1000 ml of distilled water, boil it for 1 hour over low heat, strain it through gauze, squeeze out the meat juice, and reserve the meat juice for later use. Grind the meat with a meat grinder or chop with a knife, preferably into fine grains.
(2) Add distilled water to the reserved gravy to make a total volume of 2000 ml, add 20 g of peptone Chen, 2 g of glucose, 5 g of NaCl and grated meat scraps. Place in a flask and shake well, heat to dissolve peptone.
(3) Take the upper solution and adjust the pH to 8.0, mark the height of the liquid in the flask with a marker on the wall of the flask, 1.05 ^kg/cm2, sterilize at 121.3°C for 15 minutes to replenish the amount of evaporated water, readjust the pH to 8.0, and then boil for 10-20 minutes to replenish the amount of water, and then adjust the pH to 7.4.
(4) Shake the contents of the flask well, and divide the solution and the meat residue into small test tubes; the meat residue should be used approximately, and sterile procedures should be applied to add sterilized paraffinic petroleum jelly to insulate it from oxygen.
(5) Before inoculation can be done as described above has been done in the Butcher's medium boiling 10 minutes to remove the dissolved oxygen, if covered with a layer of paraffin vaseline, need to paraffin vaseline first in the flame side of the micro-heating, so that it is dissolved. When the medium is not hot to the touch, access the Clostridium pasteurianum according to the liquid inoculation method, and then inoculate the test tube vertically, so that the paraffin petroleum jelly solidified and sealed medium. Then place it in 30℃ for incubation.
3. Anaerobic tank culture
(1) Invert the plate with meat paste peptone agar medium, after solidification and drying, take two plates, inoculate half of the side of each plate with Clostridium perfringens, and inoculate the other half with Pseudomonas fluorescens, and label them well. Take one of the inoculated petri dishes and place it on the petri dish holder of the anaerobic tank, and then put it into the anaerobic culture tank; the other inoculated petri dish was placed in the incubation room at 30℃ for cultivation.
(2) Cut open the catalyst bag, pour the catalyst into the porous catalyst box under the anaerobic tank lid, and tighten the lid of the catalyst box.
(3) Cut the gas generator bag at the shredding line and quickly place this gas generator bag on the clip of the metal rack in the tank and add approximately 10 ml of water to the bag. At the same time, with the cooperation of another person, cut open the indicator bag, expose the indicator strip and immediately put it into the tank.
(4) Quickly close the lid of the anaerobic tank, screw the fixed beam tightly, and incubate at 30℃.

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