Method for biologic breeding anaerobic microbe without need of accessorial incubator

Abstract

This invention discloses a method for culturing anaerobic microbes. The method comprises: (1) preparing culture medium solution, and adding methylene blue as the redox indicator; (2) adding antioxidant into the culture medium solution at room temperature, shaking for dissolution, loading into a culture bottle, and covering with a rubber plug and a bottle lid within 30 min; (3) drilling the rubberplug with a syringe needle, introducing nitrogen, and exhausting at the same time to remove air above the solution in the culture bottle; (4) sterilizing the culture bottle with high-temperature steam, and cooling for further use. The method has such advantages as no need for anaerobic incubator easy operation and low cost. The redox indicator is still stable even after high-temperature sterilization. The antioxidant is safe and nontoxic, and can effectively remove oxygen.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a method for cultivating anaerobic microorganisms without the assistance of an anaerobic incubator. Background technique [0002] At present, the cultivation of anaerobic microorganisms generally requires the use of an anaerobic incubator. This kind of anaerobic incubator needs to consume a large amount of pure nitrogen or carbon dioxide to remove the air in the box, and the oxygen partial pressure still cannot meet the environmental requirements of many absolute anaerobic microorganisms. The experimental operation needs to wear long-arm gloves in the box It is extremely inconvenient. [0003] Find through document retrieval to prior art, Joblin etc. have published on " Current Microbiology " (" modern microbiology ", 2002 45 volumes 46-53 pages) entitled " Degradation of FreshRyegrass by Methanogenic Co-Cultures of Ruminal Fungi Grown in the Presence or Abse...

AI Technical Summary

Problems solved by technology

This kind of anaerobic incubator needs to consume a large amount of pure nitrogen or carbon dioxide to remove the air in the box, and the oxygen partial pressure still cannot meet the environmental requirements of many absolute anaerobic microorganisms. The experimental operation needs to wear long-arm gloves in the box very inconvenient

[0003] Find through document retrieval to prior art, Joblin etc. have published on " Current Microbiology " (" modern microbiology ", 2002 45 volumes 46-53 pages) entitled " Degradation of FreshRyegrass by Methanogenic Co-Cultures of Ruminal Fungi Grown in the Presence or Absence of Fibrobacter succinogenes (“Degradation of fresh ryegrass by rumen fungal methanogenic co-cultivation in the presence or absence of Bacteroides succinoides”), proposed an anaerobic culture aided by an anaerobic incubator Bottle to cultivate the Hungate rolling tube method of anaerobic microorganism, this method utilizes resazurin as redox state indicator, utilizes cysteine ​​and sodium sulfide to remove oxygen in the bottle, the deficiency of this method is: (1) resazurin It will be destroyed during high temperature sterilization, the color will change from blue to light pink, and the color difference is weak. When the medium solution itself has color, it cannot effectively display the color difference change, so it cannot indicate whether there is trace oxygen in the bottle; ( 2) At room temperature (such as 20°C), the reaction speed of sodium sulfide and oxygen is very fast. Often, the operation of subpackaging the medium and capping the bottle is not completed, and the added sodium sulfide has been exhausted, and then the oxygen that enters the bottle Cannot be removed; (3) Sodium sulfide is a strong base, highly corrosive, and reacts with dissolved oxygen in water to generate hydrogen sulfide, which is toxic to many anaerobic microorganisms