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Isolated culture method of anaerobic bacterium

An anaerobic bacteria, separation and cultivation technology, applied in the direction of bacteria, etc., can solve the problems of anaerobic bacteria separation and cultivation, error of counting results, death of anaerobic bacteria, etc., and achieve good effect of oxygen isolation

Inactive Publication Date: 2016-01-06
田发益
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The invention provides a method for isolating and cultivating anaerobic bacteria by using the existing petri dish through the double-reverse sandwich method to solve the condition that there is no separation and culturing of anaerobic bacteria in general laboratories, and the anaerobic bacteria may die during the inspection process , or partial death leads to technical problems in the isolation, cultivation and counting of anaerobic bacteria.

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  • Isolated culture method of anaerobic bacterium
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preparation example Construction

[0024] 2. Preparation of experimental reagents:

[0025] It is prepared by adding 16g-20g of agar to 1L of 0.9% physiological saline, and it is prepared into a crystal clear sealing culture gel, which is convenient for the observation of anaerobic bacteria during the cultivation process. For the anaerobic bacteria to be inoculated, according to different types of anaerobic bacteria, adopt their respective conventional methods and requirements to prepare selective culture gel.

[0026] 3. Inoculation, sealing, cultivation and colony selection of anaerobic bacteria:

[0027] First, under aseptic conditions, first pour the sealed culture gel into the inside of the sterilized petri dish cover, after cooling and solidifying, inoculate a certain amount, and inoculate the anaerobic bacteria to be inoculated into the inside of the petri dish cover The surface that has been perfused with sealing culture gel should be evenly coated and left to stand for 5-10 minutes. If the standing ti...

Embodiment 1

[0034] It is made of 1L of 0.9% physiological saline plus 18g of agar, and is formulated into a crystal clear sealing culture gel.

[0035] Selective culture gel for screening Clostridium rumen, mainly used for the isolation and cultivation of butyrico vibrio fibrinolyticus, made from the following raw materials: meat extract 10g, yeast extract 1.5g, peptone 10g, water-soluble starch 1g, glucose 1g , sodium acetate 5g, L-cysteine ​​0.5g, distilled water 1L, after the above raw materials are fully mixed, adjust the pH value of the mixture between 7.1 and 7.2, and then sterilize, after the above culture gel is sterilized, every 50ml , add 0.5ml of 5% Na 2 S and 0.5 ml of ferric citrate with a concentration of 7% (both reagents are added to filter miscellaneous bacteria through an ultrafiltration membrane with a pore size of 0.25 μm). The above-mentioned screening of rumen Clostridium is prepared by mixing selective culture gel with rumen fluid sterilization volume ratio of 1:1 ...

Embodiment 2

[0039] It is made of 1L of 0.9% physiological saline plus 18g of agar, and is formulated into a crystal clear sealing culture gel.

[0040] Culture gel for isolating aerobic bacteria, mainly used for the isolation and cultivation of butyrico vibrio fibrinolyticus and Bacteroides, made from the following raw materials: tryptone 1g, yeast extract 0.5g, beef extract 0.2g, agar 1.5g , 7.5ml of salt solution I, 7.5ml of salt solution II, 0.1ml of 0.1% resazurin solution, 78ml of distilled water. After dissolving the above raw materials in sequence, 2 g of glucose and 1 ml of 0.07% hemin were added, and finally the pH value of the mixture was adjusted to 7. After sterilization, add 8% Na 2 CO 3 Solution 5ml, 3% cysteine ​​hydrochloride 1ml.

[0041] Wherein, the above-mentioned salt solution I is a concentration of 0.6% K 2 HPO 4 solution; the above salt solution II is made of 0.6% KH 2 PO 4 , 1.2% (NH 4 ) 2 SO 4 , 1.2% NaCl, 0.12% MgSO 4 ·7H 2 O, 0.12% CaCl 2 composed ...

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Abstract

The invention provides an isolation and culture method of an anaerobic bacterium under the condition of an ordinary laboratory. The isolation and culture method comprises the following steps: preparing transparent closed culture glue and selective culture glue; pouring the closed culture glue into the dish cover of a petri dish under an aseptic condition; after the closed culture glue is cooled and solidified, inoculating the glue with to-be-inoculated anaerobic bacterium, uniformly smearing the anaerobic bacterium on the surface of the closed culture glue, and standing; then, adding the selective culture glue until the selective culture glue totally covers the surface of the closed culture glue and slightly bulges in the middle; quickly putting the dish bottom of the sterilized petri dish downwards, slowly putting the product inside, lightly pressing, and removing bubbles; finally, sealing the periphery of the gap between the dish bottom of the petri dish and the dish cover of the petri dish, and putting the sealed product into a valve bag sterilized by 75% ethyl alcohol and an ultraviolet lamp in advance for culture. The method provided by the invention is quite easy and convenient and quite good in oxygen isolation effect, and the condition required by the anaerobic bacterium during the culture process is completely met; the closed culture glue is transparent, so that the growth condition of a bacterial colony during the culture process can be observed.

Description

technical field [0001] The invention provides a simple culture method for anaerobic bacteria, which adopts a culture dish double-reverse sandwich method to cultivate anaerobic bacteria, and this method can be applied to research processes such as separation, diagnosis and counting of most anaerobic bacteria. Background technique [0002] Bacteria exist in a mixed state in nature, and to obtain the desired strains, they must be separated from them. There are many ways to isolate and purify bacteria, but the basic principles are similar, that is, the sample to be separated is diluted or concentrated in a certain proportion, inoculated on selective culture gel, and the bacterial colonies are reproduced in a certain amount and dispersed state as much as possible during the separation and culture process. And make it grow into a single colony of pure species. [0003] The isolation and culture of bacteria is generally divided into the cultivation of aerobic bacteria, the cultiva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20
Inventor 田发益
Owner 田发益