There are many methods for disrupting tissue cells, including mechanical methods, physical methods, chemical methods, and biochemical methods. Before the crushing, the material often needs to be pretreated. For example, the animal material needs to remove the connective tissue, adipose tissue and blood stain which are not related to the experiment, and the plant seeds need to be removed, and the microbial material needs to separate the bacteria and the fermentation liquid component.
The crushing methods and conditions used are different for different experimental scales, different experimental materials and experimental requirements. Some tough tissues, such as muscles, plant roots, etc., often require strong agitation or grinding to destroy their tissue cells. The softer tissues such as the liver and brain can achieve the purpose of completely destroying the cells with a common glass homogenizer. For the same experimental material, due to the different experimental purposes (requirements), there are also some differences in the method of fragmentation, such as the nucleic acid extracted and the nucleotides extracted, the former must maintain very mild conditions to maintain the integrity of the molecule. The latter can be used in a strong way.
Mechanical method
A method of crushing tissue cells mainly by mechanical shearing force, and commonly used instruments include tissue mashers, homogenizers, mortars, grinding, and presses.
1) Tissue mincers are generally used for animal tissues, plant fleshy seeds, tender leaf buds, etc., and the rotational speed can be as high as 10,000 rpm/M or more. Due to the large mechanical shear of the rotating blade, the preparation of some larger molecules such as nucleic acids is rarely used.
2) The gap between the grinding ball of the homogenizer homogenizer and the inner wall of the glass tube is kept at a distance of a few tenths of a millimeter, and the degree of broken cells is higher than that of the tissue masher. The material of the homogenizer can be made of hard plastic, stainless steel, artificial fluorescent resin or the like in addition to glass.
3) The mortar is mostly used for bacteria or other hard plant materials. A small amount of quartz sand, glass powder or other abrasive is often added during grinding to improve the grinding effect.
4) Bacterial Mill is an improved grinder that has a larger grinding area than the mortar and has an outlet at the lower part. In the operation, the bacteria and the grinding powder are firstly mushy, and each time a small spoon is added, the bacterial cells can be completely ground by grinding for 20-30 seconds.
2. Physical law
A method of disrupting tissue cells mainly by various physical factors. Common methods used in biochemical preparation are:
1) Repeated freeze-dissolving method: The sample is deeply cooled to -15--20 degrees to freeze it, and then slowly melted, repeated many times, and used for animal materials.
2) Rapid thermal quenching method Put the material into boiling water for 85-90 minutes and rapidly cool in the water bath. This method can be used for bacteria and virus materials.
3) The ultrasonic processing frequency is generally selected from 10KC to 200KC, the power range is 200-500 watts, and the processing time ranges from several minutes to several tens of minutes, and attention is paid to cooling during processing. This method is mostly used for microbial materials.
3. Chemical and Biochemical Methods
There are autolysis, enzymatic hydrolysis and surfactant methods.
1) Self-dissolving method A method of breaking cells by using an enzyme system in a tissue cell at a certain pH and an appropriate temperature. This process takes a long time, and a small amount of preservatives such as toluene, chloroform, etc. are often used to prevent cell contamination.
2) Enzymatic dissolution method Using various hydrolases, such as lysozyme, cellulase, snail enzyme, hemicellulase, lipase, etc., the cell wall is decomposed to release the cell contents. Some bacteria are not sensitive to lysozyme, and after adding a small amount of sulfhydryl reagent or 8 moles of urea, they are converted to be sensitive to lysozyme and dissolved.
3) Surfactant treatment such as sodium lauryl sulfate, lauryl chloride chloride, and the like.
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