List of biotechnology practice knowledge points

I. Application of traditional fermentation technology

1. Principle of fruit wine production: Yeast is a facultative anaerobic microorganism. In anoxic, acidic fermentation broth, yeast can grow and multiply, while most other microorganisms are inhibited from growing. Yeast fermentation under anaerobic conditions: C6H12O6→2C2H5OH+2CO2+ small amount of energy

2. Principle of fruit vinegar production: When oxygen and sugar sources are sufficient, acetic acid bacteria will decompose the sugar in grape juice into acetic acid. In the absence of a sugar source, the acetic acid bacteria change the ethanol to acetaldehyde and then change the acetaldehyde to acetic acid.

3, fruit wine vinegar production process: pick grapes → rinse → juice → alcohol fermentation → fruit wine (→ acetic acid fermentation → fruit vinegar)

4, fermented milk production principle: a variety Microorganisms are involved in the fermentation of tofu, and the main role is mucor. Mucor is a filamentous fungus. The metabolic type is heterotrophic aerobic. Proteases produced by microorganisms such as Mucor can decompose proteins in tofu into small peptides and amino acids; lipases hydrolyze fats into glycerol and fatty acids. Under the synergy of various microorganisms, ordinary tofu is transformed into a unique flavored bean curd.

5. Process of making fermented bean curd: Let the tofu grow on the tofu → add salt and marinate → add marinated soup to bottle → seal and marinate

6. Kimchi production principle: making kimchi The microorganism used is a lactic acid bacterium whose metabolism type is heterotrophic anaerobic type. Under anaerobic conditions, glucose is broken down into lactic acid.

7. Kimchi production process:

8. The method for determining nitrite content is colorimetry.

Second, microbial culture and application

(1) Microbial culture and utilization

1. Microbial culture and separation technology

(1) Microbial growth needs to contain water, carbon source, nitrogen source, and inorganic salt.

(2) Whether the medium is qualified or not is verified by colony growth on the surface of the uninoculated medium.

(3) Commonly used aseptic techniques include 1. cleaning and disinfecting the space for the experimental operation, the operator’s clothing and hands. 2. Sterilize the vessels, inoculation devices, and culture medium used for microbial culture. 3. In order to avoid the contamination of microorganisms in the surrounding environment, the experimental operation should be carried out near the flame of the alcohol lamp. 4. During the experimental operation, the material utensils that have been sterilized should be avoided from coming into contact with the surrounding objects.

Test site refinement:

1 Sterilization refers to the use of strong physical and chemical factors to kill all microorganisms inside and outside the object, including spores and spores. Disinfection refers to the use of milder physical and chemical methods to kill only microorganisms that are harmful to the human body on the surface or internal parts of the object, excluding spores and spores.

2 sterilization methods include burning sterilization, dry heat sterilization, high pressure steam sterilization, burning sterilization for metal tools for inoculation; dry heat sterilization for high temperature resistance, Need to keep dry glassware, etc.; autoclave sterilization for medium, etc.

3 disinfection methods include boiling disinfection for liquid disinfection; pasteurization for liquids that are not resistant to high temperatures Chemical or UV disinfection for the disinfection of surfaces.

(4) Pure culture of microorganisms refers to the prevention of invasion by foreign bacteria.

(5) Steps for preparing solid medium: calculating, weighing, dissolving, sterilizing, and pouring the plate

(6) The most commonly used method for microbial inoculation is flat scribing Method and dilution coating plate method. The plate scribing method is an operation of continuously scribing a surface of an agar solid medium by an inoculating loop. The aggregated species are gradually diluted and dispersed to the surface of the medium. After several lines of scribing, the population of visible sub-cells that are propagated from one cell can be isolated, which is the purified colony. In the dilution coating method, the bacterial liquid is subjected to a series of gradient dilution, and then the bacterial liquids of different dilutions are separately applied to the surface of the agar solid medium to be cultured. It is divided into two steps: serial dilution operation and coating plate operation.

Test site refinement:

1 After the plate is condensed, if water droplets are dripped onto the medium, the bacteria will spread, so that it cannot be counted and separated. In order to prevent condensation from dripping onto the medium, the plate must be inverted when cultured.

2 Inoculation operation The inoculation ring should be burned before each scribing, because each operation should be sterilized in time to prevent contamination of the bacteria. At the end of the operation, the reason for still burning the inoculating loop is to prevent the bacteria from polluting the environment.

2. Determination of the number of specific microorganisms

(6) The method for determining the number of microorganisms is directly counted by a microscope, and the number of colonies is counted.

3. Selection of microorganisms for culture medium

(7) In microbiology, a medium that allows growth of specific types of microorganisms while inhibiting or preventing the growth of other types of microorganisms , called the selection medium. For example, in the formulation of the selection medium, urea is used as the sole nitrogen source in the medium. In principle, only microorganisms capable of using urea can grow.

Test site refinement:

1 The principle of selecting the medium configuration is to increase or decrease a certain component so that the microorganism to be selected can grow, and other microorganisms cannot grow.

The addition of penicillin to the medium can separate yeast and mold

3. The nitrogen-fixing microorganism can be isolated without adding a nitrogen source.

3. Polymerase Amplification of DNA fragments by chain reaction

1. PCR: The abbreviation for polymerase chain reaction is a technique for rapidly amplifying DNA fragments in vitro. It can replicate millions of copies of DNA in a few hours with a very small amount of DNA as a template.

2. Amplification direction: always extends from the 5′ end to the 3′ end of the daughter strand.

3. Reasons for the need for primers: DNA polymerase cannot synthesize DNA de novo, but can only extend the DNA strand from the 3′ end of the primer, so DNA replication requires primers.

4. Principle: Principle of thermal denaturation,

5. Conditions: DNA template, two primers combined with two template strands, deoxynucleotides, heat-resistant Taq DNA polymerase, while controlling temperature.

6. Process: denaturation (high temperature 94 ° C), renaturation (low temperature 55 ° C), extension (medium temperature 72 ° C)

7. RESULTS: DNA polymerase only specifically replicates the DNA sequence between the two primers, allowing the fixed length sequence to be exponentially amplified.

4. Extraction and Separation of Hemoglobin

1. Principle of Experiment: Using differences in various properties of proteins, such as the shape and size of molecules, the nature and amount of charge, and solubility. , adsorption properties and affinity for other molecules, separation of different kinds of proteins.

2. Principle of gel chromatography: An effective method for separating proteins based on the relative molecular mass. Molecules with relatively small molecular mass pass through the internal channels of the porous gel particles, which are long and slow to flow; molecules with relatively large molecular mass pass directly through the gaps of the gel particles, and the distance is short and the flow is fast.