I. General steps and methods of screening auxotrophic strains
① A large number of auxotrophic strains were induced by physical methods such as ultraviolet rays and chemical mutagens. Irradiate 15W or 20W ultraviolet lamp at 15cm ~ 30cm 10s ~ 20min. Before irradiating the tested bacteria, the lamp tube should be preheated for 20min minutes to stabilize the light. The bacterial suspension should be stirred by magnetic force, so that all single cells or monospores are evenly irradiated. Special attention should be paid to the operation under dark or infrared light. After inoculation, the apparatus should be tightly wrapped with black cloth to prevent the recovery of visible light.
② elimination of wild type
A) Antibiotic method Some antibiotics can kill growing and reproducing microorganisms, but have no killing effect on static microorganisms. For example, penicillin can inhibit the biosynthesis of peptidoglycan in bacterial cell wall, and nystatin can destroy fungal cell membrane and kill bacteria and fungi respectively.
B) Mycelium filtration is suitable for filamentous microorganisms, such as actinomycetes and molds. In the basic medium, wild-type spores can germinate and grow into mycelium, while auxotrophic spores do not grow. Inoculate the mutated spores into liquid basal medium, shake culture 10h ~ 12h to germinate the prototrophic type (as long as the hyphae can be seen by naked eyes), then filter with filter paper, cotton or glass filter funnel, and the hyphae are filtered out, so that the undegerminated defective spores can pass through smoothly, thus achieving the purpose of enriching the auxotrophic type.
③ Common methods for detecting defect types.
A) One-by-one determination method (point-seed method) is to coat and separate the bacterial liquid after mutation treatment and elimination of wild type on complete medium, and point each colony grown after culture on basic medium and another complete medium respectively. Any colony that cannot grow on the basic medium but can grow on the corresponding position of the complete medium indicates that it is auxotrophic.
B) Dilute the mutagenic bacteria solution in the sandwich culture method, mix it with the basic culture medium and pour it into a Petri dish. After solidification, add a thin layer of basic culture medium, which does not contain bacteria. After culture, the growing colonies were marked on the bottom of the Petri dish. Then add a layer of complete culture medium, and most of the new colonies after culture are auxotrophic (Figure 3-3 1). This method is suitable for facultative anaerobic bacteria.
C) The velvet is wrapped on a cylindrical table with a diameter smaller than that of a flat dish by photocopying method, fixed, used as a photocopying inoculation tool and sterilized for later use. The wild-type bacteria solution eliminated by mutation was coated on the surface of complete culture medium and cultured. When the colony grows, gently press it on this plate with a photocopying inoculation tool, and then press it on another basic medium plate. Colonies that are not long on the basic medium but grow on the corresponding part of the complete medium after culture are auxotrophic.
(4) Identification of auxotroph-growth spectrum method: Inoculate the detected auxotroph strains on complete culture medium for culture, wash the growing thalli or spores, and centrifugally wash them to prepare bacterial suspension with the concentration of 107/ml ~ 108/ml. Take 0. 1ml of this suspension and mix it with the basic culture medium, pour it into a Petri dish, concentrate it until it is slightly dry, then add circular filter paper pieces with various nutrients in different areas, culture and observe the growth reaction.
In the process of screening auxotrophs, we must pay attention to phenotypic delay. Because mutagens generally only act on single-stranded DNA, mutation can not be reflected in the phenotype, and DNA replication and cell division are needed to mutate the phenotype. This phenomenon is called phenotypic delay.
2. Enrichment and isolation methods of auxotrophic mutants of antibiotic-sensitive bacteria Adding antibiotics to prototrophic culture medium (that is, culture medium lacking nutrients required by auxotrophy) can kill the growing wild-type cells, but the mutants that cannot grow are spared.