Bacterial Culture Bacterial culture is a technique of growing and reproducing bacteria by artificial means. Bacteria are extremely widespread in nature, large in number and diverse, and can either benefit mankind or be the cause of disease. Most bacteria can be cultured artificially, i.e., they are inoculated on a culture medium and allowed to grow and multiply. The cultured bacteria are used for research, identification and applications. Bacterial culture is a complex technique.
Chinese name bacterial culture culture conditions 37 ℃ in the put 18 ~ 24 hours to grow the substance of a complex technology methods
Artificial Methods
1. Culture conditions
2. Points of Attention
3. Specific Cultivation Steps
4. Daily management and testing
5. Criteria for cultivation success or failure
6. Analysis and handling of problems
1. Cultivation conditions Cultivation should be based on the type of bacteria and purpose, such as the selection of culture methods, media, and the development of culture conditions (temperature, pH, time, the need for oxygen or not, etc.). The general procedure is to first inoculate the specimen on solid medium to do separation culture. Then the resulting individual colonies are further identified by morphological, biochemical and serological reactions. Culture medium commonly used beef broth, peptone, sodium chloride, glucose, blood and other special substances required by certain bacteria prepared into liquid, semi-solid, solid and so on. General bacteria can be placed in aerobic conditions, 37 ℃ in 18 ~ 24 hours of growth. Anaerobic bacteria need to be placed in an anaerobic environment for 2 to 3 days after growth. Individual bacteria such as tuberculosis should be cultured for as long as 1 month.
2. Points of attention due to the ubiquity of bacteria, so from the preparation of culture medium, the entire culture process must be carried out in accordance with the requirements of aseptic operation, otherwise external bacterial contamination of the specimen will lead to erroneous results; and cultivation of pathogenic bacteria once contaminated with the environment, it will cause cross-infection.
Bacterial culture carried out for the purpose of disease diagnosis should select suitable specimens (blood, urine, stool, pus, secretions, etc.), and the results obtained should be interpreted in the light of the clinical situation.
3. Specific culture steps take the method of photosynthetic bacterial culture as an example. The method of photosynthetic bacterial culture is divided into four steps in order: sterilization of containers and tools, preparation of culture medium, inoculation and culture management.
(I) Sterilization of containers and tools: refer to and omit here.
(II) Preparation of culture media
1. Cultivation water: If the cultivated photosynthetic bacteria are freshwater species, distilled water can be used for strain cultivation, and sterilized tap water (or well water) can be used for production cultivation. If the cultivated photosynthesizing bacteria are seawater species, the medium can be prepared with natural seawater, and it should be noted that when adding phosphorus into seawater, dipotassium hydrogen phosphate cannot be used, but potassium dihydrogen phosphate should be used, otherwise a large amount of precipitation will be produced.
2. Sterilization and disinfection of culture medium for strain culture should be sterilized by autoclave together with the culture container. Small productive culture can be prepared culture solution with ordinary aluminum pot or large triangular flask boiling sterilization. Large-scale production culture is the precipitation of sand filtered water with bleach (or bleach) sterilization after use.
3. medium preparation according to the nutritional needs of the cultivated species to choose the appropriate medium formula. According to the formula of the culture medium to the required substances weighed, one by one dissolved, mixed, formulated into a culture medium. Can also be prepared into the mother liquor, when used in proportion to add a certain amount can be.
(III) inoculation of culture medium should be inoculated immediately after preparation. Photosynthetic bacteria productive culture according to the amount of seed is relatively high, generally 20% to 50%, that is, the amount of strain mother liquor and the newly formulated culture solution although the ratio of 1:4 ~ 1:1, should not be less than 20%, especially micro-air culture, inoculation amount should be higher, otherwise it is difficult for photosynthesizing bacteria to take absolute advantage of the culture solution, which affects the final yield and quality of culture.
(IV) Cultivation management of photosynthetic bacteria in the process of cultivation, the management work includes three aspects: daily management operation and test, observation and inspection of growth, as well as the analysis and treatment of problems.
4 Daily management and testing
(1) Stirring and aerating: the photosynthetic bacteria must be aerated or stirred during the process of cultivation, which serves to help the precipitated photosynthetic bacteria to float up to get the light and keep the bacterial cells in good growth. Small anaerobic culture is often used to manually shake the culture container to make the bacterial cells float, at least three times a day, regular shaking. Large anaerobic cultures use mechanical stirrers or small pumps to circulate water slowly to keep the bacteria suspended. Microaerophilic culture is to help the bacteria to float by aerating, because the dissolved oxygen content in the culture solution increases, the photosynthetic bacterial reproduction is inhibited and the yield decreases, so the amount of aerating must be strictly controlled. Generally, timed intermittent inflation is used, and the inflation volume is controlled between 1 and 1.5 liters/(l-h), and the amount of dissolved oxygen is kept below 1×10-6.
(2) Adjustment of illumination: Cultivation of photosynthetic bacteria requires continuous illumination. In daily management, the light level should be adjusted frequently as needed. Sunlight can be utilized for cultivation during the daytime, while artificial light source is needed for illumination at night, or artificial light source can be utilized completely for cultivation. Artificial light sources generally use tungsten iodine lamps or incandescent bulbs. The light intensity required for different cultivation methods varies. General culture light intensity should be controlled between 2000 to 5000lx. If the photosynthetic bacteria grow and multiply quickly and the cell density is high, the light intensity should be increased to 5000 to 10000lx. The light intensity can be adjusted by adjusting the distance between the culture container and the light source or by using a controllable power box.
(3) Adjustment of temperature: Photosynthetic bacteria have a wide range of adaptability to temperature, and a boat can grow and reproduce normally within the range of 23-39℃, so it is not necessary to adjust the temperature. Cultivation at room temperature can also be adjusted to control the temperature in the most suitable range for the growth and reproduction of photosynthetic bacteria, so that the photosynthetic bacteria grow better.
(4) Determination and adjustment of acidity and alkalinity: In the process of cultivating photosynthetic bacteria, attention must be paid to the change of acidity and alkalinity. Due to the massive reproduction of photosynthetic bacteria, the pH value of the bacterial solution rises, which means that the photosynthetic bacteria are in the exponential growth period. However, when the pH value exceeds the optimal range or even the adaptive range of growth, the growth of photosynthetic bacteria reaches the peak and then the growth declines. If the acidity and alkalinity of the bacterial solution can be adjusted in time to keep the pH value in the optimal range, the photosynthetic bacteria can continue to grow and reproduce. In order to prolong the exponential growth period of photosynthetic bacteria and increase the utilization rate of the culture medium and the yield per unit of water, it is very important to measure and adjust the pH value. A boat adopts the method of adding acid to reduce the pH of the bacterial solution, acetic acid, lactic acid and hydrochloric acid department can be used, the most commonly used is acetic acid. In the daily management, must be every day or every other day to determine the pH value of the bacterial fluid, when the pH value rises beyond the optimal range, that is, add acid adjustment. If the pH is not measured and adjusted during the cultivation process, when the growth of photosynthetic bacteria reaches a certain density the pH also rises above 9, and the bacterial growth is impeded, at which time it should be harvested or re-expanded for cultivation. The final yield obtained without adjusting the pH value during the cultivation process is low.
5 Criteria for the success or failure of culture Observation and inspection of the growth situation of photosynthetic bacteria is the criterion for the success or failure of culture. In the process of cultivation, the general situation of growth and reproduction of photosynthetic bacteria can be understood by observing the color of the bacterial solution and its changes, whether the color of the bacterial solution is normal or not, and whether the color changes from light to dark after inoculation, all of which reflect whether the photosynthetic bacteria are growing and reproducing normally as well as how fast or slow the reproduction speed is. If necessary, it can be examined by microscope to understand the situation.
6 Analysis and treatment of the problem Through daily management, testing, inspection, understanding the growth of photosynthetic bacteria, it can be analyzed in conjunction with the changes in environmental conditions at the time, to find out the reasons that affect the growth and reproduction of photosynthetic bacteria, and take appropriate measures. There are many reasons affecting the growth of photosynthetic bacteria, the internal cause is whether the strain is good or not, and the external cause is light, temperature, nutrition, enemy and anaerobic degree. Temperature, light and pH value can affect the growth of photosynthetic bacteria, and temperature, light and pH value are mutually restrictive, temperature and light strength is the unity of opposites, so the optimal conditions for the growth of photosynthetic bacteria should be mutually responsive, that is, the temperature is high, the light should be weak; the temperature is low, the light should be strong. If the temperature is high, the light is strong, the pH value will rise rapidly, the culture medium produces precipitation, inhibit the growth of light table bacteria; if the temperature is low, the light is weak, the photosynthetic bacteria can not get the best energy, the growth rate is also slow. The test concluded that the optimal conditions for the growth of photosynthetic bacteria are: ① temperature 15 ~ 20 ℃, light 30,000 ~ 50,000lx, medium pH value of 7.0; ② temperature 25 ~ 30 ℃, light for 3000 ~ 5000lx, medium pH value of 7.0.
