Culture and identification of Pseudomonas aeruginosa
Pseudomonas aeruginosa (Pseudomonas aeruginosa) is an aerobic or partially anaerobic Gram-negative bacterium that is widely found in environments such as soil, water bodies and hospitals. Pseudomonas aeruginosa is usually cultured using liquid medium or solid medium, which promotes the growth and multiplication of Pseudomonas aeruginosa by providing appropriate nutrients and conditions. Methods used to determine Pseudomonas aeruginosa include biochemical tests, molecular biology methods, and fluorescent staining.
Operation method
Culture and determination of Pseudomonas aeruginosa
Materials and Instruments
Pseudomonas aeruginosa strains, liquid media, solid media, test tubes, petri dishes, biochemical reagents, fluorescent stains, etc. Move 1、Preparation of culture medium: Add appropriate amount of nutrient broth medium (BHI) or E. coli LB medium into appropriate amount of distilled water, mix well, and then heat until completely dissolved, and then autoclaved. Nutritional broth medium formula: beef paste 3.0 g, peptone 10.0 g, NaCl 5.0 g, agar 20.0 g (liquid medium does not contain), distilled water 1.0 L, pH 7.0. 2、Inoculation of medium: add the required antibiotics (if needed) to the sterilized medium, and then inoculate Pseudomonas aeruginosa into the medium using aseptic technique. 3、Setting of culture conditions: Place the inoculated medium in a 37 ℃ constant temperature incubator and incubate for 18~24 hours. 4、Observation of colonies: Observe the morphology of the colonies in the petri dish, Pseudomonas aeruginosa colonies usually show gray, green or yellow-green, irregular shape, fuzzy edge, smooth or rough surface. 5、Preparation of bacterial smear: take a certain number of colonies with a sterile wire ring, smear it on a glass slice, and then sterilize it by fire. 6、Staining of bacterial smear: use Gram stain to stain the bacterial smear. ① Initial staining: add crystal violet drops on the bacterial membrane to cover the bacterial membrane, about 1 minute later, rinse the staining solution with a fine stream of water. ② Enzyme staining: add several drops of Lugol's iodine solution, rinse gently after about 1 minute. ③ Decolorization: add 3-4 drops of 95% alcohol, shake the slide gently for a few seconds, tilt the slide to discard the alcohol, and so on for several times, then the alcohol will be almost colorless or slightly mauve, and then rinse it at last. ④ Repeat staining: add a few drops of diluted red dye solution, rinse after about 1 minute. ⑤ After staining, use absorbent paper to dry the slide, dry and process it, add drops of cedar oil and examine it microscopically. 7. Identification of bacteria: Determine the species of Pseudomonas aeruginosa based on the comprehensive judgment of the observed colony morphology, the morphology of the bacterial smear and the results of biochemical tests. 8, Appendix: (1) The colony morphology of Pseudomonas aeruginosa usually has the following characteristics: Shape: the colonies of Pseudomonas aeruginosa are irregular in shape, showing a forked, branched or reticulate morphology. Color: The color of Pseudomonas aeruginosa colonies is usually gray, green or yellow-green. Size: Pseudomonas aeruginosa colonies are usually about 2-3 millimeters in size. Edge: The edges of Pseudomonas aeruginosa colonies are usually blurred, forked, branched or reticulate. Surface: The surface of Pseudomonas aeruginosa colonies is usually smooth or rough, and sometimes it may also show an uneven pattern. 2) Oxidation/reduction test: Pseudomonas aeruginosa is a gram-negative bacillus that is oxidase positive. The redox properties of Pseudomonas aeruginosa can be determined by observing changes in the redox indicator. 3) pH test: Pseudomonas aeruginosa is usually acid-producing, and the pH of Pseudomonas aeruginosa can be detected by changes in the acid-base indicator. 4) Carbohydrate test: Pseudomonas aeruginosa can usually utilize carbohydrates such as glucose and lactose for metabolism. Media containing these carbohydrates can be used to test Pseudomonas aeruginosa. Caveat 1) The selection of Pseudomonas aeruginosa strains should be based on the experimental needs and the characteristics of Pseudomonas aeruginosa.2) Aseptic operation should be paid attention to during the cultivation process to avoid the contamination of stray bacteria and bacteria.3) The culture conditions should be set according to the growth characteristics of Pseudomonas aeruginosa strains to promote their growth and reproduction.4) Biochemical tests and molecular biology methods should be based on the experimental design to choose appropriate methods and reagents to avoid errors.5) Fluorescent staining methods should pay attention to the concentration of fluorescent stain and staining time to avoid affecting the accuracy of the results.6) Common problems include colony contamination, impure strains, too slow or too fast growth rate.7) Operators should pay attention to protective measures to avoid self-infection. For more product details, please visit Aladdin Scientific website.