Culture and identification of microorganisms
Culture and identification of microorganisms
Summary
Mycobacterium avium culture includes modified Roche culture and liquid culture (e.g. BD MGIT 960 liquid medium culture). At present, the solid Roche culture method is the most commonly used laboratory culture method for Mycobacterium tuberculosis in China, and the method is very representative.
Mycobacterium tuberculosis grows on solid culture medium, the morphology of colonies is clearly visible, and samples with more typical colony morphology can be identified by directly observing the morphology of colonies, therefore, solid Roche culture method is commonly used for isolation and cultivation, identification, preservation of strains and determination of the sensitivity of anti-tuberculosis drugs of clinically suspected tuberculosis specimens. Compared with smear examination, the positive rate of modified Roche solid culture is higher.
Principle
Under the heating condition, the mycolic acid in the cell wall of Mycobacterium can be firmly bound to the red dye solution to form a complex, and will not be decolorized after hydrochloric acid and alcohol treatment, so that Mycobacterium still shows red color after re-staining, while blue color is shown in the background and other stray bacteria.
At present, Ziehl-Neelsen sputum smear microscopy is also the most common method used in China's TB-related laboratories, with simple and quick operation, high specificity and low experimental cost.
Fluorescent pigment Auramine "O" is a kind of gold-yellow alkaline dye containing free amino group, which can produce strong fluorescent pigment and is selective, and its ions have cationic charge, while Mycobacterium tuberculosis has negative charge, therefore, Auramine "O" can be combined with negatively charged bacilli to color, and under the stimulation of violet and blue light irradiation by high-pressure mercury lamps in the fluorescence microscope, the tuberculosis bacilli stained with Auramine "O" can emit strong orange-yellow fluorescence, and when examined, it is easy to be used. Under the stimulation of high-pressure mercury lamp in the fluorescence microscope, the tuberculosis bacteria stained with Auramine "O" will emit a strong orange fluorescence, and the bright bacteria can be detected with a high magnification.
Operation method
Cultivation and identification of Mycobacterium spp.
Principle
Under the heating condition, the mycolic acid in the cell wall of Mycobacterium can be firmly bound to the red dye solution to form a complex, and will not be decolorized after hydrochloric acid and alcohol treatment, so that Mycobacterium still shows red color after re-staining, while blue color is shown in the background and other stray bacteria. At present, Ziehl-Neelsen sputum smear microscopy is also the most common method used in China's TB-related laboratories, with simple and quick operation, high specificity and low experimental cost. Fluorescent pigment Auramine "O" is a kind of gold-yellow alkaline dye containing free amino group, which can produce strong fluorescent pigment and is selective, and its ions have cationic charge, while Mycobacterium tuberculosis has negative charge, therefore, Auramine "O" can be combined with negatively charged bacilli to color, and under the stimulation of violet and blue light irradiation by high-pressure mercury lamps in the fluorescence microscope, the tuberculosis bacilli stained with Auramine "O" can emit strong orange-yellow fluorescence, and when examined, it is easy to be used. Under the stimulation of high-pressure mercury lamp in the fluorescence microscope, the tuberculosis bacteria stained with Auramine "O" will emit a strong orange fluorescence, and the bright bacteria can be detected with a high magnification.
Materials and Instruments
4% NaOH specimen pre-treatment solution, centrifuge tubes Move The procedure for culturing and identifying Mycobacterium species in sputum specimens is as follows: 1. Pick about 5 mL of sputum into a 50 mL labeled centrifuge tube. 2. Add 1~2 times of 4% NaOH specimen pre-treatment solution: vortex and shake for 1~2 min. 3. Allow to stand at room temperature for 15-20 minutes, not more than 20 minutes. 4, add sterile PBS (PH 6.8) to about 45 mL, cover tightly. 5、Centrifuge at 3000 g for 15 min, pour off the supernatant. 6. Add 1~3 mL of PBS (PH 6.8) to neutralize the PH to 6.8. 7, take 0.1 mL and inoculate into 2 medium slant culture tubes. 8. Judgment standard of specimen pre-treatment: 3~5% contamination rate is preferred. 9、Place the specimen in 37℃ incubator. 10、Observe the growth of colony once in 3 days and once in 7 days after inoculation, and identify the mycobacteria if it is found that the colony grows. Thereafter, observe once a week, record the colony growth and contamination. 11, there is colony growth need to be stained to confirm, culture to the eighth week no colony growth can be initially judged as "Mycobacterium culture negative. 12、After positive colony growth is found in the culture medium, a small number of colonies are picked from the culture medium and inoculated in Roche tubes in the biosafety cabinet, and smeared directly (the culture medium can be mixed with 5% carbolic acid solution on the slide to kill Mycobacterium), and the smears are irradiated with ultraviolet rays for 1 h before they are taken out of the biosafety cabinet. The smears should be stained with fluorescent amine O stain; 13. Positive fluorescein O staining should be further confirmed by Cecropin's antacid staining; 14. If the stain is positive, the culture fluid should be used for further Mycobacterium tuberculosis antigen gold standard testing; 15. In addition, DNA sequencing can also be used for strain identification. The operation process can be divided into genome extraction - PCR amplification - Sanger sequencing (see DNA sequencing experimental process). Caveat 1. The concentration of NaOH can be adjusted to adjust the contamination rate. 2. Use the specimen pre-treatment solution within 24 hours. For more product details, please visit Aladdin Scientific website.
Roche medium, incubator