Bacterial spore staining
(1) Experimental purpose: To learn the bacterial spore staining method
(2) Experimental principle: The bacterial spores have thick and dense walls, low permeability, and are not easy to be colored. If the general staining method can only be used to color the bacteria and the spores are not colored (spores are colorless and transparent). The spore staining method is designed according to the characteristic that the spores are difficult to stain and difficult to decolor once dyed. All spore staining methods are based on the same principle: in addition to using dyes with strong coloring power, heating is required to promote spore coloring. When staining the spores, the bacteria will also be colored, and then washed with water, the color of the spores is difficult to seep, and the bacteria will discolor. Then counter-stain the bacteria with a dye with strong contrast, so that the bacteria and spores show different colors, so that the spores can be set off more clearly for easy observation.
(3) Experimental equipment
1. Live material: Bacillus thuringiensis or Bacillus subtilis cultivated for 36 hours.
2. Staining solution and reagents: 5% malachite green aqueous solution, 0.5% samarium aqueous solution [see Appendix II (1) 6, 5].
3. Equipment: small test tube (75mm × 10mm), beaker (300mL), dropper, slide shelf, inoculation ring, lens cleaning paper, tweezers, microscope, etc.
(4) Experimental method
1. Modified Schaeffer and Fulton's staining method
(1) Preparation of bacterial solution: add 1-2 drops of sterile water to a small test tube, use the inoculation ring to pick 2-3 rings of bacteria from the inclined surface in the test tube and mix well to make a thick bacterial solution .
(2) Add staining solution: add 2-3 drops of 5% malachite green aqueous solution to the small test tube, stir with the inoculating ring to mix the dye and bacterial solution.
(3) Heating: Immerse the test tube in a boiling water bath (beaker) and heat for 15-20 minutes.
(4) Smear: Use the inoculation ring to pick up several rings of bacterial solution from the bottom of the test tube on a clean glass slide, make a coated surface, and dry it.
(5) Fixation: Pass the smear through the alcohol lamp flame 3 times.
(6) Decolorization: Wash with water until there is no malachite green color in the flowing water.
(7) Counterstaining: After dyeing with a solution of Safranin for 5 minutes, the staining solution was poured out, without washing with water, and directly blotted with absorbent paper.
(8) Microscopic examination: first low magnification, then high magnification, and finally observe with oil lens.
Results: The spores were green, and the cysts and cells were red.
2. Schaeffer and Fulton's staining method
(1) Smear: The bacteria to be tested are made into a thin smear according to the conventional method.
(2) Drying and fixing: After the smear is dried, it passes through the alcohol lamp flame 2-3 times.
(3) Dyeing:
â‘ Add staining solution: add 5% malachite green water solution to the smear (the dye is covered with smear), then place the smear on the copper plate, and use the flame of an alcohol lamp to heat the stain to start counting time Maintain about 15-20min. Add staining solution at any time during the heating process, and do not let the specimens dry up. (The temperature should not be too high when heating).
â‘¡Washing: After the slide is cooled, rinse gently with water until there is no staining solution in the outflowing water.
â‘¢ Counterstaining: staining with Safranin for 5min.
(4) Wash, dry or blot dry.
(5) Microscopic examination: first low power, then high power, and finally observe the morphology of spores and bacteria under oil microscope. .
Result: The spores were green and the bacterial cells were red.
(5) Experimental work:
Plot the morphology of the spores and bacteria of the materials used.
(6) Precautions
1. The bacterial species used for spore staining should be controlled for bacterial age.
2. The improved method is superior to the conventional smear method in saving dyes, simplifying operations, and improving the quality of specimens, and can be used preferentially.
3. When using the improved method, to get a good smear, first prepare a thick bacterial solution, and secondly, when taking the stained bacterial solution from a small test tube, it should be fully stirred with the inoculation ring before picking the bacterial solution Otherwise, the bacteria will sink to the bottom of the tube, and there will be too few bacteria when smearing.
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