Why is the magnification of the oil lens larger than that of the ordinary objective lens?
Reasons why bacteria need to be fixed before staining in microbiological experiments:
1: Kills cells, is easy to stain with dye.
2: Make the bacteria adhere to the glass slide, and it is not easy to be washed away by water.
When fixing, it should be dried slowly. If you really want to speed up, you can dry it a few times in the distance above the flame of the alcohol lamp. Don’t let the glass slide heat up, otherwise it may destroy the life form of bacteria.
Gram staining and microscopic observation of bacteria
1. Experimental principle
Gram staining principle: Bacteria respond differently to Gram staining due to the composition and structure of their cell walls. The cell wall of Gram-positive bacteria is mainly a network knot formed by peptidoglycan. When treated with ethanol, the pore size of the network structure becomes smaller due to dehydration, and the permeability decreases, so that the complex of crystal violet-iodine The substance is not easy to be eluted and retained in the cell, and the blue-purple color of the primary staining agent is still retained after decolorization and counterstaining. The peptidoglycan layer of the cell wall of Gram-negative bacteria is thin and the lipid content is high, so when the decolorization treatment is performed, the lipid is dissolved by ethanol (or acetone), and the permeability of the cell wall increases, so that the complex of crystal violet-iodine It is easier to be eluted, and after counterstaining with a counterstain, the cells are stained with the red color of the counterstain. Microscope imaging principle: Modern ordinary optical microscopes use two lens systems of eyepiece and objective lens to magnify the image, so they are often called compound microscopes. In the optical system of the microscope, the performance of the objective lens is the most critical, and the magnification of the oil lens is the largest, which is the most important for microbiological research. The main purpose of adding immersion oil between the glass slide and the lens is to increase the brightness of the illumination and increase the resolution of the microscope. Oil is used instead of air as the light transmission medium to reduce the refraction or total reflection of light and make the observed image clearer.
2. Experimental equipment
1. Colonies:
Escherichia coli 24h nutrient agar slant culture,
Staphylococcus aureus about 24h nutrient agar slant culture, Bacillus subtilis 12-18h nutrient agar slant culture.
2. Solutions or reagents:
Distilled water, iodine solution, crystal violet staining solution, 95% alcohol, safranin staining solution, cedar oil. 3. Instrument:
Microscope, glass slide, inoculation loop, alcohol lamp, tweezers, lens tissue.
3. Experimental steps
a:
1. Smear
Take out the slide, ignite the slide, burn out the alcohol on the slide and sterilize it, put a small drop of distilled water in the middle, use the inoculation loop to gently pick a small amount of bacteria on the slant medium of the test tube, During the whole process, the test tube mouth should be above the alcohol lamp, and the speed should be fast to prevent contamination of the culture medium. Then smear the small water droplets on the glass slide in a circular motion, stop when the water droplets are cloudy, and wait for them to dry and fix.
2. Primary dyeing
Drop crystal violet (it is appropriate to just cover the bacterial film) on the colony, stain for 1-2 minutes, pour off the staining solution, and rinse with fine water from the top of the slide until the eluate is colorless, and place the stain on the slide. Shake off the water on the slices.
3. Mordant
Add iodine solution dropwise to wash away residual water, cover for about 1min, and wash with water.
4. Decolorization
Shake off the water on the glass slide, tilt the glass slide, and add 95% alcohol to decolorize it under the white background, until the outflowing alcohol just does not appear blue, immediately rinse the alcohol with water, and place the glass slide Shake off the water on the slices.
5. Counterstaining
Counterstain with safranin solution for about 1-2 minutes, wash with water, and then blot dry with absorbent paper
6. Microscopic examination:
Look for the bacterial colony to be observed under the low magnification lens (keep moving the slide, if you feel a red shadow, stop immediately, adjust the magnification, if it is not a colony, continue to find), raise the lens barrel, and then switch to the oil mirror. Add a drop of cedar oil to the sample area, and carefully lower the lens barrel with the coarse adjuster so that the oil lens is immersed in the oil and almost touches the specimen. Raise the condenser to the highest position and fully open the aperture, use the coarse adjuster to raise the lens barrel slowly until the object image appears in the field of view and use the fine adjuster to make it clear and in focus.
