Bacterial cell wall is very thin, the cell wall of Gram-positive bacteria is 20-30 nm, and the cell wall of Gram-negative bacteria is 10-13 nm.The main chemical constituent of bacterial cell wall is peptidoglycan, which has poor ability to combine with dyes, and is not easy to be colored, in the process of bacterial staining, the dyes generally enter into the cells through the penetration and diffusion of the cell wall, and the cell wall itself is not stained.
Operation method
Staining of bacterial cell walls
Principle
Based on the phenomenon that bacterial cells produce a separation of the plasma wall in hypertonic solutions or after treatment with ether vapor, it is also possible to distinguish between the cell wall and the cytoplasmic membrane under an ordinary light microscope after staining.
Materials and Instruments
Bacillus megaterium Bacillus subtilis Move 1. Tannic acid method For more product details, please visit Aladdin Scientific website.
Crystalline violet aqueous solution Tannic acid (ellagic acid) aqueous solution Phosphomolybdic acid aqueous solution Methyl green aqueous solution NaCl Crystalline violet aqueous solution Bouin's fixative Sulphur cordial aqueous solution Ether
Ether Vapor Bottle
(1) Bacillus megaterium cultured for 16 to 18 hours is smeared in the usual way.
(2) Stain with 5% tannic acid for 5 minutes and then wash with water.
(3) Stain with 0.2% crystal violet for 3~5 minutes, wash with water and blow dry. Observed with oil microscope, the cell wall was purple and the cytoplasm was lavender.
2. Phosphomolybdic acid method
(1) Prepare a thick smear by immersing it in a 1% aqueous solution of phosphomolybdic acid for 3 to 5 minutes before it dries.
(2) Stain with 1% aqueous methyl green solution for 3-5 minutes.
(3) After washing with water, blow-dry and observe with an oil microscope. The cell wall is green and the cytoplasm is colorless.
3. Distinguish between cell wall and cytoplasmic membrane
(1) Ether vapor method
(1) Bacillus megaterium was applied to a coverslip and the coverslip was turned so that it was placed on the spout of an ether vial and steamed for 3 minutes.
② Remove the coverslip and place it in Bouin's fixative for 30 minutes, then remove it and wash it in water.
(iii) Staining with sulfur cordial for 30 seconds.
④Wash with water, seal with water, and place under an oil microscope for observation.
(2) NaCl method
(i) Take a drop of 25% NaCl solution on a clean slide.
② Pick a small loop of Bacillus subtilis cultured for 6 hours, spread evenly in 25% NaCl water droplets and leave to dry naturally.
(iii) Add 0.01% crystal violet dropwise on it so as to cover the bacterial part, wash with water after 30 seconds, dry, and observe the result by oil microscope.