Gram staining and special morphology observation of bacteria
Experimental principle
Dyeing method:
Gram staining is one of the most widely used differential staining methods in bacteriology. Founded in 1884 by the Danish physician Gram. The method is to first stain the bacteria with crystal violet, add a mordant (increasing the affinity of the dye and the cells), then decolorize with a decolorizing agent (alcohol or acetone), and then dye with a counterstain. If the bacteria are not discolored, the color of the original dye is preserved as Gram-positive bacteria (G+); if it is discolored, the color of the counterstain is Gram-negative (G-). This staining method can classify all bacteria with cell walls into two major categories: Gram-positive bacteria and Gram-negative bacteria.
Single staining method: only the size, shape, and cell arrangement of microorganisms can be observed, but microorganisms and their special structures cannot be identified.
Counterstaining method: dyeing with two or more kinds of dyeing liquids, which helps to identify microorganisms, is also called differential staining. Commonly used are: Gram staining, acid-fast staining, spore staining, flagella staining, etc.
Principle of dyeing: It is now believed that the different responses of bacteria to Gram staining are mainly due to the different cell wall structure and chemical composition of Gram-positive and Gram-negative bacteria. Gram-positive bacteria have thick cell walls, high peptidoglycan content, large cross-linking degree, and low lipid content. When decolorized by 95% ethanol, the peptidoglycan layer has a smaller pore size, reduced permeability, and binds to cells. The complex of crystal violet and iodine is not easily removed, so the cells retain the color at the time of initial dyeing. The cell wall of Gram-negative bacteria is thinner, contains more lipidoids, and the content of peptidoglycan is less. The outer layer of lipids is dissolved when decolorization of ethanol, which increases the permeability of cell walls and makes the initial dyed crystals. The complex of purple and iodine is prone to oozing out, and as a result, the cells are discolored, and after counterstaining, the counterstained color is dyed.
Experimental reagent
Gram staining solution, a set of crystal violet iodine solution
95% ethanol saffron red malachite green
Laboratory equipment
Microscope cover glass slide
Experimental Materials
Species: Escherichia coli, Staphylococcus aureus, Bacillus subtilis
Experimental procedure
1. Gram staining (1) Smear (2) Initial dyeing: Add ammonium oxalate crystal violet dye solution to the prepared coating surface, dye for 1 minute, pour the dye solution, and rinse until there is no purple.
(3) mordant dye: first wash the residual water with the newly-added Lu's iodine solution, and then cover the coated surface with it for 1 minute, then wash it.
(4) Decolorization: After removing residual water, 95% alcohol was added dropwise for decoloration for about 15-20 seconds, and immediately rinsed with running water.
(5) Counterstaining: Add the saffron staining solution, dye for 3-5 minutes, wash with water and blot dry with absorbent paper.
(6) Microscopic examination: observe the staining results and plot.
2. Dyeing process: smear (E. coli and Staphylococcus aureus) → dry → fixed → primary dyeing (crystallization dyeing process: purple staining 1 min) → mordant (iodine 1 min) → water wash → 95% ethanol decolorization (1 min) → complex Dyeing (saffron red staining for 1 min) → drying → microscopic examination (oil mirror)
3. Use and maintenance of the oil mirror (1) Add a drop of cedar oil to the slide on the part to be observed, then slowly turn the oil mirror. When converting the oil mirror, look at the distance between the lens and the slide horizontally from the side to make the lens Immerse in oil without crushing the slide.
(2) Observe the eyepiece with both eyes, and slowly turn the coarse adjuster until the blurred object appears, then use the fine adjuster until the object is clear.
(3) If there is no object or the target is not ideal, look for it again.
(4) After the oil mirror is used, first wipe the lens and the cedar oil on the specimen with a little ethanol, then wipe it off with a dry mirror paper.
Precautions
1. The strain should be selected from logarithmic strains;
2. The smear is not too thick, and the smear is thin and uniform;
3. Decolorization or excessive discoloration may cause false positive or false negative of Gram staining. The decolorization time depends on the thickness of the smear, room temperature, etc.
4. At the end of the experiment, please make sure the oil lens is wiped clean.
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