Staining techniques and microscopic observation of micro-organisms
1. Purpose
1.1 Learn the basic techniques of microbial smears and staining.
1.2 Master the simple staining method of bacteria.
1.3 Preliminarily understand the morphological characteristics of bacteria, and consolidate the knowledge of how to use oil lenses and aseptic operation techniques.
2 Principles
Smearing and staining of bacteria is a basic technique in microbiology experiments. Bacterial cells are small and transparent, making them difficult to identify under an ordinary light microscope; they must be stained. A single dye is used to dye bacteria, so that the stained bacteria form a clear color difference from the background, so that their morphology and structure can be observed more clearly. This method is easy to operate and suitable for observing the general shape of bacterial cells and the arrangement of bacteria. Basic dyes are commonly used for simple staining. This is because in neutral, alkaline or weakly acidic solutions, bacterial cells are usually negatively charged, and when basic dyes are ionized, the dyeing part of their molecules is positively charged, so they are alkaline. The staining part of the dye easily combines with bacteria to color them. The stained bacterial cells contrast sharply with the background, making them easier to identify under a microscope. Dyes commonly used for simple dyeing include methylene blue, crystal violet, basic fuchsin, etc. When bacteria decompose sugars and produce acid, causing the pH of the culture medium to drop, the positive charge on the bacteria increases. At this time, acidic dyes such as eosin, acid fuchsin, or Congo red can be used for staining. Bacteria must be fixed before staining. It has two purposes: one is to kill bacteria and make them adhere to the glass slide; the other is to increase their affinity for dyes. Two commonly used methods are heating and chemical fixation. When fixing, try to maintain the original shape of the cells.
3 materials
3.1 Bacteria: Bacillus subtilis 12-18h nutrient agar slant culture, Staphylococcus aureus 24h nutrient agar slant culture, Escherichia coli 24h nutrient agar slant culture, baker's yeast agar slant culture.
3.2 Dyeing agent: Lu's alkaline methylene blue dye (or ammonium oxalate crystal violet dye), carbolic acid fuchsin dye.
3.3 Instruments or other utensils: microscope, alcohol lamp, slide, inoculation loop, slide rack, double-layer bottle (containing cedar oil and xylene), lens tissue, physiological saline or distilled water, etc.
4. Process: smear → drying → fixation → staining → washing → drying → microscopic examination.
5 steps
5.1 Smear: Take two clean and oil-free glass slides. Under sterile conditions, put a small drop of normal saline (or distilled water) on each center of the glass slide. Use an inoculation loop to sterilize the bacteria from Bacillus subtilis. , Micrococcus luteus and Escherichia coli, pick a little bacterial lawn in the water droplets on the slant, mix well and apply it into a thin film. If you use a bacterial suspension (or liquid culture) smear, you can use an inoculating loop to pick 2 to 3 loops and apply them directly on the slide. Be careful not to drop too much physiological saline (distilled water) and remove bacteria and apply it evenly and not too thickly.
5.2 Drying Dry naturally at room temperature. You can also heat it slightly over an alcohol lamp with the coated side facing up to dry it. But be sure not to get too close to the flame, as too high a temperature will destroy the morphology of the bacteria.
5.3 Fixation If heated drying is used, fixation and drying are combined into one step, and the method is the same as drying. Smear, dry and heat-fix.
5.4 Staining: Place the slide flat on the slide rack, and add 1 to 2 drops of dye solution onto the smear (it is appropriate for the dye solution to just cover the smear film). Stain with Lu's basic methylene blue for 1 to 2 minutes, and stain with carbolic acid fuchsin (or ammonium oxalate crystal violet) for about 1 minute.
5.5 Washing with water: Pour off the dye solution and gently rinse with tap water from one end of the slide until the water running down from the smear is colorless. When washing with water, do not wash the coated surface directly with water. The water flow should not be too fast or too large to prevent the smear film from falling off.
5.6 Drying: Shake off the water droplets on the slide and dry naturally, blow dry with a hair dryer or absorb it with absorbent paper (be careful not to wipe off the bacteria). 5.7 Microscopic examination After the smear is dry, perform microscopic examination. The smear must be completely dry before viewing with an oil lens.
6 Results Draw the morphology of E. coli and Micrococcus luteus observed after single staining.
