Simple staining is a method of staining bacteria using a single dye. This method is easy to perform and is suitable for the observation of the general shape of the organism and the arrangement of the bacteria. Source: Laboratory Microbiology (3rd Edition)
Operation method
simple dyeing
Principle
Alkaline dyes are commonly used for simple staining because: in neutral, alkaline or weakly acidic solutions, bacterial cells are usually negatively charged, whereas alkaline dyes ionize with the staining portion of their molecules positively charged (acid dyes ionize with the staining portion of their molecules positively charged). Therefore, the staining portion of the basic dye can easily bind to the bacteria to color them. The stained bacterial cells form a sharp contrast with the background and are easier to recognize under the microscope. Dyes commonly used for simple staining include: American blue, crystal violet, alkaline complex red, etc. When the bacterial decomposition of sugar acid production, so that the pH of the culture medium decreased, the positive charge of the bacteria increased, at this time, we can use Eosin, acidic red or Congo red and other acidic dyes staining.
Materials and Instruments
Bacillus subtilis (12-18 h nutrient agar slant cultures) Micrococcus garciniae (~24 h nutrient agar slant cultures) Move 1. Smear Take two slides, each with a small drop (or 1-2 rings picked with an inoculating ring) of saline (or distilled water) in the center of the slide, and pick a small amount of bacterial moss from Bacillus subtilis and Micrococcus garciniae slant in the drop of water by aseptic operation with a jointed ring (Fig. IV-1), and mix the wells wells to form a thin film. If you use bacterial suspension (or liquid culture) to smear, you can use the inoculating ring to pick 2~3 rings and apply them directly on the slide. 2. Drying Dry naturally at room temperature. 3. Fixation Apply the smear face up and pass it through the flame 2~3 times. This procedure is called thermal fixation, and its purpose is to solidify the cytoplasm to fix the cell morphology. The purpose of this procedure is to solidify the cytoplasm so as to fix the cell morphology and make it firmly attached to the slide. 4. Staining Place the slide flat on a slide holder and add drops of staining solution to the smear (the solution should cover the smear film). Stain the slide for 1-2 min with Lübeck's Basic Mellanox Blue, and for about 1 min with Carbonic Acid Red (or Ammonium Oxalate Crystal Violet). 5. Washing Pour off the staining solution. Rinse with tap water until the water running down the smear is colorless. 6. Drying Dry naturally, or blow dry with hairdryer, or absorbent paper. 7. Microscopy Microscopic examination of the smear after drying. Caveat 1. Slides should be clean and free of grease spots, saline drops and bacteria should not be excessive, and smears should be evenly applied. It should not be too thick. 2. The temperature of heat fixation should not be too high (the back of the slide should not be hot), otherwise it will change or even destroy the cell morphology. 3. When washing, do not rinse the coated surface directly, but let the water flow down from one end of the slide. The water flow should not be too rapid, too large, so as to avoid the smear film off. 4. The smear must be completely dry before observation with an oil microscope. For more product details, please visit Aladdin Scientific website.
Lü's Basic Mellanox Blue Stain / Oxalic Acid by Crystalline Violet Stain Qi's Carbonic Acid Red Stain
Microscope, alcohol lamp, slides, inoculation rings, double bottles (with cedar oil and xylene), microscope paper, physiological saline.