Staining techniques and microscopic observation of microorganisms
Simple Staining of Bacteria
1 Objectives
1.1 To learn the basic techniques of microbial smear and staining.
1.2 To master the simple staining method of bacteria.
1.3 To get to know the morphological characteristics of bacteria, and to consolidate the learning of the use of oil mirror and aseptic operation techniques.
2 Principle The smear and staining of bacteria is a basic technique in microbiology experiments. The cells of bacteria are small and transparent, they are not easy to recognize under the ordinary light microscope, and they must be stained. The use of a single dye to stain the bacteria, so that the stained organism and the background to form a clear color difference, so that their morphology and structure can be more clearly observed. 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. Alkaline dyes are commonly used for simple staining, because in neutral, alkaline or weakly acidic solutions, bacterial cells are usually negatively charged, and when alkaline dyes are ionized, the staining portion of their molecules is positively charged, so the staining portion of alkaline dyes can easily bind to the bacteria to make the bacteria colored. The stained bacterial cells contrast sharply with the background and are easier to recognize under the microscope. Commonly used dyes for simple staining include merocyanine blue, crystal violet, and basic compound red. When the bacterial decomposition of sugar to produce acid so that the pH of the medium decreases, the positive charge of the bacteria increases, and then can be used in red, acidic red or Congo red and other acidic dyes staining. The bacteria must be fixed before staining. Its purpose is twofold: one is to kill the bacteria and make the bacteria adhere to the slide; the second is to increase its affinity for the dye. Two methods are commonly used: heating and chemical fixation. Try to maintain the original morphology of the cells when fixing.
3 Materials
3.1 Strain Bacillus subtilis 12-18h nutrient agar slant culture, Staphylococcus aureus about 24h nutrient agar slant culture, Escherichia coli 24h nutrient agar slant culture, baker's yeast agar slant culture.
3.2 Stain Lue's alkaline methylene blue stain (or ammonium oxalate crystal violet stain), carbolic acid compound red stain.
3.3 Instruments or other paraphernalia Microscope, alcohol lamp, slide, inoculation ring, slide holder, double-layered bottle (filled with cedar oil and xylene), microscope paper, physiological saline or distilled water, etc.
4 Procedure Smear → drying → fixation → staining → washing → drying → microscopic examination.
5 Steps
5.1 smear Take two clean and oil-free slides, each with a small drop of saline (or distilled water) in the center of the slide under aseptic conditions, with an inoculating ring to aseptically operate, respectively, from the Bacillus subtilis, Garcinia micrococcus and Escherichia coli slant to pick a little moss in the drop of water, mixing and coated with a film. If the bacterial suspension (or liquid culture) smear, can be used to inoculate the ring pick 2 to 3 rings directly on the slide. Note that the saline (distilled water) and take the bacteria should not be too much and spread evenly, not too thick.
5.2 Drying Dry naturally at room temperature. It can also be dried by heating slightly above an alcohol lamp with the coated side up. However, do not get too close to the flame, because the temperature is too high will destroy the morphology of the bacteria.
5.3 Fixation If heat drying is used, fixation and drying are combined into a single step in the same way as drying. Smear, dry and heat fix.
5.4 Staining Place the slide flat on the slide shelf, add 1 to 2 drops of staining solution on the smear (the staining solution just covers the smear film is appropriate). Stain the smear with Lü's Basic Mellanox Blue for 1~2min, and with Carbonate Red (or Ammonium Oxalate Crystal Violet) for about 1min.
5.5 Rinsing Pour off the staining solution and gently rinse with tap water from one end of the slide until the water running down from the smear is colorless. When rinsing, do not let the water flow directly to wash the coated surface. The water flow should not be too rapid or too large to avoid dislodging the smear film.
5.6 Drying Shake off the water droplets on the slide to dry naturally, blow dry or absorbent paper can be used to absorb dry (be careful not to wipe off the bacterial body). 5.7 Microscopic examination The smear is dried and examined by microscopy. The smear must be completely dry before it can be observed with an oil microscope.
6 Results Draw the morphology of E. coli and Micrococcus garciniae after single staining.
Gram Stain
1 Purpose
1.1 To understand the principle of Gram stain and its importance in the classification and identification of bacteria.
1.2 To learn and master the Gram staining technique and to consolidate the learning of the use of the oil lens of the light microscope.
