Observation experiments on the basic morphological structures of pathogenic organisms
Observation experiments on the basic morphological structures of pathogenic organisms
Microbial cells are small and transparent, not easy to identify under the ordinary optical microscope, they must be stained, so that the stained organisms and the background to form an obvious color difference, so that its morphology and structure can be more clearly observed. Therefore, microbial staining technique is an important means of observing the morphology and structure of microorganisms. (Note: No technique is perfect. The stained microbial specimen is dead, and the morphology and structure of microorganisms will undergo some changes during the staining process, which can not fully represent the real situation of their living cells, and must be paid attention to when staining and observing).
Operation method
Gram stain (used distinguished two different kinds of bacteria)
Principle
Microbial cells are small and transparent, not easy to identify under the ordinary optical microscope, they must be stained, so that the stained organisms and the background to form an obvious color difference, so that its morphology and structure can be more clearly observed. Therefore, microbial staining technique is an important means of observing the morphology and structure of microorganisms. (Note: No technique is perfect. The stained microbial specimen is dead, and the morphology and structure of microorganisms will undergo some changes during the staining process, which cannot fully represent the real situation of their living cells, and attention must be paid to the staining observation). The isoelectric point of bacteria is low, and the pH value is about between 2-5, so in neutral, alkaline or weakly acidic solutions, the bacterial proteins are negatively charged after ionization; and the dye ions are cathodically charged when alkaline dyes are ionized. Therefore, negatively charged bacteria often bind with positively charged alkaline dyes. Therefore, alkaline dyes are commonly used in bacteriology for staining. Other factors affecting staining are the structure of the bacterial cell and the permeability of its outer membrane, such as the permeability of the cell membrane, the size of the membrane pore and the integrity of the cellular structure, which all play a role in staining. In addition, the composition of the culture medium, the age of the bacteria, the dielectric content and pH of the staining solution, temperature, the role of drugs, etc., can also affect the staining of bacteria. Microbial staining methods are generally categorized into two types: single staining method and compound staining method. The former stains microorganisms with a single dye, but does not identify the microorganisms. Compound staining method is to use two or more than two dyes, have the role of assisting in the identification of microorganisms. Therefore, it is also called differential staining method. Commonly used compound staining method has Gram stain and antacid staining method, in addition to identify the structure of the cell parts (such as bud cells, flagella, nucleus, etc.) special staining method. Single stains and Gram stains are commonly used in microbiological tests. The Gram stain reaction is an important trait in the classification and identification of bacteria. It was created in 1884 by Gram, a Danish physician. The Gram stain not only observes the morphology of the bacteria, but also distinguishes all bacteria into two major groups: those with a bluish-purple staining reaction are known as Gram-positive bacteria, denoted by G+, and those with a reddish staining reaction (the color of the re-stain) are known as Gram-negative bacteria, denoted by G-. The different responses of bacteria to Gram staining are due to differences in the composition and structure of their cell walls. The cell wall of Gram-positive bacteria is mainly composed of a reticular structure formed by peptidoglycan, and during the staining process, when treated with ethanol, the pore size in the reticular structure becomes smaller due to dehydration, and the permeability is reduced, so that the crystal violet-iodine complex is retained in the cell and is not easily decolorized, and, therefore, shows a blue-violet color; the cell wall of Gram-negative bacteria has a low content of peptidoglycan and a high content of lipids, and when treated with When treated with ethanol, the lipids are dissolved and the permeability of the cell wall is increased, making the crystal violet-iodine complex easy to be decolorized by ethanol extraction and then stained with the color of the re-staining solution (re-staining red), therefore, showing a red color. Four different solutions are used for Gram staining: basic dye (primary stain); mordant; decolorizing agent; and counterstain. The primary dye used for Gram staining is generally basic dye primary dye crystal violet. The function of mordant is to increase the affinity or adhesion between the dye and the cells, i.e., to help the dye to fix on the cells in a certain way so that it is not easy to come off, iodine (iodine) is a commonly used mordant. Decolorizing agent is to decolorize the stained cells, different types of cells have different decolorizing reactions, some can be decolorized, some can't, decolorizing agent commonly used 95% alcohol (ethanol). Restaining solution is also a kind of alkaline dye, its color is different from the initial staining solution, the purpose of restaining is to make the decolorized cells stained with a color different from the initial staining solution, while the non-decolorized cells still maintain the color of the initial staining solution, and to distinguish the cells into two major groups, G+ and G-, and the commonly used restaining solution is the diluted carbolic acid restaining red solution.
Materials and Instruments
Bacteria Move I. Reagents and equipment Caveat 1. The key to the success or failure of Gram staining is the decolorization time; if the decolorization is excessive, Gram-positive bacteria can also be decolorized and mistaken for Gram-negative bacteria; if the decolorization time is too short, Gram-negative bacteria can also be mistaken for Gram-positive bacteria. Therefore, the decolorization time must be strictly controlled. 2. If you choose to use cultured bacteria for staining, it is appropriate to use 18-24 hours old bacteria, if the bacteria are too old, due to the death of the bacteria or autolysis often make Gram-positive bacteria turn negative. Common Problems I. Preparation of Gram staining solution 1. crystal violet solution For more product details, please visit Aladdin Scientific website.
Crystalline violet staining solution, iodine, ethanol, carbonate, carbonic acid.
Toothpick Microscope Slide Alcohol lamp Staining vat
1. Equipment: toothpicks, microscopes, slides, alcohol lamps, staining cylinders, etc.
2. Staining solution: one set of Gram stain (crystal violet stain, iodine solution, 95% ethanol, diluted carbolic acid compound red solution).
II. Production of bacterial specimen sheets
1. Smear
Place 1 drop of distilled water on a clean slide, pick a small amount of tartar with a toothpick, place it in the drop of water on the slide, mix it with the water to make a suspension and apply it in a thin layer of about 1 cm in diameter.
2. Drying
Smears are best allowed to dry naturally at room temperature. Sometimes, in order to dry them more quickly, the specimen can be placed facing upwards, holding the sides of one end of the slide, and carefully heating it slightly on high on an alcohol lamp to evaporate the water, but do not keep it close to the flame or heat it too long to prevent the specimen from withering and becoming deformed.
3. Fixed
The specimen is fixed as soon as it is dried, and the fixation serves three purposes:
(1) Kills microorganisms and immobilizes cellular structures.
(2) Ensure that the organisms adhere more firmly to the slide and prevent the specimen from being washed away by water.
(3) Change the permeability of the dye to the cell because dead protoplasm is easier to stain than living protoplasm.
Fixation often use high temperature, hand-held slide end (coated specimen distal end), specimen up, in the outer layer of the alcohol lamp flame as soon as possible back and forth through 3-4 times, a total of about 2-3 seconds, and from time to time to the back of the slide heated to touch the skin, do not feel overly hot is appropriate (not more than 60 ℃), placed to be cold, after the staining.
III. Coloring
1. Initial dyeing
Add 1 drop of crystal violet, about 1 minute, wash.
2. Mordanting
Drops of iodine solution are washed to remove residual water and covered for about 1 minute, rinsed in water.
3. Decolorization
Shake off the water on the slide and line it with a white background. Wash the slide with drops of 95% alcohol until the effluent alcohol is colorless or slightly mauve, about 20-30 seconds, and immediately rinse the alcohol with water.
4. Re-dyeing
Stain with diluted carbolic acid redox solution for 30 seconds and then wash with water.
5. Microscopy
After drying with absorbent paper, place the oil mirror to observe.
IV. Judgment of results
Gram-negative bacteria appear red and Gram-positive bacteria appear purple. The Gram stain reaction of dispersed bacteria is used as a guide; bacteria that are too densely packed often give false positives.
Crystal violet ethanol saturated solution (crystal violet 2 g dissolved in 20 mL of 95% ethanol) 20 mL, 1% ammonium oxalate aqueous solution 80 mL, the two liquids will be mixed well and filtered for 24 h. This solution is not easy to store, if precipitation occurs, it needs to be prepared again. This solution is not easy to store, if precipitation occurs, need to be reconstituted.
Iodine 1 g, potassium iodide 2 g, distilled water 300 mL. first dissolve potassium iodide in a small amount of distilled water, then add iodine to make it completely dissolved, and then add distilled water to 300 mL that is. After preparation, store in a brown bottle for spare, if it becomes light yellow, it can not be used.
Used for decolorization, after decolorization, one of the following (4) or (5) can be used for re-dyeing.
Alkaline red ethanol saturated solution (alkaline red 1 g, 95% ethanol l0 mL, 5% carbolic acid 90 mL mixed and dissolved into alkaline red ethanol saturated solution), take the carbolic acid red saturated solution of l0 mL plus 90 mL of distilled water that is made.