In microbiological testing and experimental research, Gram staining and biological microscopy are inseparable core combinations. Gram staining, as a classic bacterial identification staining technique, can classify and distinguish bacteria according to differences in bacterial cell wall structure. Biological microscopy, especially oil microscope systems, is a tool for observing staining results and identifying bacterial morphology. The two complement each other, becoming the basic core technology for preliminary identification of bacteria, clinical infection diagnosis, and basic research on microorganisms, and are used in many fields such as medical testing, food testing, and environmental monitoring.
Founded in 1884 by Danish physician Hans Christian Gramm, the Gram method is a fundamental and important differential staining method in microbiology. The core principle of this technology is based on the differences in the structure and composition of the bacterial cell wall. The bacterial cell wall is mainly composed of peptidoglycan and lipid, and the cell wall structure of gram-positive bacteria and gram-negative bacteria is very different: the cell wall peptidoglycan layer of gram-positive bacteria is thick, the lipid content is low, and the network structure is dense; while the cell wall peptidoglycan layer of gram-negative bacteria is thin and loose, and the outer layer is rich in lipid outer membrane. This difference in structure is the fundamental basis for Gram staining to be able to distinguish between the two types of bacteria.
The complete Gram dyeing operation is divided into five core steps: smear fixation, primary dyeing, mordant dyeing, decolorization, and double dyeing. Each step directly affects the final test results. First, apply the bacterial solution to the slide evenly, and fix it by flame after natural drying, which can not only kill the bacteria, but also make the bacteria firmly adhere to the slide to avoid subsequent flushing and falling off. Subsequently, ammonium oxalate crystalline violet was used for initial dyeing for one minute, so that all bacterial bacteria were dyed purple; then, Lugo iodine solution was added for mordant dyeing for one minute, and a stable crystalline violet-iodine complex was formed inside the bacteria, which was firmly bound to the bacterial cells. Among them, ethanol decolorization is the most critical and error-prone step in the entire dyeing process: dropwise addition of 95% ethanol decolorization for 20 to 30 seconds, the thick and heavy peptidoglycan layer of gram-positive bacteria will quickly dehydrate and shrink, the mesh will be closed, the purple complex will be locked in the cell, and the bacteria will remain purple; while the lipid outer membrane of gram-negative bacteria is dissolved by ethanol, the cell wall permeability will be greatly improved, the crystal violet-iodine complex will be eluted, and the bacteria will become colorless. Finally, after re-dyeing with saffron staining solution for one minute, the decolorized gram-negative bacteria will be dyed pink or red, while the gram-positive bacteria still retain the initial blue-violet color, and the two types of bacteria can be clearly distinguished.
Simple staining cannot complete bacterial identification. It is necessary to rely on a biological microscope for microscopic observation in order to visually identify the staining results, morphological size and arrangement of bacteria. Bacterial bodies are actively tiny, and the transmittance of undyed bacteria is close to that of aqueous solutions, making it difficult to observe clear contours under ordinary microscopes, which is why dyeing operations are essential. The open-field biological microscope used in conventional experiments is equipped with four-level objectives, namely 4 × scanning objectives, 10 × low-power objectives, 40 × high-power objectives, and 100 × oil-immersed objectives. Different objectives perform their duties and position the observation field step by step.
In the microscopy operation, the observation principle of "from low to high" should be followed. First, scan the slide with a 10 × low-power objective lens, find a uniform bacterial smear area and move it to the center of the field of view to judge whether the thickness of the smear is qualified; then switch the 40 × high-power objective lens to further confirm the field of view, and eliminate interference such as impurities and thick bacterial membranes. The final observation of bacterial staining results and morphology must use a 100 × oil immersion objective, which is the core configuration of bacterial microscopy. Due to the large refractive loss and insufficient resolution of the high power dry objective, it is not possible to clearly distinguish tiny bacteria, so cedar oil needs to be added to the slide specimen. The refractive index of cedar oil is close to that of slide glass, which can effectively reduce light refraction and loss, greatly improve the resolution of the microscope, and with a 10x eyepiece, it can achieve a total magnification of 1000 times, which is enough to clearly observe the color, morphology and arrangement characteristics of bacteria. After the end of the microscopy, it is necessary to wipe the oil lens with a special lens wipe paper in time to avoid residual corrosion of the cedar oil lens and affect the accuracy of the equipment.
In the practical process, the staining technology is highly linked to the error observed by the microscope, and any operational error will lead to erroneous interpretation of the results. If the smear is too thick, the bacteria will be stacked, and the decolorization of ethanol will not be sufficient, so that the gram-negative bacteria will appear false positive; if the decolorization time is too long, the cell wall of the gram-positive bacteria will be damaged, the pigment loss will occur, and the false negative will occur. At the same time, aging strains cultured for more than 24 hours will be damaged due to aging of the cell wall, and the staining results will be biased; while the oil, bubbles in the field of view, and inappropriate light and darkness of the microscope lens will also interfere with color recognition, and it is impossible to accurately distinguish purple positive bacteria from red negative bacteria. Therefore, standard staining operations, appropriate strain status, and standard microscope usage methods are the three keys to ensure the accuracy of experimental results.
The combination of Gram staining and biological microscopy has the advantages of simple operation, fast and efficient, low cost, and high practical value. In clinical medicine, the types of pathogenic bacteria can be quickly distinguished to provide a preliminary basis for the selection of clinical antibiotics. Gram-positive bacteria are often sensitive to penicillin drugs, and gram-negative bacteria are more suitable for quinolones, third-generation cephalosporins and other drugs. In food and environmental testing, the contamination of pathogens in water and food can be quickly screened. In microbiological teaching and scientific research, it is a basic means to observe bacterial morphology and classification and identification.
In summary, the Gram staining method achieves the specific color rendering of bacteria, and the biological microscope provides visualization conditions for the observation of microscopic bacteria. The two complement each other and are indispensable. Proficient in dyeing operation specifications and microscope use skills, avoiding practical errors, and being able to accurately complete preliminary identification of bacteria, is the basic ability to carry out microbiological experiments and clinical tests, and also lays an important foundation for subsequent in-depth studies such as bacterial isolation and culture, drug susceptibility tests, etc.
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