One of the principles of bacterial identification is to complete the identification with the least number of tests. Morphological examination of bacteria is the most basic, simple and rapid routine identification method. Bacterial morphology includes bacterial cell morphology and bacterial colony morphology. The correct description of bacterial morphology depends on the method, culture medium, culture time, staining reagent and so on. Different culture media and culture time will affect the morphology of bacteria. The following is my understanding of the navigation function of morphological examination in bacterial identification. Welcome to reading.
1 observation of population characteristics
Bacteria grow on the surface of solid medium to form colonies. The description of colonies should include colony size (divided into three types, namely, large, medium and small, bounded by 1mm), shape, height, edge, surface state, density (transparency), hardness (feeling when scraping colonies with inoculation ring), internal structure, color, luster, adhesion to the surface of culture medium, hemolysis, odor and pigment production of colonies. The purpose of observing colony characteristics is to preliminarily identify bacteria. Some characteristics are unique to some bacteria and have the identification value of genus or species. For example, the umbilical fossa colony of Streptococcus pneumoniae on sheep blood agar plate (as shown in figure 1), the special internal structure of Actinobacillus colony (as shown in figure 2), the hat-like colony eroding Aiken, the wrinkle-like colony of Pseudomonas stutzeri, the migratory colony of Proteus, the water-soluble pigment of Pseudomonas aeruginosa, the fat-soluble pigment of Porphyromonas (as shown in figure 3) and the red pigment of Serratia marcescens (as shown in figure 4). Indole odor produced by Staphylococcus aureus, rat hole odor produced by Haemophilus, rotten soil odor produced by Nocardia, rotten ammonia odor produced by anaerobic bacteria, yellow fluorescein produced by Pseudomonas fluorescens, brick red fluorescein produced by some anaerobic bacteria, etc. Some bacteria produce pigment under the influence of gas (Serratia marcescens does not produce color in anaerobic environment), some bacteria produce pigment under the influence of light (some fast-growing mycobacteria produce color from light or dark).
Observation and description of bacterial colony characteristics is the first step of bacterial identification, and its main purpose is embodied in the following aspects.
1. 1 Quantitative or semi-quantitative of pathogenic bacteria After quantitative or semi-quantitative inoculation of specimens, quantitative or semi-quantitative results can be obtained through colony counting. Quantitative analysis of bacteria has clinical significance in urine analysis and dynamic observation of curative effect, and semi-quantitative analysis is suitable for the result analysis of open specimens (determining dominant bacteria).
1.2 It is more likely to confirm that the open pathogen specimen is contaminated by the common flora of skin and mucosa around the inflammatory tissue. When observing the culture results, the pathogenic bacteria should be determined according to the results of direct smear microscopy and the morphological characteristics of colonies.
1.3 When interpreting the results of blood culture microscopy and analyzing the transfer results of positive blood culture bottles, we should combine the results of direct smear microscopy with positive culture solution. If the smear results are inconsistent with the culture results, operational errors or contamination should be considered.
1.4 According to the morphological characteristics of colonies on different media, combined with the results of Gram staining microscopy and rapid auxiliary tests (oxidase, catalase, coagulase, etc.), the identification direction was determined. ), the identification direction can be determined.
2 observation of bacterial morphology
Gram-stained cells morphology is the basis of bacterial taxonomy. Clinical microbiology laboratory still uses the method of cell morphology to identify bacteria. Mastering the standard methods of cell morphology can provide a reliable guarantee for accurate identification of bacteria. Observing and describing the morphological characteristics of bacterial cells is the second step of bacterial identification, and its main purpose is embodied in the following aspects.
2. 1 Explain the culture results and determine the identification direction. Whether it is liquid culture (such as blood culture, direct enrichment culture of specimens) or solid culture (agar plate inoculation), smear staining should be carried out to determine the nature of the culture, so that it can be reported quickly and point out the direction for the next identification. Fig. 5 shows the microscopic results of Gram staining of blood culture positive culture smear.
2.2 means to identify bacteria The morphology of bacteria can be combined with the morphological characteristics of colonies to identify bacteria. For example, it is easy to distinguish whether the grass green hemolytic colony growing on the selective chocolate plate (containing vancomycin) is Lactobacillus or Enterococcus (VRE) by smear microscope. The colony morphology of Acinetobacter baumannii is similar to that of Klebsiella, with the difference that the former is gram-negative cocci (proximity cocci) and the latter is gram-negative bacilli; The colony morphology of erysipelas on blood plate is very similar to that of Streptococcus viridis, but it is quite different under microscope. Figure 6 shows the morphology of erysipelas under the Gram staining microscope.
2.3 Basis for identification direction and reagent selection The identification direction of Staphylococcus and Streptococcus, Gram-positive cocci and Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli are all different, and the selection of identification reagents is also different.
2.4 Selection of drug sensitivity combinations According to CLSI literature, each bacterium has a certain combination of antibacterial drugs to choose from. In order to select the drug sensitivity combination accurately, it must be supported by accurate cell morphology results.
Observation on the special structure of three kinds of bacteria
3. 1 capsule is a layer of mucus wrapped by some bacteria outside the cell wall. Different bacteria have different capsule components, which are generally composed of polysaccharides, peptides or protein. It is necessary to observe the capsule as a virulence factor of bacteria, but capsule staining is of little value in identifying strains isolated from clinical specimens. Fig. 7 shows the results of E.coli capsule staining.
3.2 flagella observation of bacterial flagella is of great value for bacterial identification. According to the position and quantity of flagella, bacteria can be divided into polar flagella, double flagella, Pleistocene flagella, periflagella and lateral flagella. Flagella is the dynamic organ of bacteria. In most cases, the existence of bacterial flagella can be verified by dynamic test, but dynamic test can not replace flagella staining for bacterial identification. Fig. 8 shows the results of flagella staining of Vibrio cholerae.
3.3 Spore morphology includes spore shape, spore position and sporangium enlargement. The shape of spores can be divided into cylindrical, oval and spherical, and the position of spores can be divided into terminal, proximal, middle or near middle. According to the spore morphology, Bacillus and Clostridium can be distinguished. Bud staining method is not superior to Gram staining method, and spore observation by phase contrast microscope is superior to bud staining method and Gram staining method. Fig. 9 shows the staining results of Clostridium difficile spores.
3.4 metachromatic granules is mainly composed of polyphosphate, which becomes larger with the extension of bacterial age. Polyphosphate particles have a special reaction to some dyes, resulting in a different color from the dyes used, hence the name metachromatic granules. If dyed with toluidine blue and methylene blue, it is not blue but purple. Corynebacterium and some Bacillus often contain this metachromatic granules. Metachromatic granules of Corynebacterium diphtheriae is located at both ends of the cell, so it is also called polar body, which is helpful for the identification of Corynebacterium diphtheriae. Fig. 10 shows the result of metachromatic granules staining of Corynebacterium diphtheriae.
To sum up, the routine identification of bacteria in clinical microbiology laboratory can not be separated from morphological examination. It is hard to imagine that Gram staining and morphological examination errors will get correct identification results. Morphological examination plays an important role in bacterial identification. Navigation? Role, has important value, clinical microbiology laboratory workers should pay attention to.
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