![]() In 2015, HMI combined with principal component analysis (PCA) was used for the early and rapid identification of Salmonella serotypes. In 2013, HMI was used with a k-nearest neighbor (k-NN) classifier to detect and classify non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups. The HMI technology makes it possible to identify bacteria. Because the optical microscope is restricted by the diffraction limit, for small-sized bacteria, the difference between them cannot be observed from the morphology. At present, HMI technology has been widely used in the identification and analysis in biologies, such as classification of fungi, detection of nerve fibers, identification of microalgae, and location or detection of cancerous tissues/cells. Hyperspectral microscopic imaging (HMI) is a non-destructive, non-contact, and advanced detection technology that can simultaneously obtain two-dimensional morphological characteristics and one-dimensional spectral characteristics of target objects and form a three-dimensional hyperspectral data cube. Considering that this slight staining difference is difficult to observe with naked eye or ordinary color cameras, hyperspectral microscopy has been employed in the present study. This difference can be used to realize a more precise classification of Gram-stained bacteria. In fact, the slight difference in pH values causes different ionization of the auxochrome of crystal violet (CV) molecules during the Gram stain process, leading to a slight difference in the staining effect. The pH value of the intracellular environment of bacteria affects the electrostatic interaction, thereby affecting the effect of Gram stain. Considering that Gram stain is currently one of the most common staining techniques for making bacteria visible, its effectiveness will be further enhanced if bacteria can be classified more precisely through Gram stain. Because these two species of bacteria have different effects on plants, we expect that they can be labeled specifically by staining. However, they cannot be classified via Gram stain because these two species of bacteria are Gram-positive and have a similar morphology. cereus in the soil can be isolated from the roots of plants. However, this method is relatively inaccurate and is unfavorable for the subdivision of bacterial species. ![]() ![]() Therefore, bacteria can be classified as Gram-positive or Gram-negative according to their colors. Those stained purple are Gram-positive bacteria with the pH values ranging from 1.75 to 4.15, while those stained pink are Gram-negative bacteria with the pH values ranging from 2.07 to 3.65. One group of bacteria forms a strong electrostatic bond with the dye and is stained purple in the primary stain, while another group of bacteria forms a weak electrostatic bond with the dye, thereby dye-losing after decolorization, following which they are stained pink in the counterstain. This method of staining differentiates bacteria into two large groups. Conventional Gram stain involves the following four steps : primary stain, mordant, decolorization, and counterstain. This stain method utilizes the principle of electrostatic interaction between dye and microorganism to realize staining and has the advantages of a simple staining procedure and high staining efficiency. Gram stain is a microbial staining technique that was first devised by Hans Christian Gram in 1884. We believe this method can be used for other Gram-positive bacteria and Gram-negative bacteria, realizing a more elaborate classification for Gram-stained bacteria. The results show that the classification accuracy of two species of Gram-positive bacteria, B. Besides the spectral features, the spatial features were also used to improve the quality of bacterial identification. Harnessing hyperspectral microscopy can capture this subtle difference and enable precise classification. Consequently, there is a subtle difference in the absorption peak of Gram-stained bacteria. The pH-value differences in the intracellular environment of various types of bacteria can lead to different ionization of the auxochrome of crystal violet (CV) molecules during the Gram stain process. Here, we present a method to precisely classify Gram-positive bacteria via hyperspectral microscopy. cereus) have different effects on plants, nonetheless, they are both Gram-positive and difficult to be differentiated. For instance, soil-rhizosphere bacteria, Bacillus megaterium ( B. However, such an inaccurate classification is unfavorable for bacterial research. Gram stain is one of the most common techniques used to visualize bacteria under microscopy and classify bacteria into two large groups (Gram-positive and Gram-negative).
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