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Related Concept Videos

Infrared (IR) Spectroscopy: Overview01:09

Infrared (IR) Spectroscopy: Overview

When electromagnetic radiation passes through a material, atoms or molecules transition from a lower to a higher energy state by absorbing radiation corresponding to the energy difference between the two states. The absorption of infrared (IR) radiation causes transitions between vibrational energy levels in a molecule. Therefore, IR spectroscopy is a useful analytical tool for determining the molecular structure of molecules.
Different compounds display unique properties due to their...
IR Spectrum01:19

IR Spectrum

When infrared (IR) radiation passes through a molecule, the bonds stretch or bend by absorbing the radiation. This absorption creates the molecule's absorption spectrum, which is the plot of its percentage transmittance versus wavenumber.
Transmittance is defined as the ratio of the radiant power passing through a sample to that from the radiation's source. Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete...
IR Spectrometers01:25

IR Spectrometers

There are two main infrared (IR) spectrophotometers: dispersive IR spectrometers and Fourier transform infrared (FTIR) spectrometers. In a dispersive IR spectrometer, a beam of infrared radiation produced by a hot wire is divided into two parallel equal-intensity beams using mirrors. One beam passes through the sample, while another is a reference beam. The beams then move through the monochromator, which separates the radiations into a continuous spectrum of different frequencies. The...
Fixation and Sectioning01:03

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...
Differential Staining Technique01:26

Differential Staining Technique

Differential staining is an essential microbiological technique that exploits variations in cell wall structures to classify and identify microorganisms. It facilitates the distinction of bacteria, aiding in diagnostic and research applications. Two of the most widely used differential staining methods are Gram staining and acid-fast staining, both of which rely on the chemical and structural differences in bacterial cell walls.Gram Staining TechniqueGram staining differentiates bacteria by...
Special Staining Techniques01:13

Special Staining Techniques

Specialized staining techniques play a vital role in microbiology by enabling the visualization of specific bacterial structures that remain undetectable with standard microscopy methods. These techniques not only enhance the structural visualization of bacterial cells but also provide critical insights into their pathogenicity and classification. Additionally, they support diagnostic and research endeavors in microbiology by identifying key bacterial features.Capsule Staining for Virulence...

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Updated: Jul 25, 2026

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)
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Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Published on: November 1, 2017

The infrared spectroscopic Gram stain.

P L Lang1, J L Hodges, C D Keefer

  • 1Department of Chemistry, Ball State University, Muncie, IN 47306, USA. plang@gw.bsu.edu

Cellular and Molecular Biology (Noisy-Le-Grand, France)
|April 9, 2002
PubMed
Summary

Attenuated total reflectance (ATR) infrared microscopy can differentiate Gram-positive and Gram-negative bacteria directly on agar. This method offers high accuracy and better spectral information than specular reflectance for bacterial Gram staining analysis.

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Area of Science:

  • Microbiology
  • Spectroscopy
  • Analytical Chemistry

Background:

  • Accurate bacterial identification is crucial in clinical and research settings.
  • Traditional Gram staining is a fundamental microbiological technique.
  • Spectroscopic methods offer potential for rapid, non-destructive analysis of bacterial properties.

Purpose of the Study:

  • To evaluate the efficacy of attenuated total reflectance (ATR) infrared microscopy for in situ Gram stain differentiation of bacterial colonies.
  • To compare the performance of ATR spectroscopy with specular reflectance for Gram stain analysis.
  • To explore the potential of ATR spectra for assessing bacterial cell wall variability.

Main Methods:

  • In situ ATR spectra acquisition from bacterial colonies on agar using an infrared microscope.
  • Principal components regression (PCR) analysis for spectral data interpretation.
  • Cross-validation experiments using standard bacterial sets (31 and 44 species).

Main Results:

  • High correlation (0.9502 and 0.9520) between ATR spectra and Gram stain properties was achieved.
  • ATR spectroscopy demonstrated superior differentiating capabilities compared to specular reflectance.
  • Bacteria grown on blood agar also yielded reliable ATR spectra for Gram stain differentiation.
  • ATR spectral values can quantify Gram variability, showing shifts as cell walls deteriorate.

Conclusions:

  • In situ ATR infrared microscopy is a powerful, accurate technique for bacterial Gram stain differentiation directly on growth media.
  • ATR spectroscopy provides enhanced spectral information and predictive capabilities for bacterial analysis.
  • This method holds promise for rapid, non-destructive identification and characterization of bacteria.