National Institute of Cholera and Enteric Diseases

Indian Council of Medical Research
Department of Health Research, Ministry of Health and Family Welfare, Government of India
WHO Collaborating Centre for Research and Training on Diarrhoeal Diseases

Electron Microscopy

About Electron Microscopy

Electron Microscopy

Electron microscopy at the National Institute of Cholera and Enteric Diseases started in the early 80’s with the installation of a Philips state-of-the-art transmission electron microscope, model 420T. One high vacuum evaporator (shadow caster), Polaron E6100, was also installed along with the EM. Later on one LKB Nova ultramicrotome, a LKB 7800 knifemaker , one De Vere enlarger, one JEOL JEE-400 high vacuum evaporator along with a JEOL HDT400 hydrophilic treatment apparatus were also installed. At the beginning of the new millennium the laboratory was upgraded to a cryoEM laboratory. To this end, one FEI Tecnai 12 BioTwin transmission electron microscope with a Gatan cryostage, one Leica EM CPC universal cryo-workstation, one Leica Ultracut UCT ultramicrotome with FC6 cryo attachment were installed in the laboratory.

The electron microscope is used primarily for research and occasionally for diagnosis. The techniques in routine use are negative-staining analysis, Kleinschmidt’s protein monolayer technique of DNA, partial denaturation mapping of DNA, heteroduplex analysis of DNA, protein-free spreading methods of DNA and RNA, immunoelectron microscopy, ferritin labellig, ultramicrotomy, darkfield electron microscopy and electron diffraction, cryoelectron microscopy, three-dimensional image reconstruction and tomography, environmental scanning electron microscopy and atomic force microscopy.

There are several projects going on in the laboratory. Characterization of several choleraphages is being done by electron microscopy. This laboratory, for the first time, showed the filamentous nature of RS1-KmΦ phage of V. cholerae. The morphology of different choleraphages and their DNA have been characterized. This laboratory, for the first time, constructed partial denaturation maps of DNA of vibriophages which have been used for the determination of cohesive ends, circular permutation, terminal redundancy, packaging pattern of DNA in the phage head. Also for the first time, three-dimensional structure of vibriophages has been determined using cryoelectron microscopy and single-particle analysis methods. Also packaging of DNA inside these phages has been determined using cryoelectron microscopy.

Also the structure of several hemagglutinins of Vibrio cholerae and Shigella dysenteriae has been determined using negative staining methods. Now the 3-D structures of these molecules are being worked out using cryoelectron microscopy. Hydrodynamic properties of the flagella of Vibrios have been studied as well.

Fimbriae or pili play a vital role in the attachment of bacteria to the human intestinal cell wall. These have been studied extensively in different serovars of Vibrio cholerae and Escherichia coli. Mechanism of pathogenesis of V. cholerae and E. coli has also been studied. Presence of a capsular layer in a new strain of Vibrio cholerae has been confirmed with the help of ferritin labelling method.

Histopathological changes caused by different enteric pathogens have been studied by light microscopy. Surface structural changes and in-depth ulltrastructural changes are being studied using scanning and transmission electron microscopes. Few of the important enteropathogens studied so far are: Vibrio cholerae, Helicobacter pylori, Shigella and Aeromonas hydrophila.

Some collaborative research was also carried out in the laboratory. Amongst these, studies on chlorpromazine induced aggregation of normal human hemoglobin and protein folding activity of ribosomal RNA merit special mention.

Several national workshops on electron microscopy were also organized by this division with great success.

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