Abstract
The cyanobacterium Mastigocladus laminosus (M. laminosus) is one of the most morphologically complex prokaryotes. It forms long chains of cells that are connected via septal junction complexes; such complexes allow diffusion of metabolites and regulators between neighboring cells. Cellular division occurs in multiple planes, resulting in the formation of true branches, and cell differentiation leads to the formation of specialized cell types for nitrogen fixation (heterocysts) and culture dispersal (hormogonia and necridia). Here, we describe a detailed protocol for the preparation of M. laminosus for TEM in order to visualize the ultrastructural properties of the organism. The presented preparation method is based on adding potassium permanganate as fixative which has been shown to increases the contrast of membranes (Luft, 1956), making it suitable for studies in cyanobacteria where the visualization of the photosynthetic membranes is important.
Keywords: Ultrastructure, Fischerella, Heterocyst, Hormogonia, Branching cyanobacteria
Materials and Reagents
Equipment
Procedure
Note: Some chemicals used in this protocol are highly toxic and should be used only under the fume hood with adequate precautions. Please check the MSDS before using them. Preparation of samples for ultra-thin section transmission electron microscopy requires multiple days. The timeline for the different phases of preparation is shown in Table 1. Table 1. Timeline for preparation of samples for transmission electron microscopy Days 1-7 Growth of M. laminosus (A) Day 8 Fixation and pre-embedding (B); Storage for up to 3 months possible Days 9-11 Dehydration and embedding in Araldite (C); Indefinite storage possible Day 12 Sectioning and post-staining (D); Storage for up to 1 year possible Day 13 Visualization at the transmission electron microscope
Recipes
Acknowledgments
This work was supported by a college studentship of Queen Mary University of London.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.