Comparative Investigation of Alternative Negative Staining Reagents in Bacterial Morphological Study

Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. And uranium acetate is used worldwide as a general dyeing solution in this method, but it is extremely difficult to use it by a new purchase at a research institution because it falls under the nuclear regulation substance in Japan. Therefore, the authors in this study examined alternative reagents for negative staining that could replace uranium acetate through bacterial observation with an electron microscope. 

In the study, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes were examined by four stain reagents (phosphotungstic acid (PTA), EMstainer, TI blue, and uranium acetate). Pre-cultured bacteria were stained with each stain reagents on a copper grid, washed with PBS, and observed with a transmission electron microscope. In the comparison between bacterial structures, the cell wall structure and bacterial flagella could be observed well in the order of PTA, EMstainer, and uranium acetate. With TI blue staining, flagella could be observed very poorly. In comparison between bacteria, gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, could be observed well as compared with gram positive cocci such as Staphylococcus aureus and Streptococcus pyogenes. The uranium acetate looked very coarse in background particles. Since crystals tend to precipitate, TI blue also required filtering, and electron beams were absorbed by the agglomerated crystals, and the frequency of electronic burning occurred high frequency.

In conclusion, there was clear difference in the observation conditions depending on the type of bacteria and the kind of the staining reagents in this study; especially, it was confirmed that good negative staining features of Pseudomonas aeruginosa by electron microscope were obtained by PTA and EMstainer staining. So these alternative reagents are considered to be a candidate for a negative staining.

Article by Masaaki Minami and Hiroshi Takase, from Nagoya City University, Nagoya, Japan.

Full access: http://mrw.so/17LXW1

Image by Oak Ridge National Laboratory, from Flickr-cc.