TITLE:
Comparative Investigation of Alternative Negative Staining Reagents in Bacterial Morphological Study
AUTHORS:
Masaaki Minami, Hiroshi Takase
KEYWORDS:
Negative Staining, Electron Microscope, Bacterial Micromorphology, Phosphotungstic Acid, EMstainer
JOURNAL NAME:
Journal of Biosciences and Medicines,
Vol.5 No.10,
October
23,
2017
ABSTRACT: Negative staining is an effective method that can be used for electron
microscopic study to observe fine structural morphology without destruction of
bacterial structure. Although uranium acetate is used worldwide as a general
dyeing solution, 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, we examined alternative reagents for negative
staining that could replace uranium acetate through bacterial observation with
an electron microscope. 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 this study, there was clear
difference in the observation conditions depending on the type of bacteria and
the kind of the staining reagents. Especially, it was confirmed that good
negative staining features of Pseudomonas
aeruginosa by electron microscope were obtained by PTA and EMstainer
staining. These alternative reagents are considered to be a candidate for a
negative staining.