🔬 Declining Electron Microscopy

Bacteriophage Ecology Group News (BEG News) was published mostly quarterly as an online newsletter for a total of 24 issues, July 1999 through April 2005. As follows is a reprint of an article from Volume 21. The newsletter’s successors are the ongoing Phage.org website, phage-therapy.org, and the Bacteriophage Ecology Group Facebook page.

Declining Electron Microscopy

Twenty years ago there were many electron microscopes and microscopists. My medical school alone had four Philips EM 300 microscopes. There is now only one, mine. Two others, though in perfect order, were dismantled. The fourth EM was sold to the U.S.A., which says a lot about the budget cuts that Canadian science has been subjected to. The general situation is that both electron microcopes (EM) and electron microscopists are endangered species.

A Huge Drop in Quality

It also used to be that phage papers included reasonably good micrographs. No longer. In the last 10 years standards have plunged and scores of terrible pictures, graced with improbable dimensions or no dimensions at all, have been published. Gone are such fine electron microscopists as D.E. Bradley in Canada, E. Kellenberger in Switzerland, or A.S. Tikhonenko in Russia, who led by example and kept standards up. Many (most?) phage pictures in the present literature are vastly inferior to those published in 1959 by Brenner and Horne, the fathers of negative staining (1).

This decline of EM is not seen in vertebrate and plant virology, where editorial standards are still high and poor electron micrographs and poor descriptions, as they are now frequent in phage papers, would probably be rejected. What happened? The reasons for the decline are manifold: High costs of electron microscopes. Run-away costs of EM service contracts. Shifting of research interests to molecular biology (cloning, sequencing). Retirement of experienced electron microscopists. Disappearance of EM courses. Contract research ('farming out'). Soft standards of journals: a reviewer system in disrepair.

'Farming out' of EM to other laboratories is due to the rarefaction of electron microscopes and microscopists, plus the perception of administrators that electron microscopy is a simple service. Nothing could be less true. While 'farming out' to a reputed research laboratory may be acceptable, this is not so in the case of commercial laboratories. It is just unlikely that an ordinary technician, ignorant of the project in question, lacking time and supervision, and trained in, say, pathology, is able to produce good pictures of bacterial viruses. Yet these laboratories take good money for poor service. The problem is not confined to North America. I have seen terrible examples from Australia.

Since general standards have gone down, reviewers of periodicals, even reputed ones, now frequently tolerate poor pictures. Journals of the American Society of Microbiology are no exception. The situation is particularly bad in environmental microbiology journals.

Why Electron Microscopy?

EM provides instant identification of individual phages, phage families and, very often, genera and species. It cannot be replaced by molecular probes or genome sequencing. Probes will 'catch' part of a phage genome only, the sequencing and annotation of a phage genome may take a year or more, but EM identification may take as little as 1-2 minutes. The domain of EM is the description and identification of novel phages, classification, environmental research, and purity checks and identity controls of phages with practical applications (e.g., therapeutic or typing phages). EM cannot replace molecular biology and cannot be replaced by the latter. Both techniques are complementary. An EM is an expensive precision instrument that, when properly handled, produces data close to the molecular level.

EM pictures are easily archived, directly comparable, permanent documents. In contrast, scientists change institutes, countries, and jobs, retire, or die. It is even worse with phages because, as a rule, phages described more than five years ago can no longer be obtained. Thus, EM pictures are often the only permanent results of a scientist's activity and the only records of past observations. Therefore, good EM is a must.

The Disease

It is now common to see unsharp, astigmatic, low-contrast, or scratched pictures of impure phages, without scale markers or dimensions. Some phages are barely recognizable as such, especially in environmental papers, and some papers just mention 'head-tail phages' without dimensions and micrographs. It is surprising to see such a deterioration because the literature abounds in descriptions of basic electron microscopical techniques. Clearly, this literature is not consulted. Why? Because it is not on the Net?

Staining artifacts (e.g., capsid shrinkage after positive staining with uranyl acetate) are consistently ignored. Some people present capsular slime is as a 'tailed phage,' feel the need to fix phages with glutaraldehyde (useless), or stain with uranyl acetate for 15 minutes (instead of 30 seconds). More seriously, the 'Materials' sections of phage papers are generally silent on phage purification and magnification control. Together with poor pictures, this indicates to the insider that people examined crude lysates and confided into manufacturer indications on magnification (ignoring that the magnification of electron microscopes varies with the electrical current and must be controlled or adjusted).

This kind of 'science' is useless because it produces terrible data and does not help fellow scientists. Concretely, phage workers, who isolate new phages and want to compare their viruses to those of the literature, often face the problem that data from other laboratories cannot be interpreted. It follows that an individual who practices poor EM, and produces pictures which other scientists cannot use, does a disservice to the scientific community.

Diagnosis and Cure

The principal problems seem to be examination of crude or 'dirty' lysates, absence of magnification control, and poor contrast. The first can be addressed by simple washing in a buffer. Phages must be freed from proteins and sugars of the medium. No lengthy density gradient purification is necessary; it suffices to wash the phages 1-2 times in 0.1 M ammonium acetate (best) or phosphate buffer using, for reduction of time and g forces, a medium-sized centrifuge with a fixed-angle rotor; for example, 25,000 g for 1 hour are enough for all but the smallest phages. For magnification control, one must include into each film or cassette load 1-2 pictures of an internal standard. One may use T4 tails (length 113 nm). Then: please measure your phages. It takes a few minutes only. The phages must be so described so that other people can use your descriptions. This is for posterity! The contrast problem is one of basic photography and can be solved by selecting the right photographical papers and developers.

A deadly problem arose a year ago when Kodak Company (Rochester) changed abruptly its production line. Customers were not informed and no explanation was offered. Suddenly, the excellent Ektamatic paper, which lent itself to automatic processing, was no longer available. Kodak representatives could not be contacted or did not know their own products. Fortunately, fellow electron microscopists from the Armand-Frappier Institute near Montreal suggested to me the use of Kodak Polycontrast III RC paper in conjunction with the usual Dektol or the novel Polymax T developers.

My experience is that the paper provides excellent, if sometimes too strong contrast. It allows the salvage of underexposed or underdeveloped films, but grey shades may be lost. All development must be done manually because small table-top Kodak or Ilford processors can no longer be used. The development is followed by a stop bath, fixation and washing as usual, but drying and glazing in a machine are no longer necessary because the paper is resin-coated and naturally glossy, and dries in a short while. Caution: violet smears will develop if the the Dektol developer is not totally neutralized.

(1) Brenner, S. and Horne, R.W. 1959. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34:103-110.

Hans-Wolfgang Ackermann
Félix d'Hérelle Reference Center for Bacterial Viruses, Laval University, Québec, Canada

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