Legend has it that Murphy's law was formulated in 1949 by a Captain Edward A. Murphy, then at Edwards Air Force Base in California.15
There are several variants, all meant to express a perverse outcome. Murphy's law certainly applies to bacteriophage electron microscopy.
This type of investigation is a multi-step procedure that depends on expensive and complicated instruments, refined techniques, bacteriophages, and, not least, the investigator. Problems and errors beset every step. Artifacts in particular have received little attention and will be the focus of this article.
Negative staining of viruses, arguably the technically simplest and most important single method in virology, was introduced in 1956. Hall and later Brenner and Horne used phosphotungstic acid for contrasting plant viruses.8,12
This technique was extended in 1959 to coliphage T2.9
Viruses and their components stood out as white on a dark background with unprecedented clarity. Negative staining by phosphotungstates rapidly superseeded shadowing for virus visualization. Uranyl salts and ammonium molybdate were introduced later. The standards of viral electron microscopy were set in the 1970s.11,20,21
Today, negative staining is done almost exclusively with uranyl acetate (UA) and phosphotungstate (PT) salts (
and has been applied to thousands of viruses. At present, at least 6,300 bacterial and archaeal viruses have been examined in the electron microscope after negative staining.6
Figure 1.Vibrio parahemolyticus phage KFP40. The phage is very similar to coliphage T4, but has a longer head and a larger genome. Negative staining with uranyl acetate. Tail fibers are folded along the tail.
Vibrio parahemolyticus phage KFP40. The phage is very similar to coliphage T4, but has a longer head and a larger genome. Negative staining with phosphotungstate. Tail fibers are unfolded.
Transmission electron microsocpes (TEMs) fall into three categories. (1) Conventional or “manual,” also called “analogue” microscopes using mechanical devices for stage drive and aperture alignment and analog potentiometers or variable resistors for electronic controls. Images are recorded on photographic film or plates. (2) “Digital” microscopes using far fewer controls due to a computer-based system with digital potentiometers, electronic stage drives, and a digital alignment via a centralized computer interface. Objects are generally visualized on a monitor screen and images are acquired via a “charged-couple device” or CCD sensor, replacing film or plates as the recording media. (3) Hybrids or conventional microscopes equipped with a digital camera.
Since about 1985, “manual” electron microscopes were gradually replaced by “digital” instruments. These instruments were marketed almost simultanously by three major competing companies that did so with meager or no instructions relating to contrast and image quality. An additional modification by microscope manufacturers was to change the focal length of the objective pole piece (zeta or Z angle) to increase fidelity at the cost of reduced contrast. All these factors could explain a wave of poor electron microscopical images1
which, seemingly, has not abated. Unfortunately, this wave coincided with the death of several famous and highly skilled electron microscopists, such as E. Kellenberger in Switzerland, D.E. Bradley in Canada, and A.S. Tikhonenko in Russia, who could have stemmed the tide.
To assess this problem, one of us (HWA) analyzed 155 publications with phage micrographs originating in 28 countries, published between 2007 and January 2012 in over 50 journals. Two-thirds (109) were of poor quality, namely contrastless, unsharp, astigmatic, of small size and low magnification (below 150,000×). Only 46 could be considered as good or acceptable. The adjectives “poor and good” are somewhat subjective and we apologize for this. The micrographs had been obtained with a wide selection of about 50 types or models of electron microscopes, most of which were “digital.” The vast majority of articles presented several pictures. “Manual” microscopes were apparently disappearing fast. Clearly, poor electron microscopy is a global problem.