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Bacteriophage. Jul 1, 2011; 1(4): 225–227.
PMCID: PMC3448108
The first phage electron micrographs
Hans-W. Ackermann*
Department of Microbiology; Faculty of Medicine; Laval University; Quebec, QC Canada
*Correspondence to: Hans-W. Ackermann, Email: ackermann4/at/gmail.com
Abstract
The first phage electron micrographs were published in 1940 in Germany and proved the particulate nature of bacteriophages. Phages and infected bacteria were first examined raw and unstained. US American scientists introduced shadowing and freeze-drying. Phages appeared to be tailed and morphologically heterogeneous. Phage types identified by early electron microscopy include enterobacteriophages T4, T1, T7, T5, 7–11, ViI and Pseudomonas phage PB1. This paper retraces the development of early virus electron microscopy till the introduction of negative staining.
Keywords: bacteriophage, electron microscopy, history
Electron microscopes were developed in the 1930s. Many countries participated in their development, foremost Germany and the USA, but also Belgium, Canada, England, Japan, Sweden, and later France, Italy, and Czechoslovakia.1 The first electron microscope with magnetic lenses was constructed in 1931.2,3 Haguenau et al.1 present a masterly guide to the development of electron microscopy (EM) between the first theoretic foundations in 1897 amd the latest developments in 2002, covering such diverse fields as electron optics and instrumentation and the application of electron microscope in material science, human tissues, plant cells, and viruses. EM appears as the collaborative product of innumerable scientists and a truly international endeavor, that opened the door to an explosion of knowledge. A brief, very readable account of the life of H. Ruska, early virology and early virus electron microscopy may be found in an article by Krüger and coll.4
EM is heavily indebted to the brothers Ernst and Helmut Ruska. The former devoted himself to technical developments and the second concentrated on biological electron microscopy. An electron microscopical laboratory was set up by the Siemens & Halske Company in Berlin. In 1939, Siemens produced its first commercial transmission electron microscope, called the “Hypermicroscope.”5 It resembled markedly the later, wide-spread model called “Elmiskop I.”6 The new instrument was applied around 1938 to the study of bacteria and the mouse ectromelia virus, a member of the Poxviridae family, the first virus whose size and shape were shown to the scientific world.7
In 1940, H. Ruska5 and Pfankuch and Kausche,8 all working in the Siemens & Halske laboratory, published two short papers submitted the same day to the same journal and showing the first pictures of bacteriophages in the world literature. The originals of these papers, followed by an English translation, are reproduced in this issue of Bacteriophage. H. Ruska showed broth cultures of bacteria mixed with a phage suspension at a magnification of 14,000 times. Bacteria were intact or lysed and then surrounded by large numbers of round particles. The latter were destroyed by electron bombardment and then appeared empty. These images were widely and often privately distributed in Europe and caused considerable excitement. W. Szybalski (personal communication), saw several of them, sent in summer 1939, just before World War II and still unpublished, to Prof. Rudolf Weigl at the Jan Kazimierz University in Lvov, then Poland and now Ukraine. Even during the war, in 1940–44, Weigl received more phage micrographs from H. Ruska. It appears that in this case, electron microscopy prevailed over the spirits of war. Pfankuch and Kausche had their phages purified by precipitation with aluminum hydroxide and elution. Phage titers of 10–15 per ml were so obtained. Phage particles, observed at a magnification of 22–25,000 times, appeared as round particles of 60 nm in diameter. It is remarkable how close these dimensions were to those obtained today for phage T7. These early electron micrographs proved once and for all the particulate nature of bacteriophages and put all discussions on their “ferment nature” to a rest. It is reported that d'Herelle was on his death bed when the French scientist Hauduroy showed him an electron micrograph of a bacteriophage.9
Subsequently, in 1942, H. Ruska10 confirmed these observations, noted that particles seemed to be morphologically complex, proposed to name them “Herellen,” and observed club-like structures with heads of 90–110 nm and tails of 25–250 nm in length in lysates of E. coli, Shigella and Proteus bacteria. These particles were most likely T4-like phages. He also reported phage-like particles in enterococci and staphylococci and introduced the term “phage” as a short form of d'Herelle’s earlier “bacteriophage.” In the same year, Kottmann,11 also working in the Siemens & Halske laboratory, described contact preparations from lysed areas of an E. coli culture on agar. He observed bacteria and round or bacilliform particles at a magnification of 24,000 times. The bacilliform particles measured 90–120 nm × 30–45 nm and formed palissade-like rows at the periphery of bacteria. Kottmann’s bacilliform phages were later found again in a variety of enterobacteria (E. coli, Cronobacter, Proteus, Salmonella), but are exceedingly rare in the phage world. They correspond to the novel genus “7-11-like viruses” of enterobacterial phages.12 In summarizing his observations in 1943, H. Ruska reported at least four morphotypes of bacteriophages13 and proposed a morphological classification of viruses.14
In 1938, Prebus and Hillier, then at the University of Toronto, constructed the first electron microscope in North America. The instrument achieved a magnification of 7,000 times and a resolution of 6 nm.15,16 Hillier joined RCA (Radio Corporation of America) in Camden and later Princeton, NJ, where he developed a commercial electron microscope.1 One of these RCA microscopes was used by Luria and Anderson17 to study various coli and staphylococcal phages. Particles were unstained and magnifications of up to 84,000 times were obtained. Images of a phage PC, later renamed T2, clearly showed particles of the T-even type with heads of 80 nm and tails of about 130 nm in length.17 Despite World War II and its restrictions on scientific exchange, Luria and Anderson were aware of the work performed in Germany by H. Ruska5 and Pfankuch and Kausche.8
In 1943, Luria, Delbrück and Anderson18 described two coliphages, named α and γ, in considerable detail. Lysates were deposited on a grids, which were washed by dipping them into distilled water. Phage α had a head of 45–50 nm in diameter and a long, flexible tail, resembling coliphage T1. Phage γ, with an oval head of 65 × 80 nm and a straight tail of 130 x 10–15 nm, was clearly a member of the T4 group. The authors studied infected cells at various times after infection. One phage was claimed to adsorb to flagella, but the published micrograph is inconclusive.
So far, all virus micrographs depicted unstained raw specimens. A milestone was the introduction of chromium shadowing by Williams and Wyckoff19 in 1945 as a means of contrasting and measuring viruses. The authors described it as “an oblique evaporation of a thin film of metal on the preparation.” This technique was first used on influenza and tobacco mosaic viruses and not on phages. In 1948, Wyckoff applied his novel technique to coliphages of the T series (T2, T4, T6; T1; T5; T3, T7). Preparations of phages and lysed bacteria were made from plaques on agar by the replica technique or from formalin-treated lysates. Images of “developing” bacteriophages were so obtained.20,21 In the same year, a group at Stanford University in California,22 which comprised electron microscopy pioneer Marton from Belgium, published stereo micrographs of a Pseudomonas aeruginosa phage22 which seems to be, in retrospect, a member of the genus “PB1-like phages.23 Shadowing was also used to study phage ontogenesis. “Developing phages” in the form of morula-like structures or “merophages” were reported in Italy in a Mycobacterium phage of the Siphoviridae family. The structures were said to grow a tail and then were called “telophages”24,25 For Kriss in Russia, phages consisted of spherical macromolecules which formed a tight spiral, the head, which grew a free end, the tail.26,27
A further important development occurred in 1953 when Fraser and Williams28 introduced freeze-drying prior to uranium shadowing. By this technical refinement, the authors made it likely that phages T3 and T7, hitherto thought to be tailless, had a short stubby tail. In the same year, Williams and Fraser29 re-described all seven bacteriophages of the T series, showing high-contrast pictures and reporting more exact dimensions of these viruses. Phage heads now were recognized as geometrical bodies and interpreted as octahedra, bipyramidal prisms, or rhombic dodecahedra. This concluded what may be called the “heroic period” of phage EM.
This essay would be incomplete without referring to French attempts to develop an altogether different type of electron microscopes. In the 1940s, French scientists had developed electron microscopes with either magnetic or electrostatic lenses.1 The latter type, named the CSF microscope, is described in some detail in an early book on electron microscopy30 and was apparently used at the Pasteur Institute of Paris to visualize numerous bacteria and vertebrate and plant viruses.6,30 It also seems that it was applied to bacteriophages before 1947 and used to illustrate a B. subtilis phage and the T4-like coliphage C16. The latter was said to have a tail length of 750 nm,31 which is clearly an impossibility and contradicted by further observations. The published accounts of these observations are somewhat unclear. Reverting to the Ruska magnetic electron microscope, transferred as a war spoil to the Pasteur Institute of Paris, French scientists studied several Vi phages of Salmonella typhi and found that they belonged to the myovirus and siphovirus families of tailed phages.32 The early period of viral and bacterial electron microscopy was to end abruptly in 1959 with the introduction of negative staining by Brenner and Horne,33 working in Cambridge, England. This novel technique allowed to visualize viruses in unprecedented clarity and revolutionized virology and our understanding of viruses.
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