Bacteriophages are viruses that infect bacteria. Phage research led to ground-breaking discoveries illuminating how genetic information is stored1, transferred1, controlled (lysogeny)2, and protected (restriction3 and CRISPR/Cas systems4). Restriction enzymes made genetic engineering possible, whereas phage display technology5 allowed the development of target-specific antibody medicines. Phages and technologies such as phage display have an enormous potential that has not yet been fully realized in medicine, diagnostics, and materials science/engineering.
In this insight, we would like to ask a very simple question – what is the color of the phage? This question sparked our attention when we came across several papers6–8 published from 1967 through 1970 mentioning the blue phage band. Since then, the mysterious blue band of phage had been spoken about in some publications but had never been demonstrated.
To see the blue phage band, researchers of the papers, cited above, used a method called equilibrium sedimentation in density gradients introduced in 19579 and fully developed in 196310. In particular, a transparent tube is filled with 4–7 layers of cesium chloride (CsCl) solutions atop each other (typically, using Pasteur pipettes). The bottom layer has the highest density, whereas the top layer has the lowest density of CsCl. In this way, a discontinuous CsCl step density gradient is formed. Then, a sample containing phage is layered on top of this gradient, the tube is placed into a metal bucket, which is attached to a swinging-bucket rotor, which is spun for 1 hour in an ultracentrifuge at a speed of 40,000 revolutions per minute roughly corresponding to a relative centrifugal force of 200,000 g. As a result, the phage particles migrate toward the bottom of the tube and concentrate to form a band in the CsCl layer with a density equal to the (buoyant) density of the phage particles. Hence, the equilibrium sedimentation is achieved.
The photograph shows the result of such an equilibrium sedimentation experiment. Indeed, the phage particles are seen as a blue band. There are actually two blue bands. The lower, major one, contains at least 1013 infectious phage particles. The faint blue band seen above is not infectious as the capsids of the phages with this particular buoyant density are devoid of genetic material (they are empty). Finally, there is a thick pale white layer of cellular debris.
We have just demonstrated that the phage band is blue. This, however, begs two other questions related to the phage band: (1) why is it bright? and (2) why is it blue? It is clear that the presence of genetic material in the capsid (i.e., the protein shell of a phage particle surrounding the genetic material) does not affect the phage color. Thus, it is the interaction of the incident light and the surfaces of the phage particles that gives the blue color to the phage band.
The interaction of an incident light and a particle is of electromagnetic nature resulting in light scattering at a specific scattering angle, the angle (θ) between the direction of incident and scattered light (more precisely, the angle between incident and scattered electromagnetic wave). The light scattering angle depends on the size of the particle. It was demonstrated that if the particle size is in the 17–170 nm range, then the scattering intensity is high for all angles within the 0°–180° range (except at 90°) and dipole scattering is observed11–12. Thus, such particles should appear bright. The capsids of the phage particles, forming the bright blue band in the photograph, are spherical with a diameter of 60 nm (∅ 60 nm), which falls within the 17–170 nm particle range. In fact, the sizes of most of the phages described in studies6–8 mentioned above fall within the same particle range (see table below).
|SPP1 (capsid)||∅ 60|
|LPP-1 (capsid)||∅ 60|
|P22 (capsid)||∅ 60|
|T7 (capsid)||∅ 60|
|fd||∅ 7 × 880|
If the particle size, however, exceeds 1000 nm (1 μm), then high scattering intensity is observed only at scattering angles close to 0° (i.e., θ = 0°) and forward scattering is observed12. The angle of the incident light that is not scattered is θ = 0°. Thus, for particles with diameters of ≥1 μm, the direction of the scattered light coincides with the direction of the incident light. This results in a pale color. The thick pale white layer of cellular debris in the photograph strongly suggests that the pieces it is composed of have sizes ≥1 μm. The scattering of the incident light by particles of this size does not depend on the wavelength, explaining why the white color is observed.
The table above clearly shows that all phages except one are spherical and have diameters, which fall within 26–60 nm range. These phages are much smaller than the wavelength of the incident light (visible light 400–700 nm). The scattering of the incident light by these phage particles should strongly depend on the wavelength. In particular, the scattering at 400 nm (near ultraviolet) should be almost 10 times greater than the scattering at 700 nm irrespectively of equal incident intensity. Such scattering is called Rayleigh scattering, which is an approximation of Mie theory. Thus, phage particles continuously and preferentially scatter the near ultraviolet spectrum (blue) of the incident light, which explains why the observed phage band is blue.
Does it mean that all spherical particles much smaller than the wavelength of the incident light are blue? Not at all. For instance, absorbance is as important as scattering in determining the resulting particle color, which is also sometimes influenced by the background used for the observation (e.g., plasmonic nanoparticles). For the phage band in the photograph, the background (black or white) used for the observation did not affect phage band color perception. In addition, the factors that can influence the color of a particle are: particle homogeneity, numerous particles presence, particle shape, particle internal structure, etc.
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