The Phage



Engineered phages: The future of bioremediation?

In the dynamic world of scientific discovery, researchers are constantly finding innovative solutions to some of our most pressing challenges. A recent study has unveiled an approach to environmental remediation using genetically modified bacteriophages. Yes, you read that right – phages might just be the key to cleaning up heavy metal contamination in our environment.

Scientists from the Biosensor Group and Environmental Safety Group at the Korea Institute of Science and Technology Europe Forschungsgesellschaft mbH, along with researchers from the Bioprogrammable Materials Group at the INM – Leibniz Institute for New Materials in Saarbrücken, Germany, conducted a study on genetically modified fd bacteriophages for potential applications in bioremediation studies.

Published in a peer-reviewed scientific journal, this study focuses on fd bacteriophages, which are filamentous viruses that exclusively target bacteria. Researchers took these viruses and gave them a superhero-like upgrade by genetically modifying them to display a unique peptide sequence designed to bind specifically to copper ions (Cu(II)).

The visual aspect of these engineered viruses is fascinating. Atomic force microscopy (AFM) images reveal the typical filamentous structures of these virus constructs, each with a thickness of around 2-3 nanometers. Imagine tiny, flexible wires at the nanoscale – that’s the beauty of these engineered viruses.

But why the interest in copper ions? Copper, while essential in small amounts, can pose environmental risks when present in excess. The goal here is to create a biological tool that can selectively bind to and remove copper ions from the environment.

The researchers conducted a series of experiments to explore the capabilities of these engineered viruses. When exposed to a copper-rich environment, the phages displayed remarkable changes. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analyses showed that the engineered viruses formed elongated structures with a mineral layer after copper exposure, unlike their untreated counterparts.

In simpler terms, these phages acted like tiny sponges, soaking up copper ions and transforming into elongated structures with a visible coating – a visual testament to their newfound capabilities.

The study also delves into the nuts and bolts of the genetic modifications. The viruses were engineered to display different versions of a copper-binding peptide, each with varying lengths. The results? The longer the displayed peptide, the larger and more intricate the virus assemblies became after exposure to copper.

Imagine these engineered viruses as nature’s nanoscale clean-up crew. They selectively bind to copper ions, forming larger structures that can potentially be harnessed for environmental remediation. The study hints at the possibility of using these engineered viruses in bioremediation efforts to target metal species in environmental samples.

This is not just a laboratory experiment; it’s a glimpse into the future of sustainable technologies. While the study involves complex scientific processes, the core message is simple – scientists are exploring creative, nature-inspired solutions to tackle environmental challenges.

As we envision a future where technology and nature collaborate for the greater good, these engineered viruses stand as tiny ambassadors of innovation, demonstrating the potential for biologically driven solutions to real-world problems. Who would have thought that bacteriophages could be our allies in the quest for a cleaner, healthier planet?

In a world where scientific breakthroughs often feel distant and esoteric, this study brings us a step closer to a future where even the tiniest organisms can play a big role in safeguarding our environment. Nature, it seems, has more tricks up its sleeve than we could have ever imagined.

Read the full published article on Korkmaz, N., Himawan, S., Usman, M., Baik, S., Kim, M., Bacteriophage Engineering for Improved Copper Ion Binding. Macromol. Biosci. 2023, 2300354.

Increased Temperature Facilitates Rapid Genome Ejection in Phages

In a groundbreaking study published in the Proceedings of the National Academy of Sciences in 2023, researchers from Lund University, Sweden in collaboration with their counterparts at the NIST Synchrotron Facility in the United States, mapped the atomic-level processes occurring within a bacteriophage particle when subjected to changes in temperature. This investigation delves into a critical aspect of viral (phage) replication: the structural arrangement of a viral genome within its capsid and its impact on the dynamics of genome release during infection.

The study builds upon previous research that had identified a temperature-induced transition in the mechanical properties of the packaged λ-genome, shifting from a solid-like to a fluid-like state. This transition facilitated rapid DNA ejection, a process central to viral replication. Nevertheless, prior to this study, the precise structural origins of this transition remained elusive.

To understand these structural changes, the scientists used a method called Small-Angle Neutron Scattering (SANS). They came up with a new way to do this by matching the scattering signal from the virus’s outer shell with a special liquid called deuterated buffer. This helped them see how the genetic material (double-stranded DNA or dsDNA) is packed inside the virus’s shell. To be extra sure they got it right, they also used Small-Angle X-ray Scattering and special microscope images (cryoelectron microscopy) to figure out the starting details about the DNA inside the virus.

The results showed that when the temperature increased, something interesting happened inside the virus. Inside the virus, the genetic material had two different parts. One part was tightly packed and ordered like a hexagon on the outer edge of the virus, while the other part in the center was less ordered and not as tightly packed.

As the temperature rose from 20°C to 40°C, the inner part of the genetic material went through a change. This change happened at a temperature close to the normal temperature of infection, around 37°C. During this change, the genetic material in the core became less tightly packed, and it had fewer defects in its structure. This change made the genetic material more mobile, which is essential for it to be quickly released from the virus into a host cell, causing an infection.

The insights gained from this study reconcile with prior observations regarding the mechanical transition of DNA within the phage λ capsid. They shed light on the atomic-level processes underlying temperature-induced DNA density transitions in viral capsids and provide a deeper understanding of how viruses orchestrate their infection mechanisms. These findings hold significant implications for the development of antiviral strategies and therapeutics.

Reference: Villanueva Valencia, J. R., Tsimtsirakis, E., Krueger, S., & Evilevitch, A. (2023). Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS. Proceedings of the National Academy of Sciences120(45), e2220518120.

cover photo courtesy of Rob Cot, T2Q.

Unlocking the Secrets of Phages: Revolutionizing Biotechnology with New Structural Insights

Phages, the intriguing viruses that target bacteria, have long held the key to a variety of applications in biotechnology and medicine. In a groundbreaking collaboration, researchers from the University of Exeter (United Kingdom), Massey University, and Nanophage Technologies in New Zealand have achieved a significant milestone by unraveling the enigmatic structure of a widely utilized form of phage. This breakthrough discovery promises to empower scientists with the knowledge needed to design more advanced applications for these viral workhorses.

One of the primary applications of phages lies in a technique called phage display, which plays a vital role in drug discovery. Phage display involves fusing a gene fragment of interest with a specific phage gene responsible for producing one of the phage’s coat proteins. This fusion results in the display of the protein of interest on the surface of the phage, allowing scientists to assess its biological activity and conduct targeted experiments.

While numerous phage variants exist, filamentous phages, named for their long and slender morphology, are frequently employed in phage display and other applications due to their ability to accommodate the display of multiple proteins across their surface. Despite their practical utility, researchers have long grappled with the lack of comprehensive knowledge regarding the structure of filamentous phages.

In a groundbreaking publication in the esteemed journal Nature Communications, Dr. Vicki Gold of the University of Exeter presents the first-ever elucidation of the structure of a filamentous phage.

“Phages constitute a captivating and rapidly expanding field of research, offering a range of current and potential applications. Until now, however, we have lacked a complete understanding of what filamentous phages truly look like. With our recent breakthrough, we have provided the initial glimpse into this mysterious world, paving the way for enhanced phage applications in the future.”

Dr. Vicki

One of the main challenges in capturing the structure of filamentous phages lies in their considerable length, which has hindered previous attempts to obtain full images. To overcome this hurdle, researchers ingeniously created miniaturized versions of the phages that were approximately ten times shorter, resembling straight nanorods rather than the entangled spaghetti-like filaments of their larger counterparts. This downscaled model allowed for comprehensive imaging using cutting-edge cryo-electron microscopy techniques, enabling researchers to visualize the phage’s structure in its entirety.

The newfound knowledge of filamentous phage structure opens up a multitude of possibilities for scientists in the field of biotechnology. By understanding the fundamental architecture and characteristics of these viruses, researchers can now optimize their applications in drug discovery, diagnostic tools, and targeted therapies. The ability to engineer specific coat proteins on the surface of filamentous phages holds immense potential for developing novel treatments and interventions, tailored to combat specific diseases or deliver therapeutic agents with exceptional precision.

Furthermore, this breakthrough offers insights into the vast diversity of phages that inhabit our world. With billions of phage types yet to be fully explored, scientists can now leverage this newfound structural understanding to delve deeper into the extraordinary world of phages. Unlocking the secrets of these viral entities may unveil groundbreaking solutions for challenges in various fields, from biotechnology and medicine to environmental and agricultural applications.

As the realms of biotechnology and medicine continue to evolve, phages stand as captivating agents of change. With each breakthrough, scientists move closer to harnessing the immense potential of these viral superheroes. The groundbreaking research conducted by Dr. Vicki Gold and her collaborators ushers in a new era of phage-based biotechnology, with far-reaching implications for human health and our understanding of the microbial world. As the mysteries of phages unfold, the future holds great promise for their transformative role in shaping the world of biotechnology.

Featured photo adapted from the original research paper

Phage Visualization Under Microscope: The Types, Techniques, and Importance

Bacteriophage research has seen a resurgence in recent years due to its potential for treating antibiotic-resistant bacteria. One of the critical ways to study phages is through visualization under a microscope. Microscopy techniques have come a long way in recent years, and there are many different types of microscopes that can be used to visualize phages. The type of microscope used will depend on the specific research question and the properties of the phage being studied. For example, electron microscopes provide high-resolution images of phages, but they require special preparation and handling, whereas light microscopes are less invasive and can be used to study phages in their natural environment.

In this article, we will take a closer look at the different types of microscopes that can be used for phage visualization, the techniques employed, and the importance of studying phages in this manner. We will explore the advantages and limitations of each type of microscope and the circumstances under which they are used. Additionally, we will delve into the latest advances in phage microscopy and their implications for future research.

Types of Microscopes for Phage Visualization

There are several types of microscopes that can be used to visualize bacteriophages, each with its own advantages and limitations. The most common types of microscopes used for phage visualization include:

Light Microscopy

Light Microscopy: Light microscopy is the most widely used method for visualizing bacteriophages. It can be done using bright-field, phase contrast, or fluorescence techniques. Bright-field microscopy is the simplest method, where a beam of light is passed through the specimen and the image is viewed directly. This method is useful for observing the overall shape and size of the phages but may not provide the highest resolution. Phase contrast microscopy is similar to bright-field microscopy, but it uses a special condenser to enhance the contrast of the image.

Ligh microscopy can provide better visibility of phages in solution and is commonly used for studying the behavior of phages in liquid cultures. Fluorescence microscopy uses a special filter to excite specific dyes in the specimen, resulting in a brightly colored image. This method is useful for identifying specific components of phages, such as viral proteins, but requires the use of fluorescent dyes and specialized equipment.

Transmission Electron Microscopy (TEM)

Transmission Electron Microscopy (TEM): TEM is a powerful technique for visualizing bacteriophages at high resolution. It involves passing a beam of electrons through a thin section of the specimen and observing the resulting image on a screen. TEM can provide images at resolutions of a few nanometers and is particularly useful for studying the structure of phages. However, this method requires the use of a vacuum chamber and can be destructive to the specimen. Additionally, samples must be prepared in a specific way, such as being embedded in resin and thinly sectioned, before they can be viewed under TEM.

Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM): SEM is similar to TEM but uses a beam of electrons to scan the surface of the specimen rather than passing through it. This results in images that show the surface features of the specimen in great detail. SEM is particularly useful for studying the surface morphology of phages and can be used to examine the surface features of phages in their natural state. However, this method also requires the use of a vacuum chamber and can be destructive to the specimen.

Atomic Force Microscopy (AFM)

Atomic Force Microscopy (AFM): AFM is a type of scanning probe microscopy that uses a small probe to scan the surface of the specimen. The probe is moved over the surface of the specimen, and the forces between the probe and the specimen are measured. AFM can provide images at resolutions of a few nanometers and is useful for studying the surface properties of phages. This method is particularly useful for studying the mechanical properties of phages and can be used to examine phages in their natural state without the need for a vacuum chamber. However, AFM is a relatively new technique and may not be as widely available as other methods.

Techniques for Phage Visualization

Once the type of microscope is chosen, there are various techniques that can be used to visualize phages. Some of the most commonly used techniques include:


This involves the use of special dyes that can be used to color the phages for better visualization. Some standard staining methods include negative staining, which uses a dark background to highlight the phages, and positive staining, which uses a bright background to highlight the phages.


This involves the use of special proteins or other molecules that can be added to the phages to make them visible under the microscope. These include fluorescent proteins, which can be used to label phages for fluorescence microscopy, or gold nanoparticles, which can be used to label phages for electron microscopy.


This involves growing phages in a bacterial culture and observing them as they infect and lyse the bacteria. This technique is particularly useful for studying the life cycle of phages and their interactions with bacteria.

Phage microscopy gallery

This image captures the results of using a fluorescence microscope to examine phage-QD complexes bound to cells. The sample was exposed to low levels of biotinylated phage-QD complexes and illuminated with both white light and fluorescence excitation. The resulting images show two different "blinking states" of a single QD, one "off" (a) and one "on" (b). A higher concentration of phage-QD complexes can be seen in image (c) and compared to the bright-field transmission image (d) of the same sample area. It's worth noting that while some cells appear immobilized on the substrate, others appear mobile in solution, which can result in out-of-focus fluorescence images when the focus is maintained on cells on a substrate surface. The scale bars in the image are 1 micrometer (a and b) and 2 micrometers (c and d)."
These images extracted from a study done by Edgar et al (2006) capture the results of using a fluorescence microscope to examine phage-QD complexes bound to cells. The sample was exposed to low levels of biotinylated phage-QD complexes and illuminated with both white light and fluorescence excitation. The resulting images show two different “blinking states” of a single QD, one “off” (a) and one “on” (b). A higher concentration of phage-QD complexes can be seen in image (c) and compared to the bright-field transmission image (d) of the same sample area. It’s worth noting that while some cells appear immobilized on the substrate, others appear mobile in solution, which can result in out-of-focus fluorescence images when the focus is maintained on cells on a substrate surface. The scale bars in the image are 1 micrometer (a and b) and 2 micrometers (c and d).
Atomic force microscopy showing bacteriophages
In (a) through (e) are AFM images of individual phage particles that exhibit the 3 + 1 tail fibers, most originating from base plates. In (d) the phage head has burst under osmotic shock to release a bolus of DNA, but the 3 + 1 fiber remains intact. In (f) is a higher magnification AFM image of a 5 + 0 tassel that is just beginning to disintegrate and display some degree of the substructure. Scan areas are (a) 1 um × 1 um, (b) 1 um × 1 um, (c) 1 um × 1 um, (d) 0.5 × 0.5 um, (e) 1 um × 1um and (f) 0.5 um × 0.5 um. Study done by Kunetsov et al (2013)
Scanning electron microscopy showing bacteriophages on the surface of bacteria
A study done by Lappanen, et al (2017) presented a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). This method is an improved version of traditional scanning electron microscopy
Transmission electron microscopy showing T shaped phage bacteriophages
Transmission electron micrographs of three bacteriophages used in the study done by Gelzinis, et al (2015): (a) Vb-AphaS-CL131, (b) T4 and (c) Vb-AphaM-CL132. Scale bars¼ 50 nm.

Importance of Phage Visualization

The ability to visualize phages under a microscope is crucial for understanding their biology and potential applications. Some of the key reasons why phage visualization is important include:

  • Understanding Phage Structure: By visualizing phages under a microscope, scientists can study the physical structure of these viruses in great detail. This information is crucial for understanding how phages infect and replicate within their host cells.
  • Identifying Phage-Bacteria Interactions: By observing phages in their natural habitat, scientists can study how these viruses

ICTV and morphological classification of bacteriophages

In 2022, ICTV changed the classification of some bacteriophages based on their morphological taxa. Prior to that, microscopy was a valuable tool for classification. The changes impacted the morphology-based families Myoviridae, Podoviridae, and Siphoviridae, and the order Caudovirales was removed and replaced by the class Caudoviricetes to group all tailed-viruses with icosahedral capsids and double-stranded DNA genomes, both of bacterial and archaeal origin. Click here to read the full ICTV article.


The choice of a microscopy technique depends on the research question and the sample. Light microscopy is the simplest and most widely used method for visualizing bacteriophages, but it has its limitations. TEM, SEM, and AFM provide much higher-resolution images, but they are more complex and expensive. As technology advances, researchers will have access to new and more powerful tools for studying bacteriophages.


  1. “Phage Microscopy” by David M. White, in “Phages: Methods and Protocols” edited by Margarita Salas, Springer, 2010.
  2. “Phage imaging: from electron microscopy to superresolution fluorescence” by T. J. Foster and R. W. Hendrix, in “Phages: Biology and Applications” edited by R. Calendar, CRC Press, 2013.
  3. “Scanning electron microscopy and atomic force microscopy of bacteriophages” by J. A. McEwan, in “Phages: Methods and Protocols” edited by Margarita Salas, Springer, 2010.
  4. “Bacteriophage Research: A Revitalized Approach to Antibiotics” by J. Soothill, Microbiology Today, vol. 36, pp. 122-125, 2009.
  5. “Phage Microscopy: Techniques and Applications” by K. M. Keiler, J. Bacteriol., vol. 193, pp. 727-735, 2011.
  6. “Visualizing Bacteriophages Using Transmission Electron Microscopy” by L. J. Black and R. J. Doyle, Methods Enzymol., vol. 504, pp. 3-23, 2012.
  7. “Scanning Electron Microscopy of Bacteriophages” by P. D. R. Moineau, J. Virol. Methods, vol. 128, pp. 97-106, 2005.
  8. “Atomic Force Microscopy of Bacteriophages” by J. R. Parsek and E. P. Greenberg, Nat. Rev. Microbiol., vol. 2, pp. 801-811, 2004.

Can genomics/meta-genomics replace transimission electron microscopy (TEM)?

Can genomics replace electron microscopy?

next-generation sequencing
next-generation sequencing

This might be suggested by the rise of rapid sequencing and the ensuing increased availability of wholly sequenced virus genomes. It is indeed advocated in discussions by unconditional partisans of genomics. The answer is round ‘‘no.’’ Genomics gives us the genome and genes, thus the elementary building blocks of a virus. It also provides gene order and direction of transcription. It identifies genes coding for proteins with homology to known enzymes or virion components, restriction-modification enzymes, capsid protein size, or the length of tape measure proteins. Further, genomics indicates horizontal gene transfer or gene swapping, may reveal relationships between virus groups and individual viruses, and allow quantifying connections and constructing phylogenetic trees. This provides unprecedented insights into virus evolution and is a precious help in phage classification.

However, electron microscopy provides information on virion structure. At the same time, genomics does not show the whole virus, gives no single dimension, provides no information on virus structure and physicochemical properties, identifies unusual bases such as 5-hydroxymethylcytosine, and predicts only some biological properties, such as a lysogenic nature. No sequence can indicate simple things such as the size of phage capsids, their geometry, or the number of capsomers. If, as likely, the length of phage tails depends on the length of ruler protein genes (Katsura and Hendrix, 1984; Pedulla et al., 2003), this must be ascertained by measuring many phage tails under strict magnification control. Unfortunately, this has not been the case. If, as pretended, a genome contains all information on a virus, we have not yet found the instruction manual to read it. 
Concerning virus identification, genomics generally does not indicate to which virus family a tailed phage belongs; for example, there are no sequences specific to Myo-, Sipho-, or Podoviridae. Only in the case of small polyhedral or filamentous phages (Micro-, Levi, and Inoviridae) does genomics allow for identifying virus families (Ackermann and Kropinski, 2007). Similarly, a Bacillus tectivirus from the earthworm gut was identified by genomics alone without the benefit of electron microscopy (Schuch et al., 2010). However, in general, investigation of a complete virus sequence may take months and is infinitely slower and more labor-intensive than electron microscopy. 

Can metagenomics replace electron microscopy?

 The answer is ‘‘no’’ again. For virus identification, metagenomics relies totally on known and identified genes and genomes, which, in turn, belong to viruses known and characterized by electron microscopy. In other terms, the vast majority of numerous genes detected by metagenomics can be identified only to the extent as they belong to known sequences from known viruses. Further, metagenomics will not tell whether any detected lines belong to complete, infectious virions or not. 

Can electron microscopy replace genomics?

 The answer is ‘‘yes,’’ but only when it comes to identifying high-level taxonomic categories. Clearly, electron microscopy and genomics (or metagenomics) are not alternatives but complementary. Both of them answer different questions and appear as other fingers of the same hand.
(Read more about bacteriophage here)

Problems of electron microscopy (TEM) for bacteriophages

Transmission electron microscopy history

Transmission electron microscope
Transmission electron microscope

Electron microscopy has always had problems with imaging and interpretation, but the rise of digital electron microscopy and CCD cameras in the 1990s created a novel situation. In a general way, it appears that the quality of phage electron microscopy (read more about phages here) has slipped and that many present-day phage electron micrographs are far inferior in quality to the first images of negatively stained phages taken in the late 1950s (Brenner et al., 1959). A peak in phage electron microscopy was reached in the 1970s (see Dalton and Haguenau, 1973), but this seems to be forgotten. For example, in a confidential survey of about 130 phage papers since 2006, which described novel phages by mostly digital TEM, 70 featured low-contrast, unsharp, astigmatic, poor to inferior pictures. Some ‘‘phage descriptions’’ reported neither phage dimensions nor stains and did not specify the electron microscopes used. Only some 20 papers showed good-quality figures. The decline of phage electron microscopy may be linked to personal factors, namely the loss of great electron microscopists such as Eduard Kellenberger or Tom Anderson, their replacement by inexperienced investigators, and perceived leniency even of reputed journals to accept substandard micrographs. Indeed, poor micrographs can be associated with an inadequate technique, whether in specimen processing or imaging, regardless of the electron microscope used. Digital TEMs and CCD cameras are here to stay. CCD cameras have largely obviated darkroom photography and are wildly popular with inexperienced microscopists who fear working in the darkroom. 

  1. Compared to conventional TEMs, digital electron microscopes appear to be more expensive, cannot be maintained typically by users, and need costly service contracts. 
  2. Their life span remains to be seen, and they are more challenging to control than ‘‘manual’’ TEMs concerning contrast and magnification. However, conventional photographic chemicals and papers may be challenging to find because the market has shrunk. Bacteriophage Electron Microscopy 23 
  3. The relative quality of the various digital TEMs and CCD cameras is challenging to evaluate in the absence of comparative studies. It seems that present top-grade TEMs, whether produced by FEI, JEOL, or Hitachi and concomitant CCD cameras, are roughly equivalent concerning resolution. The instruments are improved continuously. For example, TEMs manufactured by the FEI Company (Hillsboro, OR), which acquired the Philips Electron Optics Division, produce micrographs of exceptional quality. 
  4. With ‘‘manual’’ TEMs, contrast is controlled in the darkroom through graded filters and papers. In the case of digital TEMs, one can obtain high-resolution and high-contrast pictures by adjusting pixel intensities with CCD camera software (Tiekotter and Ackermann, 2009). Unfortunately, the manufacturers of electron microscopes have seemingly neglected to issue guidelines for contrast enhancement, leaving users to fend for themselves. 
  5. With both ‘‘manual’’ and digital TEMs, magnification is controlled utilizing test specimens, for example, catalase crystals (Luftig, 1967) or T4 phage tails. Latex spheres or diffraction grating replicas are suitable for low magnification only (10–30,000x). With ‘‘manual’’ microscopes, magnification can be corrected in the darkroom within minutes. The elaboration of digital electron microscopes usually is set by the installer and cannot, or only with great difficulty, be adjusted by the user. The user must photograph test specimens and define correction factors by calculation to control magnification. 
Practically, it is recommended that 
  1. TEM manufacturers publish instructions for contrast enhancement. 
  2. All specimens are purified before the examination (read about purification here). Crude lysates are to be banished. Purification is achieved most easily by differential centrifugation and washing in the buffer. 
  3. Improve contrast of digital microscopes via Photoshop technology. 
  4. Control magnification regularly using test specimens.

In a general way, it appears that the quality of phage electron microscopy has slipped. Many present-day phage electron micrographs are far inferior in quality to the first images of negatively stained phages taken in the late 1950s (Brenner et al., 1959). Credit to Ackerman et al