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Viruses under the Microscope

Characteristics, Morphology & Life Cycle 

Taking a look at viruses under the microscope. Unlike other unicellular organisms like bacteria, viruses are commonly referred to as particles rather than cells. These particles (virion) are also not alive given that they are unable to grow or multiply on their own. Their size also makes a majority of viruses impossible to see under a light microscope.

* A virus is between 100and 500 times smaller than bacteria

* Researchers suggest that all living organisms (including unicellular organisms) are host to at least a single virus particle


Virus Microscopy 


Virions are very small ranging between 400nm (mimivirus, poxviruses) to about 25nm (polio virus) given that the resolution of conventional compound microscopes is limited to half the wavelength of radiation that is typically used for imaging (200 nm) they only allow for users to view the general morphology of "giant viruses".

Some of the techniques that have been suggested for viewing viruses include:


Fluorescence Microscopy Technique


This is a relatively new technique that is used to track viral DNA in the cells of the host. For this technique, ethyl-modified nucleosides are used to label the DNA of such viruses as adenovirus, herpes virus and vaccinia virus that have infected the cells of the host. The cell samples are then viewed under a confocal microscope.

The method therefore simply involves using fluorochromes, which allows the organism to fluoresce when viewed. Essentially, fluorochromes only fluoresce when they attach to the target organism/object. In the event that the virus of interest is present in a sample, this allows for the attachment to be seen indicating the presence of the virus.

* Fluorescence microscopy technique is useful for the quantitative estimation. It helps show the concentration of viral particles in a given sample.


Total Internal Reflection Dark-Field Microscopy (TIRDFM)


TIRDFM is a label-free imaging technique that can be used to view larger virus particles. As such, no labeling is required for this method. This system can simply be achieved by using a perforated mirror in place of a dichroic mirror that is commonly used in conventional objective-type TIRDFM.

Sample preparation for TIRDFM is a relatively complex procedure that involves the following steps:

  • Inoculation of Influenza A/Puerto Rico/8/34 (H1N1) virus into the allantoic fluid of an 11 day embryonated egg at 37 degrees centigrade for 3 days
  • Centrifugation of the allantoic fluid solution at 2,500g for about 20 minutes at 4 degree
  • Pass the supernatant through a membrane filter (pore size 0.45um)
  • Subject the resulting solution to 10-60 percent sucrose density gradient centrifugation for about 2 hours at 120,000g and at 4 degree in a swing bucket rotor
  • Obtain 2mL aliquots of the gradient
  • Analyze the fractions using SDS polyacrylamide gels and centrifuge for 2 hours at 120,000 g at 4 degrees centigrade
  • Suspend the pellet in phosphate buffered saline


Observation

When viewed under TIRDFM, it is possible to see spots (blight spots) with a dark background.

See more on Total Internal Reflection.


Transmission Electron Microscopy




A transmission electron microscope allows for about 1000 times higher resolution compared to typical light microscopes. It allows for better observation of virions.

For a TEM, higher resolution is achieved because the large wavelength light beam is replaced with an electron beam with 0.005nm wavelength. Here, the electron beams are produced by tungsten, LaB6 filaments or a field emission gun thereby replacing visible light. The resultant monochromatic beam is then accelerated in vacuum through a 40-100kV voltage before going through magnet fields that work as lenses.

Sample preparation

The sample to be viewed using transmission electron microscope has to be carefully prepared for observation. This involves the following steps:

  • Apply 10um of the suspension to a hydrophobic surface of a parafilm square in a Petri dish
  • Float a formvar-coated grid on to the drop for about one minute (make sure the formvar side is in contact with the liquid sample)
  • Float the grid in a drop of 1.5 percent phosphotungstic acid (2% ammonium molybdate or 1% aqueous uranyl acetate can be used depending on the type of specimen being prepared) - these serve as stains


Observation

When viewed under the transmission electron microscope, virions can be seen as small particles inside the cell (in the cells they have infected).



Cryo-Electron Microscopy



In order to get a better view of the structure of a virus, researchers have been using a number of techniques including electron tomography, immunoelectron microscopy and cryo-electron microscopy.

These have also been shown to be important techniques for showing how viruses attach to cells, how they assimilate during replication as well as their their association with various cellular mechanisms during replication.

In cryo-electron microscopy, the sample is frozen using liquid nitrogen and observed under a transmission electron microscope that is equipped with a cryo-stage. However, the images may be reconstructed in order to obtain a three dimension of the virus.


Basic Virus Characteristics


  • A majority of viruses are very small (about 100 times smaller compared to bacteria)
  • They are dependent on the host to reproduce
  • They have a receptor-binding protein that allow them to attach to the host cell surface


Morphology of Virus Particles


When the structure of a virus is viewed under a powerful microscope, it may be icosahedral or helical. These particles are composed of a protein coat that envelopes the genetic material and is typically referred to as a nucleocapsid.

The shape and size of the capsid is largely dependent on the family to which the virus belongs. They are made up of subunits of proteins referred to as capsomeres (it is the arrangement of these protein subunits that determines the shape of the capsid).

Although viruses are typically very small, recent studies have discovered that some viruses are large in size. A good example of this is the mimivirus that has been described as a giant virus. With a diameter of about 700nm, the mimivirus has been shown to be larger in comparison to some bacteria.

Virions carry their genome (DNA/RNA) inside the capsid. Depending on the virus, viruses either carry DNA or RNA which is useful for the production of new viruses. Whereas ordinary viruses carry between 2 and 100 genes inside their capsid, giant viruses like mimivirus may contain as many as 1,000 genes.

Unlike other types of unicellular organisms like bacteria, viruses lack other types of essential organelles such as ribosome and mitochondria among others that would make it possible for them to manufacture and store important molecules like proteins for energy generation. As a result, they are unable to exist (grow and reproduce) freely and can only do so when they infect the cells of a host.

Once they infect the cells of the host, viruses will start using the organelle of the said host to thrive. However, this is done at the expense of the host's cells that are destroyed in the process. Here, infection of the cell will result in new viral particles, which then go on to infect new cells. 

At any given time, there are about 1031 viruses that exist


Life Cycle of a Virus


Because viruses are not alive, their life cycle is largely dependent on the host.

It involves the following phases/steps:

Attachment

Once the virus gets into the body, it uses specific attachment proteins to bind receptors on the surface of the cell of the host.

These attachment proteins are important and determine the type of cell the virus will attach on. For this reason, some viruses are only capable of infecting a few hosts as they can only infect a few cells.

*Example: Rabies virus use glycoprotein as the attachment molecule. These molecules can attach to such receptors as acetylcholine receptor present on neurons. This therefore makes it possible for the virus to attach to and infect cells present on neurons.

Penetration

Penetration is the process through which the virus injects its genetic material in to the cell of the host. This process may either take place through endocytosis or direct membrane fusion. In endocytosis, the virus attaches onto the receptors at the coated pits. Here, the pits pinch off and forms coated vesicles. These vesicles then fuse with endocytic vesicles before fusing with lysosomes. However, the genome of the virus fuses with the membrane in the presence of fusion proteins to avoid being destroyed by lysosome enzymes.

Ultimately, the RNA is able to reach the cytoplasm without being destroyed. On the other hand, the virus will use the plasma membrane as an enveloped virus having been induced by binding to the receptor. This is different from endocytosis, which is induced by changes in pH.


Uncoating and Targeting

While some viruses release their genome in the cell after penetration (process known as uncoating), replication cycle starts in the capsid with some viruses like retroviruses. This is made possible by the conformational changes of the capsid

Gene expression and replication- During this phase, new viral genomes and virion components are produced and the genome of the virus gets into the DNA of the host.

Virus assembly and release - This is the last phase that involves the assembly of new virions. Newly formed genomes are inserted into a capsid forming new nucleocapsids that repeat the cycle. 

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References

Dorothy H. Crawford (2011) Viruses: A Very Short Introduction

https://msu.edu/course/mmg/569/lifecycles.htm


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