How do bacteriophages grow in labs?
For bacteriophages, cultures are grown by infecting bacterial cells. The phage can then be isolated from the resulting plaques in a lawn of bacteria on a plate. Bacteriophages infecting a bacteria: Virus or phage cultures require host cells in which to multiply.
What is phage assay?
Principle of Phage Plaque Assay When a suspension of an infective phage (e.g. T4 phage) is spread over the lawn of susceptible bacterial cells (e.g. Escherichia coli), the phage attaches the bacterial cell, replicate inside it, and kills it during its lytic release. plaque-forming units (PFU) in a sample.
What two structures are found in all viruses?
All viruses contain nucleic acid, either DNA or RNA (but not both), and a protein coat, which encases the nucleic acid. Some viruses are also enclosed by an envelope of fat and protein molecules. In its infective form, outside the cell, a virus particle is called a virion.
What is the hypothesis for the phage infection experiment?
In their experiments, Hershey and Chase showed that when bacteriophages, which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not. Hershey and Chase and subsequent discoveries all served to prove that DNA is the hereditary material.
Why did Hershey and Chase use bacteriophages?
Alfred Hershey and Martha Chase used the bacteriophages because of their connection to DNA. In one batch, the phages (short for bacteriophages) were grown with radioactive phosphorous, which means it was incorporated into phage DNA. The radioactivity in the pellet was measured and it was also measured in the liquid.
What is the name of the procedure used to dilute out the bacteriophage?
The process of determining phage concentration by dilution and plating with susceptible cells is called titering or the plaque assay. This method determines the number of viable phage particles in a stock suspension. A bacteriophage capable of productively infecting a cell is called a plaque-forming unit (PFU).
What is CCID50?
Cell culture infectious dose 50% (CCID50): the amount of a virus sufficient to cause a cytopathic effect in 50% of inoculated replicate cell cultures, as determined in an end-point dilution assay in monolayer cell cultures.
How is TCID calculated?
- Calculate Proportionate Distance (PD) between the two dilutions in between 50%
- Calculate 50 % end point. Log lower dilution= dilution in which position is next.
- Add PD and Log lower dilution. Example above: -6 + .375 =-6.375.
- Calculate TCID 50/ml. Divide by the ml of viral innoculum added to row A.
- Calculate PFU/ml.
What does each plaque represent?
Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a plaque-forming unit (PFU) and is used to quantitate the number of infective phage particles in the culture.
Is there an ideal environment for collecting samples that contain bacteriophages?
Thus, soil that is not conducive to bacterial growth (e.g., cold, dry, acidic) is less likely to yield a phage. Conversely, areas where high bacterial growth might be (damp, aerated, warm, decaying soil) are good places to collect samples.
What is plaque assay technique?
The plaque assay can be used to purify a clonal population of virus or to determine viral titer as plaque-forming units per ml (pfu/ml) so that known amounts of virus can be used to infect cells during subsequent work. Each group of infected cells is referred to as a plaque. …
How do you concentrate your Phage?
My method is 1. inoculate host bacterium in broth (250 ml) to get a OD=0.2-0.3; 2. add 2ml phage lysate (108pfu), then culture for 18-24h (sometimes the culture is quite clear); 3. Centrifuge and filtre to remove bacterial cells and debris.
What is in a clear plaque?
Plaques are clear zones formed in a lawn of cells due to lysis by phage. A clear plaque will be formed if the host is completely susceptible to the phage. (Often a clear plaque will be slightly turbid at the edge because the cells at the edge of the plaque are not yet fully lysed.)
Can virus be cultured in lab?
Cultivation of Viruses. Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate).
What is TCID?
The TCID50 (Median Tissue Culture Infectious Dose) assay is one method used to verify the viral titer of a testing virus. Host tissue cells are cultured on a well plate titer, and then varying dilutions of the testing viral fluid are added to the wells.