Why do you need to dilute bacteria to count them?
We use serial dilutions to create decreasing concentrations of the original sample that are then plated so that a plate will be created with a low enough number of bacteria that we can count individual colonies. Therefore, we need to dilute our original sample before plating.
How do you calculate PFU ml?
Then use the formula: (total PFU needed) / (PFU/ml) = total ml of virus needed to reach your desired dose. For example: You have a virus with a titer of 1.3×1011 PFU/ml and a well that contains 1.8×106 cells.
What does the plaque assay determine quizlet?
What is the point of Bacteriophage Plaque Assay for Phage Titer? demonstrates the ability of viruses to replicate inside a susceptible host cell. Each plaque represents the lysis of a phage-infected bacterial cell.
What is a 1 to 4 dilution?
A 1:4 dilution ratio means that a simple dilution contains one part concentrated solution or solute and four parts of the solvent, which is usually water.
What is a 1 to 2 dilution?
The ratio 1:2 is a 50% solution, so let’s say 1:2 is in respect to substances A : B. This means that if you have solvent e.g. water as B and Substance as A: you must add X amount of A and twice that amount of B. By taking one volume of the original solution and diluting it to two volumes.
What is the difference between dilution and dilution factor?
Dilution is the process of diluting or mixing two or more substances or even compounds. Dilution is also a term for reducing the concentration of a formula. Dilution factor or DF, on the other hand, is a term used to describe the ratio of the final volume over the aliquot volume.
What is a 1 to 3 dilution?
If you have a 1:3 dilution, i.e. a 1:3 dilution ratio, this means that you add 1 unit volume of solute (e.g., concentrate) to 3 unit volumes of the solvent (e.g., water), which will give a total of 4 units of volume. You may already be using the dilution ratio in your everyday life without knowing it!
How do you do a plaque assay?
To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers.
What is hard agar?
The hard agar is the substrate for bacterial growth. The soft agar is used to mix the bacteria and phage dilutions which is then spread over the hard agar. The bacteria form a lawn with clear zones cause by viral growth.
How do you calculate the dilution factor of a serial dilution?
In serial dilutions, you multiply the dilution factors for each step. The dilution factor or the dilution is the initial volume divided by the final volume. For example, if you add a 1 mL sample to 9 mL of diluent to get 10 mL of solution, DF=ViVf = 1mL10mL=110 .
How do you dilute a water sample?
Use a sterile pipet to add 11 mL of sample into the dilution water bottle. 5. Put the cap on the dilution water bottle and invert for 30 seconds (25 times). This is a 10x dilution (sample is diluted by a factor of 10).
What is plaque assay used for?
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.
What is a 1 to 100 dilution?
For a 1:100 dilution, one part of the solution is mixed with 99 parts new solvent. The final volume of the diluted sample is 1000 µL (1 mL), and the concentration is 1/10 that of the original solution. A 1:10 dilution is also called a 10x dilution.
Why is it necessary to have a top and a bottom agar layer in the plaque assay?
The bottom layer solid layer is crucial for an even bacteria lawn, which the phage will grow on. The softer, semi-solid media allows for (like Victor mentioned) better diffusion, or an easier medium for the phage to interact with the bacteria.
What is an infectivity assay?
The infectivity assay is used to titrate virus-containing clarified culture supernatant fluids to determine the 5O%-tissue culture infective dose (TCIDSO) of HIV-1 per ml of original fluid.
Why is soft agar used in plaque assay?
Plaques do not continue to spread indefinitely. The medium used in phage plaque assays has a relatively low percentage of agar and therefore is called soft agar; it permits diffusion of phage to nearby uninfected cells but does not permit new phages to move to remote parts of the plate.
What is responsible for the shape of a virion?
The amount and arrangement of the proteins and nucleic acid of viruses determine their size and shape. The nucleic acid and proteins of each class of viruses assemble themselves into a structure called a nucleoprotein, or nucleocapsid.
What is the purpose of performing a serial dilution on a sample?
Dilution is the process of making a solution weaker or less concentrated. In microbiology, serial dilutions (log dilutions) are used to decrease a bacterial concentration to a required concentration for a specific test method, or to a concentration which is easier to count when plated to an agar plate.
How do you count phages?
The number of phage particles contained in the original stock phage culture is determined by counting the number of plaques formed on the seeded agar plate and multiplying this by the dilution factor. For a valid phage count, the number of plaques per plate should not exceed 300 nor be less than 30.
What is a 1 in 10 dilution?
For example, to make a 1:10 dilution of a 1M NaCl solution, you would mix one “part” of the 1M solution with nine “parts” of solvent (probably water), for a total of ten “parts.” Therefore, 1:10 dilution means 1 part + 9 parts of water (or other diluent).
What is the meaning of dilution factor?
In chemistry and biology, the dilution ratio is the ratio of solute to solvent. This is often confused with “dilution factor” which is an expression which describes the ratio of the aliquot volume to the final volume. Dilution factor is a notation often used in commercial assays.
Why do we need to dilute the sample?
What is the purpose of dilution? A dilution can be performed not only to lower the concentration of the analyte that is being tested, so that it is in range, but also to help eliminate interferences from other substances that may be present in the sample that can artificially alter the analysis.
What is the plaque method quizlet?
Plaque Method. method in which a sample of bacteriophage is mixed with host bacteria and melter agar, poured into a, petri dish, and following several viral multiplication cycles, all of the bacteria in the area surrounding the virus are destroyed. Plaques.
What does 5% dilution mean?
Answer: 1:5 dilution = 1/5 dilution = 1 part sample and 4 parts diluent in a total of 5 parts. If you need 10 ml, final volume, then you need 1/5 of 10 ml = 2 ml sample. To bring this 2 ml sample up to a total volume of 10 ml, you must add 10 ml – 2 ml = 8 ml diluent. 2.
What is the basic principle behind serial dilution?
Serial dilution is a process through which the concentration of an organism, bacteria in this example, is systematically reduced through successive resuspension in fixed volumes of liquid diluent. Usually the volume of the diluent is a multiple of 10 to facilitate logarithmic reduction of the sample organism.
What is the purpose of top agar?
Top agar preparations contain lower concentrations of agar (7 g/L) than normal solutions used to prepare agar plates (15 g/L). The low agar concentration allows progeny phage from lysed cells to diffuse through the media and infect neighbouring bacterial cells.
What is a 7 1 dilution?
Now lets do a 7:1 dilution for a 32oz bottle. Again, change the dilution ratio numbers to addition like this: 7+1=8. Then we divide 32oz by 8 and we get 4oz. So put 4 ounces of chemical into the bottle and fill the rest with water for a 7:1 dilution.