Report Plaque count, average plaque area, and total plaque area are automatically calculated and reported for each sample. Incorporating the BioStack Microplate Stacker enables processing and analysis of up to 50 plates. Plaque Assay. Get more info. The titer of the virus stock is therefore 1. Dulbecco, R. Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Dear Sir , we are facing problem with viral plaque assay that always our cell lines die after poring the mixture of agrose..
Due to time constraints it is not feasible to order pre-made in. Any suggestions welcome, thanks. Skip to main content Skip to primary sidebar One of the most important procedures in virology is measuring the virus titer — the concentration of viruses in a sample. Comments Dear Sir , we are facing problem with viral plaque assay that always our cell lines die after poring the mixture of agrose..
Here, the average number of plaques formed to the 10 to the minus three and 10 to the minus four dilution plates, were divided by their respective dilution factors to obtain the number of plaque forming units, or PFUs, in microliters of phage mixture. To convert the value to PFU per milliliter, multiply the generated values by 10, as only microliters of phage dilution mix was used during the bacteriophage overlay preparation step, producing an additional dilution factor of Finally, calculate the average of the values obtained from the different dilution plates.
This will give the average number of PFUs per milliliter. The number of PFUs corresponds to the number of infective phage particles in the original sample. Subscription Required. Please recommend JoVE to your librarian. Despite multiple technological advances, plaque assays remain the gold standard for determination of viral titer as PFU and essential for isolation of pure bacteriophage populations. Susceptible host cells are cultivated in the top coat of a two layered agar-plate, forming a homogenous bed enabling viral replication.
The initial event where an isolated bacteriophage in lytic lifecycle infects a cell, replicates within it, and eventually lyses it, is too small to observe.
However, the virions released will infect adjacent cells, subsequently giving rise to a clearing zone, or plaque, denoting the presence of a single PFU.
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Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. Log in or Start trial to access full content. Set-up Before commencing any work involving microbes, make sure that the work space is sterilized e. Always wear a lab coat and gloves, keep long hair tied back, and ensure that any wounds are particularly well protected.
Protocol LB Media preparation Note: Depending on the host bacterial strain and the bacteriophage, a different a liquid medium may be more suitable for the initial culturing of the host bacterial strain or a different solid medium may be more suitable for subsequent growth. Lysogeny broth LB is used in this protocol for the broth and the agar.
Mix 4 g LB in mL distilled water, in triplicate, for the LB broth, the solid bottom agar, and the soft top agar. All solutions should be prepared in containers able to hold twice the final volume to prevent overflow while autoclaving.
Note: If performing the assay in triplicate, prepare double the amount of LB bottom agar. Adjust the pH of all three solutions to 7. Add 3 g of agar power to the bottom agar bottle to make a 1. Close the caps as soon the run is finished to prevent contamination. Pour 15 mL aliquots of the solid agar medium into sterile Petri dishes avoid shaking to prevent foaming and allow the agar to solidify for a few hours or overnight at room temperature Figure 4 A.
The morning of the assay, add 0. Note: If you are performing the assay in triplicate, prepare double the amount of this culture. This can be determined spectrophotometrically by an OD of 0. Keep the culture at room temperature until the bacteria are to be added to the top LB agar. To do this, prepare additional dilutions of the bacteriophage in the second and third rows of the plate.
Mix the sample well by pipetting and down. This will create a dilution range of 10 -1 Plating Label the base of the Petri dishes previously prepared in step 2.
It should be hot enough to remain in liquid form, but cool enough to not kill added bacteria. Mix 4 mL bacterial culture from step 2. Swirl to evenly distribute the cells but avoid shaking to prevent foaming Figure 4 A.
Label one sterile test tube for each of the serial dilution steps and one for the control sample, for a total of 7 labeled test tubes. Work quickly through step 2. Discard the used pipette tip and transfer the same volume from each of the serially diluted bacteriophage samples step 2. Immediately transfer the 5 mL mixtures onto the labelled, pre-heated, solid agar plates Figure 4 A. Gently swirl the plates to even spread the mixtures.
Note: If you are performing the assay in triplicate, repeat steps 2. Data Analysis and Results Counting plaques Ensure that no plaques are visible in the plates marked "control", as this would indicate viral contamination. Begin with the plates labelled 10 -6 , containing the most diluted bacteriophage sample. Count the plaques without removing the lid, marking them with a pen as you go to indicate which plaques have already been counted.
Count the remaining plates. Some plates may have too few or too many plaques to count. Use the plates with plaques for further analysis. Calculating PFU Divide the number of plaques by the dilution factor, ex. Note: If performing the assay in triplicate, use the average number of plaques from the three plates.
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