Calculate PFU per mL
The plaque assay from overlay to stained plate, the countable range, controls, worked examples of the PFU/mL formula, MOI, and troubleshooting.
Chapters
- 0:15 Where the plaque assay came from
- 1:30 What a plaque is
- 2:42 Why one PFU is one infectious unit
- 3:58 How PFU compares with particle count
- 5:07 Run a phage double agar overlay
- 6:33 Choose the cells for an animal virus
- 7:34 Why the overlay is semi-solid
- 8:47 Adsorb, overlay, fix and stain
- 10:01 Make a tenfold serial dilution
- 11:12 Pick the plates you may count
- 12:28 Controls that give the count meaning
- 13:28 The PFU formula and its units
- 14:42 Worked example with a phage overlay
- 15:56 Worked example with wells and a different volume
- 17:08 Check the numbers with a calculator
- 17:46 TCID50 and focus-forming units
- 19:14 Work out the MOI from a titer
- 20:25 Troubleshoot plates with nothing to count
- 21:43 Troubleshoot plaques that look wrong
- 23:33 Tell the faults apart
- 24:25 Safety and record keeping
- 25:02 Summary and practice question
Transcript
0:15 Where the plaque assay came from
Felix d'Herelle described bacteriophage, the viruses that infect bacteria, in 1917. He was a French-Canadian microbiologist. In the same work he described how to isolate phage from the clear zones they leave in a growing bacterial culture, where the phage have killed the bacteria. Counting those clearings measures the concentration of infectious phage in a sample. Frederick Twort published his own description of the same phenomenon in 1915. Textbooks credit both men with the discovery. The soft agar overlay that microbiology teaching laboratories use today comes from work on counting phage in the 1930s and 1940s. For thirty-five years the method applied to phage only. In 1952 Renato Dulbecco produced the same kind of clearing in monolayer tissue cultures, starting from single particles of an animal virus. The plate held a continuous sheet of cultured cells instead of a lawn of bacteria. Two years later Dulbecco applied the same approach to poliovirus and isolated pure lines of virus.
1:30 What a plaque is
The layer of cells comes first, because the localized clearings, or plaques, are a feature of that layer. A lawn of bacteria, or a sheet of cultured cells, grows as one continuous layer across the surface of the plate. A plaque is a localized area of infected or dead cells inside that continuous growth. Anything that kills a patch of the layer leaves a hole you can see without a microscope. Infection then spreads cell by cell, and it stays local. Adsorption is the step where a phage attaches to the surface of a cell. One phage adsorbs to one host cell, and that cell then releases new phage particles, the progeny of the first phage. Those progeny infect the neighboring cells in the lawn. After enough rounds, the dead patch is wide enough to show as a clearing. The clearing stays a disc because the infection moves outward from one point at about the same rate in each direction. The plate needs time for those rounds to run. On a bacterial lawn, plaques appear in about 6 to 24 hours, depending on the phage. In the monolayer format you stain the sheet at the end, and each plaque shows as a pale hole in a colored layer.
2:42 Why one PFU is one infectious unit
Counting plaques gives a titer only once the unit of that count is fixed. A plaque-forming unit, or PFU, is the amount of virus that produces one plaque, which is one infectious unit. At the dilutions you use for counting, one plaque starts from one infectious particle and its progeny. The number of plaques then equals the number of infectious units in the volume you put on the plate. Titer names the concentration of infectious units in a suspension, and you report it as PFU/mL. This unit counts only the particles that form plaques under the conditions of the assay. A particle that cannot complete a replication cycle in the cells you chose produces no plaque, so it never enters the count. One plaque corresponds to one infectious unit, except when several particles infect the same cell. Measurements on marked poliovirus stocks found more than one parental virus in 5 to 7 percent of plaques, even at very low numbers of particles per cell. Virus stocks hold aggregates as well as single particles. Several particles stuck together infect one cell and give one plaque. One plaque is therefore one infectious unit rather than strictly one particle. Each plaque therefore marks at least one infection event.
3:58 How PFU compares with particle count
A plaque assay counts infectious units. Electron microscopy and genome copy methods count physical particles, whether or not they can start an infection. The particle-to-PFU ratio is the total number of particles in a sample divided by the infectious units in that same sample. Reported ratios are 1 or 2 for bacteriophages and vaccinia virus. Herpes virus sits near 10, influenza between 20 and 50, and poliovirus between 30 and 1000. A ratio above one means that some particles in the preparation do not form a plaque, and a ratio of 30 or more means that most of them do not. Damage and incomplete assembly account for part of that, and a particle that lands on a cell which does not support it gives no plaque either. Aggregation also lowers the count, because several particles stuck together infect one cell and give one plaque. The ratio also depends on how you measure each side. A PFU/mL figure belongs with the cell line and the assay that produced the count. A ratio near 1 for a phage says that almost all of its particles can infect a host cell.
5:07 Run a phage double agar overlay
The phage format is the double agar overlay, a plate with two agar layers, and it measures the concentration of infectious phage particles in a sample. The plate carries two layers. A base of 1.5 percent hard agar holds the nutrients. On top of that you pour about 3 mL of 0.7 percent soft agar, which you keep molten in a water bath at 50 °C. The soft layer carries the host cells and the phage. You add about 0.3 mL of a host culture in early log phase, the stage of rapid division soon after growth starts, at roughly 10⁸ cells per mL. Then you add 0.1 mL of the diluted phage. You roll the tube between your palms for two or three seconds, then pour it onto a warm, dry base plate. A quick, gentle figure eight spreads the soft layer without tearing the surface of the base. Soft agar lets the phage diffuse through the layer, which gives more consistent plaques. It also removes the uneven absorption into hard agar that direct plating produced. You then incubate the plate inverted, for example at 35 °C. The host strain has to be sensitive to the phage. Phages are highly host-specific, and with the wrong strain you see no plaques at all. A drop version of the same assay puts drops of each dilution onto a set lawn, which handles large numbers of samples faster than the classical overlay.
6:33 Choose the cells for an animal virus
For an animal virus the lawn is a monolayer of cultured cells, and the assay counts holes in that sheet. A permissive cell line is one in which the virus completes a full replication cycle, so an infected cell produces progeny that infect its neighbors. Whether a cell line is permissive depends on the virus. Protocols therefore tabulate host cells for each virus rather than naming one line for all work. You start from a monolayer with normal cell shape, covering about 90 to 100 percent of the growth surface. Confluency is that fraction of the surface covered by cells. You check the sheet under a microscope before you plate, and you set aside wells that are patchy. A gap in the sheet looks like a plaque after staining, and a thin sheet lets the infection spread too far. Titrating the same stock on two cell lines can give two different titers. You report the cell line with the titer, because the number belongs to that combination. Influenza assays use MDCK cells, for example.
7:34 Why the overlay is semi-solid
Without an overlay the progeny move through the liquid medium and infect cells across the well, so no discrete plaque forms. A semi-solid overlay is agarose, methylcellulose or a suspension of microcrystalline cellulose laid over the monolayer. It restricts spread to the cells immediately around each infection, which allows discrete countable plaques. The overlay also sets how large the plaques become, because plaque size increases as the overlay becomes less viscous. A stiff overlay therefore holds each plaque small. In a direct comparison, a cellulose overlay gave larger plaques than agar or methylcellulose. Low-viscosity overlays are easier to handle, particularly in the small wells of a 96-well plate, and you can apply one of them without removing the inoculum first. Some viruses need an additive in the overlay before they spread at all. Influenza assays on MDCK cells include trypsin in the overlay at about 1 microgram per mL. Each plaque on the plate then represents one infection that stayed in one place. You keep one overlay type for a whole series, because plaque size differs between overlay types.
8:47 Adsorb, overlay, fix and stain
You put a measured volume of each dilution onto the monolayer and rock the plate at 10 to 15 minute intervals while the virus attaches. Rocking also stops the sheet from drying out. You then remove the inoculum and cover the cells with the overlay. The cells release progeny after that point, and the progeny stay near the cell of origin. You equilibrate an agarose or cellulose overlay at 56 °C and use it warm but not hot. Hot overlay kills cells and lowers the titer, and in one comparison more plaques formed under a cellulose overlay than under heated agar. Agarose begins to set below 42 °C, so you work in small batches. After incubation you fix the cells, for example in 10 percent formaldehyde for 30 minutes or longer. Fixation also inactivates the virus before you handle the plate on the bench. You then stain, for example with 1 percent crystal violet in 20 percent ethanol for about 15 minutes, and rinse. The stain colors the surviving cells and leaves each plaque as a pale hole. Neutral red is another stain for this step. You can apply it early and it stays in contact with the overlay, although it stains less distinctly than crystal violet.
10:01 Make a tenfold serial dilution
A stock contains more infectious units than one plate can resolve as separate plaques, so you dilute it in a tenfold series before plating. Each step moves one volume of sample into nine volumes of sterile diluent, for example 0.1 mL into 0.9 mL. The tube you have just made supplies the sample for the next tube. Each tube carries one tenth of the concentration of the previous tube. The accuracy of the whole series rests on the mixing step, because an unmixed tube passes the wrong concentration down the rest of the series. You mix each tube by pipetting in and out of the meniscus, the curved liquid surface at the top of the tube, before you take the next volume. You change to a fresh sterile tip at each transfer, so a stronger dilution does not carry over into the next tube. You plate a range of consecutive dilutions, for example 10⁻³ through 10⁻⁶. You find out which dilution landed in the countable range only after incubation. Aim the series at that window rather than at a single plate. For the phage overlay, protocols aim for 100 to 250 plaques on the countable plate.
11:12 Pick the plates you may count
After incubation the series gives plates with very different numbers of plaques, so a counting rule decides which plate carries the result. For the phage overlay you count the plates that hold 30 to 300 plaques. Below 30 plaques the random sampling error is large compared with the count, so the estimate is unreliable. Above 300 the plaques merge, and over-incubation enlarges them further until the lawn clears completely. Complete clearing of the lawn, or confluent lysis, gives no count at all. On a monolayer each protocol sets its own window per well. One protocol discounts wells with fewer than 5 or more than 100 plaques. The counts should also fall tenfold for each tenfold dilution, because plaque number varies linearly with the dilution you plate. A series that does not behave that way points at the pipetting rather than at the biology. You plate at least two wells or plates per dilution. You average the replicates that fall inside the window before you calculate. A plate with 9 plaques sits outside the window, so its count stays out of the average even when the rest of the series looks sound. The countable window differs between formats, so you check the window for the assay you are running before you count anything.
12:28 Controls that give the count meaning
A plate of host cells with no virus added tells you whether the clearings on the other plates belong to your virus. Clearings on that plate point at contamination, or at a phage that is already present in the culture. A plate of the diluent alone checks the diluent and the tips. A contaminated diluent produces growth across the whole series. The host and virus pair is itself a control. With a strain the phage does not recognize you get no plaques, whatever the titer of the stock. For phage work, one standard pair is E. coli B with one of the T-even phages, the group that includes T2, T4 and T6. You set the control plates up at the same time as the dilution series, so they go through the same incubation. A second count of the same plate by someone else catches counting differences before they look like a disagreement between replicates. The control plates carry no count of their own. They tell you whether you can rely on the plates you did count.
13:28 The PFU formula and its units
With the countable plate chosen and the control plates clean, one calculation turns the count into a titer. PFU/mL equals the number of plaques divided by the product of the dilution and the volume of that dilution you plated, in mL. Read the denominator as a volume. The dilution multiplied by the plated volume is the volume of the original stock that reached the plate. The formula is then the count divided by that volume. Write the dilution as the fraction it is. A 10⁻⁵ dilution is the fraction one over one hundred thousand, so dividing by it multiplies by one hundred thousand. Keep the units through the calculation. Plaques divided by mL of original stock gives PFU/mL. Scientific notation keeps the exponents straight. You write the number with one digit before the decimal point, multiplied by ten raised to a whole-number exponent. The dilution and the volume each carry an exponent, and you add them separately from the count. For 0.1 mL plated, the volume contributes a factor of ten, and that factor belongs in the answer. A plate count carries about two significant figures, so you round the answer to two.
14:42 Worked example with a phage overlay
The first example is a phage overlay. You plated 0.1 mL of each dilution in duplicate. The 10⁻⁴ plates show confluent lysis, so they hold no count. The two 10⁻⁵ plates hold 142 and 154 plaques, and their mean is 148. The two 10⁻⁶ plates hold 12 and 19 plaques. Only the 10⁻⁵ plates fall between 30 and 300, so they alone carry the result. Take the denominator first. 10⁻⁵ multiplied by 0.1 mL is 10⁻⁶ mL. 10⁻⁶ mL is the volume of original stock each plate received. Then divide. 148 plaques divided by 10⁻⁶ mL gives 1.48 × 10⁸ PFU/mL. A plate count carries two significant figures, so you report 1.5 × 10⁸ PFU/mL. Check the result against the neighboring plate. Twelve plaques at 10⁻⁶ would give 1.2 × 10⁸ PFU/mL, the same order of magnitude, although that plate sits below the window.
15:56 Worked example with wells and a different volume
The second example changes the plated volume. You put 0.2 mL of each dilution into a well of a six-well plate, in duplicate, under an agarose overlay, and you fixed and stained the plate afterwards. The 10⁻³ wells hold about 400 plaques each, above the limit for a well, so you leave those wells out of the count. The two 10⁻⁴ wells hold 36 and 44 plaques, both inside the window, and their mean is 40. Work out the denominator before the division. 10⁻⁴ multiplied by 0.2 mL is 2 × 10⁻⁵ mL. 40 plaques divided by 2 × 10⁻⁵ mL gives 2.0 × 10⁶ PFU/mL. Compare the volumes. The same 40 plaques from 0.1 mL would give 4 × 10⁶ PFU/mL, twice as high. The plated volume sits in the denominator, so halving it doubles the titer. The two dilutions also behave as they should. 400 plaques at 10⁻³ against 40 at 10⁻⁴ is a tenfold drop for a tenfold dilution.
17:08 Check the numbers with a calculator
A calculator repeats the same arithmetic in one step. You enter the mean plaque count, 40, then the dilution as its exponent, 4, which stands for 10⁻⁴. You enter the inoculum volume in mL, 0.2. It returns 2.00 × 10⁶ PFU/mL, the number the hand calculation gave. You also enter the number of cells seeded in the well, one million here. The panel reports 0.40 infectious units added per cell for that inoculum. A line under the result reports that the count sits inside the 30 to 300 window.
17:46 TCID50 and focus-forming units
Some viruses do not kill cells quickly or completely enough to leave a readable hole, so the assay changes the endpoint instead. The 50 percent tissue culture infectious dose, or TCID50, is the dilution of the virus that infects half the inoculated wells. You read each well as infected or not, rather than counting plaques. The 50 percent endpoint comes from the pattern of positive and negative wells across the series, by the Spearman-Karber or the Reed and Muench calculation. Both of those take statistical work, and simpler endpoint formulas exist for the same purpose. The theoretical relation between the two units is about 0.69 PFU for each TCID50, and it comes from the Poisson distribution, which gives how often each number of independent events occurs. A stock at 1 × 10⁵ TCID50/mL therefore predicts roughly 7 × 10⁴ PFU/mL. The relation can fail even for the same virus and cell combination, because the two assays use different set-ups. You do not swap the units freely. A focus-forming assay uses the same dilution series under a semi-solid overlay, and finds infected cells by antibody staining instead of by lysis, so you count each stained cluster of cells, a focus, in place of a plaque. It reports focus-forming units per mL, and it suits viruses that do not break the cell membrane.
19:14 Work out the MOI from a titer
An experiment starts from the number of infectious units you want per cell. Multiplicity of infection, abbreviated MOI, is the infectious units you add divided by the number of cells in the vessel. MOI is an average over the whole vessel. The number of particles that reach one cell follows that same Poisson distribution, because the particles arrive independently of one another. At an MOI below one, many cells receive nothing and a few receive more than one particle. Work the arithmetic in the direction you need. Take a stock at 2 × 10⁶ PFU/mL and a flask holding 5 × 10⁵ cells. An MOI of 0.1 needs 5 × 10⁴ PFU. Dividing those PFU by the titer gives 0.025 mL, or 25 µL of stock. In the other direction, 0.2 mL of that stock on one million cells delivers 4 × 10⁵ PFU, an MOI of 0.4. A low MOI keeps each plaque independent, which is the condition the plaque assay itself needs.
20:25 Troubleshoot plates with nothing to count
A series with no plaques on any plate has several possible causes, and you can separate them without repeating the whole assay. The host or the cell line may not support the virus. Phages are highly host-specific, and a host strain the phage does not recognize gives no plaques whatever the titer of the stock. The overlay may have killed the cells or the virus. An overlay used too hot inactivates virus, and in one comparison fewer plaques formed under a heated agar overlay than under a cellulose overlay applied without heating. The overlay may be missing an additive the virus needs. Influenza plaques need trypsin in the overlay, and without it you see nothing at a high titer. The series may have gone too far. Plate a lower dilution alongside, and plate a larger volume of that dilution. A count below the window still produces a number, and that number is unreliable. Repeat with a lower dilution instead of reporting the count, because random sampling error dominates there. Confluent lysis is the opposite fault. Too much virus on the plate, or incubation long enough for plaques to grow together, clears the whole lawn and leaves nothing to count. Read the plates while the plaques are still discrete, and take the count from a higher dilution in the same series.
21:43 Troubleshoot plaques that look wrong
When the plaques are small, check the overlay before you suspect the virus. Plaque size increases as the overlay gets less viscous, so a stiffer overlay gives smaller plaques. Short incubation also gives small plaques. You separate the two by leaving one plate in the incubator longer. If its plaques grow larger at the same count, the cause was short incubation, and if they stay the same size, the overlay is limiting their size. Fuzzy or irregular outlines are not automatically a fault. Plaques are not necessarily round, and their appearance depends on the phage, on the medium and on the conditions the plate was grown in. Indistinct plaques can also come from the stain, because neutral red stains less distinctly than crystal violet. Smeared plaques on a monolayer come from movement of the overlay across the sheet. An overlay that slides across the sheet during incubation drags the infection with it, so you move the plate as little as possible once the overlay is on. A monolayer that lifts or shows holes before staining points at overlay that went on too hot, or at a sheet that dried out during adsorption. Equilibrate the overlay at 56 °C, apply it warm, and rock the plate during attachment. Bubbles in soft agar look like plaques. Pour the top layer without introducing air bubbles. Judge a doubtful clearing by its edge, because a bubble has a sharp regular rim and the lawn around a bubble does not thin. Growth that is not a plaque is contamination. Discard the series when you see colonies or fungal growth on a plate, or clearings on the virus-free control. Replicates that disagree widely point at a pipetting step, so check that you mixed each tube before you drew the next volume. Mix each tube, change the tip at each step, and average only the replicates inside the window.
23:33 Tell the faults apart
Faults are easier to name in a fixed order, because each question rules out a group of causes. Look at the control plates first. Growth on the virus-free plate makes the other readings unreliable until you have dealt with the contamination. Then look at the series rather than at one plate. Counts that fall tenfold for each tenfold dilution point at a sound series, so a single odd plate is a plate problem. Counts that do not fall tenfold point at the dilutions, so repeat the series with fresh diluent and deliberate mixing at each step. Then look at the lawn or the monolayer under a microscope before you count, because a patchy sheet accounts for both false plaques and missing ones. Last, look at the plaques themselves. Compare their size and their edges against the overlay you chose and the time the plate spent in the incubator.
24:25 Safety and record keeping
Virus and phage suspensions travel as aerosols, so you open tubes and plate dilutions in a cabinet suited to the agent, at the biosafety level set for that virus. The formaldehyde step also inactivates the virus, so you can stain and count the plate on the bench. Teaching laboratories use phage with a host strain that is safe to handle, which is why students meet the double agar overlay before any animal virus. Write the dilution and the plated volume on each plate as you prepare the plate. A plate with no dilution and no volume written on it cannot enter the calculation.
25:02 Summary and practice question
A plaque is a localized area of killed cells in a continuous lawn or monolayer. One PFU is one infectious unit, and a single particle can fail to form a plaque. The two formats are the phage double agar overlay and the monolayer assay. In the monolayer assay a semi-solid overlay holds each infection in place on a sheet of cultured cells. Count only the plates inside the countable window, and average the replicates. PFU/mL is the plaque count divided by the product of the dilution and the plated volume in mL. The same titer gives the MOI for an experiment, from the infectious units you add and the number of cells. Two wells at the 10⁻⁵ dilution each received 0.5 mL of inoculum and hold 38 and 42 plaques. Work out the mean count, the volume of original stock each well received, and the titer in PFU/mL.