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It was adapted from the Wikipedia page Virtual_colony_count and contains some or all of that page's content licensed under a CC BY-SA license. Post-publication review comments or direct edits can be left at the version as it appears on Wikipedia.
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DOI: 10.15347/wjs/2020.003
QID: Q86161728
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Bryan Ericksen (16 February 2020). "Virtual colony count". WikiJournal of Science 3 (1): 3. doi:10.15347/WJS/2020.003. Wikidata Q86161728. ISSN 2470-6345. https://upload.wikimedia.org/wikiversity/en/a/a3/Virtual_colony_count.pdf.
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Wikipedia: This work is adapted from the Wikipedia article Virtual colony count (CC BY-SA). Content has also subsequently been used to update that same Wikipedia article Virtual colony count.
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This is an open access article distributed under the Creative Commons Attribution ShareAlike License, which permits unrestricted use, distribution, and reproduction, provided the original author and source are credited.
Thomas Shafee 
(handling editor) contact
Jennifer Payne
Article information
Plagiarism check
Pass. Report from WMF copyvios tool: 0% overlap detected. T.Shafee(Evo﹠Evo)talk 07:35, 26 July 2019 (UTC)
First peer review
Review by anonymous peer reviewer , Researcher in antimicrobial peptide activity in vitro and in vivo
These assessment comments were submitted on , and refer to this previous version of the article
- Introductory paragraph
Is the technique limited to antimicrobial peptides or can it also be used for other antimicrobial molecules?
Thus far the technique has been limited to antimicrobial peptides. It could potentially work with other antimicrobial agents, as long as the activity of the agent is abrogated at the time twice-concentrated Mueller Hinton Broth is added. The previous two sentences have been added to the end of the section, Virtual colony count. I should mention that I have conducted an experiment with an antimicrobial agent that was not completely abrogated by the addition of broth, vancomycin, and the kinetics of surviving batch cultures was greatly affected; in other words, the growth curves were no longer parallel.
The comments on the lack of activity of antimicrobial peptides in broth is not applicable to all mediums. That is there are a number of culture media that are amenable to antimicrobial activity assays using peptides. This nullifies the argument that MIC assays don’t work for peptides. The authors either need to specify why the it is important that the particular broth they are referring to (I assume it is LB or RPMI) needs to be used for the MIC assays. This is a repeated problem throughout the article. The use of low salt growth media to assess the activity of antimicrobial peptides is very common in the literature.
In the Minimum inhibitory concentration section, rich broth is specified. I have further specified "such as Mueller-Hinton Broth". I also further specified standardized MIC methods using MHB are not applicable to defensins.
There is an obvious parallel with ΔΔCT measurements in qPCR assays that should come up much sooner than it does.
Although a detailed discussion of qPCR might interest some readers, it is somewhat off the topic of this publication. Besides, Ct delays in qPCR and Tt delays in VCC are caused by unrelated phenomena. Therefore, I did not mention this parallel in the article.
- Antimicrobial susceptibility testing
Defensins are found in many organisms other than mammals, please amend this statement.
The word mammals has been replaced by many organisms
- VCC
There needs to be some mention that this method is limited identifying microbicidal molecules and will not detect microbistatic activities.
Actually the VCC method can detect either bactericidal or bacteriostatic activity. Its limitation is that it cannot distinguish between them. However, bacteriostatic activity can be quantified by measuring the difference in threshold times between the "input" and "output" controls. This information has been added to the end of the VCC section. In addition, a description of the "input" and "output" controls has been added to the General laboratory procedure section.
What happens if this assay is used with a salt tolerant antimicrobial peptide?
Vancomycin was tested using VCC as noted above; its activity was not abrogated by the addition of twice concentrated MHB, and growth curves of survivors were not parallel, especially at the highest concentration of vancomycin that produced survivors. It is not my intent to publish the vancomycin experiment.
The statement “Virtual colony forming units, or CFUv, were held constant among different strains tested, so that the amount of cell membrane was roughly equal, as described in the original VCC publication” is unclear. Please elaborate.
The sentences "Virtual colony forming units, or CFUv, is defined in the original VCC publication. CFUv was held constant among the six strains tested so that the turbidity, and thus the amount of cell membrane, in each experiment was roughly equal." have been added.
“Because CFUv was standardized to the CFU of Escherichia coli ATCC 25922, CFU, not CFUv, can be reported with this strain.” I think it would be more appropriate to list this as CFU equivalents as opposed to actual CFU.
The number of colonies produced by a certain optical density reading of this strain was measured by an actual colony counting experiment. Therefore, it is appropriate to use actual CFU with this strain.
- General laboratory procedure for use in VCC assays
The order of sentences seems to be off. Why is the addition of TSB for the 2 h incubation mentioned before the 2 h incubation
"Added to" has been replaced by "present in".
How did the authors decide when to add the 1% tryptic soy broth during the 2 h incubation?
"Added to" has been replaced by "present in".
Why switch between OD an cell count as a measure of culture density halfway through the method? Why not just have the data is OD in stead of CFUv and compare to the amount of time it takes an untreated control to reach the OD threshold? If you just use OD you can eliminate setting up the standard curve, which in itself is a potential source of error/variability in the experiment.
I am unsure the source of confusion in this comment, since the threshold is set at an OD of 0.02, not a CFUv value. The standard curve is required in order to convert optical density readings to threshold times and virtual survival.
The entire parafilm discussion is unnecessary, at the very least it needs tidying up.
A further explanation has been added, "It should be noted that in the initially published VCC experiments only the internal 60 wells of the plate were used, since evaporation changed the volume of the edge wells during the 12-hour incubation." The addition of parafilm to the assay is actually a major improvement, since it allows using all of the 96-well plate for the experimental portion of the assay.
- Quantitative growth kinetics
Adhering cells that are not in the light path will affect the OD reading. It means there is a concentration of cells that are not in suspension, not affecting the ability of light to pass through the solution and therefore the OD reading is lower than it should be. This is kind of addressed in the cohesion point but is not obvious.
This sentence has been changed to "Unless these cells happen to be directly in the light path, they cannot be directly detected by the plate reader."
Second peer review
reviewer-annotated pdf
Review by Jen Payne , Biochemistry & Molecular Biology, Monash University
These assessment comments were submitted on , and refer to this previous version of the article
An article that requires work to help insure it reaches the audience. Currently readability could be improved in sections. Figures require improvement to the legends to adequately describe the figures and additionally would benefit from having descriptions present rather than just well numbers in figures. For more detailed comments on each section see the attached document.
Reviewer comments below copied from the annotated pdf, alongside author responses:
In the Antimicrobial susceptibility section: “strongly inhibited by physiological salt concentrations. In order to measure the antimicrobial activity of HNPs, they had to be incubated with cells in a low salt buffer as a separate initial step,” Please add to this description. As currently there is a logical jump in that if HNPs are inhibited by physiological salt conditions, how are these antimicrobial activities determined with this VCC assay relevant to their biological activity in vivo? Therefore, if these results are not providing an understand of the peptide that is biologically relevant what is the advantage of this assay.
There is no way to assay peptides such as the defensin HNP1 in the presence of physiologically relevant salt concentrations without using an overwhelmingly high defensin concentration. Therefore, any assay that measures HNP1 activity uses conditions that are different from those present in the body. A sentence has been added to this effect. It should be noted that very high defensin concentrations are present in vivo. In the neutrophil granules, the defensin concentration is above 10 millimolar.
Has a physiologically relevant media been tested in this VCC assay instead of MHB? Has activity been monitored with the addition of serum or albumin?
No, physiologically relevant media have not been tested, but since activity is measured in the low salt buffer, not in the presence of MHB, the use of physiologically relevant media instead of MHB would not make the assay more physiologically relevant. Activity has not been monitored with the addition of serum or albumin. Some activities of defensins are inhibited by serum, while others are not.[1]
In the MIC section: “ rich broth such as Mueller-Hinton broth (MHB)” is this the cation adjusted version? Cations can also have a big impact on the antimicrobial activity measured. Perhaps mentioning this effect would be worthwhile?
Non-cation-adjusted MHB has been used for the majority of VCC assays. Cation-adjusted MHB (CAMHB) has also been tested, and it can be used interchangably with non-cation-adjusted MHB. It is important to emphasize that the assay measures activity in the low salt phosphate buffer, not in the presence of MHB, so adjusting the cation concentration of the MHB does not affect the defensin activity measured by the assay. Defensins are inhibited by the addition of either MHB or CAMHB. The sentence, "Either cation-adjusted or non-cation-adjusted MHB may be used." has been added to the "General laboratory procedure for use in VCC assays" section.
“because defensins must be incubated in a low salt buffer, not rich broth, in order to
measure their activity.” What is the difference in salt concentration between these two?
The low salt buffer contains 10 mM sodium phosphate. According to one reference, Mueller Hinton Broth (apparently without cation adjustment) contains 0.37 mM Mg2+, 0.15 mM Ca2+, 97 mM Na+, 6.0 mM K+, and 77 mM Cl-.[2]
In VCC section: “by simply adding an equal volume of twice-concentrated broth after the two hour incubation in the low salt buffer, as long as there is a way to determine how many cells survived at the end of the incubation period using batch cultures.” This sentence is not easy to understand, please rewrite and clarify.
This sentence has been divided into two sentences for clarity.
“turbidity, or optical density, of the 96-well plate” it is not the 96-well plate that changes in optical density over time but the bacterial culture contained within. Change this wording to clarify.
The words "batch culture within the" have been added for clarity.
“ of the exponentially growing cells is known, then the number of cells originally present in the inoculum can be calculated. This inoculum” Inoculum is not the right word to use here. Change to ….number of cells originally present in the culture can be calculated. This starting number of cells”
"Inoculum" has been replaced by "starting number of cells".
“is defined in the original VCC publication” please define it here as well
CFUv was defined in the subsequent two sentences, as the text now indicates.
“as long as the activity of the agent is abrogated at the time twice-concentrated Mueller Hinton Broth is added” do you mean…. as long as the twice-concentrated Mueller Hinton Broth inactivates the antimicrobial activity of the agent?
The text has been changed to read, "as long as the twice-concentrated Mueller Hinton Broth inactivates the antimicrobial activity of the agent".
“CFUv was held constant among the six strains tested so that the turbidity, and thus the amount of cell membrane, in each experiment was roughly equal. Because CFUv was standardized to the CFU of Escherichia coli ATCC 25922, CFU, not CFUv, can be reported with this strain.” This does not belong in the general VCC description as it is specific for the experiments carried out that have not been introduced yet. This needs to be moved and incorporated below.
This text has been moved to the section, General laboratory procedure for use in VCC assays
In the section: General laboratory procedure for use in VCC assays “antimicrobial peptides are diluted on a 96-well plate (Costar 3595)”- “96-well plate” please define what type of plastic. Peptides will stick to different types of plastics effecting the amount of peptide available for killing bacteria. Tissue culture treated plates will also effect growth curves.
The text has been changed to reflect the fact that Costar 3595 plates are tissue culture treated.
“Parafilm M six squares long by one half square” could this be something like the Breathe easy membrane instead? (https://www.sigmaaldrich.com/catalog/product/sigma/z380059?lang=en®ion=AU)
This product has not been tested. The advantage of using Parafilm to seal the edge of the plate is that the Parafilm does not cover the portion of the lid in the light path of the plate reader, so optical density readings are not affected.
“threshold optical density of 0.02.” How was this threshold determined? When would this change?
This question has already been answered in the section, "Results: calibration": "0.02 was initially chosen as the lowest threshold that cleared all noise in the initial dataset. A higher threshold may be desirable if there is noise above the 0.02 threshold, as occurred in one well of the 96-well plate in the example experiment.
In the section: Quantitative growth kinetics “It relates the kinetic time taken for the turbidity of a bacterial batch microbiological culture in a well of a 96-well microplate to reach a threshold difference in turbidity to a 10- fold dilution series of calibration growth curves.” This definition seems verbose and meaning is lost because of it. Perhaps something like this instead- It relates the time taken for the turbidity of a bacterial culture in a well of a microplate to reach a threshold in relation to calibration growth curves.
This change has been made as suggested.
Figure 1- Change the key in this figure to label the 6 replicates for each of the concentration
of the calibration curves. Add more information to the legend to ensure this figure is stand
alone. Information currently in legend regarding plating should be included as main text
instead.
The legend has been changed as suggested.
Figure one is not mentioned in the main text.
A sentence introducing Figure 1 has been added in the QGK section.
In the section: Bacteria studied in the history of VCC assays “six strains of E. coli, S. aureus, Bacillus cereus, and Enterobacter aerogenes. [1] “ Use the full names of the bacteria here.
Escherichia and Staphylococcus have been spelled out.
In the section: Antimicrobial peptides studied in the history of VCC assays “ A conserved glycine in a beta bulge in HNP2 was replaced with a series of D-amino acids resulting in VCC activity proportional to side chain hydrophobicity and charge.[12] VCC showed that N-terminally acetylated and/or C-terminally amidated HNP2 activity is proportional to electrostatic charge.[13] VCC results were again proportional to charge for a series of salt bridge-disrupting mutants, suggesting that the salt bridge is not required for HNP2 function.[14] VCC measured the importance of N-terminal natural and artificial pro segments of the propeptide HNP1, dramatically altering activity against Escherichia coli and Staphylococcus aureus. [15][16] “ In this section the effect of these changes is not fully described. Define what is meant by proportional and altering? Do these changes result in a decrease on increase in antimicrobial activity?
The word proportional has been replaced by increase and altering has been replaced by decrease.
In the section: Inoculum effect “An inoculum effect has been previously described for many antimicrobial agents” Provide references for this statement.
The review reference has been moved so that it follows this statement.
Change inoculum to the following underlined in this sentence. “such that the agent is less effective when more bacteria are added to the assay.”
This change has been made.
“Because the inoculum of bacteria was 20-fold greater in the VCC assay compared to the standardized traditional colony count protocol used, the difference could have been due to an inoculum effect, although the effect would have been the reverse of the inoculum effect normally seen with other antimicrobial agents, since the higher inoculum showed more activity. This possibility was investigated in a series of experiments mainly focusing on the defensin HNP1 and the bacterial strains E. coli, S. aureus and B. cereus.” This section currently reads as if the experiment detailed below should be a comparison of VCC assay and the traditional colony count in respect to a range of inoculum.
VCC experiments have been specified.
In the section: Example VCC experiment “HNP1 was synthesized on an ABI 433A synthesizer using an optimized HBTU activation/DIEA in situ neutralization protocol developed by Kent and coworkers for Boc chemistry solid phase peptide synthesis (SPPS)” What was the yield? How was this peptide purified? How was the peptide mass quantified? How did you ensure that you had successfully made and purified this peptide?
"The high performance liquid chromatography purification and electrospray ionization mass spectrometry confirmation of the correct mass of the purified peptide have been detailed elsewhere" has been added and referenced.
Why is this NaPiT buffer different from the one listed above in section “General laboratory procedure for use in VCC assays?” Define how the TSB enhances the defensin activity?
"It is thought that allowing the cells to grow during this step makes cell walls and cell membranes more vulnerable to disruption by defensins" has been added.
“to a cell concentration of 2x108CFU/mL” how did you determine these cultures were at this CFU/mL? Do you know what absorbance equals a particular CFU/mL? If this is by absorbance to you check the CFU counts each time you perform the assay?
This determination was made at the time of the original VCC publication (Ericksen et al. 2005).
“and then the calibration dilutions were done in row G as described in the caption of Figure 2.” The description of the calibration dilutions is better suited in the main text. Please remove from the figure legend and place here.
This information has been moved to the main text.
“Input controls were added to wells B1-B1” What volumes are added? What are the in the input controls?
The text was changed to: "The input controls consisted of the cell suspension incubated on ice during the 2-hour incubation step, then added to the plate at the same inoculum as wells 2-11 in the same row." The text "50 μL of cell suspension were added for both input and output controls." was added.
“and then 100 μL of twice-concentrated MHB was added to all wells except row G.” Add a description of what is in row G and how this curve was prepared.
The detailed description of Row G has been moved from the caption of Figure 2 to the main text, as noted above. The words "the calibration curve in" were added before "Row G".
“Reading every 5 minutes and shaking 10s in a linear fashion before reading.” State the Absorbance that the plate is read at.
The text, "the optical density at 650 nm" was added.
“Rows 36-134 of this spreadsheet were copied and pasted in rows 37-135 of spreadsheet "raw".” Add to this sentence to describe why this step was done.
The text, "in order to add the raw data in the proper position to be read by the macro", was added.
In the section: Results: observation of the 96-well plate “precipitates were visible as white specks” do you have images of these precipitates?
What do you think makes up these precipitates? Is this to do with bacterial growth or the peptide falling out of solution? Does this happen with other peptides?
These precipitates were not photographed in this experiment. Other peptides were not tested. The sentence, "Presumably these precipitates were cell clumps" was added.
In the section: Results: calibration “evenly spaced and parallel up to a change in optical density of about 0.3,” Explain why they lose this at this point?
The sentence, "As cells exit from exponential growth and enter stationary phase, slight variations in growth characteristics cause deviations in growth above this region" has been added.
“The calibration curve was linear with an R2 value of 0.992.” Is there a figure for this?
"The text, "as is apparent from the linear regression curve fit shown in the spreadsheet "calibration" in the Excel file" has been added.
Figure 3. Provide not just the well numbers but also the starting E. coli concentrations in
these wells.
Figure 3 has been updated with the starting cell concentrations in the legend.
Results: calculation of virtual survival “It is calculated according to Brewster.” Provide a brief summary of how this is performed.
“Virtual survival values are plotted in Figure 4.” Please provide description of these results and what they mean here.
The text, "Virtual survival is a measure of antimicrobial activity that relates the measured threshold times of the experimental wells to the threshold times of the calibration curve. For example, a virtual survival value of 1 x 10-3 corresponds to a threshold time delay equal to a relative cell dilution of 1 x 10-3 CFU/mL in the calibration curve in row G, as calculated using linear regression" has been added
Color for the different bacterial concentrations in figure 4 would make this figure easier to interpret.
Color has been added to Figure 4.
Results and discussion: a pronounced inoculum effect In figure 7 & 8, include the well number along with the HNP-1 concentration for all wells.
The HNP-1 concentration of each well has been added to the legends of Figures 7 and 8.
“These results unequivocally rule out the interpretation of the previously published experiments[2] comparing traditional colony count results with VCC results that would have attributed the difference to an inoculum effect.” A side by side comparison of the two assays would be required.
The reference does present a side-by-side comparison of the two assays. To emphasize this fact, "side-by-side" has been added.
Replicate data and other strains How much variation is there between experiments? This is a single biological replicate, that does not contain any technical replicates. This experiment needs to be repeated and replicate data shown.
Six replicates of this experiment were performed, which differed only in the cell concentrations used. An additional seven figures showing the virtual survival plots from the other five replicates, a composite virtual survival plot showing the mean curves from all six experiments, and a virtual lethal dose table showing standard error will be added. These figures have been prepared and will be added following the addition of the figures requested by the third reviewer.
- ↑ "Multifaceted mechanisms of HIV-1 entry inhibition by human α-defensin". J Biol Chem. 287 (34): 28821-38. 2012. doi:10.1074/jbc.M112.375949. PMID 22733823. PMC 3436536. //www.ncbi.nlm.nih.gov/pmc/articles/PMC3436536/.
- ↑ "Effect of salt concentration on the apparent in-vitro susceptibility of Pseudomonas and other gram-negative bacilli to gentamicin". J Infect Dis. 124 (Suppl): S59-64. 1971. PMID 5001630.
Third peer review
Review by anonymous peer reviewer , My lab works on antimicrobial peptides and we routinely perform antifungal and antibacterial assays.
These assessment comments were submitted on , and refer to this previous version of the article
This article reviews the published literature on the VCC experiments. It describes the advantages of VCC over the traditional colony count. It also outlines the protocol for VCC assay using the antibacterial peptide and its target bacterial pathogen. Importantly, it presents five factors that influence quantitative growth kinetics which relates the kinetic time taken for the turbidity of a bacterial culture in a well of a 96-well microplate to reach a threshold difference in turbidity to a 10-fold dilution series of calibration growth curves. Finally, it describes the results of a VCC experiment investigating the effect of varying the inoculum of bacterial cells when assayed against HNP1 defensin. The results demonstrated strong inoculum effect especially at high inocula.
This review is of interest to all scientists who use in vitro antibacterial assays to assess the antibacterial activity of antimicrobial peptides.
I suggest that author makes use of flowchart diagrams to present various steps involved in VCC assays. Also, an easy-to-understand table showing the potential technical difficulties and artifacts that may be encountered in VCC and ways to address them would be helpful. Finally, is it possible to assess antibacterial susceptibility using fluorescence-based determination of bacterial metabolism instead of relying on monitoring bacterial growth by turbidity in VCC assays?
A flowchart diagram has been added (Figure 1) and a table showing technical difficulties and artifacts has been added (Figure 8). Yes, it is possible to assess antibacterial activity by fluorescence. I have seen several published methods using resazurin. A recent report [1] describes a rapid method in picoliter format that provides results after a one hour incubation, which is a significant improvement over VCC, which requires a two hour incubation plus a significant enough portion of the 12 hour incubation time to see a real time difference in threshold times. A detailed discussion of fluorescence-based methods is beyond the scope of this article.