Virtual colony count
Virtual colony count (VCC) is a kinetic, 96-well microbiological assay originally developed to measure the activity of defensins.
[2] It utilizes a method of enumerating bacteria called quantitative growth kinetics, which compares the time taken for a bacterial batch culture to reach a threshold optical density with that of a series of calibration curves.
The name VCC has also been used to describe the application of quantitative growth kinetics to enumerate bacteria in cell culture infection models.
The time taken for each growth curve to reach a threshold change in optical density is then converted into virtual survival values, which serve as a measure of antimicrobial activity.
However, a complication arose when researchers wished to study the antibacterial activity of some antimicrobial peptides, because they are inhibited by rich media, whether supplied on an agar plate or in broth.
They are divided into several structural classes including alpha, beta, and theta, based on the pattern of disulfide bonding.
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, before rich media was added allowing the enumeration of survivors.
One method commonly employed to measure antimicrobial activity in liquid is to expose the antimicrobial agent to cells during an incubation time such as two hours, and then enumerate survivors by diluting the mixture and then spreading a portion of the liquid on an agar plate containing rich media.
After spreading, the agar plates are then incubated overnight and the number of colony forming units (CFU) is counted the next day.
The traditional colony count method could be modified to measure antimicrobial activity in the 96-well plate without the need for sampling the wells and spreading surviving cells on agar plates by simply adding an equal volume of twice-concentrated broth after the two hour incubation in the low salt buffer.
Meanwhile, antimicrobial peptides are diluted on a 96-well plate (Costar 3595, which are tissue culture-treated) in 10 mM sodium phosphate pH 7.4 such that the final volume is 90 microliters.
A 10-fold dilution series of this suspension was made ranging from 107 to 100 CFUv/ml in 200 μl total volume of PMH, occupying eight wells of the 96-well plate.
Raw data is imported into Microsoft Excel, where the macro VCC Calculate is run to determine the time required for each growth curve to reach a threshold optical density of 0.02.
[12] The initial virtual colony count study measured the activity of all six human alpha defensins concurrently on the same 96-well plate: HNP1, HNP2, HNP3, HNP4, HD5, and HD6.
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.
[15] 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.
[16][17] Enantiomer forms of HNP1, HNP4, HD5 and Beta-defensin 2 composed entirely of D-amino acids suggested differing mechanisms of defensin activity against Gram-positive and Gram-negative bacteria.
This possibility was investigated in a series of VCC experiments mainly focusing on the defensin HNP1 and the bacterial strains E. coli, S. aureus and B. cereus.
[26] VCC users are cautioned to transfer cells in a small volume such as 10 microliters beneath a larger volume such as 90 microliters, similar to the QGK calibration curves shown above and the calibration curves reported in the initial VCC publication,[1] but unlike the experimental procedure used to test defensin activity in that same paper.
[10] The original method published in 2005 involved the transfer of 50 microliters of cell suspensions to 50 microliters of liquid, which generates froth, bubbles and turbidity that is incompatible with a turbidimetric method when cells are transferred directly to the bottoms of the wells beneath the phosphate buffer solutions.
[27] Bioaerosols of hazardous bacteria can also pose safety risks that can be reduced by conducting experiments within a biosafety cabinet.
