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Quantification of Polysaccharides Fixed to Gram Stained Slides F1000Research 2015, 4:1 Last updated: 16 MAY 2019 METHOD ARTICLE Quantification of polysaccharides fixed to Gram stained slides using lactophenol cotton blue and digital image processing [version 1; peer review: awaiting peer review] Bryan Ericksen Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA First published: 05 Jan 2015, 4:1 ( Open Peer Review v1 https://doi.org/10.12688/f1000research.5779.1) Second version: 13 Apr 2015, 4:1 ( https://doi.org/10.12688/f1000research.5779.2) Reviewer Status Third version: 15 May 2017, 4:1 ( https://doi.org/10.12688/f1000research.5779.3) Invited Reviewers Fourth version: 13 Jul 2017, 4:1 ( 1 2 3 https://doi.org/10.12688/f1000research.5779.4) Latest published: 06 Dec 2017, 4:1 ( https://doi.org/10.12688/f1000research.5779.5) version 5 report published Abstract 06 Dec 2017 I discovered indigo rings and circles in Escherichia coli ATCC® 25922™ cultures when I added the non-specific polysaccharide stain lactophenol cotton blue to Gram stained slides sampled from 96-well plates used to version 4 report measure quantitative growth kinetics (QGK) in virtual colony count published antimicrobial assays. I attribute the dark blue staining to the presence of 13 Jul 2017 capsular polysaccharides and bacterial slime associated with clumps of cells. Since all bacterial cells are glycosylated, the majority of cells stain light blue. The contrast between dark and light staining is sufficient to version 3 report report enable a digital image processing thresholding technique to be quantitative published for circular or ring-shaped structures that imply the presence of slime fixed 15 May 2017 to the glass. These polysaccharides indicate a possible mechanism of resistance to antimicrobial peptides such as defensins, lectins with high affinity for polysaccharides and glycosylated proteins. Adding lactophenol version 2 cotton blue to Gram stained slides is a quick and inexpensive way to screen published 13 Apr 2015 cell cultures for bacterial slime, clumps and biofilms, revealing details of polysaccharide secretion that are missed using the Gram stain alone. version 1 Combined with QGK threshold times, the lactophenol cotton blue Gram published stain followed by digital image processing provides quantitative information 05 Jan 2015 useful for quality control, environmental monitoring and detection of clumping environmental factors. 1 Klaus Kayser, Charité-Universitätsmedizin Keywords Berlin, Berlin, Germany Antimicrobials, Biofilms, E. coli, Environmental 2 Venkataramana Kandi, Pratima Institute of Medical Sciences, Karimnagar, India 3 William R. Jacobs, Albert Einstein College of Medicine, New York, USA Page 1 of 9 F1000Research 2015, 4:1 Last updated: 16 MAY 2019 Any reports and responses or comments on the article can be found at the end of the article. Corresponding author: Bryan Ericksen ([email protected]) Competing interests: No competing interests were disclosed. Grant information: The author acknowledges Peprotech, Inc. for funding this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2015 Ericksen B. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Ericksen B. Quantification of polysaccharides fixed to Gram stained slides using lactophenol cotton blue and digital image processing [version 1; peer review: awaiting peer review] F1000Research 2015, 4:1 ( https://doi.org/10.12688/f1000research.5779.1) First published: 05 Jan 2015, 4:1 (https://doi.org/10.12688/f1000research.5779.1) Page 2 of 9 F1000Research 2015, 4:1 Last updated: 16 MAY 2019 Introduction so 2×MHB was chosen as the outgrowth media in VCC studies The virtual colony count (VCC) microbiological assay has been of E. coli ATCC® 25922™ at UMB, published between 2005 and used for over a decade to measure the effect of antimicrobial pep- 2013 (Ericksen et al., 2005; Zhao et al., 2013). tides such as defensins and LL-37 against a variety of bacteria. The VCC procedure first exposes bacterial cells to the active antimicro- The method of enumeration (Brewster, 2003) of surviving cells bial agent in 10 mM sodium phosphate pH 7.4 for two hours, then used by VCC is termed quantitative growth kinetics (QGK). It a twice-concentrated broth is added to simultaneously inhibit the relates the time taken for the turbidity of a microbiological culture antimicrobial activity and induce exponential growth. The method in a well of a 96-well microplate to reach a threshold difference in was initially developed by Robert I. Lehrer of the University of turbidity to a 10-fold dilution series, conducted as a separate cali- California, Los Angeles (UCLA) using twice-concentrated tryptic bration experiment (Figure 1A). Quantification of the number of soy broth (2×TSB) as the outgrowth media. However, the com- viable cells follows an algorithm mathematically identical to quan- monly used antimicrobial susceptibility testing positive control titative real-time polymerase chain reaction (QPCR) (Heid et al., strain E. coli ATCC® 25922™ formed macroscopic clumps in TSB 1996), except that with QGK the cells, rather than copies of PCR up to several millimeters in diameter when grown in the in 125 mL products, grow exponentially. The time taken to reach the thresh- disposable Erlenmeyer flasks with 0.22 micron filters in the caps old is called the “threshold time”, Tt, which is equivalent to the at 37°C shaking 250 rpm (Institute of Human Virology building QPCR value “cycle time” or Ct. While the calibration curve linear at the University of Maryland Baltimore [UMB]). These clumps regression R2 values were often >0.999 (Figure 1B), scatter below were not present in a clinical laboratory at UMB that routinely uses a ΔOD650 of 0.01 (Figure 1A, green, lavender and blue points and ATCC® 25922™ grown in TSB for antimicrobial susceptibility circles) suggested the presence of bacterial clumps. Gram stains testing, nor were they evident using E. coli ATCC® 43827™ (ML- (Gram, 1884) of both TSB and MHB cultures revealed far fewer 35) in the IHV building. ATCC® 25922™ formed an apparently clumps than expected, given that a broad size distribution should homogeneous suspension with no clumps visible to the unaided accompany the intermediate steps leading to macroscopic clumps. eye when Mueller-Hinton Broth (MHB) was substituted for TSB, Apparently, most clumps were not retained on the glass during the 1 Escherichia coli ATCC 25922 calibrations extuplicate growth curves A1 A2 A3 A A4 A5 A6 B 600 E. coli ATCC 25922 calibration curve A7 A8 A9 A10 A11 A12 B1 B2 B3 y = -68.142x + 590.59 2 ) B4 B5 B6 0 500 R = 0.9999 65 B7 B8 B9 0.1 B10 B11 B12 C1 C2 C3 107 106 105 104 103 102 101 100 CFU/mL C4 C5 C6 C7 C8 C9 400 C10 C11 C12 threshold = 0.02 D1 D2 D3 0.02 D4 D5 D6 T T T T T T T t t t t t t t D7 D8 D9 300 0.01 D10 D11 D12 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 200 F1 F2 F3 F4 F5 F6 F7 F8 F9 Threshold time Tt (mintues) 0.001 F10 F11 F12 G1 G2 G3 100 G4 G5 G6 change In optical density at 650 nm (∆OD G7 G8 G9 G10 G11 G12 H1 H2 H3 0 H4 H5 H6 H7 H8 H9 0 1 2 3 4 5 6 7 0.0001 H10 H11 H12 0 100 200 300 400 500 600 700 Log (CFU/mL) Time (minutes) C Figure 1. Quantitative growth kinetics (QGK) and the lactophenol cotton blue Gram stain. A: Escherichia coli ATCC 25922 was diluted 10-fold in Mueller-Hinton Broth and growth kinetics was monitored for 12h using a Tecan infinite M1000 plate reader according to the UMB procedure (Ericksen et al., 2005). QGK was used to calculate threshold times, Tt. B: Calibration curve relating Tt to the logarithm of cell concentration in a separate experiment. Both experiments shown in A and B were conducted in November, 2011 by Le Zhao. C: Lactophenol Cotton Blue Gram Stain Procedure. Overnight steps allowed for equilibration to the ambient humidity during summer months in the IHV building at UMB, which ranged from 40–60%. Water content and temperature may be important factors for the caramelization process to be quantitatively reproducible. Page 3 of 9 F1000Research 2015, 4:1 Last updated: 16 MAY 2019 Gram stain procedure, whether fixed to the slide by heat or metha- a single large macroscopic clump (up to 1 cm long, equal to the nol. The application of lactophenol cotton blue, ordinarily used to cuvette width) at the base of the cuvette. OD650 plummeted up to visualize fungi by staining cell wall polysaccharides such as chitin, 2% per minute, reaching equilibrium after a 10–20% decrease revealed circles and rings consistent with the caramelized residue when placed in a room temperature HPLC detector, as cells in of polysaccharides, which presumably included capsular polysac- suspension joined the clump beneath the light path. The optical charides and slime secreted concomitantly with clump and biofilm density readings declined so rapidly that only the first two digits formation. These indigo circles and rings could be consistent either of the four reported by the Waters 600 detector could be recorded. with a heterogeneous subpopulation of E. coli or with slight con- Macroscopic clumping in the batch culture or cuvette outside the tamination with a second strain. detector was no longer observed after four changes: 1. using a small HEPA-filtered air purifier, 2. replacing in-house deionized Materials and methods Milli-Q water with purchased molecular biology grade water, 3.
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