Mar 07, 2023
Version 1
Compound Screening and Growth Curves V.1
- 1North Carolina State University
External link: http://go.ncsu.edu/htdprotocols
Protocol Citation: Carlos Carlos Goller 2023. Compound Screening and Growth Curves. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4qzzgmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 06, 2023
Last Modified: February 28, 2024
Protocol Integer ID: 78210
Keywords: growth, MIC, IC50
Funders Acknowledgement:
Biotechnology Program (BIT) at NC State
Grant ID: 1
Disclaimer
This protocol was created for the BIT 479/579 High-throughput Discovery course and for use with students. It has been used by and optimized for students.
Abstract
Overview
The bacterial genus Delftia was named for the city of Delft in the Netherlands, where the species type was isolated and for the Delft research groups that had a critical role in the early development of bacteriology. Species of Delftia have since been isolated all over the world in different environments and have been known to cause infections in humans.1Delftia are gram-negative rod-shaped aerobes. Species of Delftia are often resistant to several compounds, including the common disinfectant chlorhexidine,2 most antibiotics in the aminoglycoside group,3 and heavy metals.4 One of Delftia’s most surprising talents is its ability to concentrate heavy metals like gold.5 Biofilms found on gold deposits suggest that these bacteria are responsible for their formation.6 Known species of Delftia include D. acidovorans, D. tsuruhatensis, D. deserti, D. lucustris, and D. litopenaei. Refer to Figure 1 for an example of Delftia acidovorans colonies growing on tryptic soy agar (TSA).
Figure 1.Delftia acidovorans colonies growing on tryptic soy agar (TSA)
Where are Delftia spp.found?
Delftia spp. are commonly isolated from aquatic environments such as lakes7 and wastewater8 and are thought to be ubiquitous. Because of their affinity for aquatic environments, Delftia have been identified in organisms and objects frequently found in these environments, such as mosquitoes9 and birds’ feathers.10Delftia aren’t just aquatic microbes, though. These bacteria have been identified in unexpected places like desert soil11 and contact lens cases.12
An Opportunistic Pathogen
Species of Delftia have been known to cause various infections in healthy and immunocompromised people. Many of these infections are (IV) line-related,13,14 but other infections have occurred due to intravenous drug use.3Delftia were also implicated in eye inflammation related to contact lens use.12 NC State students and staff have even found Delftia DNA in surveys of kitchen sinks performed by students in the BIT 477/577 Metagenomics lab module!
Our Project
We will set up a series of high-throughput (HT) assays to test whether different gold chloride concentrations inhibit the growth of different Delftia strains. Gold chloride has been explored for its antibacterial properties15. However, Delftia spp. have a unique non-ribosomal peptide that helps relieve gold toxicity by producing delftibactin5.
Bacterial strains will be grown in 96-well plates to test multiple concentrations of gold chloride and antibiotics and the effect of miniaturization of the assay. We will set up plates in the first lab session to test several strains at multiple doses with numerous replicates. Plates will incubate for one day at 30ºC before assessing cell viability using a stain for metabolic activity, PrestoBlue®. This approach can be adapted to screen active compounds from a high-throughput screening (HTS) campaign. For this first compound screen, we will focus on the effects of automation and miniaturization on assay reproducibility.
We will test gold chloride concentrations in a twelve-point dose-response curve. At the end of this experiment, you can determine which concentrations are inhibitory by calculating the percent growth inhibition and IC50. We will compare the susceptibility of various Delftia strains.
Image Attribution
Delftia acidovorans ATCC 13751 colonies grown on tryptic soy agar (TSA). NC State University Biotechnology Program (BIT). William DeFoor, BIT SURE 2014.
Guidelines
Additional Resource : PrestoBlue Cell Viability Reagent Protocol
Materials
Materials
- Overnight cultures of Delftia acidovorans SPH-1, Delftia acidovorans Cs1-4, and other Delftia spp. isolates in 2.5 ml of Tryptic Soy Broth (TSB, 30ºC with shaking 16-18 hrs)
- Teknova™Tryptic Soy Broth (TSB)
- epMotion 5075 and 50 and 300 filter tips (Eppendorf)
- Teknova antibiotic stocks
Equipment
- Eppendorf epMotion 5075 liquid handler
- Eppendorf BioSpec
- Biotek/Agilent LogPhase 600 plate reader
Safety warnings
While Delftia spp. are ubiquitous, we will treat them as potential sources of infection and abide by BSL-2 standards. We will minimize contact with cultures, use hoods, and submit waste for disinfection/autoclaving. Please use the clear biohazard bags at your stations and the biohazard bin next to the sink.
Before start
Please put on your personal protective equipment (PPE) supplied by your instructors. PPE for this lab includes gloves, disposable lab coats, and eye protection.
Before you start, clean bench surfaces and pipettors with ethanol.
Plate Setup and Incubation
Plate Setup and Incubation
1d 0h 30m
1d 0h 30m
Read the procedure listed below. Discuss with your lab partner the key steps to program the liquid handler to successfully complete this task. Pay attention to the reagents, tube format, and equipment we have. Remember the goal: reproducibly testing compounds (with replicates) and including negative and positive controls.
Figure 1. Schematic of procedure to use liquid handler to seed plates with bacteria and treatments. A set of Delftia spp. Bacterial isolates were grown on Tryptic Soy Agar (TSA) and then liquid broth (Tryptic Soy Broth) before diluting to a specific OD600. The eqMotion 5075 liquid handler was used to seed plates with bacteria and treatments: water/buffer control (negative control), antibiotic (positive control), and gold chloride (test compound). Plates were incubated with shaking in a LogPhase 600 for 24 hours with OD600 readings obtained every 10 min. After 24 hours, Invitrogen PrestoBlue viability stain was added to all wells to quantify viability. Each condition was tested in triplicate wells for calculations of inhibitory concentrations. Created with BioRender.com
As a group, we will use a common script and the liquid handler to prepare sets of plates for analyses. Each bay will set up one plate.
Grow Overnight cultures of Delftia spp. in 2.5 mL of TSB, shaking at 300 rpm and incubating at 30 °C
For each strain, subculture the bacteria to approximately 5 x 105 CFU in 25 mL of fresh TSB by performing the following:
Measure the OD600 of the overnight culture (1:10 dilution in TSB);
Plug this number into the formula at the bottom of the page and multiply by 10 to ensure we have enough bacteria in each well.
Take the calculated amount (μL) from the overnight culture and add to 25 mL of fresh tryptic soy broth (TSB); mix well.
Add 100 µL of diluted bacterial suspension into each well of rows 2-12.
Add 194.88 µL of subcultured bacterial suspension into each well of row 1.
Add gold chloride (5.12 µL from a stock solution) to wells B1, C1, and D1.
Add antibiotic solution (5.12 µL from a stock solution) to E1, F1, and G1.
Add water (5.12 µL of water) to A1 and H1 (to ensure the diluent is not toxic to the bacteria)
Thoroughly mix the wells in column 1 and remove 100 µL and add to the wells in column 2.
Thoroughly mix the wells in column 2 and remove 100 µL and add to the wells in column 3.
Continue the serial dilution down the 96-well plate and discard the last 100 μL from column 12 (Note: There should be 100 μL in each well now).
Incubate at 30 °C for 24:00:00 with 300 rpm LogPhase600 setting in the LogPhase 600 plate reader. Take A600 readings every 10 min.
1d
Inspect the 96-well plate for growth. The MIC (Minimum Inhibitory Concentration) is the first concentration at which no growth is observed. (Note: there should be growth in rows A and H).
Read OD600 and add 10 µL of PrestoBlue to each well.
Incubate at 30 °C for 00:30:00 . Read OD570 and OD600 for normalization. Review the information provided in the PrestoBlue resource.
30m
Note the concentration at which each compound inhibits the growth of the bacteria. Record data in your electronic lab notebook.
Note
[We will complete steps 14-16 for you and send you the data.]
Plate Layout
Figure 2. Plate layout for growth inhibition curves. Each row of the 96-well plate will be seeded with bacteria treated with water, antibiotic, or gold chloride. The columns of the plate will be used to dilute the concentration of the treatment to determine inhibitory concentrations. Created with BioRender.com
Note
Formula: μL of overnight culture to take = (100/OD600)*0.0005*25 ml * 10
Note
Analyses
- Explain how the viability stain PrestoBlue works.
- Calculate each stain’s mean and CV normalized PrestoBlue absorbance.
- Calculate the percent inhibition produced by each treatment with respect to the water control.
- Calculate the % inhibition produced by each compound with respect to the water controls.
- Calculate IC50 for each treatment you tested.
- Summarize your results in a table that includes: IC50, treatment, and observations for all plates.
Formula
μL of overnight culture to take = (100/OD600)*0.0005*25ml
Protocol references
Acknowledgements:
Michelle Musante, an undergraduate senior, helped write the background to this lab as part of her BIO 493 research experience. She is interested in the natural ecology of Delftia in water fountains and gutters.
Image:
Delftia acidovorans ATCC 13751 colonies grown on tryptic soy agar (TSA). NC State University Biotechnology Program (BIT). William DeFoor, BIT SURE 2014.
References
- Wen A, Fegan M, Hayward C, Chakraborty S, Sly LI. Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. Nov., comb. Nov. International Journal of Systematic Bacteriology. 1999;49(2):567–576. doi:10.1099/00207713-49-2-567.
- Rema T, Medihala P, Lawrence JR, et al. Proteomic analyses of Chlorhexidine tolerance mechanisms in Delftia acidovorans Biofilms. mSphere. 2016;1(1):e00017–15. doi:10.1128/msphere.00017-15.
- Mahmood S, Taylor KE, Overman TL, McCormick MI. Acute Infective Endocarditis caused by Delftia acidovorans, a rare Pathogen complicating intravenous drug use. Journal of Clinical Microbiology. 2012;50(11):3799–3800. doi:10.1128/jcm.00553-12.
- Bautista-Hernández DA, Ramírez-Burgos LI, Duran-Páramo E, Fernández-Linares L. Zinc and lead Biosorption by Delftia tsuruhatensis: A bacterial strain resistant to metals isolated from mine Tailings. Journal of Water Resource and Protection. 2012;04(04):207–216. doi:10.4236/jwarp.2012.44023.
- Johnston CW, Wyatt MA, Li X, et al. Gold biomineralization by a metallophore from a gold-associated microbe. Nature Chemical Biology. 2013;9(4):241–243. doi:10.1038/nchembio.1179.
- Reith F, Lengke M, Falconer D, Craw D, Southam G. The geomicrobiology of gold. The ISME Journal. 2007;1(7):567–584.
- Zaffar RM, Ganai BA. Bacterial biota of Nigeen lake waters (Kashmir valley). Microbial Pathogenesis. 2016;97:52–58. doi:10.1016/j.micpath.2016.05.005.
- Stolze Y, Eikmeyer F, Wibberg D, et al. IncP-1 plasmids of Comamonas sp. and Delftia sp. Strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet. Microbiology. 2012;158(Pt_8):2060–2072. doi:10.1099/mic.0.059220-0.
- Chavshin A, Oshaghi M, Vatandoost H, Pourmand M, Raeisi A, Terenius O. Isolation and identification of culturable bacteria from wild Anopheles culicifacies, a first step in a paratransgenesis approach. Parasites & Vectors. 2014;7(1):419. doi:10.1186/1756-3305-7-419.
- Herzog B, Overy DP, Haltli B, Kerr RG. Discovery of keratinases using bacteria isolated from marine environments. Systematic and Applied Microbiology. 2016;39(1):49–57. doi:10.1016/j.syapm.2015.10.004.
- Li C-T, Yan Z-F, Chu X, et al. Delftia deserti sp. Nov., isolated from a desert soil sample. Antonie van Leeuwenhoek. 2015;107(6):1445–1450. doi:10.1007/s10482-015-0440-4.
- Wiley L, Bridge DR, Odom JV, Elliott T, Olson JC. Bacterial Biofilm diversity in contact lens-related disease: Emerging role of Achromobacter , Stenotrophomonas , and Delftia. Investigative Opthalmology & Visual Science. 2012;53(7):3896. doi:10.1167/iovs.11-8762.
- Lang KJ, Chinzowu T, Cann KJ. Delftia acidovorans as an unusual causative organism in line-related sepsis. Indian Journal of Microbiology. 2011;52(1):102–103. doi:10.1007/s12088-011-0221-3.
- Chotikanatis K, Bäcker M, Rosas-Garcia G, Hammerschlag MR. Recurrent Intravascular-Catheter-Related Bacteremia caused by Delftia acidovorans in a Hemodialysis patient. Journal of Clinical Microbiology. 2011;49(9):3418–3421. doi:10.1128/jcm.00625-11.
- Shareena Dasari TP, Zhang Y, Yu H. Antibacterial Activity and Cytotoxicity of Gold (I) and (III) Ions and Gold Nanoparticles. Biochem Pharmacol (Los Angel). 2015 Dec;4(6):199. doi: 10.4172/2167-0501.1000199. Epub 2015 Dec 20. PMID: 27019770; PMCID: PMC4807878.