Editor
Simab Kanwal
  • Research Associate, Mahidol University
Research fields
  • Biochemistry, Pharmaceutical compounds, Analytical Chemistry, Chromatography, Phytochemistry
A Novel and Robust Method for Investigating Fungal Biofilm
Authors:  Biswambhar Biswas, Shumaiza Asif, Rekha Puria and Anil Thakur, date: 01/05/2025, view: 67, Q&A: 0

Candida auris, labeled an urgent threat by the CDC, shows significant resilience to treatments and disinfectants via biofilm formation, complicating treatment/disease management. The inconsistencies in biofilm architecture observed across studies hinder the understanding of its role in pathogenesis. Our novel in vitro technique cultivates C. auris biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other Candida species like C. glabrata and C. albicans, is cost-effective and mimics the niche of biofilm formation. It is suitable for high-throughput drug screening and repurposing efforts, aiding in the development of new therapeutics. Our technique represents a significant advancement in Candida biofilm research, addressing the need for consistent, reproducible biofilm models. We detail a step-by-step procedure for creating a substratum for biofilm growth and measuring biofilm thickness using confocal laser scanning microscopy (CLSM) and ultrastructure by scanning electron microscopy (SEM). This method provides consistent outcomes across various Candida species.

Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate
Authors:  Peng Wan, Meiyue Tao, Yumeng Zhou, Wenjun Han, Jianxia Wang and Jinghan Wang, date: 12/20/2024, view: 209, Q&A: 0

Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples. This protocol describes a step-by-step cryo-scanning electron microscopy (cryo-SEM) experimental procedure with Chlorella sorokiniana as the subject. By employing filter paper as the sample substrate, we propose a simple and reliable method for cryo-fixation and freeze-fracture of Chlorella sorokiniana in water suspension. The advantage of using filter paper as a substrate lies in its ability to support a thin film of sample, enabling a cold knife to make a cut effortlessly and produce a clean freeze-fractured surface for SEM investigation. By following the approach described in this protocol, both the internal structure and surface morphology of Chlorella sorokiniana can be easily resolved with high quality. This protocol is highly versatile and can be applied to samples dispersed in water or solvents, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto filter paper.

Seed Collection in Temperate Trees—Clean, Fast, and Effective Extraction of Populus Seeds for Laboratory Use and Long-term Storage
Authors:  Naima Bhutta, Oscar F. Nunez-Martinez, Carmen Mei and Katharina Bräutigam, date: 02/05/2024, view: 647, Q&A: 0

Seeds ensure the growth of a new generation of plants and are thus central to maintaining plant populations and ecosystem processes. Nevertheless, much remains to be learned about seed biology and responses of germinated seedlings to environmental challenges. Experiments aiming to close these knowledge gaps critically depend on the availability of healthy, viable seeds. Here, we report a protocol for the collection of seeds from plants in the genus Populus. This genus comprises trees with a wide distribution in temperate forests and with economic relevance, used as scientific models for perennial plants. As seed characteristics can vary drastically between taxonomic groups, protocols need to be tailored carefully. Our protocol takes the delicate nature of Populus seeds into account. It uses P. deltoides as an example and provides a template to optimize bulk seed extraction for other Populus species and plants with similar seed characteristics. The protocol is designed to only use items available in most labs and households and that can be sterilized easily. The unique characteristics of this protocol allow for the fast and effective extraction of high-quality seeds. Here, we report on seed collection, extraction, cleaning, storage, and viability tests. Moreover, extracted seeds are well suited for tissue culture and experiments under sterile conditions. Seed material obtained with this protocol can be used to further our understanding of tree seed biology, seedling performance under climate change, or diversity of forest genetic resources.


Key features

Populus species produce seeds that are small, delicate, non-dormant, with plenty of seed hair. Collection of seed material needs to be timed properly.

• Processing, seed extraction, seed cleaning, and storage using simple, sterilizable laboratory and household items only. Obtained seeds are pure, high quality, close to 100% viability.

• Seeds work well in tissue culture and in experiments under sterile conditions.

• Extractability, speed, and seed germination were studied and confirmed for Populus deltoides as an example.

• Can also serve as template for bulk seed collection from other Populus species and plant groups that produce delicate seeds (with no or little modifications).


Graphical overview


Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates
Authors:  Sven Reichardt, Annick Stintzi and Andreas Schaller, date: 02/05/2023, view: 456, Q&A: 0

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor–processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage.


Graphical abstract


A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient.


Graphic abstract:



Figure 1. Satellite cell isolation using the ice-cold treatment method.


Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
Authors:  Gareth Howell and Robert-Jan Bleichrodt, date: 04/20/2021, view: 3564, Q&A: 0

Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.

Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria
Authors:  Marcus Cheng, Siew-Ging Gong and Céline M. Lévesque, date: 11/20/2020, view: 4697, Q&A: 0
Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in liquid medium, we developed a simple and inexpensive peptide extraction procedure using a semi-solid nutrient-rich agar medium. We hereby present a detailed procedure that leads to the rapid extraction of secreted bioactive bacteriocin peptides from the oral species Streptococcus mutans, a prolific bacteriocin-producing species, and its potential application for bacteriocin extraction from other LAB (e.g., Streptococcus, Lactococcus, Enterococcus). We also present a simple method for the detection of bacteriocin activity from the purified extracellular peptide extract.
Construction and Validation of A Low-cost, Small-scale, Multiplex Continuous Culturing System for Microorganisms
Authors:  Lilit Tonoyan, Freddy Guihéneuf, Ruari Friel and Vincent O’Flaherty, date: 11/05/2020, view: 2732, Q&A: 0

This protocol details the construction of a simple, low-cost, small-scale, multiplex chemostat system designed for the continuous cultivation of microorganisms in suspension (i.e., bacteria, yeast, microalgae). The continuous culture device can operate at a working volume of 25 ml and allows the run of 8 chemostats in parallel by a single person. It provides a platform for parallel, long-term studies of evolution and adaptation of microorganisms under the stress of antimicrobial agents and/or toxic pollutants. The system complies with the varied needs of researchers for an accessible, highly-throughput and reliable tool that is nevertheless easy to construct, use and operate, and not demanding of space, materials, medium supply and workload. Here, we also validate the use of this system to generate de novo resistance towards a novel antimicrobial and a commonly used antibiotic in an antimicrobial-sensitive model organism. We believe that this "Do It Yourself" (DIY) system may constitute a useful tool to address the global problem of antibiotic resistance and to develop non-antibiotic based therapies.

Gas Chromatography Detection Protocol of Short-chain Fatty Acids in Mice Feces
Authors:  Chaozheng Zhang, An Liu, Tianshuang Zhang, Yi Li and Hua Zhao, date: 07/05/2020, view: 5130, Q&A: 0
Short-chain fatty acids (SCFAs), which are formed mainly by bacteria fermenting undigested carbohydrates in the colon, they are based on the number of carbon atoms in the carbon chain. Organic fatty acids with less than 6 carbon atoms are called short-chain fatty acids. SCFAs are closely related to various aspects of the human body, so more and more researchers concentrate on SCFAs. This protocol describes, a direct injection gas chromatography detection method with a pretreatment method for extracting SCFA from mice feces by combining acidification. The corresponding sample limit of quantization (LOQ) and limit of detection (LOD) are 0.8-1.0 mg/L and 0.5-0.8 mg/L, respectively. The correlation coefficient of calibration curve is greater than 0.999. The recovery rate of the spiked standard is 80%-102%. This method can be used to analyze and determine SCFAs in mice feces. Therefore, this is an economical, effective and reproducible method for SCFAs measurement in mice samples.
1-MCP (1-methylcyclopropene) Treatment Protocol for Fruit or Vegetables
Authors:  Gamrasni Dan, Goldway Martin, Stern Yosi, Breitel Dario and Aharoni Asaph, date: 05/20/2017, view: 15474, Q&A: 4
1-MCP (1-methylcyclopropene) is a simple synthetic hydrocarbon molecule that interacts with the ethylene receptor and inhibits the response of fruit or plant to ethylene. 1-MCP has opened new opportunities in handling harvested crops and serves as a powerful tool to learn about plant response to ethylene (Watkins and Miller, 2006). 1-MCP is manufactured by Agrofresh and known by its commercial name SmartfreshSM.
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