Name: isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture
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Description: Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Dr. Stephen Alderman, Dr. David Leaf, and Erin Macri are gratefully acknowledged for help with microscopy. This research was funded through a grant from the NIFA Specialty Crops Research Initiative, USDA SCRI-SREP Grant Award No. 2009-02484. Briana Kinash, Kevin Kinloch, Megan Leonhard Joseph McCollum, Maria McSharry and Nicole Sallee provided excellent technical assistance and thoughtful discussions.

This procedure requires at least several months for incubation of BDM films in soil, and several more months for sequential bioassays both on agar plates and in agar-free, chemically defined broth to assess colonization and degradation. Individual methods are listed in the order they will be performed.
1. Incubation of BDM Films in Soil
Incorporate BDM films into soil under conditions mimicking those under which they will be expected to degrade. Acquire 400 g (dry wei...

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The procedure described herein represents a first-pass technique for isolating potential BDM degraders from soil, and was successfully used to isolate fungi from BDMs buried in soil for seven months. Fungi grew when reinoculated onto fresh BDM material of the same type, indicating that the isolated fungi were indeed colonizers, and that the films were not inhibitory to fungal growth. Isolation of plastic-degrading fungi and bacteria potentially could lead to their use, individually or in combinations, for amendments to s...

Degradation has historically been considered an undesirable attribute of plastic polymers, because breakdown shortens product life span and durability. Recently, awareness of the environmental problems presented by plastic waste in the natural environment1,2,3 has made biodegradable plastics an attractive alternative to conventional plastic materials. Degradation (defined as structural changes, fragmentation, and reduction in molecular weight, integrity, and strength4,5) occurs via a series of events, including both abiotic processes (thermal stress, photo-oxidation, hydrolysis, erosion and mechanical stress), and biological degradation6. While abiotic processes can change the fragment size and characteristics of plastics, microorganisms are required for their ultimate mineralization to water and carbon dioxide (in aerobic conditions) and/or methane (under anaerobic conditions).
A substantial niche for biodegradable plastics exists in agriculture, where plastic mulches are used to prevent weed growth, to retain soil moisture and to increase soil temperatures7,8 . Hundreds of thousands of acres in the United States alone are covered with plastic mulches9, including mulches composed of biodegradable plastic. Following a crop growing season, the options for disposing of biodegradable mulches (BDMs) include disposal in a landfill, incineration for energy recovery10, degradation via composting, or degradation in the soil after tillage11. Of these, the least labor-intensive fate is plowing BDMs into the soil, but without efficient degradation and mineralization during non-crop months (generally in the winter), plastic fragments could remain and interfere with agricultural equipment during spring tillage and planting, and persist in the environment where they significantly impact wildlife, plant life, and microbiota1,2,3,10.
Although many plastic products, including agricultural mulch films, bear the label "biodegradable" or "compostable", in practice, degradation and mineralization may be too inefficient and/or too incomplete for in-soil decomposition to be a viable alternative for disposal of these products. For example, oxo-biodegradable polyethylenes achieved only 12.4% mineralization after one year of weathering and three subsequent months in a 58 &deg;C compost, and less than half that amount of mineralization occurred when the compost temperature was 25 &deg;C12. In the winter, soil temperatures at most locations would be lower than either of these temperatures, presumably resulting in even lower microbial activity and consequently, less mineralization. In addition to slow degradation rates, misuse of the term "biodegradable" has led to distrust of these products by consumers13,14, including those in the agricultural industry. Biodegradation is the conversion of polymers to carbon dioxide (and/or methane) and water14 by naturally-occurring microorganisms4. Therefore, biodegradation must be measured chemically; the physical association of microorganisms with a substrate does not imply microbial degradation of that material.
As part of an effort to examine sustainable use of BDMs in agriculture, this study focused on discovering microorganisms native to agricultural soils that colonize and degrade commercially-available BDMs. Standard test methods have been published for chemically measuring the breakdown of biodegradable plastics by abiotic and biological means15,16,17. However, these methods do not address degradation of plastics by individual microbial species, or provide methods for their isolation. The methodology herein more closely resembles standard methods designed to evaluate plastics for resistance to microbial breakdown after inoculating specimens with fungal spores18,19.
When formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones13. This method has been used previously to identify microorganisms that degrade polymers such as polyurethane20, poly-(butylene succinate-co-adipate)21, and poly(lactic acid)22. A similar method involves suspending pure powdered plastic in liquid medium where the plastic is the sole carbon source20,23. While these methods have the advantage of a defined system, they poorly mimic in situ degradation of BDMs. First, the surface area is distributed differently because BDMs are not dispersed in small particles throughout the soil matrix, but rather, sold and used as films. Second, the chemical makeup of BDMs is different from pure polymers. BDMs generally contain additives such as fillers, plasticizers, and colorants, and these additives may affect microbial growth and thereby, the rate of mineralization. For this reason, and because the composition of certain commercial films in this study were proprietary, plastic film in its field-ready form was utilized to isolate fungi and bacteria. For simplicity, the methods below are described only for fungi, with modifications noted where appropriate for bacterial isolations.
In a recent study24, three commercially-available BDMs and one experimental film were used at agricultural sites in three different regions of the United States for one growing season, and subsequently placed in mesh (250 micron) bags and buried for one winter in soil at the same sites. The 250 micron mesh openings allow fungal hyphae to penetrate while excluding roots and most soil fauna, and minimizing soil encroachment25,26. Nylon materials prevent bag degradation in soil. Following excavation, fungal isolates were recovered from BDM pieces and assessed for growth on minimal medium without a source of carbon except for the agar and a 5 cm x 5 cm surface-disinfested square of new, unused BDM film that was pre-disinfested. Most plastics used as films cannot be autoclaved without loss of integrity, so UV light was used to kill any microbial cells residing on the plastics. ISO 84619 recommends surface-disinfesting in 70% ethanol and subsequent drying, but if using this method, one must ensure that no component or additive of the film is adversely affected by the ethanol. Since BDMs presumably are manufactured to withstand sunlight, UV was chosen as a decontamination method.
Isolates that grew on BDM pieces better than on minimal medium alone were selected for further study. Agar, a polysaccharide produced by marine algae, is used to solidify microbial media because it is typically not utilized metabolically by agriculturally and medically notable microorganisms; however, agar-hydrolyzing enzymes have been isolated from marine bacteria27 and agar-hydrolyzing bacteria also have been isolated from soil28. BDM polymers and agar are both expected to be rare substrates for enzymes secreted by soil fungi, which have not evolved in environments that contain these polymers as potential nutrient sources, but both substrates are present in the plate bioassay described herein (Step 7). Fungi that use BDMs but not agar as a carbon source can be differentiated from fungi that use agar only, by comparing growth on agar-solidified medium containing i) no added carbon source except agar (negative control), ii) BDM films (experimental) and iii) glucose (positive control). Growth of all isolates is expected on minimal medium plus glucose; fungi not arising on glucose-containing plates may not be capable of growth on the particular minimal medium used in the experiment. Potential BDM degraders should grow on agar-solidified minimal medium + BDM film better than they grow on agar-solidified minimal medium alone. Fungi growing on minimal medium plates are agar-degraders or oligotrophs, and are also expected to grow on the agar associated with BDM films in bioassay plates, but not on the films themselves (unless they serendipitously also degrade BDM polymers).
To eliminate the possibility of seeing microbial growth due to utilization of agar and not of BDMs, we followed our initial assay for BDM colonization on agar plates with a bioassay in defined broth medium (Step 9). BDM pieces represented the only known carbon source in the bioassay tubes.
After the initial screening, and upon reviving glycerol stocks of the isolates, some formed scant but visible mycelia in liquid defined medium containing no known carbon source. These results suggested that some of the acquired isolates were oligotrophs - organisms that grow by scavenging very small amounts of carbon, nitrogen, and other nutrients dissolved either in the aqueous environment or existing as volatiles in the air29,30,31. Species identification via 18S ribosomal DNA analysis supported this view, as many of the isolates matched fungal genera previously reported to exhibit oligotrophy32. Oligotrophs, which are commonly saprophytes, require a broad range of metabolic capabilities for substrate utilization in a range of environments30. Thus, it is not surprising that the same fungi we isolated from BDMs (presumably requiring unusual enzymatic capabilities) demonstrated oligotrophic capacities, and were able to grow on trace contaminants such as skin oils from fingerprints, dust, or trace volatiles in the air. Due to the isolation of oligotrophs, we concluded that growth on a BDM surface alone could not be used to infer BDM breakdown. The methods described herein reflect our efforts to screen native BDM colonizers from agricultural soils for bona fide BDM breakdown.

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

In a recent study24, four replicates each of three commercially-available BDMs labeled "biodegradable", plus an experimental film and a conventional plastic control, were placed over soil as mulch for tomato production in the spring of 2010 at Mount Vernon, WA, Knoxville, TN, and Lubbock, TX. In the fall of 2010, BDM film squares were cut from each weathered mulch in four replicate plots, and native soil was removed from directly beneath the area where the mulch sample had been excised. Each weathered BDM squa...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

isolation-native-soil-microorganisms-with-potential-for-breaking-down

Research

JoVE Journal

Biology

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...