Name: isolation and screening from soil biodiversity for fungi involved in the degradation of recalcitrant materials
Uploaded: 2022-05-16T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials at JoVE.com

We acknowledge Scuola di Alta Formazione Dottorale (SAFD) of the University of Pavia and Professor Solveig Tosi for providing the opportunity for this work.

1. Selection of fungal strains able to degrade recalcitrant materials from soil
<ol>
	<li>Preparation of antibiotic solution.
	<ol>
		<li>Put penicillin (50 mg/L), streptomycin (40 mg/L), chlortetracycline (40 mg/L), neomycin (100 mg/L), and chloramphenicol (100 mg/L) into 250 mL of deionized sterile water.</li>
		<li>Before adding chloramphenicol to the antibiotic solution, dissolve it into 3 mL of &#8805;99% ethanol.</li>
		<li>Place the antibiotic solution on a magnetic stirrer (no heat) with a magn...

The rich biodiversity of soil is an abundant source of fungi that possess numerous metabolic abilities, some of which could be potential candidates for bioremediation. Selective media tests (Section 1 of the protocol) are easy-to-perform and effective methods for isolating fungi able to grow on natural complex polymers as their sole carbon source. Fungi can produce extracellular, non-specific hydrolases and oxidoreductases30 such as the ligninolytic enzymes laccases and peroxidases<sup class='xref...

Soil is a fundamental component of life on Earth and is the basis of many ecosystems. The minerals, organic matter, and microorganisms in the soil can be considered as one system, with close associations and interactions occurring between them. The interactions of these compounds have an important impact on terrestrial processes, environmental quality, and ecosystem health1. Soil pollution poses serious environmental problems worldwide. The indiscriminate, long-term, and excessive application of recalcitrant and toxic substances, such as pesticides, petroleum products, plastics, and other chemicals, has serious effects on soil ecology and, as a result, can alter soil microbiota. Microbial communities in soils are composed of a wide range of organisms in different physiological states, with the majority being bacteria and fungi. Many of the contaminants in soils have medium- to long-term stability, and their persistence can lead to the development of adaptive mechanisms that allow the microorganisms to utilize recalcitrant substances as nutrients2,3. These microorganisms can, therefore, be considered for bioremediation techniques.
Bioremediation tries to mitigate the effects of pollution by using microorganisms and their enzymes for the degradation or transformation of waste into less toxic or non-toxic compounds. Various species of archaea, bacteria, algae, and fungi possess this bioremediation ability4. As a result of their particular biodegradative actions, fungi are especially promising organisms for bioremediation. They can attack different substrates using their hyphal network, enabling them to penetrate the soil matrix more efficiently than other microorganisms. Additionally, they can reach inaccessible interstices where contaminants are difficult to remove5, and they can also survive low moisture levels6. Moreover, fungi synthesize different cassettes of unspecific enzymes, usually to degrade natural recalcitrant substances such as cellulose, lignin, and humic acids. Those that lack the target substrate can be involved in the degradation of a wide range of recalcitrant pollutants, such as hydrocarbons, plastics, and pesticides7,8,9,10. Therefore, although many fungal species have already been reported as bioremediation agents, there is increasing interest in exploring species that have not yet been studied to select candidates for the bioremediation of recalcitrant contaminating substances. The species already known to have bioremediation properties belong to the phyla Ascomycota11,12,13, Basidiomycota14,15,&#160;and Mucoromycota. For example, the genera Penicillium and Aspergillus are well known to be involved in the degradation of aliphatic hydrocarbons13, different plastic polymers16,17,18, heavy metals19, and dyes20. Similarly, studies carried out on basidiomycetes fungi, such as Phanerochaete chrysosporium and Trametes versicolor, have revealed their involvement in the oxidation of recalcitrant materials such as aromatic hydrocarbons13 and plastics21. Another example of fungi involved in the biodegradation processes are the zygomycetes Rhizopus spp., Mucor spp., and Cunninghamella spp.22,23. In particular, Cunninghamella is able to oxidase aromatic hydrocarbons and is considered a model organism for studying the detoxification of products from a wide range of xenobiotics13.
There are several fungal enzymes involved in the major degradative processes of recalcitrant materials24,25, such as esterase, laccase, peroxidase, and protease. Laccases are copper-containing oxidases produced in the cell and subsequently secreted, that allow the oxidation of a variety of phenolic and aromatic compounds. They can degrade ortho and para diphenols, the amino group-containing phenols, lignin, and the aryl group-containing diamines26. Peroxidases use hydrogen peroxide as a mediator to degrade lignin and other aromatic compounds. There are many different peroxidases, but the ones with the greatest potential to degrade toxic substances are lignin peroxidase and manganese peroxidase27.
Esterases and proteases belong to the group of extra- or ecto-cellular enzymes, which act outside their cells of origin but are still bound to them. These enzymes can catalyze the hydrolysis of large recalcitrant molecules into smaller ones. Due to their low substrate specificity, these enzymes can play a key role in the bioremediation of various pollutants, such as textile dyes, effluents released from the pulp and paper industries and leather tanning, petroleum products, plastics, and pesticides28,29,30.
A number of screening methods to select for bioremediative fungal strains have already been published. For example, straw-based agar medium has been used to screen for white-rot fungi with high potential in the polycyclic aromatic hydrocarbons (PAH) degradation31; and small pieces of rotting wood have been placed onto malt extract agar (MEA) to isolate wood-rotting fungi32. However, most of the methods that have already been proposed select very specific fungi for their activity of interest. This research proposes a wider approach for selecting soil fungi with a broader range of action. The method relies on the initial plating of serial dilutions of soil samples onto a medium amended with humic acids or lignocellulose mixed with antibiotics to select fungi with the ability to degrade these natural recalcitrant substances. Humic acids and lignocellulose, in fact, are substances that are extremely resistant to biodegradation since they have very complex molecular structures, and this allows them to be excellent indicators of the degradative ability of the tested fungi33,34. Subsequently, the fungi selected in the first tests are screened to identify those with the potential to degrade specific pollutants such as petrolatum, used engine oil, and plastics. Finally, qualitative enzymatic tests are performed to detect fungal strains able to produce enzymes involved in the biodegradation processes of recalcitrant substances. For this purpose, protease and esterase tests are conducted, while gallic acid and guaiacol are used as indicators of laccase and other ligninolytic enzyme production35,36. These substrates are used because a strong correlation has been found between the ability of fungi to oxidize them to their brown-colored form and the possession of ligninolytic ability37,38,39.
Through these protocols, it is possible to isolate fungal strains with high degradative potential and a broad spectrum of action directly from soil samples. The isolation of these fungal strains could help find new candidates for bioremediation purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 microwell plate</td>
			<td>Greiner bio-one</td>
			<td>650185</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>84609.05</td>
			<td></td>
		</tr>
		<tr>
			<td>Bushnell-Haas Broth</td>
			<td>Fluka</td>
			<td>B5051</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroamphenicol</td>
			<td>Eurobio</td>
			<td>GABCRL006Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlortetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001451</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl2&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C3279</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20821.296</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter 0.2 &#181;m</td>
			<td>Whatman</td>
			<td>10462200</td>
			<td></td>
		</tr>
		<tr>
			<td>gallic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7384</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slips</td>
			<td>Biosigma</td>
			<td>VBS634</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials 15 mL</td>
			<td>SciLabware</td>
			<td>P35467</td>
			<td></td>
		</tr>
		<tr>
			<td>guaiacol</td>
			<td>Sigma-Aldrich</td>
			<td>G5502</td>
			<td></td>
		</tr>
		<tr>
			<td>High-density polyethylene (HDPE)</td>
			<td>Sigma-Aldrich</td>
			<td>434272</td>
			<td></td>
		</tr>
		<tr>
			<td>Humic acids</td>
			<td>Aldrich Chemistry</td>
			<td>53680</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocellulose</td>
			<td>/</td>
			<td>/</td>
			<td>Sterilized bioethanol production waste</td>
		</tr>
		<tr>
			<td>L-shaped cell spreader</td>
			<td>Laboindustria S.p.a</td>
			<td>21133</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stirrer</td>
			<td>A.C.E.F</td>
			<td>8235</td>
			<td></td>
		</tr>
		<tr>
			<td>Malt Extract Broth</td>
			<td>Sigma-Aldrich</td>
			<td>70146</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 1000 &#956;L</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 200&#160; &#956;L</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#183;4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1651</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Sigma-Aldrich</td>
			<td>N0401000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>1504489</td>
			<td></td>
		</tr>
		<tr>
			<td>peptone</td>
			<td>Sigma-Aldrich</td>
			<td>83059</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET)</td>
			<td>Goodfellow</td>
			<td>ES306031</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Laboindustria S.p.a</td>
			<td>21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Petrolatum (Paraffin liquid)</td>
			<td>A.C.E.F</td>
			<td>009661</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td>Sigma-Aldrich</td>
			<td>P6685</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>331651</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane (PUR)</td>
			<td>Sigma-Aldrich</td>
			<td>GF20677923</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl chloride (PVC)</td>
			<td>Sigma-Aldrich</td>
			<td>81388</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile falcon tube</td>
			<td>Greiner bio-one</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile glass vials 20 mL</td>
			<td>Sigma-Aldrich</td>
			<td>SU860051</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile point 1000&#160; &#956;L</td>
			<td>Gilson</td>
			<td>F172511</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile point 200&#160; &#956;L</td>
			<td>Gilson</td>
			<td>F172311</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile polyethylene bags</td>
			<td>WHIRL-PAK</td>
			<td>B01018</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile syringe</td>
			<td>Rays</td>
			<td>5523CM25</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S-6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Used engine oil</td>
			<td>/</td>
			<td>/</td>
			<td>complex mixture of hydrocarbons, engine additives, and metals, provided by an Italian private company</td>
		</tr>
		<tr>
			<td>Vials 50 mL</td>
			<td>Sigma-Aldrich</td>
			<td>33108-U</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Z0152</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and screening from soil biodiversity for fungi involved in the degradation of recalcitrant materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

The selective media methods (Section 1 of the protocol) allowed the rich biodiversity of soil to be screened and the fungi with high bioremediation potential to be selected. With the humic acid and lignocellulose media, more than 100 fungal strains were isolated. These fungi produced enzymes involved in the biodegradation of natural recalcitrant materials, which have a chemical structure resembling many pollutants. However, the fungal strains isolated with the selective media needed further screening. Specifically, the s...

Watch this Scientific Journal Video about Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials at JoVE.com

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

This protocol is significant because it isolates fungal strains with high degradative potential directly from soil samples to be considered as candidates for bioremediation purposes. This protocol is a low-cost screening to find fungi with bioremediation potential. Moreover, it gives results that are easy to interpret.Since this technique involves handling microorganisms, it is very important to work in sterility using a laminar flow cabinet or a Bunsen burner to avoid contamination. To begin, sample the soil of interest and sieve the soil using a two-millimeter mesh to remove any roots and plant debris. Under a laminar flow cabinet, add one gram of the sieved soil into a sterile 15-milliliter tube.Then, add 10 milliliters of sterile deionized water to make a 10 to the minus one dilution. Shake the tube horizontally for 30 minutes. Before the suspension settles, take one milliliter of 10 to the minus one dilution with a sterile pipette and transfer it to a nine-milliliter of deionized water blank to make 10 to the minus two dilution.Vortex the suspension thoroughly. Similarly, make a 10 to the minus three dilution from a 10 to the minus two dilution tube. For plating soil dilutions on selective media, collect 100 microliters of the 10 to the minus three dilution using a micropipette with a sterile point and transfer it onto a humic agar Petri dish.Distribute the dilution evenly on the dish surface using a sterile, disposable, L-shaped cell spreader. Prepare four to five such dishes. Similarly, prepare lignocellulose dishes from a 10 to the minus three dilution tube.Allow the dishes to air dry for 10 to 15 minutes before incubating at 25 degrees Celsius in the dark for 15 days. Check all the fungal colonies present for similarities. If necessary, prepare a slide to be observed under a light microscope.Under a laminar flow cabinet or at a Bunsen burner, isolate each chosen fungal strain by gently removing a small part of the mycelium from the colony with a sterile inoculation needle. Transfer the isolated colony to a new malt extract agar, or MEA, dish. Transfer 20 milliliters of Bushnell Haas, or BH medium, into 50-milliliter glass vials.Transfer seven milliliters of deionized water into 15-milliliter vials and add pieces of broken glass cover slips into each vial. Sterilize the vials by autoclaving at 121 degrees Celsius for 20 minutes. To test growth on petrolatum, add one milliliter of petrolatum to each 50-milliliter BH vial using a sterile pipette under the laminar flow cabinet.Keep some vials with only BH as the negative control. Remove the mycelium on the surface of the medium from the MEA plate with the chosen fungal strain using a sterile needle and transfer it to a 15-milliliter glass vial with the broken cover slips. Agitate the vial on a vortex mixer for two minutes, allowing the broken cover slips to cut the cube of the fungal colony, making a suspension.Inoculate each petrolatum vial with 200 microliters suspension. Make two replicates for each fungal strain. For the negative control, add 200 microliters of fungal suspension into the vial containing only BH medium.Also, keep the vial with petrolatum without any fungal inoculation to check for contamination in the medium. Incubate the vials at 25 degrees Celsius in the dark and observe them after 15 and 30 days from the inoculum. To test growth on used engine oil, transfer 500 microliters of used engine oil onto the BHA Petri dish and distribute the oil evenly.Then, inoculate the plate with fungal strain by collecting the mycelium from MEA plates. To test growth on plastics, transfer 200 microliters of fungal suspension into a 96-microwell plate. Make three replicates of the fungal strain-plastic combination.Add 10 milligrams of a different plastic powder to each well with the fungal suspension. For the negative control, add 200 microliters of BH medium to the well with the fungal suspension. Moreover, for each plastic powder, fill a well with 200 microliters of BH solution and 10 milligrams of each plastic dust to check for contamination.Incubate the plates at 25 degrees Celsius in the dark and observe them after 15 and 30 days. For qualitative laccases calorimetric test, prepare one liter of potato dextrose agar, or PDA, and autoclave for 20 minutes. After autoclaving, when the medium has cooled down but is still liquid, add 400 microliters of guaiacol under a laminar flow hood and plate it into Petri dishes.Inoculate the PDA guaiacol plate with fungal strain by collecting the mycelium from the MEA plate. For negative controls, prepare PDA plates with each fungal strain and one plate with PDA and guaiacol. Incubate the plates at 25 degrees Celsius in the dark and observe them after seven days.For the qualitative esterases test, prepare one liter of Tween 80 medium. Then, place the medium on a magnetic stirrer without heating with a stirring bar inside the medium. After 10 minutes, when everything is melted, transfer five milliliters of the medium into 20-milliliter sterile vials and autoclave.Add 200 microliters of fungal suspension to the Tween 80 medium sterilized vial. For the negative control, add five milliliters of autoclaved BH medium into a 20-milliliter vial. Then, add 200 microliters of fungal suspension to the vial.Incubate the vials at 25 degrees Celsius in the dark for 21 days and observe them every seven days. The percentage of fungal strains able to grow on petrolatum, used engine oil, and plastic powders as their sole carbon source with different degrees of success was evaluated. The ability of fungi to exploit petrolatum was assessed as the difference in colony growth between controlled BH and containing petrolatum.Almost 75%of the tested strains were able to use petrolatum as their sole carbon source and 21%exhibited maximum growth. The growth for plates containing only BHA and in the presence of engine oil was also monitored. Almost 90%of the strains grew on used engine oil, with 39%showing maximum growth.The formation of a red-brownish halo around the fungal colony indicated higher production of laccases and other ligninolytic enzymes. Approximately 60%of the fungi were able to produce laccases. Moreover, 17%of the strains produced an intense dark halo around the colony in the guaiacol medium.Esterases activity was assessed by the formation of a white precipitate in Tween 80 medium. Almost 60%of strains showed esterases activity and 13%demonstrated higher precipitate formation. This protocol can be tailored to screen for fungi able to degrade any pollutant by adding the recalcitrant substance of interest to a minimal culture medium.

isolation-screening-from-soil-biodiversity-for-fungi-involved

Research

JoVE Journal

Biology

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 ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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.

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 ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.