Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms’ thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.     

The isolation of large quantities of undamaged cellular organelles and cytosolic enzymes using a low-shear continuous tissue homogenizer

There is often a need to isolate large quantities of subcellular components such as membrane-coated organelles (e.g., nuclei, lysosomes, and mitochondria), cell membranes, and soluble (cytosolic) proteins. Instruments which can homogenize relatively large masses of tissue, primarily those with rapidly rotating blades and cylinders, are excessively vigorous, often resulting in damaged and/or low yields of the subcellular components. This paper describes procedures for obtaining high yields of undamaged subcellular components using a continuous bulk tissue homogenizer which performs with low shear (the low-shear continuous homogenizer or LSC). This homogenizer is simple in operation, durable and can be used with a variety of tissues. Fibrous tissues are more difficult to homogenize using this instrumentation and require a premincing to small pieces (0.2 to 1.0-cm diam) followed by filtration through 2-4 mesh (two to four apertures per inch). Methods for bulk preparations with enhanced recoveries of undamaged nuclei, and a typical soluble multimeric enzyme, phosphofructokinase, are presented. Electron microscope views of the homogenates show the preserved state of the other subcellular components. The LSC homogenizer requires less physical effort with no "hands on" operation and thus is safer. This homogenizer requires less homogenization time compared to the smaller, hand-held Potter-Elvehjem-type homogenizers. Operations requiring low temperature can be performed at room temperature as long as the continuously passing homogenate solutions are kept chilled.     

Optimal operation of high-pressure homogenization for intracellular product recovery

An optimal control methodology for the homogenization of bacterial cells to recover intracellular products is presented. A Fluent computational fluid dynamics (CFD) model is used to quantify the hydrodynamic forces present in the homogenizer, and empirical models are used to relate these forces to experimentally obtained cell disruption and product recovery data. The optimal homogenizer operation, in terms of either constant cell breakage or maximum intracellular product recovery, is determined using these empirical models. We illustrate this methodology with an Escherichia coli bacterial system used to produce DNA plasmids. Homogenization is performed using an industrial APV–Gaulin high-pressure homogenizer. The modeling and optimization results for this E. coli–DNA plasmid system show good agreement with the experimental data.     

Mechanical disintegration of microorganisms in an industrial homogenizer

Disintegration of microorganisms in a continuously working industrial homogenizer has been studied. The homogenizer consists of rotating discs in a cylinder filled with glass beads. Different parameters for disintegration of baker's yeast were investigated. The disintegration process is a first-order reaction and it is influenced by the flow rate of the suspension and by the agitator speed. At a flow rate of 200 liters/hr about 85% of the yeast cells can be disrupted in a single pass through the disintegrator. This type of disintegrator can be used for disruption of cells in order to produce single-cell protein, active enzymes and other valuable cell components.     

Preparation and properties of immobilized methanol oxidase

Methanol oxidase produced by the yeast Hansenula polymorpha DL-1 was used for the enzymatic oxidation of methanol to formaldehyde. The kinetics of enzyme and protein release during cell desruption were studied at the laboratory scale with a Braun homogenizer and the pilot plant scale with a Manton–Gaulin homogenizer. Conditions were defined for maximum release and retention of high activity in cell-free extracts. Methanol oxidase was immobilized by adsorption on DEAE-cellulose from enzymes in cell-free extracts or from ammonium sulfate purified purified fractions. The kinetics of formaldehyde formation with both soluble and immobilized enzyme was studied in batch and continuous reactors.     

Influence of broth dilution on the disruption of Escherichia coli

Summary The effect of cell concentration (5 to 150 g/L wet wt after broth dilution) on homogenizer disruption efficiency and homogenate viscosity is reported for E. coli. Broth dilution increases homogenizer efficiency and decreases feed and homogenate viscosity. However, this increase in disruption efficiency is not sufficient to warrant dilution of the broth prior to homogenization. The optimal feed concentration is the maximum possible that does not lead to practical handling difficulties due to high viscosity.     

A method for isolating intact mitochondria and nuclei from the same homogenate,and the influence of mitochondrial destruction on the properties of cell nuclei

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0-6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl(2) is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The alpha-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0-6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Improved tissue and cell homogenizer

Modification of an existing tissue homogenizer is described, which converts to a milk-like consistency even difficult to macerate tissues such as cartilage, hair, and small bones.     

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

Although protein kinase F_A/GSK-3α (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase F_A/GSK-3α is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32M sucrose, approximately 40% of the total F_A/GSK-3α was found in the cytosol. However, when brain was homogenized in buffer containing 0.32M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total F_A/GSK-3α was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase F_A/GSK-3α in high recovery and in a substantial amount from brain tissue. The data explain why kinase F_A/GSK-3α cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase F_A/GSK-3α from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase F_A/GSK-3α in the regulation of brain functions especially in the brain particulate membrane fractions.     

Rapid preparation of a noncultured skin cell suspension that promotes wound healing

Autologous skin cell suspensions have been used for wound healing in patients with burns and against normal pigmentation in vitiligo. To separate cells and the extracellular matrix from skin tissue, most researchers use enzymatic digestion. Therefore, this process is difficult to perform during a routine surgical procedure. We aimed to prepare a suspension of noncultured autologous skin cells (NCSCs) using a tissue homogenizer as a new method instead of harsh biochemical reagents. The potential clinical applicability of NCSCs was analyzed using a nude-rat model of burn healing. After optimization of the homogenizer settings, cell viability ranged from 52 to 89%. Scanning electron microscopy showed evidence of keratinocyte-like cell morphology, and several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the NCSCs. The rat model revealed that NCSCs accelerated skin regeneration. NCSCs could be generated using a tissue homogenizer for enhancement of wound healing in vivo. In the NCSC group of wounds, on day 7 of epithelialization, granulation was observed, whereas on day 14, there was a significant increase in skin adnexa regeneration as compared to the control group (PBS treatment; p < 0.05). This study suggests that the proposed process is rapid and does not require the use of biochemical agents. Thus, we recommend a combination of surgical treatment with the new therapy for a burn as an effective method.     

A hydrodynamic mechanism for the disintegration of Saccharomyces cerevisiae in an industrial homogenizer

Amongst the commercial type of homogenizers the Manton-GaulinAPV homogenizer (APV Company Ltd., Crawley, Surrey, England) which is generally being used for other purposes than cell disintegration processes, has recently been proved to be effective for the breakage of yeast cells. To understand fully the disintegration process occurring in such machines it becomes necessary to describe their functions through mathematical expressionsbased on a realistic hydrodynamic model. A mathematical expression describing the protein release at an applied pressure has been derived from an energy balance in the homogenizer combined with the size distribution function of yeast cell population. This expression has been confirmed experimentally under conditions where it shows that turbulence is the controlling factor in the system. Furthermore it indicates the area where more investigations are needed to improve the efficiency of the process of disintegration.     

The effect of different media and culture techniques on plating efficiency of strawberry mesophyll cells in culture

Mesophyll cells of strawberry were isolated mechanically by a hand homogenizer, One g healthy, fully-expanded leaves yielded 10⁷ cells. The agar culture method was the only culture technique found suitable to obtain any quantitative data.     

Simulation of particle size distribution changes occurring during high-pressure disruption of bakers' yeast

Measurements of size distributions are provided for the breakage of commercial packed bakers' yeast cells as a function of operating pressure and number of passes through a Manton Gaulin high-pressure homogenizer. A two parameter model was developed, based upon the use of a Boltzmann function, to simulate the changes in size distribution that accompany the cell breakage process. The effects of operating pressure and number of passes are incorporated in the model and the result is used to simulate the particle size distribution of the cell homogenate. The results show that there is little breakage below a threshold pressure of 115 bar and above which breakage is critically dependent upon the pressure and number of passes through the homogenizer. The analysis provides a means of studying the efficiency of centrifugation that may follow cell disruption and provides the basis for further studies of size distribution changes accompanying cell disruption. (c) 1996 John Wiley & Sons, Inc.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Breakage of yeast: a method for processing multiple samples.

A procedure is described for the rapid preparation of mitochondria and the soluble cell fraction of yeast. The method makes use of an adaptor for the Braun homogenizer which allows 16 samples to be processed at once.     

Optimization of Nanoemulsion Fabrication Using Microfluidization: Role of Surfactant Concentration on Formation and Stability

Nanoemulsions have some important potential advantages over conventional emulsions for certain commercial applications due to their optical clarity, high physical stability, and ability to increase the bioavailability of lipophilic bioactives. In this study, the factors influencing droplet size and stability in nanoemulsions fabricated from a hydrocarbon oil and an anionic surfactant were examined. Octadecane oil-in-water nanoemulsions were produced by a high pressure homogenizer (microfluidizer) using sodium dodecyl sulfate (SDS) as a model anionic surfactant. The influence of homogenization pressure, number of passes, and surfactant concentration was examined. The droplet size decreased with increasing homogenization pressure, number of passes, and surfactant concentration. Nanoemulsions with low turbidity and small droplet diameters (≈62 nm) could be produced under optimized conditions. Interestingly, nanoemulsions containing relatively high surfactant levels were highly susceptible to creaming when they were only passed through the homogenizer a few times, which was attributed to depletion flocculation. These results show the importance of optimizing surfactant levels to produce small droplets that are also stable to creaming.     

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE,AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl₂ is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Critical analysis of quantitative indicators of cell disruption applied to Saccharomyces cerevisiae processed with an industrial high pressure homogenizer

A comparison of quantification techniques was performed on suspensions of Saccharomyces cerevisiae which had been disrupted with a high pressure homogenizer. The quantification techniques included cell counting, monitoring protein release, UV absorbance, turbidity, sample mass loss analysis, variations in viscosity and measuring the particle size distribution of the homogenate. It was found that all quantification techniques resulted in similar relationships between the measured extent of disruption and number of passes through the homogenizer. The data from all techniques (except particle sizing) could be fitted to simple exponential decay models at various homogenization pressures. Turbidity, particle sizing and UV absorbance generally gave more conservative estimates of the extent of cell disruption compared to protein release and cell counting. Measuring both the turbidity and monitoring the release of cellular metabolites using UV absorbance gave simple, reliable and reproducible measures of disruption and were identified as being the most applicable to on-line disruption monitoring.     

Gaulin homogenization: a mechanistic study

Free radical-based oxidation has been detected in the normal operating regime of the Gaulin homogenizer, demonstrating that cavitation occurs in this important industrial bioprocessing equipment. Free radical generation is suppressed by imposition of back pressure, proving that such cavitation occurs in the impingement section. The calculated value of the cavitation number is consistent with submerged jet cavitation, wherein a high-speed jet exiting from the valve gap accelerates fluid in the impingement region, creating the vacuum conditions for cavitation. Using polysaccharides as a model shear-sensitive compound, their breakage pattern in the homogenizer was characterized by molecular size and polydispersity and compared to those of fluid shear flows in capillary tubes and cavitating flow from a sonic horn. The results indicate that breakage occurs primarily by fluid shear, although a contribution by cavitation is also apparent when back pressure is applied. Because biological molecules can readily react with free radicals and the alterations caused thereby are subtle in nature, a thorough evaluation of the impact of free radicals in upstream homogenization is warranted.     

Protein Synthesis During Fungal Spore Germination II. Aminoacyl-soluble Ribonucleic Acid Synthetase Activities During Germination of Botryodiplodia theobromae Spores

The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores.