Advances in cellular fractionation germinated, quite accidentally, the field of molecular biology, and many years later, more purposefully initiated the modern era of cell biology. In 1869, Friedrich Miescher began investigating the nucleus, at a time when cell theory was still in its infancy. He isolated neutrophils from pus on bandages with saline washes, then separated nuclei from cytoplasm by alkaline extraction. The resulting precipitates contained a substance with phosphorous in novel stoichiometric ratios. He named it nuclein, and its function as the carrier of heritable information would not be appreciated or even hypothesized for several decades. In the 1940s, Albert Claude, interested in obtaining a functional picture of a living cell, pioneered the use of differential centrifugation, and combining it with nascent electron microscopy, identified several subcellular organelles, and isolated viral particles after infection. Later improvements to tissue fractionation led to seminal discoveries in oxidative phosphorylation, intracellular digestion, protein synthesis, transport and secretion, organelle biosynthesis, and the study of metabolites.
An unsung hero in these landmarks is cell disruption via homogenization. Throughout the early years, however, homogenization was limited to the ancient technology of the mortar and pestle, or to the destructive power of the Waring blender. In countless experiments, fractions that were deemed insoluble in homogenization steps were simply discarded, until the realization that these seemingly impenetrable residues contained compartments, particles, and molecules of great interest. Although mortars and pestles and Douncers are still widely used for applications ranging from RNA extraction to disruption of organs and embryos, modern mechanical homogenizers are usually more reliable, especially when throughput is a concern. Bead mill homogenizers facilitate high-throughput disruption of hard tissues in small volumes, and highpressure homogenizers can create emulsifications and cell lysates at industrial scales. Both are less versatile and ubiquitous, however, than rotor-stators and sonicators.
A rotor-stator works by rapid rotation of an inner rotor within a stationary sheath, disrupting cells or tissue by mechanical tearing, shear forces, or cavitation, a shock wave produced by rapidly collapsing bubbles in solution. A sonicator consists of a generator and a transducer, which uses a piezoelectric charge to impart its resonant frequency to an ultrasonic probe. The probe’s resulting vibrations cause shear and shock waves in the liquid sample, resulting in an emulsion. For both types of homogenizer, the probe tip diameter determines the appropriate sample volume, and the vessel in which disruption or emulsification can take place. Therefore, there is a wide variety of probe sizes, and a corresponding range of samples that a laboratory homogenizer can handle, from less than 200 microliters to more than five liters.
Companies such as Omni International supply rotor-stator generator probes that vary in size and have different types of tips. Samples of the smallest volumes and softest consistencies use the narrowest probes with flat bottoms. The incorporation of saw-tooth blades in medium-sized probes allows disruption of larger and tougher samples, while large probes with open-slotted bottoms can be used for larger volumes and thicker samples in beakers and flasks. For sonicators, BioLogics and others provide solid or tapped ultrasonic tips that correspond to larger volumes, necessitating wider diameters, lower radiation of ultrasonic energy, and a wider cavitation field. Solid tips are more appropriate for organic solvents, unlike tapped tips, which have threaded ends to accept disposable attachments for high-throughput applications. Microtips for smaller volumes transfer a greater amount of energy with narrower cavitation. Stepped and tapered microtips are similar in the way they impart ultrasonic vibrations, although stepped microtips are longer and can reach into deeper vessels, potentially mitigating sample loss due to splashing or evaporation through heat. Regardless of whether your protocol calls for mechanical disruption or ultrasonic emulsification, there’s a probe for that.