Not long after the Waring blender was introduced, the Dounce homogenizer was developed. Although this apparatus resembles a ground glass homogenizer, it operates by forcing a sample between the tube’s walls and pestle. As the sample and liquid are squeezed past the pestle, shearing forces are generated.
The Dounce homogenizer is particularly effective in lysing tissue culture cells and finely diced tissue to yield lysates containing subcellular particles that are still intact. If membrane fragments and organelles are required, the Dounce homogenizer is a useful instrument. After placing the sample in the tube, the pestle is inserted, crushed, and then removed.
This up and down motion is repeated, resulting in repeated shearing of the sample. The shearing force can be regulated to some extent by employing pestles of varying sizes. The pestle with the bigger diameter fits more snugly and produces a stronger shear, while the opposite is true for the smaller pestle.
What is Dounce Homogenizer?
A Dounce homogenizer is a mechanical device used for homogenizing biological samples, such as cells, tissues, and bacteria. It consists of a glass or stainless steel pestle that is manually rotated in a cylindrical container (known as a Dounce homogenizer) to break up and evenly distribute the sample.
The importance of the Dounce homogenizer is that it allows for efficient and consistent homogenization of biological samples, which is crucial for many laboratory procedures, such as the isolation and analysis of subcellular components, the extraction of DNA or RNA, and the preparation of samples for microscopy. Additionally, Dounce homogenizer can be used to prepare sample for downstream application such as western blot and ELISA. The manual operation allows for a high degree of control over the homogenization process, enabling the researcher to adjust the level of homogenization as needed.
The Dounce homogenizer was invented by Dr. Theodore Dounce in the 1940s. Dr. Dounce was a biochemist who developed the homogenizer while working at the Carnegie Institution for Science in Washington, D.C. He designed the homogenizer as a simple and efficient tool for breaking up biological samples, such as cells and tissues, for the purpose of isolating and analyzing their components.
The original Dounce homogenizer consisted of a glass pestle and cylinder, which were manually rotated to break up the sample. This manual design was improved upon over the years, but the basic principle of using a pestle to homogenize samples remains the same.
The Dounce homogenizer quickly became a popular tool in many laboratories, particularly in the fields of biochemistry and cell biology. Due to its ease of use, reproducibility and manual operation, it is still widely used in many laboratories today.
In recent years, many new homogenizing devices have been developed and become popular, such as bead mill homogenizer, rotor-stator homogenizer, and ultrasonic homogenizer. However, Dounce homogenizer is still a valuable tool for some applications, particularly for homogenizing delicate or small samples.
Working Principle of Dounce Homogenizer
In conjunction with hypotonic buffers, Dounce homogenization improves the lysis of adherent and suspension cells. The addition of hypotonic buffers causes the cytoplasm of a cell to enlarge, allowing for the mechanical rupture of cell membranes. The cytoplasmic proteins liberated by Dounce homogenization can be processed separately from the remaining intact nuclei, which can undergo high-salt extraction for detergent-free nuclear protein extraction. In this approach, cells are initially made vulnerable to dounce lysis by incubation in a hypotonic solution. Continue double-chopping until the majority of cells are lysed, leaving intact nuclei from which nuclear proteins can be isolated. This approach does not facilitate the isolation of histones, but it is excellent for isolating transcription factors and other chromatin-bound proteins. To maintain high protein concentrations, it is necessary to keep buffer volumes to a minimum. After hypotonic and high-salt extraction, cytoplasmic and nuclear fractions are subjected to dialysis to achieve physiological salt conditions prior to further usage.
The dounce homogenizer from Active Motif is perfect for preparing cell lysates or chromatin, especially if the cell and tissue samples are resistant to lysis under standard detergent conditions. There are two sizes of dounces for small and big sample preparations. Each dounce includes two pestles with varying diameters. One pestle fits snugly within the shaft of the dounce to maximise friction and cell rupture, while the other pestle is great for producing a homogeneous sample. Since it is composed of glass, the dounce homogenizer is simple to clean and sanitise between usage.
Prats of Dounce Homogenizer
The main parts of a Dounce homogenizer are:
- Pestle: This is the part of the homogenizer that is used to crush and grind the sample. It is typically made of glass or stainless steel and is manually rotated to break up the sample.
- Cylinder: This is the container that holds the sample. It is typically made of glass or stainless steel and is designed to be compatible with the pestle.
- Teflon or stainless steel adapter: It is used to adapt the pestle to the cylinder and also to prevent the pestle from sticking to the cylinder.
- Pestle handle: This is the handle that is used to manually rotate the pestle in the cylinder.
- O-ring: it is used to seal the cylinder and prevent leaks.
- Pestle stop: it is used to set the depth of the pestle in the cylinder
- Pestle lock: it is used to keep the pestle in place during homogenization.
Some Dounce homogenizer may have additional parts, such as a lid for the cylinder, or a holder for the pestle handle, depending on the specific model or manufacturer.
Operating Procedure of Dounce Homogenizer
The operating procedure of a Dounce homogenizer typically includes the following steps:
- Assemble the homogenizer: This includes attaching the pestle to the cylinder with the Teflon or stainless steel adapter, and making sure that the O-ring is properly in place to prevent leaks.
- Prepare the sample: The sample should be prepared according to the specific requirements of the application. It should be cut into small pieces and placed in the cylinder.
- Homogenize the sample: The pestle is manually rotated in the cylinder to break up the sample and achieve the desired level of homogenization. The operator can use the pestle stop to set the depth of the pestle in the cylinder, and the pestle lock to keep the pestle in place.
- Repeat homogenization as needed: Depending on the application, the operator may need to repeat the homogenization process several times to achieve the desired level of homogenization.
- Clean the homogenizer: After homogenization, the homogenizer should be disassembled and cleaned thoroughly to remove any remaining sample.
Note that the above procedure is a general guideline and may vary depending on the specific type and model of the Dounce homogenizer, and the sample being homogenized. It is always recommended to follow the manufacturer’s instructions for use and safety precautions.
Types of Dounce Homogenizer
There are two main types of Dounce homogenizers:
- Type A Dounce homogenizer: This type has a tight clearance between the pestle and cylinder, which is suitable for homogenizing soft samples such as cells and tissues.
- Type B Dounce homogenizer: This type has a wider clearance between the pestle and cylinder, which is suitable for homogenizing tougher samples such as seeds and plant tissues.
Additionally, Dounce homogenizer can be made of glass or stainless steel, glass is fragile and can’t withstand high speed and pressure, stainless steel is more durable and can withstand high speed and pressure, but it is more expensive than glass.
It is worth noting that some manufacturers may have variations or different names for their Dounce homogenizers, but the basic principle of using a pestle to homogenize samples remains the same. The choice of type depends on the characteristics of the samples, and the type of downstream application. It is always recommended to follow the manufacturer’s instructions for use and safety precautions.
Applications of Dounce Homogenizer
Dounce homogenizer is used in a wide range of applications, particularly in the fields of biochemistry, cell biology, and molecular biology. Some common applications include:
- Isolation of subcellular components: Dounce homogenizer can be used to break up cells and tissues to isolate different organelles, such as nuclei, mitochondria, and lysosomes, for further analysis.
- Extraction of DNA and RNA: Dounce homogenizer can be used to homogenize cells and tissues to extract DNA and RNA for downstream applications such as PCR, sequencing and cloning.
- Preparation of samples for microscopy: Dounce homogenizer can be used to homogenize samples and prepare them for observation under a microscope.
- Preparation of samples for western blot and ELISA: Dounce homogenizer can be used to homogenize samples and prepare them for downstream applications such as western blot and ELISA.
- Enzyme assays: Dounce homogenizer can be used to homogenize samples for the extraction of enzymes for activity assays.
- Bacteria and yeast: Dounce homogenizer can be used to homogenize bacteria and yeast for downstream application such as plasmid isolation and protein isolation.
Dounce homogenizer is particularly useful for homogenizing delicate or small samples, as it allows for a high degree of control over the homogenization process and is relatively gentle on the sample.
Advantages of Dounce Homogenizer
Dounce homogenizer offers several advantages over other homogenization methods:
- Control: Dounce homogenizer is a manual operation, this allows the operator to have a high degree of control over the homogenization process, and adjust the level of homogenization as needed.
- Gentle on samples: Dounce homogenizer is relatively gentle on delicate or small samples, and is less likely to cause damage compared to other methods such as high-pressure homogenizers.
- Versatility: Dounce homogenizer can be used to homogenize a wide range of samples, including cells, tissues, and bacteria.
- Reproducibility: Dounce homogenizer provides reproducible results, which is important for many laboratory procedures.
- Cost-effective: Dounce homogenizer is relatively simple and inexpensive compared to other homogenization methods, such as high-pressure homogenizers or bead mill homogenizers.
- Easy to clean: Dounce homogenizer is easy to disassemble and clean, which is important for maintaining the integrity of the samples and preventing cross-contamination.
- Manual operation: Dounce homogenizer does not require electricity, this can be useful in some laboratory settings, such as remote locations or fieldwork.
It is worth noting that Dounce homogenizer has some limitations, such as lower homogenization efficiency compared to other methods, and the need for manual labor. Therefore, it is important to consider the specific requirements of the application and compare the advantages and limitations of Dounce homogenizer with other homogenization methods before making a decision.
Disadvantages of Dounce Homogenizer
Dounce homogenizer has several disadvantages, including:
- Low efficiency: Dounce homogenizer has a lower homogenization efficiency compared to other methods, such as high-pressure homogenizers or bead mill homogenizers, which can result in longer homogenization times and the need for multiple passes.
- Manual labor: Dounce homogenizer requires manual operation, which can be time-consuming and labor-intensive.
- Limited sample size: Dounce homogenizer is limited in the amount of sample it can process, which can be a limitation for larger samples or batches.
- Risk of contamination: Dounce homogenizer has the risk of contamination if not cleaned properly.
- Limited versatility: Dounce homogenizer is limited in its ability to homogenize certain types of samples such as heat-sensitive samples or samples with high viscosity.
- Risk of breakage: Dounce homogenizer is made of glass or stainless steel, which can be fragile and prone to breaking, particularly if not handled properly.
- Limited scalability: Dounce homogenizer is not easily scalable for large scale processing or industrial applications.
It is worth noting that these disadvantages are relative to the specific application and sample type, and that Dounce homogenizer may still be the most appropriate method for some applications, particularly for homogenizing delicate or small samples.
When using a Dounce homogenizer, it is important to take the following precautions to ensure safe and effective operation:
- Wear personal protective equipment (PPE): Always wear gloves, safety goggles, and a lab coat to protect yourself from potential exposure to hazardous samples.
- Use appropriate samples: Dounce homogenizer is not suitable for samples that are highly viscous, heat-sensitive, or contain large particles.
- Keep the homogenizer clean: Always clean the homogenizer thoroughly after use to prevent cross-contamination between samples.
- Handle the homogenizer with care: The glass or stainless steel components of the homogenizer can be fragile, so handle it with care to avoid breakage.
- Use the correct size pestle: Use the correct size pestle for your sample size and application.
- Follow the manufacturer’s instructions: Always read and follow the manufacturer’s instructions for use and safety precautions.
- Use a pestle lock: Always use the pestle lock to keep the pestle in place during homogenization to prevent injuries.
- Use a pestle stop: Always use the pestle stop to set the depth of the pestle in the cylinder to prevent the pestle from hitting the bottom of the cylinder
- Keep the cylinder in the vertical position: Always keep the cylinder in the vertical position to prevent the pestle from coming out of the cylinder and causing injuries.
- Work in a safe environment: Always work in a safe environment and follow laboratory safety protocols.
By following these precautions, you can ensure safe and effective operation of the Dounce homogenizer and protect yourself and others from potential hazards.
- Burden, David. (2012). Guide to the disruption of biological samples – 2012. Random Primers. 12.