Preparing biological samples such as bones, plants, muscles, or sputum for analysis of DNA, RNA, proteins or metabolites can be a challenge. RETSCH provides laboratory mills and grinders that pulverize and homogenize solid samples but are also suitable for cell disruption. RETSCH mills are used in areas such as biotechnology, diagnostics, forensics, agriculture, and microbiology.
Cell disruption is frequently the method of choice to extract cellular components from bacteria, yeast, fungi, or microalgae and is carried out either chemically or mechanically. Mechanical methods are better suited for cells with tough walls, or if the chemicals might affect the extraction and therefore need to be avoided. A common and effective method is bead beating which uses beads to shear the cells in a suspension. Bead beating can be done on a small or large scale, using different types of vials and tubes. One way is to mix the suspension with beads and use a vortex mixer. However, this procedure is slow and inconsistent, especially with a large number of samples or long disruption times. A better way is to use RETSCH Mixer Mills with adapters which automate the process, making it fast, efficient and reproducible.
MM 400 malom - élesztősejt feltárása*
*The video shows the previous model with identical functional principle.
Increased reproducibility of total protein concentration after cell disruption, 7 min in
MM 400, 12 min Vortex.
Error bars: % standard deviation
The MM 400 processes up to 20 samples in 1.5 or 2 ml Eppendorf tubes without cross contamination which saves time for the operator. Additionally, an adapter is available to accommodate up to eight 50 ml Falcon tubes. The optimal bead size for cell disruption varies based on the cell type; for bacteria and yeast, glass beads ranging from 0.75 to 1.5 mm are recommended, while smaller beads within the range of 0.1 to 0.5 mm are more suitable for fungi and microalgae.
For DNA or RNA extraction, smaller single-use tubes up to 2 ml are ideal, whereas larger vials like the 50 ml Falcon tubes are well-suited for processing proteins or metabolites. The optimum bead beating parameters vary according to cell type. It may take some experimenting to find the best results. Usually, 30 s (most microalgae) to 7 min (yeasts in general) of bead beating are required to fully disrupt the cells.
By accepting up to fifty 2 ml single-use vials, the Mixer Mill MM 500 vario effectively increases sample throughput.
Cells of Phaeodactylum tricornutum before (left) and after cell disruption (right) with the Mixer Mill MM 400 in combination with the Falcon tube adapter.
Temperature plays a crucial role especially for applications where temperature-sensitive proteins are involved. One solution is cryogenic grinding (see corresponding section), the other one is cooling the cell suspension. For the MM 400 it was shown that the heat increase in 2 ml tubes is moderate even at 30 Hz; a simple way is to interrupt disruption times >1 min with cooling the adapter with the vials in an ice bath for one minute. In this way, the temperature will stay below 12-15°C, depending on the used bead size.
Temperature increase during cell disruption in the MM 400 at 30 Hz, or with a Vortexer, cooling on ice after each minute of cell disruption
A way to control the temperature development in bead beating is using the Mixer Mill MM 500 control. A special adapter accommodates eighteen 2 ml single-use vials per batch. The machine is connected to a chiller with 4°C cooled water which in turn cools the adapter holding the vials. In this way, the temperature of the suspension is kept at around 13°C, but without the inconvenient manual interruption phases in an ice bath. While the MM 500 control does not accept the single-use falcon tube adapter, it can be used with stainless steel grinding jars in volumes 50, 80 or 125 ml (see example on the left: P. pastoris).
If the mill is used with liquid nitrogen and the CryoPad and set to a temperature of 0°C, the temperature of the cell suspension in 2 ml single-use tubes can even be kept at 0 °C without freezing the suspension, so that effective bead beating is still possible.
Some biological samples, such as sputum from cystic fibrosis patients or tissue specimens like liver, lung or tumors, are sometimes more difficult to fully homogenize. The 2 ml single-use tubes are often too small to fit the entire sample volume, so the sample has to be split and recombined after the homogenization process, which adds extra work and time to the lab routine.
Larger grinding jars made of stainless steel, for example, can hold the whole sample volume, but they need to be cleaned after each use. An adapter for the Mixer Mill MM 400 solves this problem by allowing the use of 5 x 5 ml single-use tubes, which have more capacity and no cleaning requirement. Per batch, 10 samples can be homogenized simultaneously. 50 ml falcon tubes can also be used to homogenize tissue samples. Here, 8 sample per batch can be processed within only a few minutes.
Liver sample before and after homogenization in the MM 400
Some biological samples, such as fibrous plants, tough veins, fingernails, or certain animal or tumor tissues, are difficult to homogenize in a buffer system. They are either too soft or too hard or too fibrous to be pulverized effectively in suspension. A better alternative for these samples is cryogenic grinding, which involves freezing them with liquid nitrogen before or during the grinding process. This technique makes the samples brittle and easy to crush into homogeneous powders. Cryogenic grinding also has the benefit of preserving the integrity of e. g. proteins or volatile compounds that might degrade or evaporate at higher temperatures. Moreover, cryogenic grinding can break the intracellular organelles of some organisms, such as yeast.
For samples that are sticky, like berries, cryogenic grinding is often the only feasible method to obtain a uniform sample. The table lists some examples of sample materials that have been successfully processed by cryogenic grinding in the Mixer Mill MM 400 or the CryoMill. 2 ml steel tubes can be used for cryogenic grinding in the MM 400. Similar effects can be obtained in the MM 500 control with grinding jars up to 125 ml for larger sample volumes. Here the CryoPad mus be used to work with liquid nitrogen. In all mentioned mills, 2 ml stainless steel tubes and the corresponding adapters are also available for smaller sample volumes.
2 ml stainless steel tubes
| Sample | Kiegésztők | Feed quantity | Grinding time | Speed | Max. végfinomság (d90)/ |
| E. coli bacteria |
|
2 x 10 ml frozen cell pellets | 2 min | 30 Hz | complete cell disruption |
| muscle tissue |
|
10 g | 4 min | 25 Hz | <150 µm |
| pine needles |
|
3 min | 30 Hz | Reproducible RNA extraction of 20 samples in one step | |
| berries |
|
2 g | 40 secs | 20 Hz | <200 µm |
| finger nails |
|
1 finger nail per vial | 2 min | 25 Hz | <200 µm |
| rat gut |
|
1.8 g | 2 min | 30 Hz | <150 µm |
Pieces of meat before and after cryogenic grinding
Sticky berries
before and after cryogenic grinding
Pine needles
before and after cryogenic grinding
Agarose gel for RNA separation from pulverized pine needles showing good reproducibility and quantity of prepared RNA
Forensic samples such as hair, bones and teeth are mostly brittle and therefore usually do not need cooling before pulverization. To achieve the desired analytical fineness, the samples may have to undergo preliminary crushing in a Jaw Crusher or Cutting Mill to reduce their particle sizes to below 10 mm for further processing in a Ball Mill. Cutting Mills are used for pre-crushing bones that may be fresh and therefore not completely dry and may even contain meat residues.
RETSCH offers a range of cutting mills for primary size reduction of soft, medium-hard, elastic, tough and fibrous sample materials. The wide range of accessories allows for optimal adaptation to various applications. The SM 300 can be fitted with three different rotors and bottom sieves from 0.25 mm to 20 mm. Unlike fresh and fatty bones, dry bones can be reduced to a size of less than 0.25 mm in one or two steps. The SM 300 features a variable speed from 100 to 3,000 rpm. Pulverization of bones or teeth or hair is mostly conducted in ball mills using grinding balls > 5 mm made of steel, zirconium oxide or tungsten carbide.
Bone sample before and after grinding in a cutting mill
Human hair before and after fine grinding in a mixer mill
One of the potential complications of joint replacement surgery, such as for elbows or knees, is the infection of the surrounding tissue by various bacteria. These infections, called prosthetic joint infections (PJI), can occur anytime from a few days to several years after the surgery. They are difficult to treat because different types of bacteria can cause them, and they are not always detected by conventional methods. Therefore, it is necessary to isolate the bacteria from the tissue samples in a way that preserves their viability and allows their identification and cultivation.
This is where the Mixer Mill MM 400 can help with a simple procedure: The samples are mixed with 20 ml of sterile demineralized water and 5 ml of 1 mm glass beads in a sterilized steel jar. Disposable 30 ml wide mouth bottles can be used. Up to 8 bottles are shaken for 3.5 min at 30 Hz to remove the bacteria from the samples without destroying them. The bacteria can then be easily grown on an agar plate for further analysis. This method has a high detection rate (A.-L. Roux et. al 2010) and can be applied to any solid infected tissue sample, even if it contains implanted material.
MM 400 Rázó-golyós malom
CryoMill
MM 500 control Rázó-golyós malom
MM 500 nano Rázó-golyós malom
MM 500 vario Rázó-golyós malom
