Sample Preparation for Acid Digestion via Sample Disruption using a QIAGEN TissueLyser III ball mill
1. Introduction
This standard operating procedure (SOP) describes a procedure for sample disruption using the QIAGEN TissueLyser III ball mill. A known weight of the sample is placed in the PTFE (Teflon) Grinding Jar and a PTFE Grinding Ball is added. The sample and the Grinding Jar can be frozen in liquid nitrogen, or at room temperature1 prior to sample disruption. The sample is then simultaneously disrupted and homogenized by high-speed shaking (bead milling). The resulting powder/crumbs are then quantitatively transferred into a plastic weighing dish with a plastic spoon as a feedstock for subsequent acid digestion. Note that the sample can only come in contact with plastic (e.g. Teflon) materials to avoid metal contamination.
2. Scope
This procedure may be applied to any organic sample that is amenable to sample disruption. This includes several commodities like coffee, soy and cocoa beans.
3. Materials
| Chemical/Solution | Description | Storage Location |
|---|---|---|
| Sample | Organic sample of commodity that will be analysed | / |
| Liquid N2 | Solvent for freezing the sample for improved sample disruption | Room 225 (Freezer Room), F7 |
| Ethanol (>70%) | Cleaning solvent for the QIAGEN TissueLyser III ball mill, QIAGEN Grinding Jar Set and Grinding Ball | Room 218b (Main lab), A17 |
| MilliQ water | Millipore grade water for general cleaning | Room 218b (Main lab), A17 |
4. Equipment
| Equipment | Description | Storage Location |
|---|---|---|
| PPE | Personal Protective Equipment, which includes safety glasses, gloves and a laboratory coat | Room 218b (Main lab) |
| Ball mill | A QIAGEN TissueLyser III ball mill designed for rapid and efficient disruption of organic samples (Figure 1) | Room 218b (Main lab), A4 |
| (Analytical) Balance | Analytical Balance Sartorius CP 224 S or Regular Balance VWR SE 300 D | Room 218b (Main lab), A7 |
| Grinding Jar Set | PTFE Grinding Jars with appropriate PTFE caps and PTFE Grinding Balls (Figure 2) | Room 218b (Main lab), usually A8 |
| Liquid N2 accessories | Insulated gloves, insulated box and flask tongs to freeze sample with liquid N2 (Figure 3) | Room 218b (Main lab), A4 |
| Ultra Low Temperature Freezer (-70°C) | Freezer that can cool samples to up to -70 °C | Room 223, C1 |
| Pestle and mortar (ceramic) | Non-metallic pestle and mortar to grind tough samples like cocoa beans | |
| Weighing dish | Plastic weighing dish to transfer grinded sample into |
*
* 
Figure 1: QIAGEN TissueLyser III Figure 2: Grinding Jar Set made out of Teflon
Figure 3: Accessories for handling liquid nitrogen.
From left to right: insulated gloves, thermos, flask thongs and insulated box
5. Safety
5.1 H- and P-phrases for Liquid Nitrogen
5.2 Safety Summary : Liquid Nitrogen
Inhalation
- Risk of oxygen displacement and oxygen deficiency
\=> Ensure proper ventilation and air monitoring. Close doors and windows of adjacent premises. In case of spills: consider evacuation or self-contained breathing apparatus
Skin
- May cause cryogenic burns or injury.
\=> Wear appropriate protective equipment (safety glasses, gloves, lab coat, safety shoes) + Cold insulating gloves (EN 511)
Fire
\=> Keep away from naked flames/heat. In case of exposure: consider evacuation
5.3 H- and P-phrases for Ethanol (96%)
5.4 Safety Summary: Ethanol (96%)
Fire
Highly flammable
\=> Keep away from naked flames/heat. In case of exposure: consider evacuation
5.5 Before starting sample disruption
- Always wear safety glasses, gloves, and a lab coat.
- Always check the power cord and the plug for damage before usage
- The low temperature of liquid nitrogen can change the mechanical properties of materials. Nitrogen cooled plastic vessels can become brittle and break when used in the TissueLyser III. Only use liquid nitrogen with vessels that have been cleared for such use in the TissueLyser III. This is the case for the Grinding Jar set from QIAGEN.
- Avoid spilling water or chemicals onto the TissueLyser III as this may cause damage. Avoid spills by always keeping vessels upright.
5.6 During Sample Disruption
Do not operate the TissueLyser III with any covers or parts removed.
Do not leave the TissueLyser III unattended during operation.
The sound level may be high depending on the type of material, the number of Grinding Balls used, the set grinding frequency, and the grinding time. Excess noise in terms of intensity and duration can lead to impairments or permanent damage to hearing. Consider wearing hearing protection.
If liquid has spilled inside the TissueLyser III, switch off the TissueLyser III, disconnect it from the power outlet, and contact QIAGEN Technical Services
Depending on the sample and the disruption parameters, grinding jars and samples can heat up considerably during the disruption process. Only touch the grinding jars with protective gloves and wait for equilibration to room temperature before opening the jars
6. Sample Disruption
6.1 Preparing the Sample in the Grinding Jar
1) For tough samples, take 0,3-2,0 g of sample per jar. Ensure that both Grinding Jars are of similar weight. Do not operate the TissueLyser III with only a single grinding jar installed. There should always be a second grinding jar containing sample and its own grinding ball
2) Add the Grinding Ball
3) Fit the cap upright on the Grinding Jar and screw fingertight. Make sure to not screw slantwise as this may damage the inner thread
4) In general:
- Plant tissues and lyophilized tissues can be disrupted and homogenized at room temperature without a lysis buffer.
- Alternatively, most fresh tissues can be disrupted with a lysis buffer at room temperature.
- If previous methods do not work or if no lysis buffer is available, fresh or frozen tissues can be disrupted without a lysis buffer if the Grinding Jar containing the sample is frozen in liquid nitrogen for 1 min. Lysis buffer must not be added if processing frozen Grinding Jar sets.
To acquire a grain size of **\<250 μm**:
- Cocoa can be grinded with pestle and mortar
- Roasted coffee beans can be disrupted at room temperature
- Timber can be disrupted after freezing at -70 °C
- Green coffee and soy beans can be disrupted after freezing with liquid N2.
5) Freezing with liquid N2 goes as follows:
1) Retrieve liquid N2 from stock with insulated gloves and in a thermos (Figure 4)
Figure 4: Retrieving liquid N2 from stock with insulated gloves and in a thermos
2) Place the jars containing the sample in the insulated box and pour the liquid N2 onto the jars and into the box
3) You will notice the liquid will start bubbling when in contact with the jars. Slowly turn over and roll the jars with flask thongs (like sausages on a grill) so they are frozen homogenically (Figure 5)
*Figure 5: Rolling around the jars with a flask thong*
4) When the liquid stops bubbling, both the jars and the liquid are about the same temperature and the jars are ready for disruption. If not needed anymore, let the liquid nitrogen evaporate.
6.2 Attaching the Grinding Jar Set
Attach a TissueLyser Grinding Jar Set to the TissueLyser III as follows:
- If the locking pin isn’t already unlocked; pull the locking pin (A) upward out of its slot, and rotate it by 90° (Figure 6). This releases the locking device.
- Turn the handwheel (B) counterclockwise until the maximum clamping range is reached.
- Insert the grinding jar, and press it lightly into the indentation in the clamp (C).
- Rotate back the locking pin (A) by 90° until it engages with its slot.
- Turn the handwheel (B) clockwise with two fingers until the grinding jar is seated in the clamp and level and does not move freely. Continue to rotate the handwheel clockwise until six to eight easily audible clicks are heard; the locking pin (A) rises and falls with each audible click.
Figure 6: Left-hand clamp of the TissueLyser III. A Locking pin. B Handwheel. C Indentation in clamp
| ! Damage or personal injury can occur if the TissueLyser Grinding Jars are not securely attached |
|---|
6.3 Running an experiment
The TissueLyser III provides high-speed shaking with oscillation frequencies of 3–30 Hz (180−1800 oscillations per minute).
1) Switch on the instrument at the main power switch on the back. The display turns on and the rotary knob is illuminated (Figure 7)
2) Open and close the instrument once to initiate the safety sensor. The instrument is now ready for usage.
Figure 7: Display and illuminated rotary knob
The instrument is controlled with the integrated touchscreen located on top of the instrument in combination with the rotary knob. The different symbols and their meanings are described in Appendix A.
3) Choose one of the following methods to disrupt the sample:
- Direct parameter entry on the home screen. The oscillation frequency can be adjusted in steps of 1 Hz (Figure 8). Disruption time, from 10 seconds up to a maximum of 8 hours, can also be set using the touchscreen. The disruption parameters for a particular application can be stored in the memory of the TissueLyser III as a program or a program cycle. For recommended settings, see later.
- Selection of a disruption program
- Selection of a disruption program cycle
Figure 8: A Home B Programs C Program Cycles D System Settings E Disruption time F Disruption
Keep in mind that Grinding Jars and samples can heat up considerably during the disruption process. This is why rest periods should be added if creating a new program cycle.
Recommended settings are as follows:
- 2 min at 30 Hz for roasted coffee beans
- 2 min at 30 Hz for timber that has been frozen at -70 °C
- 1 min at 30 Hz for green coffee and soy beans that have been frozen in liquid N2
4) Press the “START” key on the touch screen. The TissueLyser III will now run with the specified settings. If a program cycle with pause is run, the instrument will automatically pause the run after half of the cycles. It will only continue the disruption when “START” is pressed again. Listen carefully for changes in sound as these may indicate that the Grinding Jars are not properly attached. In this case, take out the Grinding Jars and reattach them.
| ! To pause the TissueLyser III before reaching the end of the run, press the “PAUSE” key on the keypad. To continue operation of the TissueLyser III from the time remaining, press the “START” key. To cancel operation of the TissueLyser III, press the “STOP” key. When the TissueLyser III is no longer in operation or in case of emergency, switch it off with the power switch. |
|---|
5) When the run is finished, remove the Grinding Jars (with protective gloves). You can do this by lifting the locking pin (A) and turning the handwheel (B) in the opposite direction. If the locking pin cannot be pulled upward to release it, do not force it upward using a hammer or other tool, as damage may occur. To release the locking pin, turn the handwheel slightly clockwise: the locking pin should then be freely moveable.
6) Wait for equilibration to room temperature before opening the jars.
7) Transfer the content quantitatively into a weighing dish and proceed according to your task at hand. You can use a pipet tip to scrape the sample into the weighing dish but use different weighing dishes and pipet tips for different samples to avoid cross-contamination.
8) Clean and dry the Grinding Jar Set and TissueLyser III instrument and inspect all parts for damage as described in 7. Cleaning, maintenance and storage.
7. Cleaning, maintenance and storage
Perform the following regular maintenance procedures (i.e. after each daily use) to ensure reliable operation of the TissueLyser III.
7.1 Cleaning
7.1.1 TissueLyser III
If solvents or saline, acidic, or alkaline solutions are spilt on the TissueLyser III, wipe them away immediately.
Only perform cleaning with liquids after disconnecting the instrument from the power grid.
The TissueLyser III should be wiped after use using a soft cloth moistened with a suitable cleaning agent (e.g. Ethanol, 70% for general cleaning). Use liquids only on cloth, never free-flowing (Do not use spray bottles to spray cleaning or disinfectant liquids onto surfaces of the TissueLyser III). Do not use bleach to clean or disinfect used labware. Bleach in contact with salts from the buffers used can produce toxic fumes. To remove residual cleaning agent and avoid instrument damage, the TissueLyser III should then be immediately wiped with a soft cloth moistened with water.
7.1.2 Grinding Jar Set
After each use, the components of the TissueLyser Grinding Jar Set should be rinsed 1x with DI water (left sink), rinsed 3x with MilliQ water, rinsed 1x with Ethanol (\<70%) and dried with a paper towel. Try to catch the ethanol rinse water in a separate waste container as this can not go into the sink (Figure 9). Try to remove any stubborn stains. Spray bottles can be used for the TissueLyser Grinding Jar Sets. Do not autoclave the Grinding Jar Set. The Grinder Jar Set must be dry before it is reused.
Figure 9: Catch the ethanol rinse water
7.2 Maintenance
7.2.1 TissueLyser III
If the TissueLyser III is not in use, switch it off at the power switch. If the TissueLyser III will not be used for a prolonged period of time, unplug the power cord from the power outlet.
Every 6 months, the limit switch and the brake should be checked for correct operation. To check the limit switch and the brake: Install a TissueLyser Grinding Jar Set in the instrument according to instructions. Switch on the TissueLyser III. Set the oscillation frequency to 30 Hz. Press the “START” key. Raise the hood. The drive motor should switch itself off, and “E50 SECURITY CIRCUIT” should appear in the display. Lower the hood. The drive motor should not switch itself on again. If there is a defect, contact QIAGEN
Pull the power cord before replacement of the fuses. Replacement of the fuses without removal of the power cord can lead to life threatening electrical shock. For the procedure of how to replace the fuses, please refer to the User Manual
7.2.2 Grinding Jar Set
TissueLyser Grinding Jar Sets may become worn, depending on the frequency of use and the samples disrupted. This may lead to damage to the clamps of the TissueLyser III. Check the TissueLyser Grinding Jar Sets regularly for wear and tear, and replace if necessary
8. Waste disposal
Any non-hazardous organic material that has not been in contact with chemicals can be disposed of in the sink.
Anything containing liquid nitrogen, however, should not be disposed of in the sink to prevent spreading in the sewers. Provide a separate waste bottle.
Ethanol rinse water should be disposed of into special a ethanol waste container
9. Appendix
- Different symbols on the TissueLyser III display screen
-
with or without a QIAGEN lysis buffer ↩