PM1PY2 Essential Laboratory Skills Pre-Reading Material PDF

PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera PM1PY2 Practical: Essential Laboratory Skills Learning Outcomes ✓ In this element of the course you will learn how to use a pipettor, a balance, a vortex, a spectrophotometer, a centrifuge...

PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera PM1PY2 Practical: Essential Laboratory Skills Learning Outcomes ✓ In this element of the course you will learn how to use a pipettor, a balance, a vortex, a spectrophotometer, a centrifuge and a microscope. ✓ This element, in conjunction with a face-to-face practical will help you to develop ESSENTIAL practical skills that will be assessed in an in-class practical test and will also be used in future practicals in other modules throughout your programme. Using a Pipettor Pipettors are typically used to dispense small volumes of liquid ranging from 0.2 µL to 5 mL (Note: 1 mL=1000 µL). Within the laboratory you will find a range of different pipettors. Each pipettor is specifically used to dispense a particular volume range and should NOT be used for volumes outside of the specified range for each pipette. A schematic of a typical pipettor showing the major part and functions of each component is shown in figure 1. Figure 1. Schematic diagram of a pipettor Please note that there are different brands of pipette that may differ in their appearance. The brand that you will normally use in practical sessions is Eppendorf. Images of actual pipettors are shown below in Figures 2-4. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Figure 2. Pipetman P5000: This pipettor is used to dispense volumes from 500-5000 µL. This pipettor has a purple plunger button. Figure 3. Pipetman P1000: This pipettor is used to dispense volumes from 100-1000 µL. This pipettor has a blue plunger button. Figure 4. Pipetman P200: This pipettor is used to dispense volumes from 20-200 µL. This pipettor has a yellow plunger button. The Pipetman P20 is also has a yellow plunger button, similar to the P200 and is used to dispense volumes from 2-20 µL. Each pipettor is designed to use a specific size of pipette tip. The boxes of tips are colour-coded to match the pipettor. For example, the tips in the blue box shown below should only be used with the Pipetman P1000. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Figure 5. Images of boxes of tips used for pipettors When using a pipettor: Treat pipettors very carefully as they are expensive precision instruments. Keep upright when in use to prevent liquids running inside the shaft of the pipette. Not leave pipettes lying on the workbench where they can be knocked off and damaged. Place back on the carousel. Do not set the pipettor to dispense a volume higher or lower than maximum recommended volumes as this will damage the pipette. Please view the information in the links below, as this will help you to improve your pipetting technique and increase your learning at practical sessions. A short video showing how a pipettor should be used can be viewed here: Additional information on good pipetting practice can be found here: PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Use of a Balance In the laboratory a balance is a device used for weighing chemicals. A balance that you will find in the teaching laboratory is pictured below. Figure 6. An electronic balance How to switch it on First, the balance must be switched on, both at the socket on the wall and by pressing the ‘On/Off’ button. How to get started Chemicals must NOT be weighed directly onto the metal pan. A plastic weighing boat or a receptacle must first be placed onto the metal pan. Before adding the chemical that you would like to weigh, you must press the ‘Re-zero’ or ‘Tare’ button, this will set the reading to zero. Pipette the liquid or scoop the powder onto the weighing boat. Do NOT touch the balance, lean or bang on the bench during the procedure, as this will affect your reading. You should also use the ‘Interactive Lab Skills’ content to learn more out balances. This content can be found and accessed within the PM1PY2 Essential Laboratory Skills folder under Interactive Lab Skills>Basic Lab Skills>Weighing by taring. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Use of a Vortex Mixer In the laboratory a vortex is used to ensure that small volumes of liquids are properly mixed or make speed up the solubilisation of chemicals. To use the vortex simply push (and keep hold of) the receptacle that contains the compounds you intend to mix into the holder on top of the vortex. Figure 7. Image of a vortex and important parts PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Use of a Spectrophotometer A spectrophotometer is used in the laboratory to measure the transmissive or reflective properties of solutions as a functional of the wavelength of light. Below is an image of a spectrophotometer found in the teaching laboratories Figure 8. A spectrophotometer Typically you will use this piece of equipment to measure the absorbance of light at a particular wavelength i.e. a solutions transmissive properties. Solutions are placed into special containers called ‘cuvettes’ (see image below). Figure 9. A cuvette Cuvettes are placed into the holder situated under the blue lid (see image below). The arrowhead on the cuvette must be in the same direction as the direction of light. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Figure 10. Image inside the spectrophotometer showing the cuvette holder and direction of the beam of light How to get started Before measuring the absorbance of a solution we must first set the parameters on the spectrophotometer. Below is an image of the user interface of the spectrophotometer. Figure 11. Image of the user interface of the spectrophotometer This spectrophotometer is set up to measure absorbance (note the arrowhead under the ABS) using a wavelength of light at 660 nm. The wavelength of light can be altered using the up and down arrow keys. How to blank the spectrophotometer It is important to first blank i.e., set to zero the machine before use using an identical solution that has been used to ‘dilute’ your sample. We can refer to this as a ‘solution only’ sample. e.g., Imagine that we are trying to determine the absorbance of a suspension of yeast that has been grown in a special solution called ‘yeast broth’. In order to blank the machine, insert a cuvette containing ‘yeast broth’ that does not contain any yeast. Then press the ‘CAL’ button on the user interface. The screen should then look like the image below. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Figure 12. Image of the user interface showing the absorbance value after following the blanking procedure How to measure absorbance The spectrophotometer is now ready to read the absorbance of a solution. To do this, remove the cuvette containing the ‘solution only’ sample e.g., ‘yeast broth’ only and replace it with a cuvette containing the solution you wish to measure the absorbance of. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Use of a Benchtop Centrifuge Typically these centrifuges are used to separate soluble particles from insoluble particles. Centrifuges spin (rotate) at extremely fast speeds and must be loaded correctly to prevent damage to the machine and for the safety of the operator. How to get started Firstly, make sure that the centrifuge is switched on at the wall and using the power button located at the rear of the machine (left image). The centrifuge will now show lights on at the front (right image). Figure 13. Images of the back and front of a benchtop centrifuge Next open the lid of the centrifuge by pressing the “open lid” button as indicated in the image below. Figure 14. Image of the control panel of a benchtop centrifuge PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Next take off the “inside lid” by pressing in the red button and lifting the lid off (image below). Figure 15. Image of the inside of the benchtop centrifuge showing the inside lid. How to load the centrifuge Now we are ready to correctly ‘load’ the centrifuge. What do we mean by correct loading? This refers to the position and weight of the tubes that will be added to the centrifuge. For example, if you put a microcentrifuge tube (sample 1) with a weight of 1 g in position 1, then you MUST put another microcentrifuge tube (Sample 2) of the same weight directly opposite i.e. in position 13. (see image below). Figure 16. Image showing were two tubes should be placed for correct loading If the microcentrifuge tube is not the same weight or not directly opposite then the centrifuge is said to be “unbalanced”. Running an unbalanced PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera centrifuge can be extremely dangerous and have disastrous consequences. Below is a link to a short video showing the consequences of using an unbalanced machine: An unbalanced washing machine How to adjust the time and the speed of a centrifuge Once the centrifuge is loaded, replace the inside lid and press down until you hear a “click”, then close the outside lid and you will be ready to set the time and speed of the centrifuge. The centrifuges that you will use operate in minutes. Adjust the time using the arrow buttons (see image below). Figure 17. Image showing the control panel to adjust the time You are now ready to adjust the speed of the centrifuge. The centrifuge operates in two modes. You can either operate the centrifuge in revolutions per min (rpm or min-1) or in centrifugal force or g. You can toggle between min-1 or g using the button with the double arrow (see image below). The speed of each mode of operation is controlled by the individual arrow buttons. Please note that if you set the number to 2.5 that actually means either 2500 min-1 or 2500 g. Once everything is set, you press the start button and the centrifuge will begin to rotate your samples. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Figure 18. Image showing the control panel to adjust the speed IMPORTANT: Always wait until the centrifuge has reached your desired speed before leaving the machine unattended. PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Use of a Light (Compound) Microscope Light microscopes allow for magnification of an object approximately up to 400-1000 times depending on whether the high power or oil immersion objective is used. Furthermore, using a light microscopes 2 objects being as close as 200 nm can be recognised as such (resolution). This magnification and resolution is sufficient to visualise yeast cells (5 micrometres or µm) or single bacteria with a size of approximately 1 µm (diameter of human hair: 15~150 µm). IMPORTANT: Do not touch the glass part of the objectives with your fingers. Use only special lens cleaning paper to clean the objectives. Always keep your microscope covered when not in use. Always carry a microscope with both hands. Grasp the frame with one hand and place the other hand under the base for support. Figure 19. Image showing the important parts of a microscope PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera Important Parts of the Microscope Eyepiece (Ocular Lens) The eyepiece is used to view the specimen and contributes to magnifying the specimen. Illuminator An illuminator is a source of light usually situated at the bottom/base of the microscope. It is a LED lamp (or halogen lamp in older microscopes) to provide light to the sample. Stage A stage is a flat surface where the slide with the specimen is placed. A mechanical stage is a stage used when working with higher magnifications. The stage is moved by using the knobs, as even the slightest movement can affect the results. Objectives Objectives are the part of microscope responsible for magnifying the image of specimen. Usually there are five objective lenses in a standard microscope of 4X, 10X, 20X, 40X and 100X (IMPORTANT: The 100X objective requires immersion oil). Objective Turret The objective turret holds the objectives. The turret is turned to change the objective. This can be turned either clockwise or anti-clockwise. Condenser Lens The function of the condenser lens is to collect the light from the illuminator and focus it on the specimen. Iris Diaphragm The diaphragm is used to control the amount of light reaching the specimen. In a student scope it is a rotating disk under the stage and above the condenser. There are various holes in the diaphragm in order to facilitate the variants in the experiments carried on. Coarse and fine adjustment knobs It is a part of the microscope responsible for adjusting and determining the distance between the objective lens and the specimen. A coarse and fine adjustment knobs are present on the arm of a microscope. The function of these knobs is to move the specimen back or forth to adjust the slide, containing specimen, in order to bring it to focus. The coarse and fine PM1PY2 Fundamentals of Cell Biology G. S. Cottrell, S. Amadesi, D. Widera adjustment knobs should be carefully moved to avoid damage to the specimen and the objectives. A short video showing how to use a light or compound microscope can be found here: