Welcome to the Clinical Lab Instrumentation in the Lab Definitions While you’re looking at these slides, watch out for the underlined words. Do you know what they mean? If you don’t, stop and look them up. Introduction List of most common instruments that you’ll want to be aware of: Centrifuge Microscope Spectrophotometer Balance pH meter Autoclave Laboratory Glassware Safety Cabinets and Hoods Do you want a footer? The Centrifuge The centrifuge is an instrument designed to spin test tubes, with their bottoms tilted outward, around a central axis. The centrifugal force this creates pushes the heavier matter in the tube toward the bottom. This step is necessary to, for example, separate the components of blood in order to test it. Do you want a footer? The Centrifuge (cont.) Centrifugation is a process that involves the use of centrifugal force to separate two components of liquids. More-dense components of the mixture drop to the bottom and are called the precipitate. The less-dense components of the mixture migrate towards the top of the tube are is called the supernate. So, if we consider whole blood (basically cells, and serum) which component will drop to the bottom of a centrifuged tube? Do you want a footer? The Centrifuge (cont) The main setting on a centrifuge is the one that determines how fast it spins. This can be measured by the amount of acceleration to be applied to the sample (RCF), rather than specifying a rotational (RPM). This distinction is important because a larger centrifuge will apply more acceleration to a sample than a small centrifuge spinning at the same rotations per minute. So, two centrifuges with different diameters running at the same rotational speed will subject samples to different accelerations. RPM: revolutions per minute; is the speed expression. Radius of Rotation: (r) is the distance from the center of rotation to the tubes spinning, expressed in cm RCF: relative centrifugal field; force acting on the sample(s) 2 • RCF= 11.2 X R ( ) This is important because some tests ask for a particular centrifugal force while others require certain RPMs. To be able to go back and forth between the two measurements makes you a good laboratory scientist! Do you want a footer? The Centrifuge (cont.) Safe use of a centrifuge There are two very important realities when safely using a centrifuge 1) The tubes being spun must be balanced For tubes to be balanced, their weight must be placed equally around the circumference of bucket The Centrifuge (cont.) 2) The lid must be closed A closed lid is so important that a latch on the lid has become a required safety feature for centrifuges. Do you want a footer? The Centrifuge (cont.) Below is a microfuge which generally holds tubes 0.5 - 2 mL (much less volume than normal tubes) of liquid, and are spun at maximum speeds of 12,000–13,000 rpm ( the centrifuge on the left spins 40-50 times slower). This means that they can create pellets at the bottom of the tube, not just a more dense layer of liquid. This centrifuge, though larger than the microfuge on the right, spins slower. It’s advantage though is that it has “boats” which hold multiple tubes. Do you want a footer? The Centrifuge (cont.) Please watch this short video on how to use a centrifuge! • SAFE USE OF A CENTRIFUGE Do you want a footer? The Microscope The goal of using a microscope is to obtain increased magnification, resolution and contrast of a specimen. There are several types, but we’ll focus on the binocular (pun!) Do you want a footer? The Microscope continued The microscope you use may not be exactly like this one, but all the parts should be close to this diagram. On the next slides the parts of the microscope that are critical to efficient use will be highlighted. Do you want a footer? The Parts of a Microscope – refer to the preceding picture The oculars are the two lens at the top that you look through. They are usually 10x or 15x (magnify the image 10 or 15 times its normal size) Illuminator – light source Condenser lens focus the light onto the specimen The diaphragm (rotating disc under the stage) has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use. Do you want a footer? The Parts of a Microscope (cont.) The objective lenses are located on the nose piece above the stage. Each objective lens is labeled with its magnification power The low power objective lens is the shortest of these lenses. Higher power objective lenses are longer, and the longest of these is often an oil immersion lens that provides maximum magnification. Do you want a footer? Microscope Can you label this microscope? Do you want a footer? Using a Microscope 1) While looking through the eyepiece of the microscope, lower the stage to bring the image of the specimen into focus by turning the coarse adjustment knob. 2) Move the location of the slide on the stage to bring an item of interest into the field of view. 3) Adjust the condenser so that the light is sharply focused on the specimen, then adjust the diaphragm to control the brightness of the light. Do you want a footer? The Spectrophotometer The Spectrophotometer allows one to measure the concentration of a substance within a test sample. It does this by measuring the amount of light of a specified wavelength which passes through a medium. This all works because of a principle called Beer's law, the amount of light absorbed by a chemical in solution is proportional to the concentration of the absorbing material or chemical present. Do you want a footer? Spectrophotometer (cont.) Spectrophotometer can be used for tests like: determining how much glucose is in a specimen of blood, or how much cardiac enzyme is present after a heart attack, or how much bacteria is in spinal fluid Do you want a footer? Please watch this video on how to use the spectrophotometer. Spectrophotometer Video Do you want a footer? Top-Loading Scale Although the terms mass and weight are often used interchangeably, they are different properties. Mass is a measurement of the amount of matter something contains. Weight is the pull of gravity on an object’s mass. Practically, we use scales to measure both mass and weight, but technically, the raw numbers they give us are for weight alone. Simply, a scale measures weight and works by measuring the downward pressure on its pan. Do you want a footer? Top-Loading Scale (cont.) Scales are available with a variety of maximum capacities and with different precisions, sensitivities and tolerances. The top-loading scales in your labs are used to determine masses to ±0.01 g Analytical scales are sensitive to ± 0.0001 g. In general, top-loading balances can measure materials with masses up to several hundred grams whereas the analytical balances are limited to a maximum mass of one hundred grams. Do you want a footer? Top-Loading Scale (cont.) The sensitivity of the laboratory scales makes it necessary to adhere to some strict rules when making measurements. All scales are affected by vibrations of the bench top and by the movement of air currents around the balance pan. Top-loading scales are usually used with the balance pan open to the air. It is necessary to place these scales out of the direct path of room ventilation. They cannot be used in a hood because the drafts are too strong causing the measurements to fluctuate significantly. Do you want a footer? Precautions when using the top-loading scale 1. DO NOT ATTEMPT TO MAKE ADJUSTMENTS TO THE SCALE YOURSELF. If the scale is not functioning properly, notify your instructor. 2. Be sure the scale pan is clean. If not, remove any solid debris with a soft brush. If a liquid spill occurs, notify the instructor immediately. 3. Do not lean on, rest hands or arms on, or write lab reports on the balance tables. 4. Determine the weight of clean, dry, room-temperature objects only. 5. Clean up after yourself! Sweep off the top pan with the brush. 7. Re-zero the balances between uses and turn off the balances when all work is completed Do you want a footer? How to use the top-loading balance video How to Use a Top-Loading Balance Do you want a footer? pH Meter Ph Meters quickly determine the pH of a solution. A pH meter measures a voltage created by the ions in the solution that pass through the meter's membrane, and associate this voltage with a particular pH. pH probe pH Meter Most pH meters are calibrated by using a set of standard solutions that are guaranteed to be at a particular pH. Three of these points are the minimum number needed to establish a calibration curve with which the meter will work to associate each voltage reading with a particular pH. Watch the videos on the following slide. Take good notes! Refer to your specific pH meter for detailed tips for use. First video: How to calibrate a pH meter Calibrating a pH Meter Second video: How to use a pH meter Correct use of a pH Meter The Autoclave Autoclaves sterilize equipment and supplies, killing biological contamination and denaturing proteins. Autoclaves will not remove chemical contamination like acids or stains. Autoclave (cont.) Autoclaves generally have two cycles: 1. Fast Exhaust cycle - For dry goods, glassware, etc. This cycle charges the chamber with steam and holds it at a set temperature for a set period of time. At the end of the cycle a valve opens and the chamber rapidly returns to atmospheric pressure. 2. Slow exhaust cycle - Used to prevent sterilized liquids from boiling, steam is exhausted slowly at the end of the cycle, allowing the liquids (which will be super-heated) to cool. Sterilization requires • temperatures of at least 121-124 C • pressure of 15 psi • time of 15 minutes Safe Use of the Autoclave Not all plastics can be autoclaved. Polypropylene and polycarbonate will survive, but polyethylene and high density polyethylene will not. Different types of plastic can be identified by looking for initials imprinted on the bottom of containers. PP=polypropylene PC=polycarbonate PE=polyethlylene HDPE=high density polyethylene) If you are unsure about a new container, place it in an autoclave safe container the first time. General Precautions for Autoclave Use Do not open any autoclave until the pressure gauge labeled “chamber” is at zero, stand back and allow steam to escape through the open door before reaching in. Never open an autoclave set for “slow exhaust” until the cycle is complete. Superheated liquids can boil over, possibly damaging both autoclave and operator. After the cycle is complete, let liquids stand 10 minutes Pyrex bottles, empty or full, should have their caps placed on loosely, to prevent explosion due to expansion. Use tinfoil to cover non-safety-glass bottles (non-Pyrex). How to Use an Autoclave Video Safe Use of an Autoclave Biological Safety Cabinets and Hoods Biological Safety Cabinet (BSC) is an enclosed, HEPA filter ventilated workspace used to: safely work with materials that maybe susceptible to contamination with other external elements. create a barrier between a pathogenic substance and the laboratory personnel. BSCs and Hoods continued The safety level designation (BSL2, BSL3, BSL4) of the laboratory will determine the type of BSC used. These safety levels were created to protect the scientists working with different dangerous materials as well as those outside this environment (It wouldn’t be safe to ventilate some viruses into the atmosphere. These safety levels address that!) The levels are explained In following slides. BSL-2 Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. Laboratory personnel have specific training in handling pathogenic agents Access to the laboratory is restricted when work is being conducted Materials used in BSL-2s must be handled and disposed of as biohazards BSL-3 Biosafety Level 3 is much the same as BSL-2, but the infectious agents studied will cause serious or lethal diseases Laboratory workers need special training to work in these safety cabinets Waste is considered hazardous BSL-4 BSL-4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, and which cause severe to fatal disease in humans for which vaccines or other treatments are not available This is the environment you see on the movies where the scientists are wearing suits and air packs. Ebola virus, Marburg virus, various other hemorrhagic diseases are studied here Great Video on BSL-4 Labs Fume Hood Hoods or fume hoods are also designed to prevent contamination, but are not totally enclosed. They are used when potentially harmful reagents are made, poured, and discarded so that aerosols and/or fumes are not released into the laboratory environment. Laboratory Glassware Glassware should always be cleaned with caution as to not break, crack, chip or scratch the glassware. Glassware should be properly cleaned and sanitized according to the level of cleanliness required for a specific test. If your test requires all procedures to be done within the hood then you should use autoclaved glassware. Always rinse your glassware prior to autoclaving. Cover the top with foil. If it is a bottle, make sure the cap is not tight or cover top with foil. Always use autoclaved tape to insure the glassware was properly autoclaved. Date your autoclaved glassware. If your test does not require any procedures be done in a hood then you can use aseptic washed glassware. All glassware must be rinsed. Soak in a 10% bleach bath for at least one hour. Washed with a cleaning solution, rinsed thoroughly and dried upside down on a clean drying rack. Laboratory Grade Water Water is probably the most common laboratory reagent, so its quality is very important. Water is processed to reduce cells, bacteria, minerals, chemicals that in contaminate tests or instruments . Milli-Q water is water that has been sterilized by passing it through a filter with VERY small pores (like 2 microns in diameter). It can still have minerals and/or chemicals in it. Dionized water (DI water) has been converted into steam by heating it. The steam is then condensed back into water. This removes dissolved materials as well as killing any bacteria, resulting in pure water. Laboratory Grade Water (cont.) There are four types of laboratory water. Type 1: highest class of purity i.e mili-q or deionized water. Type 2: has impurities in the water but is still used in lab assays. Type 3 and Type 4: not pure and are used for glassware or laboratory cleaning of non sterile surfaces. NOTE: Do not consume water processed for the laboratory. Review Questions List two safety rules for centrifuges. What is the difference between a microfuge and a regular bench-top centrifuge? When thinking about a microscope, what is the name of the lens you look through? When thinking about a microscope, what is the name of the lens at the bottom of the turret – the ones that can be turned? When thinking about top-loading scales, that does it mean to tare? True or false: All plastics will melt in the autoclave. Review Questions (2) True or false: Autoclaves even remove chemical stains Why are Biosafety cabinets used? Are they the same as fume hoods? Is DI water potable water?