Chapter 2 Measurement in Biology PDF

Practical Techniques in Biology Chapter 2 Measurements in Microscope BIOL341 Measurements Biologists use the metric system for measurement. This system is based on metre, kilogram and litre. Measurement of length: Basic units used to measure length are: 1) Metres which the lar...

Practical Techniques in Biology Chapter 2 Measurements in Microscope BIOL341 Measurements Biologists use the metric system for measurement. This system is based on metre, kilogram and litre. Measurement of length: Basic units used to measure length are: 1) Metres which the largest unit of measurement in biology. 2) Centimeters (cm) which equals 10-2m 3) Millimeter (mm) which is 10-3m 4) Micrometer which is 10-6m 5) Nanometer which is 10-9m. It is the smallest unit in biology BIOL341 DR. MAHA Weight: It is measured in microgram, milligram (mg), gram (g) and kilogram (kg). 1 kg equals 1000g, 1g equals 1000mg and 1mg equals 1000 microgram. Weights are usually measured using sensitive or digital scale. Volume: Three units are used to measure volume, these are: Litre (l) equals 1000ml, Millilitre (ml) equals 1000 micro litre (μl). BIOL341 DR. MAHA Temperature: It is measured using a number of ways. The most commonly used method in biological laboratories is the centigrade thermometer and the temperature is expressed as degree Celsius (°C). BIOL341 DR. MAHA  Techniques in Microscopy An unaided eye cannot detect anything smaller than 0.1mm in diameter. Therefore cells, tissues and many smaller organisms are beyond our visual capability. A light microscope extends our vision a thousand times, so that objects as small as 0.2 micrometers in diameter can be seen. BIOL341 DR. MAHA  The electron microscope further extends our viewing capilibility down to 1 nanometer. Microscope quality depends upon the capacity to resolve, not magnify, objects. Resolution: is the capacity of an optical instrument to distinguish two points that are close together. High resolution allow us to see separate points while low resolution blurs close-together points into one image rather than showing them as separate ones. BIOL341 DR. MAHA  Modern microscopes increase both magnification and resolution by matching the properties of the light source and precision lens components. Today’s light microscopes are limited to practical magnification in the range of 1000x to 2000x and to resolving power of 0.2 micrometer. Most student microscopes have magnification power to 450x or possibly 980x and resolving BIOL341 DR. MAHA  Microscopists improve contrast by using stains that bind to cellular structures and absorb light to provide contrast. Some stains are specific for certain chemicals and bind only structures composed of those chemicals. Other are non specific and stain all structures. Types of Microscopes: There are different types of microscopes such as: 1) Compound light microscopes 2) Electron microscopes BIOL341 DR. MAHA Compound Microscope: A compound microscope is a microscope which uses multiple lenses to collect light from the sample and then separate set of lenses to focus the light into the eye or camera. Examples of this compound microscopes are: a) Student microscope b) Inverted microscope c) Phase-contrast microscope d) Fluorescence microscope. BIOL341 DR. MAHA Parts of Compound Microscope: Ocular lens: is the lens you look through. Microscopes are either monocular, having one lens, or binocular, having two lenses. Ocular lens is a system of several lenses that may include a pointer and a measuring scale. Body tube: is a hollow housing through which light travels to the ocular. BIOL341 DR. MAHA BIOL341 DR. MAHA  The objective lenses are a set of 3 to 4 lenses mounted on a rotating turret at the bottom of the body tube. The objectives gather light from specimens and projects it into the body tube. Stage: a horizontal surface on which the slide is placed. It may be equipped with simple clips for holding the slide in place or with a mechanical stage, a geared device for precisely moving the slide. Two knobs, either on top or under the stage, move the mechanical stage. BIOL341 DR. MAHA  Sub stage condenser: is located immediately under the stage, focuses light on the specimen. The diaphragm: is an adjustable light barrier built into the condenser. Light source: has an off/on switch and may have adjustable lamp intensities and colour filters. Base and arm: are heavy cast metal parts. BIOL341 DR. MAHA  Coarse focus adjustment: is a device that raises and lowers the body tube or the stage to focus the optics of specimen. The fine adjustment: changes the specimen to objective distance very slightly with each turn of the knob. It is used for all focusing of the 40x objective. Total magnification: Objective x Ocular lens lens. BIOL341 DR. MAHA  Rules for Using the microscope: Use two hands to carry a microscope, one hand holding the arm and the other holding the base. Only use cleaning lens paper and water to clean lenses. Always start off with the lowest magnification objective and work up to higher power one objective at a time. Always observe objective as it is rotated into place to ensure it does not crash into the slide. BIOL341 DR. MAHA BIOL341 DR. MAHA  Lower the stage to its lowest point before placing a slide on it. Be sure the scanning objective is in a place. Raise the stage to its highest point without crashing the slide into the objective. Focus initially by lowering the stage using the coarse focus adjustment knob After getting a close focus with the coarse adjustment knob, improve it with the fine adjustment focus knob. BIOL341 DR. MAHA  Rules for putting away the microscope 1) Rotate the scanning objective into place 2) Lower the stage to its lowest point 3) Clean lens, stage body, and base if needed. 4) Place protective cover over microscope BIOL341 DR. MAHA Preparation of Temporary mount: To prepare a wet preparation for whole mount, a small drop of water, glycerol, alcohol or iodine is put on a glass slide. The specimen is then placed on this drop and is then covered with a slide coverslip. Usually glycerol and paraffin oil are used for slides needed for a longer period of time. BIOL341 DR. MAHA The Permanent Mount: Permanent mounts are prepared in the same way as temporary mounts. However the coverslips are sealed with colourless nail paints to prevent drying of the slides. Permanent slides are either: Whole mounts or Sections: Transverse sections (T.S.), Longitudinal sections (L.S.), Horizontal sections (H.S.) and Vertical sections (V.S.) BIOL341 DR. MAHA Stereoscopic dissecting Microscopes: Stereoscopic or dissecting microscope differs from student compound microscope in that it has two objective lenses for each magnification. It is essentially two microscopes in one. Magnification on this type of microscope usually range from 4x to 50x. Stereoscopic microscopes are often used BIOL341 DR. MAHA Electron microscopes: It is a vertical television tube with electron gun at the top and the fluorescent screen at the bottom. Biological specimen must be carefully treated and prepared before being viewed by transmission electron microscope BIOL341 DR. MAHA  This treatment includes fixing the tissue to preserve structures. After the cells are fixed and the structures stabilized, it is infiltrated and embedded in hard epoxy plastic. Epoxy plastics are the principal embedding media for the preservation of tissues to be sectioned and examined by TEM The epoxy plastic may be sliced thinly. The obtained sections are stained with heavy metal salts and finally placed in the microscope for study. BIOL341 DR. MAHA Comparison between light and electron microscopes: Light Microscope Electron Microscope Lens Type Glass Electromagnetic Illumination Light beam Beam of electrons Resolution 200nm 0.1nm Magnification 2000x 100,000x specimen Dead or alive Always dead BIOL341 DR. MAHA  Using Quantitative techniques and Statistics Weighing, pipetting and spectrophotometer are daily performed in modern biological research laboratories and are needed skills. Both data and statistical analyses are commonly used in experimental biology. Two fundamental questions are always asked when using quantitative techniques. These are what precision is necessary and appropriate?and are the measurements accurate? Precision: measure how close the results are to one another. Accuracy: measures how close the results are to BIOL341 DR. MAHA the true or known value.  Precision has to do with exactness. Accuracy has nothing to do with precision. The only way to know whether an instrument is accurate is to standardize it. This is usually done by measuring a standard and calibrating the instrument. BIOL341 DR. MAHA How to pipette: Pipettes are used to measure small volume of liquid quickly and accurately. For years glass pipettes were used as a standard measuring device in research laboratories. Recently, pipetting device called micropipettes that have disposable plastic tips. BIOL341 DR. MAHA  To use a pipette, the tip is immersed in the appropriate fluid which is drawn up beyond the zero mark using a suction device. Any drops hanging from the tip of the pipette are removed by touching the tip to the inside of the beaker from which the solution was drawn. To use a micropipette, first the volume to be measured is set using the pipette dial. BIOL341 DR. MAHA BIOL341 DR. MAHA  The plunger is de-pressed to its first stop and while holding it in that position, it is submerged in the fluid to be drawn up. The plunger is slowly released to draw the fluid up. BIOL341 DR. MAHA  Spectrophotometry A spectrophotometer is an instrument designed to detect the amount of radiant energy absorbed by molecules in solution. Spectrophotometer have five basic components which are a light source, a diffraction grating, a slit, a detector (photoelectric tube) and a read out to display the output of the phototube. BIOL341 DR. MAHA BIOL341 DR. MAHA  If a current is attached to the phototube, the electric current output can be measured and displayed on a meter or a digital readout. The meter scale is usually calibrated in 2 ways: 1) Percent transmittance which runs on a scale from 0 to 100 2) Absorbance which runs from 0-2 in most practical application. BIOL341 DR. MAHA  The readout should indicate 100% transmittance, or 0 absorbance, depending on which scale being used. If it does not, a adjustment must be made. This standardizes the spectrophotometer. The amount absorbed will be proportional to the number of the dye molecules per unit volume (or concentration) of the solution. BIOL341 DR. MAHA Percent transmittance = Intensity of light through sample x 100 Intensity of light through blank Absorbance = log10 (1/T) BIOL341 DR. MAHA  Making Graphs Graphs are used to summarize data- to show the relationship between to variables. Graphs are easier to remember than numbers in a table and are used extensively in science. Graphs are either: 2) 1) Line graphs Histograms. BIOL341 DR. MAHA  Line graphs show the relationship between two variables, such as the amount of oxygen consumed by a tadpole over an extended period of time. Histograms are bar graphs used to represent frequency data that is data in which measurements are repeated and the counts are recorded. Example the values obtained when an object is weighed several times. BIOL341 DR. MAHA Rules for making line graphs: 1)Decide which variable is the dependent variable and which one is the independent variable. a)A dependent variable: is the one obtained after making experiment measurements. b)An independent variable: is the one known before the start of an experiment. 2) Always place the independent variable on the x-axis (horizontal one) and the dependent variable on the y- axis (vertical one) BIOL341 DR. MAHA 3)Always label the axes with a few words describing the variable, and always put the units of the variables in parentheses after the variable description. 4)Choose an appropriate scale for the dependent and independent variables so that the highest value of each will fit on the graph paper. BIOL341 DR. MAHA 5)Plot the data set. Make the plotted points dark enough to be seen. Always use pencil not pen in case you need to erase. If two or more data sets are to be plotted on the same coordinates, use different blotting symbols for each data set. 6)Draw smooth curves or straight lines to fit the values plotted for any one data set. BIOL341 DR. MAHA  Every graph should have a legend, a sentence explaining what the graph is about. Histograms: In making histograms, the count data are always on the y-axis. The categories in which the data fall are on the x-axis. Data are plotted as bars showing the frequencies of measurements. Bars are neatly drawn with a pencil and a BIOL341 DR. MAHA BIOL341 DR. MAHA  10 % mid1 10 % mid2 5% Quiz 1 5% Quiz 2 Lab quizes 5% Final practical 20% Final exam 40% Assignment 5% BIOL341 DR. MAHA