Experiment 1 Use of light microscope and stereomicroscope: measuring microscopic objects 1.1 Introduction The microscope is a major tool used by biologists, which was invented about 350 years ago. It is a device that allows observation of minute organisms and various cell types too small to be seen by the unaided eye. Since an unaided eye cannot detect anything smaller than 0.1 mm, a light microscope can extend our view a thousand times so that objects as small as 0.2 micrometer (2 x 10-7 m) can be seen. Whereas an electron microscope can further extend our viewing capability down to one nanometer (1 x 10-9 m). With the advance of technology, such as light scattering technique, today we can view the outlines of protein and nucleic acid molecule, which are in the range of angstrom (1 x 10-10 m). Good microscopy involves three main factors: resolution, magnification, and contrast. The resolution, or resolving power of a microscope, which will increase when the wavelength of the light source is reduced. Different microscopes will come with a range of magnifications, such as light microscope ranging from 40 to 1000 and the stereomicroscope from 7 to 30. Staining with suitable stains will increase the contrast of a specimen to be observed under a microscope. 1.2 Objectives 1. To learn the parts of a light microscope and a stereomicroscope 2. To learn how to use them 3. To learn how to properly prepare slides for microbiological examination 4. To learn how to measure microscopic objects 1.3 Materials required Light microscope (compound microscope, binocular microscope) Stereomicroscope (dissecting microscope) Glass slides and cover slips Ocular and stage micrometers Prepared slides Lens paper 9
Resolving power of different microscopy techniques 10
1.4 Parts of a light microscope (compound microscope) 11
1.5 Parts of a stereomicroscope Label the following diagram from A to F 12
1.6 Electron microscope (EM) EM was invented about 50 years ago, which makes use of electrons rather than light as the illumination source. The theoretical resolution of EM would be 100,000 times greater than that of the light microscope as electrons have a wavelength of 0.005 nm. Transmission electron microscope (TEM) Scanning electron microscope (SEM) A section of a bacterial cell of Bacillus subtilus, scale bar 200 nm Mosquito head 200X Transmission electron micrograph Scanning electron micrpgraph 13
1.7 Handling and use of a microscope Microscopes are expensive and precision optical instruments with many delicate working parts. So they must be handled carefully at all times. When moved from one place to another, the microscope must be in an upright position with the base supported. Never touch the lens of the microscope with your hands. For cleaning, use only special lens paper. When viewing a prepared slide, always have the cover slip on the upper side. Never focus downward while looking through the eyepiece, which might break the glass slide. Rather, look from the side and lower the objective to slightly touch the slide but without breaking it, then focus upward. Always locate the object to be studied with the low power objective before using the high power ones. Turn on the light source and fix your slide on the stage with the clips Bring the substage light condenser up to the highest position and close down the iris diaphragm until the light is diffused evenly across the entire field of vision. Use the coarse adjustment knob first to bring the object into focus and then use fine adjustment knob to view more details. You might need to adjust the iris diaphragm size to give better object resolution and contrast. Adjust the angle of the two eyepieces so that only one image can be viewed comfortably. One of the eyepieces can be adjusted if the focus of your two eyes is not equal. 1.8 Preparation of a Wet-mount Slide (a) Add a drop of water to a slide. (b) Place the specimen (letter e) in the water. (c) Place the edge of a coverslip on the slide so that it touches the edge of the water. (d) Slowly lower the coverslip to prevent forming and trapping air bubbles. 14
1.9 Measuring microscopic objects 1.9.1 Determination of the magnification of microscopic objects This method does not actually determine the size of the objects but it is convenient. This is done by multiplying the ocular lens magnification by the objective lens magnification, for example, if the ocular lens is 10X and objective lens 40X, then total magnification is 10 x 40 = 400X. Exercise 1.9.1 What are the minimum and maximum magnification readings that the light microscope and stereomicroscope in the lab can give? Light microscope: Minimum: ocular lens ( ) x objective lens ( ) = Maximum: ocular lens ( ) x objective lens ( ) = Stereomicroscope: ocular lens ( ) x objective lens ( ) = Minimum: ocular lens ( ) x objective lens ( ) = Maximum: ocular lens ( ) x objective lens ( ) = 15
1.9.2 The use of a pair of ocular micrometer and stage micrometer This is the most accurate method of measuring microscopic objects. The ocular micrometer is a glass disc with lines uniformly etched on it and the stage micrometer is a glass slide with etched lines 0.01 mm apart. The ocular micrometer will serve as a ruler against which the specimen under observation will be measured, after it has been calibrated against a stage micrometer. How to calibrate: 1. Match the first line of the ocular disc with the first line of the stage micrometer. Call this line 1 2. Next look for a second line on the ocular micrometer which lines up with another line on the stage micrometer. Call this line 2. 3. Count the number of spaces on the stage micrometer between line 1 and line 2. Use this number as the numerator for the equation below. Count the number of spaces on the ocular micrometer included between lines 1 and 2. Use this number as the denominator. Multiply the numerator with 0.01 mm since micrometer stage space is equal to 0.01 mm. To obtain the value of one ocular micrometer space, use the following equation: 1 ocular micrometer space = (No. of stage micrometer spaces) x 0.01 mm No. of ocular spaces Sample calculation Line 1: line 0 of the ocular micrometer and line 0 of the stage micrometer Line2: line 10 of the ocular micrometer and line 7 of the stage micrometer No. of ocular spaces = 10 No. of stage micrometer spaces = 7 Hence, 1 ocular micrometer space = (7 x 0.01 mm)/10 = 0.007 mm Exercise 1.9.2 Calibrate for each of the objectives on your microscope and record below: 1. 4X = 2. 10X = 3. 40X = 16
Calibrating an Ocular Micrometer. 17
1.10 Lab Exercise 1. Complete the following on units of measurement: Unit Abbreviation Value 1 centimeter cm 10-2 m 1 millimeter mm 10-6 m 1 nanometer 10-9 m 1 angstrom 10-10 m 2. What effect does closing the diaphram have on the image brightness? 3. Which objective lens on the light microscope in our lab has the (a) shortest working distance and (b) longest working distance? 1.11 References Microscopy: www.southwestschools.org/jsfaculty/microscopes/microscopeparts.html Vernier scale: http://biology.clc.uc.edu/fankhauser/labs/anatomy_&_physiology/a&p203/circulatory_ System/Vernier_scale/Vernier_Scales.htm 18