Exercise C.Brightfield Microscopy 1.Compound microscope Since all the living forms(microbes)with which we will be dealing are invisible to the naked eye,a microscope is an essential tool in any laboratory course in microbiology.The compound microscope employs two lens systems:the ocular or eyepiece and the objective lens.These two lens systems are separated so that the ocular lens can magnify the image formed y the objective lens.An object which is magnified 45 times by the high-dry objective lens(45x)is actually magnified 450 times when combined with the magnification provided by the ocular lens which is usually 10x.A bacterial cell with a linear diameter of 0.001 mm(1 u)can thus be magnified to the point of being visible in the microscope(0.001 mm times 450 equals 0.45 mm). The resolving power of the compound microscope is approximately 0.2u (0.0002 mm).This means that objects which are smaller than 0.2 u be distinguished clearly by the lenses and these will appear as small blurs in the field.Increased magnification will only make the blurs larger.Objects which are less than 0.2 u in diameter thus cannot be resolved by the optical microscope. 2.Use and Care of the Microscope a.Never look through eyepiece when turning adjustment knobs downward You should always begin focusing upward.Otherwise,the objective lens will go through the glass slide,thereby scratching and damaging the expensive lenses of the high-dry and oil-immersion objectives. b.Lenses should be cleaned with a lens cleaning solution and lens paper each day before and after use.Xylene can be used to remove any oil film buildup on the lenses,but it should be used sparingly and must be removed by wiping with lens tissue (xylene is a solvent which can dissolve the cement that holds the lens in the objective). 3.Oil immersion Technique a.Place a drop of immersion oil over the specimen that has been previously fixed onto a glass slide b.Using the course adjustment knob,move the oil-immersion objective (100x)into the oil and gently make contact with the slide(watch from side of scope during this procedure). c.Focusing of specimen is accomplished by turning the fine adjustment knob in a direction which makes the objective travel upward! d.If your microscope is parfocal,you can focus a specimen using the high dry objective,and then place a drop of oil on the specimen,switch to the crob Lab We 2001/Exercise1/e> 1b.htm(4of6)[831/20024:29:54PMC. Brightfield Microscopy 1. Compound microscope Since all the living forms (microbes) with which we will be dealing are invisible to the naked eye, a microscope is an essential tool in any laboratory course in microbiology. The compound microscope employs two lens systems: the ocular or eyepiece and the objective lens. These two lens systems are separated so that the ocular lens can magnify the image formed by the objective lens. An object which is magnified 45 times by the high-dry objective lens (45x) is actually magnified 450 times when combined with the magnification provided by the ocular lens which is usually 10x. A bacterial cell with a linear diameter of 0.001 mm (1 u) can thus be magnified to the point of being visible in the microscope (0.001 mm times 450 equals 0.45 mm). The resolving power of the compound microscope is approximately 0.2u (0.0002 mm). This means that objects which are smaller than 0.2 u cannot be distinguished clearly by the lenses and these will appear as small blurs in the field. Increased magnification will only make the blurs larger. Objects which are less than 0.2 u in diameter thus cannot be resolved by the optical microscope. 2. Use and Care of the Microscope a. Never look through eyepiece when turning adjustment knobs downward. You should always begin focusing upward. Otherwise, the objective lens will go through the glass slide, thereby scratching and damaging the expensive lenses of the high-dry and oil-immersion objectives. b. Lenses should be cleaned with a lens cleaning solution and lens paper each day before and after use. Xylene can be used to remove any oil film buildup on the lenses, but it should be used sparingly and must be removed by wiping with lens tissue (xylene is a solvent which can dissolve the cement that holds the lens in the objective). 3. Oil immersion Technique a. Place a drop of immersion oil over the specimen that has been previously fixed onto a glass slide. b. Using the course adjustment knob, move the oil-immersion objective (100x) into the oil and gently make contact with the slide (watch from side of scope during this procedure). c. Focusing of specimen is accomplished by turning the fine adjustment knob in a direction which makes the objective travel upward! d. If your microscope is parfocal, you can focus a specimen using the high dry objective, and then place a drop of oil on the specimen, switch to the Exercise 1 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise1/exercise1b.htm (4 of 6) [8/31/2002 4:29:54 PM]