Microscopic Techni Table Troubleshooting the Bright-Field Light Microscope Common Problem Pessible Correction No light passing theough the ocul icked in place take are that the hiab-dry chi 8.After you are finished with the microscope,place the stage micrometer would appear as illustrated 2 cenrteicr tube until the lines of the ocular micrometer are its proper storage place. parallel with those of the stage micrometer(figure 4d- Principles of Microscopic Measurement tch the lines at the left edges of the two It frequently is necessary to accurately measure the size between the lines of the ocular micrometer by observing how many spaces of the stage d with is generally expressed in metric units and is determined by the use of a m greatest accuracy in calibration if you use more ced iter ocular micrometer spaces to match with stage micrometer lines ranging from0to 100,are etched.The ocular microme ter is inserted into the cur of the microscope and then (figure 1.46).you can calibrate the ocular om时ypcdeo The】 micrometer using the following: 10 spaces on the ocular micrometer=Yspaces and 0.1 millimeter graduations.The ocular micrometer on the stage micrometer. image the slage micrometer by aligning the isms in dried fixed.or stained smears tend to be reduced as much as 10 to20%from the dimensions of the living microor ganisms.Consequently.if the actual dimer es o the micrometer For example.if 10 spaces on the ocular Procedure micrometer=6 spaces on the stage micrometer. then lar mic I ocular without the stage micrometer in place.it would appear as shown in figure 1.4a.In like manner. 1 ocular space =0.006 mm or 6.0 um. Bright-Field Light Microscope and Microscopic Measurement of Organisms
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 Bright-Field Light Microscope and Microscopic Measurement of Organisms 5 Table 1.1 Troubleshooting the Bright-Field Light Microscope Common Problem Possible Correction No light passing through the ocular Check to ensure that the microscope is completely plugged into a good receptacle Check to ensure that the power switch to the microscope is turned on Make sure the objective is locked or clicked in place Make sure the iris diaphragm is open Insufficient light passing through the ocular Raise the condenser as high as possible Open the iris diaphragm completely Make sure the objective is locked or clicked in place Lint, dust, eyelashes interferring with view Clean ocular with lens paper and cleaner Particles seem to move in hazy visual field Air bubbles in immersion oil; add more oil or make certain that oil immersion objective is in the oil Make sure that the high-dry objective is not being used with oil Make sure a temporary coverslip is not being used with oil. Oil causes the coverslip to float since the coverslip sticks to the oil and not the slide, making viewing very hazy or impossible the stage micrometer would appear as illustrated in figure 1.4b. 2. When in place, the two micrometers appear as shown in figure 1.4c. Turn the ocular in the body tube until the lines of the ocular micrometer are parallel with those of the stage micrometer (figure 1.4d). Match the lines at the left edges of the two micrometers by moving the stage micrometer. 3. Calculate the actual distance in millimeters between the lines of the ocular micrometer by observing how many spaces of the stage micrometer are included within a given number of spaces on the ocular micrometer. You will get the greatest accuracy in calibration if you use more ocular micrometer spaces to match with stage micrometer lines. Because the smallest space on the stage micrometer equals 0.01 millimeter or 10 m (figure 1.4b), you can calibrate the ocular micrometer using the following: 10 spaces on the ocular micrometer = Y spaces on the stage micrometer. Since the smallest space on a stage micrometer = 0.01 mm, then 10 spaces on the ocular micrometer = Y spaces on the stage micrometer × 0.01 mm, and 1 space on the ocular micrometer = Y spaces on the stage micrometer × 0.01 mm . 10 For example, if 10 spaces on the ocular micrometer = 6 spaces on the stage micrometer, then 1 ocular space = 6 × 0.01 mm , 10 1 ocular space = 0.006 mm or 6.0 m. 8. After you are finished with the microscope, place the low-power objective in line with the ocular, lower the tube to its lowest position, clean the oil from the oil immersion lens with lens paper and lens cleaner, cover, and return the microscope to its proper storage place. Principles of Microscopic Measurement It frequently is necessary to accurately measure the size of the microorganism one is viewing. For example, size determinations are often indispensable in the identification of a bacterial unknown. The size of microorganisms is generally expressed in metric units and is determined by the use of a microscope equipped with an ocular micrometer. An ocular micrometer is a small glass disk on which uniformly spaced lines of unknown distance, ranging from 0 to 100, are etched. The ocular micrometer is inserted into the ocular of the microscope and then calibrated against a stage micrometer, which has uniformly spaced lines of known distance etched on it. The stage micrometer is usually divided into 0.01 millimeter and 0.1 millimeter graduations. The ocular micrometer is calibrated using the stage micrometer by aligning the images at the left edge of the scales. The dimensions of microorganisms in dried, fixed, or stained smears tend to be reduced as much as 10 to 20% from the dimensions of the living microorganisms. Consequently, if the actual dimensions of a microorganism are required, measurements should be made in a wet-mount. Procedure Calibrating an Ocular Micrometer 1. If you were to observe the ocular micrometer without the stage micrometer in place, it would appear as shown in figure 1.4a. In like manner
1.Bright-FieldLight sed your calculations in the space following the table:also show your calculations to your instructor. Low power (1 jective) igure 1.4 Calibrating an Ocu ih-ry power(xobjective】 I ocular space =mm Cil inmersion (90x sbictive) ope beyond their gente stopping points cn r the magn on.the less light should b ent kno in on the high po hen when the I atch the who vear eves veen the an ocular mic 7040 the of the ire inserting.With stained prep rations such ell is the cvtop will measure closer to their actual size. Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 This numerical value holds only for the specific objective-ocular lens combination used and may vary with different microscopes. 6 Microscopic Techniques Stage micrometer Superposition of scales allows calibration of ocular scales (10 ocular units = 0.07 mm) (d) (c) Ocular micrometer Image of ocular micrometer with uniformly spaced lines Image of stage micrometer with uniform lines at standard known intervals Space = 0.01 mm 0.1 mm 0 100 20 40 60 80 0 20 40 60 80 0 100 20 40 60 80 (a) (b) Figure 1.4 Calibrating an Ocular Micrometer. HINTS AND PRECAUTIONS (1) Forcing the fine or coarse adjustment knobs on the microscope beyond their gentle stopping points can render the microscope useless. (2) A general rule for you to note is that the lower the magnification, the less light should be directed upon the object. (3) The fine adjustment knob on the microscope should be centered prior to use to allow for maximum adjustment in either direction. (4) If a slide is inadvertently placed upside down on the microscope stage, you will have no difficulty focusing the object under low and high power. However, when progressing to oil immersion, you will find it impossible to bring the object into focus. (5) Slides should always be placed on and removed from the stage when the low-power (4× or 10×) objective is in place. Removing a slide when the higher objectives are in position may scratch the lenses. (6) A note about wearing eyeglasses. A microscope can be focused; therefore, it is capable of correcting for near- or farsightedness. Individuals who wear eyeglasses that correct for near- or farsightedness do not have to wear their glasses. The microscope cannot correct for astigmatism; thus, these individuals must wear their glasses. If eyeglasses are worn, they should not touch the oculars for proper viewing. If you touch the oculars with your glasses, they may scratch either the glasses or the oculars. (7) Because lens cleaner can be harmful to objectives, be sure not to use too much cleaner or leave it on too long. The distance between the lines of an ocular micrometer is an arbitrary measurement that has meaning only if the ocular micrometer is calibrated for the specific objective being used. If it is necessary to insert an ocular micrometer in your eyepiece (ocular), ask your instructor whether it is to be inserted below the bottom lens or placed between the two lenses. Make sure that the etched graduations are on the upper surface of the glass disk that you are inserting. With stained preparations such as Gram-stained bacteria, the bacteria may measure smaller than they normally are if only the stained portion of the cell is the cytoplasm (gram-negative bacteria), whereas those whose walls are stained (gram-positive bacteria) will measure closer to their actual size. Calibrate for each of the objectives on your microscope and record below. Show all calculations in the space following the table; also show your calculations to your instructor. Low power (10× objective) 1 ocular space = _ mm High-dry power (40× objective) 1 ocular space = _ mm Oil immersion (90× objective) 1 ocular space = _ mm
Microscopic Techniques T8aa Laboratory Report 1 Name Date: Lab Section: Bright-Field Light Microscope (Basic Microscopy） Parts of a Compound Microscope 1.Your microscope may have all or most of the features described below and illustrated in figure 2.3 in you textbook.By studying this figure and reading your textbook,label the compound microscope in figure LR1.1 on the next page.Locate the indicated parts of your microscope and answer the following questions. a What is the magnification stamped on the housing of the ocularson your microscope? b.What are the magnifications of each of the objectives on your microscope? c. Calculate the total magnification for each ocular/objective combination on your microscope Objective List the magnification and numerical aperture for each objective on your microscope. Magnification of Ojective Numerical Aperture (NA) e.With some compound microscopes,loosening a lock screw allows you to rotate the body tube 18 What is the advantage of being able to rotate the body tube? f Note the horizontal and vertical scales on the mechanical stage what is the function of these scales? Where is the diaphragm on your microscope located?
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 7 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 1 Bright-Field Light Microscope (Basic Microscopy) Parts of a Compound Microscope 1. Your microscope may have all or most of the features described below and illustrated in figure 2.3 in your textbook. By studying this figure and reading your textbook, label the compound microscope in figure LR1.1 on the next page. Locate the indicated parts of your microscope and answer the following questions. a. What is the magnification stamped on the housing of the oculars on your microscope? _ b. What are the magnifications of each of the objectives on your microscope? _ _ c. Calculate the total magnification for each ocular/objective combination on your microscope. Ocular × Objective = Total Magnification _ _ _ _ _ _ _ _ _ _ _ _ d. List the magnification and numerical aperture for each objective on your microscope. Magnification of Objective Numerical Aperture (NA) _ _ _ _ _ _ _ _ e. With some compound microscopes, loosening a lock screw allows you to rotate the body tube 180°. What is the advantage of being able to rotate the body tube? _ _ f. Note the horizontal and vertical scales on the mechanical stage. What is the function of these scales? _ g. Where is the diaphragm on your microscope located? _ _
1.Bright-Field Light Figure LR1.1 Modem Bright-Field Compound Micr 8 Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 Figure LR1.1 Modern Bright-Field Compound Microscope. 8 Microscopic Techniques
Microscopic Techniques 8a me How can you regulate the diaphragm? h. Locate the substage condenser on your microscope.What is its function,and how can it be regulated? i.Can the light intensity of your microscope be regulated?Explain. Microscopic Measurement of Microorganisms 2.After your ocular micrometer has been calibrated,determine the dimensions of the prepared slides of the following microorganisms. Length width Magnification Bacterium Fungus nam Alga name 3.Draw and label,as completely as possible.the microorganisms that you measured Genus and species Genus and species Magnification:x Magnification: Genus and species Genus and species: Magnification:x Magnification:x Bright-Field Light Microscope (Basic Microscopy)
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 How can you regulate the diaphragm? _ _ h. Locate the substage condenser on your microscope. What is its function, and how can it be regulated? _ _ i. Can the light intensity of your microscope be regulated? Explain. _ _ Microscopic Measurement of Microorganisms 2. After your ocular micrometer has been calibrated, determine the dimensions of the prepared slides of the following microorganisms. Microorganism Length Width Magnification Bacterium name _ _ _ _ Fungus name _ _ _ _ Alga name _ _ _ _ Protozoan name_ _ _ _ 3. Draw and label, as completely as possible, the microorganisms that you measured. Genus and species: _ Genus and species: _ Magnification: _ Magnification: _ Genus and species: _ Genus and species: _ Magnification: _ Magnification: _ Bright-Field Light Microscope (Basic Microscopy) 9 × × × ×