BENGUET STATE UNIVERSITY, Collegeof As end Sciences Department of Biology Exercise No. 1 THE COMPOUND LIGHT MICROSCOPE AND OTHER INSTRUMENTS IN MICROBIOLOGY Microbiology deals of living organisms that cannot be viewed (seen) in any detail by the human eye. A Microbiology laboratory should be equipped with the necessary instruments needed to grow and observe such microbes. Magnifying systems used for viewing microbes come in two general forms; simple magnifying glasses or lenses that consist of a single lens of polished, rounded glass or plastic and compound microscopes that use two lens, an objective lens and an eyepiece lens to magnify the sample. Besides microscopes, equipment and supplies needed for growing microbes as well as preparing their nutritional requirements should be available, OBJECTIVES: Al the end of the activity, the students will be able to: 1. identify the different parts of the compound light microscope and determine the corresponding functions. 2. properly manipulate or operate the microscope. 3. calibrate a compound microscope and accurately measure the size of different cells 4, leam the proper care and use or handling of the microscope. MATERIALS: To be borrowed from the preparatory room: ¥ Compound microscope ¥ Immersion oll ¥ Ocular micrometer v Xylol ¥ Stage micrometer ¥ Microorganisms to be ¥ Lens paper or tissue paper studied (protozoa, fungi spores, bacteria} PROCEDURES: A. PARTS OF THE COMPOUND LIGHT MICROSCOPE 1. Examine the microscope and identify its different parts. Be guided by the given description below. Draw and label the parts of the microscope assigned to you in the worksheet provided, a. EYEPIECE: also known as ocular; where you look through to see magnified specimen; the inscribed number indicates the magnification aBENGUET STATE UNIVERSITY, College of Ans and Sciences Department of Biology. , BODY TUBE: long tube that holds the eyepiece and connects it to the objectives C. NOSEPIECE: rotating part at the oottom of the body tube: it holds the objectives d. OBJECTIVE LENS: microscope lens that first produces magnification of the specimen in a compound microscope; usually, there are 3 or 4 objective lenses on a microscope: (i) Scanner: shortest objective with 4x- 5x magnification {i) Low Power Objective (LPO): with 10x or 12.5x magnification + (ii) High Power Objective (HPO): with 40x to 60x magnification (iv) Oil immersion Objective (O10): with 100x magnification ©. ARM: part of the microscope that connects the tube to the base; when Carrying microscope, one hand grips this while the other supports the base f. COARSE ADJUSTMENT KNOB: large, round knob on the side of the microscope used to coarsely move either the stage or the upper part of the microscope to focus specimen 9. FINE ADJUSTMENT KNOB: small, round knob on the side of the microscope used to finely move the stage or upper part of the microscope to focus specimen after using the coarse adjustment knob h. STAGE: large, flat area under the objectives where the specimen to be viewed is anchored; it has a hole (see aperture) that allows light fo pass through i. STAGE CLIPS: clips on top of the stage which hold the slide in place j. APERTURE: hole in the stage that allows light through to better view the specimen k. DIAPHRAGM: controls amount of light passing through the aperture |. CONDENSER: a lens that concentrates light from the illuminator Mm. ILLUMINATOR: a steady light source (110 volts); other microscopes use a MIRROR, to reflect light from on external light source up through the botiom of the stage. B. ALIGNMENT OF LENSES 1. Focus the low power objective on any of the prepared slides 2. Adjust the mirror to the position that gives the brighter and the most evenly illuminate field of vision. 3. Check the condenser fo see ifit has been raised to its highest position. = ©BENGUET STATE UNIVERSITY, College of Ars and Scenoes Department of Biology 4. Bring the objective into focus by manipulating the coarse adjustment knob. 5. Manipulate the diaphragm lever to reduce or increase the light intensity to produce the clearest, sharpest image. C. IMMERSION OIL |. Secure a slide of a stained bacterial smear and focus first under LPO. 2. without moving the course adjustment, rotate the revolving nose piece so thal the specimen falls between the objectives (Not right below] 3. Place a drop of oll on the midale of the coverslip. 4. Carefully tum the objective lens until the 100X lens contacts the oil and snaps into position: 5. Examine the lens; it should almost touch the coverslip and the oll should fil the space between the coverslip and the ler 6. Ifit fouches the coverslip carefully back the lens off until it is about 1 mm above the coverslip. 7. While looking through the eyepiece use the FINE ADJUSTMENT knob to move the lens up and down in finy increments: this action should bring the organisms into focus. D. Calibration of Micrometer and Measurement of Specimen The unit of measurement used for microorganisms is the micrometer. One micrometers equal to 1/1000 of a milimeter. Microorganisms can be measured by using and ocular micrometer. This is a glass disc with mounted scales. in order to Use this, it must be calibrated first with the stage micrometer. A slage micrometer '5 G glass slide with graduations of known interval. The length of one small division i 0.01 mm or 10 um, whereas one large division is 0.1 mm or 100 Lm. |. Replace the eyepiece of your microscope with an ocular micrometer. 2. Place the slage micrometer on the stage and focus on ils scale, 3. Superimpose the two scales. 4, To calibrate the low power objective, move the stage micrometer laterally 50 that one line coincides with a line on the scale. 5. Look for another line on the ocular micrometer that coincides with one on the stage micrometer, then count the number of ocular spaces equivalent fo the number of stage spaces between the coincident lines at bath edges. 6. To calibrate the oil immersion lens, place a drop of immersion oil on the sage micrometer, shift to the oil immersion objective and bring the lines into focus; then proceed as in siep #5. Pees ee ts an @ —Hecti and Parco Later MaBENGUET STATE UNIVERSITY, Colage of Ais and Sciences Department of Biology 7. Calculate the calibration constant (value of one ocular micrometer division) of the microscope as follows: 8. No. of stage micrometer = No. of stage division x 10 um Calibration Constant (um/div) = no. of stage micrometer No. of ocular divisions 9. Record calibration. With calibrated ocular micrometer, focus on the specimens given and compute for the actual size. Actual Size = no. of divisions in the ocular um x calibration constant IMPORTANT: Clean the oil immersion objective and the stage micrometer immediately after use with lens paper and xylol. E, Other tools/ instruments in Microbiology With your laboratory instructor, go around the Microbiology laboratory and take note of the location, features, principles of operation and functions of the some common Microbiology laboratory equipment and iristruments. Fill in the necessary information in your activity sheet. QUESTIONS FOR RESEARCH 30 ee | 1. How does a microscope magnily an objeci@ $ 2. Define the following: \9 1. Focal Length |. Working distance Ill, Resolving Power IV, Numerical aperture V. Parfocal 3. What is the function of the immersion oil in the microscopic examination of microorganisms? _g 4. Whyis an inverted image seen under a compound microscope? Draw the path of light in a compound microscope indicating how an image is formed. 4 iar on Poa aay Nana e