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Intro Microbiology

by: Miss Meaghan Feil

Intro Microbiology MCB 2000

Miss Meaghan Feil
GPA 3.61

John Makemson

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John Makemson
Class Notes
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This 5 page Class Notes was uploaded by Miss Meaghan Feil on Monday October 12, 2015. The Class Notes belongs to MCB 2000 at Florida International University taught by John Makemson in Fall. Since its upload, it has received 16 views. For similar materials see /class/221795/mcb-2000-florida-international-university in Microbiology at Florida International University.


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Date Created: 10/12/15
THE COMPOUND MICROSCOPE In microbiology the microscope plays an important role in allowing us to see tiny objects that are normally invisible to the naked eye It is essential for students to learn how to use the microscope in a Skill ll manner Success il microscopy requires the student to a Be patient b Know the basic principles of microscopy c Take care of the microscope d Understand the nature of material observed For success il microscopy then it is necessary to understand how to control the variable microscopic factors To do this the student needs to understand the relationships between magni cation resolution and contrast PARTS OF THE MICROSCOPE Refer to the Figure on next page 1 Illuminator Most modern microscopes contain an built in base illuminator with a facility to vary the intensity of light by varying the voltage to the lamp light intensity as well as an iris diaphragm in the condenser For optimal results the proper amount of illumination should be obtained fields that are too bright or too dim will not allow you to see the details of the preparation you are examining and can lead to eyefatigue Be sure you are familiar with both the iris diaphragm mechanical and the light intensity adjustment electrical 2 Objective Lenses These are mounted on the revolving nosepiece Each lens is marked with its Magnificatioan umerical Aperture and Focal Distance Be sure you can distinguish between these numbers Most standard pathology microscopes have 4X 10X 40X and 100X oil immersion lenses The first three are used with only air between the lens and the slide the highest power lens 100X is used with a drop of immersion oil between the lens and the slide That is the light passing through the slide material being viewed passes through only oil not air as it is transmitted to the lens system The objective lenses must be kept clean Use lens tissue or Kimwipes to clean the outer exposed surfaces of the lenses Although the oil immersion lens is designed to work in oil the oil must be removed after use and before you put the microscope away at the end of the period All the other lenses do not operate with oil on them If oil gets on the air lenses it will ruin their mounts and surfaces oil must never contact the air lenses 3 Ocular LGszs Thenculaxlmses usuallymagnfywx Thusthetntalmagn cannn back and myth unm a 0110qu eld 1 wewad mm bath eyes npen Admnnnany many an the nmlmmses axe adjustedtn ynux eyes Evcv mam mama sund Memmm mm Condenser 17th cnmesy nfOlymles Cnrpnra nn Platinum Instrument Dmsmn Lake Success NY 4 Condenser Lens Below the stage is the condenser lens This focuses light onto the object and is not involved in the magni cation The focusing adjustment is a rack and pinion movement to permit vertical movement of the condenser Clear images are obtained only when the condenser lens is in proper focus when the cone of rays illuminating the object is equal to that observed by the objective lens If you have a blurry object it could well be that the condenser is out of focusadjust both the iris diaphragm and condenser focus adjustments With time your patience will be rewarded by clear crisp images 5 Coarse and Fine Adjustment Wheels These are used to raise and lower the body tube or stage depending upon the manufacture of the microscope The coarse adjustment is used to first bring the object into approximate focus starting rst with the stage as close as possible to the objective lens without touching Then move the coarse adjustment so that the stage moves away from the lens until the object is in relative focus If this is always done in this way there is no possibility that the lens gets jammed into the slidedamagjng both After the object is in relative focus it can then be brought into sharp critical focus with the fine adjustment knob MAGNIFICATION AND RESOLVING DISTANCE It is relatively easy to think of the microscope in terms of magni cation the importance of which is without dispute However the importance of magni cation is meaningless without a clear crisp image and resolution is a measure of clarity Resolution is defined by resolving distance Resolving distance is the smallest distance between two points that allows the observer to see those points as distinctly separate The most limiting factor in obtaining good resolution is the wavelength of light used for illumination Bear in mind that the bacteriologist observes objects whose own dimensions are of the same order of magnitude as the wavelength of light Blue light 360 nm to 420 nm permits greater resolution than red light 650 nm to 800 nm Sometimes it is possible to magnify an image beyond the smallest resolving distance of the lens system this is termed quotempty magni cationquot because although the image is magnified it is not distinct but blurry and can not be seen as well as if it were magni ed to a lesser amount within the resolving distance of the objective Another factor that determines resolving distance is the refractive index of the medium through which the light rays pass The refractive index of glass is 152 compared to air N 100 Light rays passing through a glass slide using the highdry lens will pass through air and be bent before reaching the objective lens Less bending will occur with water than air and even less with oil The maximal angular aperture of the lens the angle of greatest divergence of light rays that the objective lens can collect will not be realized with air With an oil immersion lens the air space between the glass slide and the lens is replaced with oil that has a refractive index very close to that of glass These factors are combined in the Numerical Aperture of the lens The numerical aperture of a lens is defined as Numerical Aperture N sin a where quotNquot is the refractive index of the material between the object and the objective lens and quotaquot is one half of the angular aperture of the objective lens If a highdry lens has an angular aperture of 111 4839sin a 0828 its numerical aperture working in air N 100 will be NA 100x0828 083 If that same lens could be used in oil most likely it couldn39tit would have a numerical aperture of NA 152x0828 126 assuming a refractive index of 152 for the oil These calculations are important because of their relationship to the resolving distance The formula for Resolving Distance is RD wavelength2 NA The reason the numerical aperture is multiplied by two is that two numerical apertures are involved that of the objective lens and that of the condenser When the condenser is in perfect focus it has the numerical aperture of the objective The denominator of the Resolving Distance equation is really the numerical aperture of the objective plus the numerical aperture of the condenser If one assumes that quotaverage blue lightquot is being used wavelength 400 nm using an oil lens of numerical aperture of 125 will allow you to resolve distinctly objects as small as the resolving distance RD 400 nm2 x 125 160 nm or 016 pm This means that under the most ideal conditions this lens is capable of distinguishing two objects as separate if they are 016 um or greater apart If the two objects are less than 016 um apart say 010 um then they will be blurred together at the point where they are 010 um apart RULES FOR USE AND CARE OR THE MICROSCOPE 1 Use only lens paper or Kimwipes to clean the optical parts of the microscope Do not use paper towels lab coat tails handkerchiefs or other such to clean the lenses P Never immerse the 10X or 40X lenses in oil LN When done each day wipe off oil from the 100X oil immersion lens 4 When done each day clean stage condenser lens the other objective lenses and ocular lenses U Do not attempt to clean the inside of the microscope or a lens 0 Keep the microscope upright when taking or returning the microscope to the cabinet When the microscope is put away the lowest power lens should be in place YOU ARE RESPONSIBLE FOR THE WELL BEING OF YOUR MICROSCOPE


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