Microbiology Roadmap: Helping you Navigate On the RoadAs you read this chapter, ask yourself…What units are used to measure something so much smaller than the unassisted human eye can detect? MicronsWhat principles of microscopy must be considered to achieve optimal magnification? Resolution, etcWhat are the major advantages, disadvantages, and uses of the various types of microscopes and microscopytechniques?What are some of the techniques required to visualize samples under the microscope?What are the commonly used microbiological differential staining techniques and what are they used to detect? Mile marker 1Or perhaps more correctly termed ‘kilometer’ marker 1? Scientists worldwide use the metric system (meters,liters, and Celsius, instead of yards, gallons, and Fahrenheit). The use of a standardized system allowscomparison of results and aids in the repetition of experiments so essential to the scientific method. 1 meter isapproximately 1.1 yards (a little over 3 feet). If you look at a metric ruler, the large divisions are centimeters(cm), which are 1/100th of a meter (m), the smaller divisions are millimeters (mm), which are 1/1000th of a meter.When microbiologists look into a microscope, they often measure things using micrometers (or microns) (µm); 1µm is 1/1000000th of a meter (or 1/1000th of a millimeter)! This means that 1000 things that are 1 micrometerlong can line up end-to-end in 1 millimeter. (1) Look at Figure 3.2 Relative sizes of objects. If you assumed a bacterial length of 2 µm and a doglength of 1.5 m, how many bacteria could line-up end-to-end from the tip of your dog’s nose to thetip of her tail?a. Note that the use of scientific notation makes this kind of calculation easier. If we assign thevalue of 1 x 100 (which is equal to 1) to a meter (m), then a micrometer (µm) is equal to 1 x10-6 m. In this way, even smaller units can be described without the zeroes becomingcumbersome. For instance, a nanometer (nm) is 1 x 10-9 m and an angstrom (Å) is equivalentto 1 x 10-10 m. That certainly is easier to use than 1/1,000,000,000 and 1/10,000,000,000!(see also Table 3.1)1,500,000(2) Return to Figure 3.2. You are probably most familiar with a compound light microscope; you mayhave used them in a general biology course or in the lab that accompanies this course. Lightmicroscopes are suitable for viewing all protozoa, and most or all bacteria. Is a light microscopesufficient to view an average size virus? What type of microscope might you need to view a virus?By the end of this chapter you should understand what the differences between the major types ofmicroscopes are. No viruses are smaller than bacteria and bacteria can only be seen on the highest magnification ofa compound microscope1Road Stop (principles of microscopy)Define the following terms:Microscopy- field of using microscopesResolution- distance between objects to distinguish them as separateTransmission -electrons sent through specimenAbsorption- fluorescent substances absorb UV light and emit visible lightFluorescence -visible radiation emitted caused by shorter wavelength of UVrefraction (particularly as it relates to the use of immersion oil)- bending of light waves in oil = glass Road Stop (Light and Electron microscopy) Define or explain the function(s) of the following terms: (helpful for your lab class)Compound microscope uses ocular lens and objective lensCondenser- focuses light on specimenIris diaphragm -circular apertureObjective lens -closest to specimen Ocular lens- where your eyes go 10X magnificationParfocal- remains in focus when lens changesOcular micrometer- measures objects viewedTransmission electron microscope (TEM) - views internal structuresScanning electron microscope (SEM) - views surfaces of specimen Micrograph- photograph taken with microscope Reading the map: The Compound Light Microscope (figure 3.12) Figure 3.12. The Compound Light Microscope.2Find the light source. Follow the path of light (yellow) from the source to the observer’s eye.(1) What happens to the light as it passes through the condenser?Focused on specimen(2) How many lenses does the light pass through as it travels between the specimen and the observer?2(3) If the objective lens magnifies the appearance of an object 40 times and the ocular lens magnifies theappearance of an object 10 times, what is the final magnification of the image? (for help see page 58)400(4) Most compound microscopes today are parfocal. Given this, when you switch between two objective lenses,you should only have to use the coarse/fine (pick one) adjustment knob when refocusing the image.fine At a crossroads: Comparing types of light microscopy (see also Table 3.2)In this chapter a number of different types of light microscopy have been discussed. Among these are standard lightmicroscopy (sometimes called bright-field microscopy), dark-field microscopy, fluorescence microscopy, and confocalmicroscopy. Each of these differs in their application and in the image of the specimen they provide; some of them differin the source of their light.(1) For each of the following queries, indicate which of the above light microscopes would be appropriate to use(note that there may be more than one answer, but in many cases, there likely is one better answer).3a. To view a thicker section of fluorescent organisms or tissues using a UV laser; potentially allowsthree-dimensional modeling of microbial communities.confocalb. To view a light-sensitive organism or a live spirochete that lacks contrast with its background.darkfieldc. To view a fixed, stained bacterial sample in which morphology, arrangement, and size are to beevaluated.brightfieldd. To view a thin sample of individual organisms dyed with acridine orange or another type offluorochrome using UV light.fluorescent Road Stop (Techniques of light microscopy) Define or describe the following proceduresWet mounts (hanging drop)___view living organisms, special slide glassSmears- uses loopfulHeat-fixation- kills bacteria, adheres to slideStain (cationic/basic vs anionic/acidic) cationic is positive, bonds to negative cellsStaining (simple vs differential)simple uses one dye, differential uses 2+Gram stain- differential stain using differences in cell wall to distinguishZiehl-Neelsen acid-fast stain- differential stain useful for mycobacteriumSchaeffer-Fulton endospore