Supplemental Lecture (97/03/25 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Microscopes
    1. A list of vocabulary words is found toward the end of this document $ lecture: Microscopes
  2. Scale of microorganisms
    1. Micrometer (µm) = 10-6 meters <= bacteria cell (0.1 µm ~ viruses); nanometer (nm) = 10-9 meters ~ an amino acid; angstrom (Å) = 10-10 meters ~ an atom.
  3. Microscope
    1. a device used to make enlarged images of minute objects.
  4. Light microscopy
    1. A microscope which uses visible light to form the observed enlarged image. Contrast with electron microscopy.
    2. Note that light microscopes are unable to visualize viruses.
  5. Simple microscope
    1. A very minimal light microscope having only a single lens.
  6. Compound microscope
    1. A light microscope consisting of multiple lenses.
    2. A contemporary compound microscope is capable of magnification of up to 2000x and resolution of up to 0.2 µm.
    3. Critical parts of a compound microscope include
      1. the ocular
      2. the objective lenses
      3. the stage
      4. the condenser
      5. the diaphragm
      6. the illuminator
      7. one or more focusing knobs.
    4. See fig 3.1, p. 54, Tortora et al., 1995.
  7. Magnification
    1. The perceived increase in size of an object upon viewing through a microscope.
    2. Total magnification obtained with a compound microscope is equal to the product of the magnification supplied by the ocular and that supplied by the objective lenses (e.g., 10X * 100X = 1000X).
    3. Note that for all but the best light microscopes, a magnification of 1000X is the best that can be achieved.
  8. Resolution [resolving power]
    1. The ability of a microscope to present a magnified image of two closely spaced points as separate points.
    2. A microscope with poor resolving power may present an image in which two closely spaced points do not appear as two separate images.
    3. The resolution of a microscope is measured in units of distance between these two hypothetical points.
    4. A microscope is only as useful as it is able to resolve the structures you are interested in viewing.
  9. Ocular
    1. The last lens or eyepiece of a compound microscope.
    2. The ocular imparts the final magnification of the image observed.
    3. It often magnifies on the order of 10X.
  10. Objective lens
    1. The first lens of a compound microscope.
    2. This is the lens closest to the specimen being observed.
    3. Different objective lenses are used in order to impart difference degrees of magnification.
    4. Magnification imparted by the objective lens often ranges from 10X, to 40X, to 100X or even 200X (the two latter magnifications achieved with oil immersion).
  11. Focal length
    1. Engraved on the side of objective lenses is both the power of the lens (e.g., 10x) and the focal length (e.g., 16 mm).
    2. Note that the focal length indicates how close the objective lens being used should be to the sample in order to bring the sample into sharp focus.
  12. Stage
    1. The often movable platform which holds the specimen.
  13. Condenser
    1. Lenses found after the illuminator but prior to the specimen.
    2. The condenser focuses the light passing through the specimen and into the objective lens.
  14. Diaphragm
    1. Adjusts to control the amount of light coming from the illuminator which enters the rest of the microscope through the condenser.
  15. Illuminator
    1. The light source for the compound microscope.
    2. This is usually an incandescent light bulb which is located at the bottom of the compound microscope.
  16. Focusing knobs
    1. These knobs move the stage up and down so that the relevant horizontal plane in the specimen is brought into focus.
    2. Often a compound microscope will have both a coarse and a fine focusing knob.
  17. Refraction [refraction index]
    1. Deflection of light from a straight line occurring upon passing from one substance to another through a slanting interface (so long as the refractive indices of the two substances are different).
    2. Refraction causes a loss of light and therefore darkening.
    3. Such darkening is seen as images using the compound microscope.
    4. Staining and other means (e.g., phase-contrast microscopy) are employed to increase the refraction displayed by specimens and therefore their visibility.
  18. Oil immersion
    1. A technique employed to increase resolution at higher magnifications.
    2. Oil with the same refractive index as the glass slide holding the specimen is placed between the specimen and the objective lens.
    3. This prevents light from refracting between the specimen and the objective lens and images are consequently sharper.
  19. Brightfield microscopy
    1. Use of a compound microscope such that the image observed is the product of light that has passed through the specimen.
    2. It is called brightfield because where there is no specimen the light observed is brightest.
    3. Thus, the background is brighter than the specimen.
    4. Because brightfield microscopy relies solely on refraction, most specimens are difficult to see without staining.
    5. See fig 3.4a, p. 56, Tortora et al., 1995.
  20. Darkfield microscopy
    1. In darkfield microscopy there is an opaque disk in the condenser which blocks all light passing in a straight line from the condenser, through the specimen, into the objective lens.
    2. As a consequence, only scattered and reflected light is observed and the background is dark.
    3. This is one way of observing microorganisms without staining and therefore is useful for observing bacteria which are not easily stained.
    4. It necessarily gives a different image from that seen when employing brightfield microscopy and staining.
    5. Darkfield microscopy is somewhat archaic since today more sophisticated methods are available for visualize stain-resistant bacteria.
  21. Phase-contrast microscopy
    1. A way of viewing free, unfixed microorganisms with good detail.
    2. Phase-contrast microscopy relies on the differential diffraction (caused by refraction) of the light passing through the specimen, its different component parts, and the suspending medium.
    3. The instrument is adjusted such that all light passing through the medium (background) remains in phase and light passing through the specimen does not thus resulting in a specimen that is darker than the background.
    4. As better put by Burrells, 1977: "A phase microscope is a devise which causes difference of refractive index between an object and its surrounding medium to be made visible in the form of an ordinary black and white image.
    5. The term ‘phase contrast’ has come to be used because the differences in phase of some rays in the light bundle passing through the system are used to give the necessary ‘contrast’ between the objective and the background."
  22. Electron microscopy (EM)
    1. A microscope employing electrons instead of light to image specimens. Electron microscopy, or EM, is capable of much better resolution than light microscopes because the wavelength of electrons is much smaller than that of the photons of visible light.
  23. Transmission electron microscopy (TEM)
    1. A type of EM used to examine very thin sections of fixed, stained, and dehydrated specimens.
    2. Gives magnifications ranging from 10,000X to 100,000X.
  24. Scanning electron microscopy (SEM)
    1. A type of EM used to examine the surfaces of intact specimens.
    2. You’ve all seen what the 3-D-looking SEM images look like, such as photographs that appear to be extreme close-ups of insects.
    3. SEM is capable of magnifying in the range of 1000X to 10,000X and resolving objects as close together as 20 nm.
  25. Staining with biologic dyes
    1. The coloring of microorganisms with dyes (chromophoric ions) that bind specifically to various cell structures.
  26. Fixation
    1. The binding of microorganisms to the microscope slide. This prevents them from being washed off the slide during staining. Fixation is usually accomplished by spreading (smearing) the organism onto the slide, drying, and then flaming the slide lightly. Fixation usually results in the death of the attached microorganisms.
  27. Basic dyes
    1. A stain that binds to acidic substances such as bacteria.
  28. Acidic dyes
    1. A stain that binds to basic substances. These tend to be the background and acidic dyes thus tend to give negative staining.
  29. Negative staining
    1. A staining method whereby the specimen is left unstained but visible against a stained background.
  30. Simple stain
    1. A stain employed to highlight the entire microorganism (as opposed to differentiating its component parts) using only a single type of dye.
    2. Contrasts with differential staining.
  31. Mordant
    1. A substance used to increase the ability of a stain to highlight the microorganism or its component parts.
  32. Differential stains
    1. A staining method employed to distinguish among different kinds of bacteria.
    2. That is, different types of bacteria are stained differently.
    3. Differential stains are not simple because at least two stains are necessary to differentiate two organisms while simultaneously assuring that both organisms are stained by at least some dye.
  33. Gram stain
    1. A method of differentiating bacteria by staining and brightfield microscopy into two broad, phylogenetically relevant groups: gram-negative bacteria and gram-positive bacteria.
    2. Gram stains are performed as follows:
      1. Bacteria are heat-fixed to a slide.
      2. A purple basic dye such as crystal violet is applied to the fixed smear as the primary stain.
      3. After a short period the primary stain is washed off with distilled water. The cells are now stained purple.
      4. The washed fixed smear is then covered with an iodine mordant.
      5. The iodine is then washed off with distilled water. The cells are now stained darker purple.
      6. The washed fixed smear is then decolorized by further washing with either ethanol or a solution consisting of ethanol and acetone. This washing removes the purple color (primary stain) from gram-negative bacteria but not from gram-positive bacteria.
      7. The decolorizing agent is washed off with distilled water.
      8. The decolorized and washed fixed smear is then counterstained with safranin, a red basic dye.
      9. The safranin stained fixed smear is again washed with distilled water. Gram-positive bacteria retain the primary stain as above and remain purple but gram-negative bacteria do not retain the primary stain following decolorizing and are consequently stained only by the counterstain. Gram-negative bacteria are thus stained red following safranin counterstaining.
      10. The slide is blotted dry and examined by brightfield microscopy. Note that gram staining is best done using growing (exponential phase) bacteria and that some bacteria are resistant to staining and thus cannot be successfully gram typed.
  34. Primary stain
    1. A simple stain employed in the course of differential staining.
    2. The function of the primary stain is to impart a color to the cell present regardless of their potential for accepting the differential stain.
  35. Counterstains
    1. A stain with a contrasting color to a primary stain.
  36. Acid-fast stain
    1. A method of differential staining that detects the presence of waxy cell wall material.
    2. Acid-fast staining is useful for bacteria which are not readily gram stained and particularly for identifying Mycobacterium spp.
  37. Special stains
    1. Stains used to view specific cellular structures such as capsules, endospores, and flagella.
  38. Vocabulary
    1. Acid-fast stain
    2. Acidic dye
    3. Basic dye
    4. Brightfield microscopy
    5. Compound microscope
    6. Condenser
    7. Darkfield microscopy
    8. Diaphragm
    9. Differential staining
    10. Fixation
    11. Focusing knobs
    12. Gram stain
    13. Illuminator
    14. Mordant
    15. Negative staining
    16. Objective lens
    17. Ocular
    18. Oil immersion
    19. Phase-contrast microscopy
    20. Primary stain
    21. Resolution
    22. Resolving power
    23. Scanning electron microscopy (SEM)
    24. Simple stain
    25. Stage
    26. Transmission electron microscopy (TEM)
  39. Practice questions
    1. Using a good compound light microscope with a resolving power of 0.3 µm, a 10X ocular lens, and a 100x oil immersion lens, would you be able to discern two objects separated by 3 µm? 0.3 µm? 300 nm? 3000 Å? [PEEK]
    2. Acidic dyes stain the __________ in a smear and are used for ___________ staining (fill in the blanks and don’t use the term simple for the second blank). [PEEK]
    3. What is the purpose of the counterstain in staining techniques such as the gram stain? [PEEK]
    4. Name a reason for using phase contrast microscopy rather than brightfield microscopy ? [PEEK]
    5. Describe why in gram staining only gram-positive cells retain the primary stain? [PEEK]
  40. Practice question answers
    1. You would be able to discern two objects separated by the four distances given because each is equal to or greater than the resolving power of the microscope.
    2. In this order the blanks should be filled with: (i) background, (ii) negative.
    3. to visualize otherwise unstained microorganisms
    4. Viewing living, unfixed, unstained cells.
    5. The key word is retain. The primary stain is retained by gram-positives because it makes an intracellular complex with the iodine mordant that stays in place so long as the cell envelope is reasonably intact. The decolorizing agent breaks down the cell envelope of the gram-negative (but not gram positive) cells sufficiently that the primary stain-mordant complex is lost (washed out). The difference between cell types is that the gram negative cell envelope is thin and includes the outer membrane, the latter which is particular susceptible to the decolorizing solvents. The answer: The primary stain-mordant complex is retained in the gram-positive cell but not gram negative cells because of differences in cell envelopes, was sufficient.
  41. References
    1. Doe, A. B., Smith, Y. Z. (1996). Intro to Everything. Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 39-42.