Studying Microbes: Bacteria, Magnification, and Resolution

1. How we study microbes Chapter 3

2. Bacteria were the first microorganisms to be studied Small in size 1000 X magnification Most about 200 nm to 1 mm • Robert Hooke • Used the term “cell” • 1665 • Anton van Leeuwenhoek • First to visualize bacteria & protozoa • 1678 • Beginning of the end of “spontaneous generation”

3. Fundamentals of Light Microscopy • Ultimate goal is to obtain information • Two factors affect amount of attainable information • Magnification • Resolution

4. Magnification - How Does a Lens Magnify? Air and glass have different refractive indices, so light refracts (bends) as it passes through a lens. Image is magnified as once parallel rays now diverge. Virtual Image Reaches the eye E E

5. Pathway of Light in Compound Microscope Virtual Image formed by ocular lens Mistake- Blue and red should be reversed here (page 75) Magnified primary image This lens not for magnification- concentrates light

6. Magnification Defined as amount of enlargement of specimen Picture of Anton van Leeuwenhoek 1X 5X 20X 100X • Magnification can be infinite • At some point, no more information can be gathered • Have reached the limiting factor of microscopy - RESOLUTION

7. Resolution • Minimum distance at which two objects can be • distinguished (the smaller the number, the better the resolution) • Several factors affect resolution • Wavelength (l) of light • Refractive index (h) of medium between lens and • specimen • Distance between lens and specimen • Contrast

8. l = wavelength of light h = refractive index q = angle between most divergent light ray gathered by lens and the center of the lens l 2hsinq Resolution = Numerical Aperture Mathematical constant that describes the efficiency of a lens 0.1 in a lowest powered lens (4 X) 1.25 in the highest power lens (100 X) Higher numerical aperture =Better Resolution

9. l 2 N.A. Resolution = l of Light Used l = wavelength of light 500 nm ---------- = 200 nm 2 x 1.25 • Different colors of light have different l • The shorter the wavelength, the higher the resolution- • 400-500 nm is excellent • Thus many light microscopes use filters to select color of light • blue-green l

10. Refractive Index of Medium Between Specimen and Lens Light path without oil Light path with oil l 2hsinq Resolution= h = refractive index • Refractive index - ability to bend light • Glass from the slide bends light more than air • Light is bent away from lens as it passes through specimen • Immersion oil has similar refractive index as glass • More light is gathered by the lens, more information

11. Oil Immersion Lens • = 1 for air • = 1.5 for glass • = 1.52 immersion oil

12. The Distance Between the Lens and the Specimen • The further the lens from the • sample, the more light is lost • The closer the lens, more light • gathered • More light means more • information and higher resolution • q = angle between most divergent • light ray gathered by lens and the • center of the lens Lens far from specimen > Q Lens close to specimen

13. l 2hsinq Resolution = Numerical Aperture Website micro.magnet.fsu.edu/primer/java/nuaperture/ Visit this website

14. The Parts of a Light Microscope • Two lenses, ocular and • objective • Total magnification is • obtained by multiplying • magnification power of all • lenses i. e. Objective lens - 10X Ocular lens - 40X Total magnification - 400X

15. Contrast The maximum intensity difference between the darkest and lightest points in an image • Too much or too little and you lose information • Microscopy has several methods to add contrast

16. Types of Light Microscopy Bright-field Dark-field Phase contrast Fluorescence Differential Interference Contrast (DIC) (Also known as Nemarski Optics) Confocal

17. Bright-Field Microscopy • Condenser creates a bright white background against which to see specimens. • Useful for live, unstained material, preserved & stained material (better). • Disadvantage - cells and organelles within them are often times transparent. • Great for colored materials

18. Dark-Field Microscopy • An opaque disk over middle • of light source creates a • donut-shaped beam. • The “stop” blocks all light • from entering the objective • lens except peripheral light • that is reflected off the sides of • the specimen. • Specimen appears light • against a dark background. • Great for observing inclusions • in living cells

19. Phase-Contrast • Light is diffracted due to the fact that various intra- and extra- cellular structures have different refractive indexes. • Transforms subtle changes in light waves passing through the specimen into differences in light intensity creating great contrast- background is often grey • Excellent for internal cellular structures. Phase contrast microscopy of a urine specimen. The urine contains epithelial cells, red and white blood cells and baccilli shaped bacteria.

20. 3 Views of a Cell • Bright-field • Dark-field • Phase-contrast

21. Fluorescence • A substance fluoresces when it absorbs light at one wavelength, and emits it at another. • Fluorescent stains the cell to produce a bright object on dark background. • Stains can be highly specific by conjugating fluorescent species to antibodies. • Distinguish between live and dead cells. • Diagnosis of infections.

22. Differential Interference Contrast (Nemarski) • Uses prisms in light path to control direction of light • Great for internal structures • Gives a “3-D” effect…

23. Confocal Microscopy • Uses fluorescent stains for contrast. • A laser illuminates a very thin section of the specimen. • Produces a series of very clear images. • Can construct 3-D images by reconstructing optical sections using a computer.

24. Electron Microscopy page 76 • l of electrons smaller than photons • Uses electron beam instead of light • Uses magnets to focus instead of lenses • This results in better resolution • 0.5 nm • Magnification • 100,000 to 1,000,000 X

25. Scanning Electron Microscopy - 100,000X • Scans and magnifies external surface of specimen producing a 3-D image • Coat with metal stain • Electrons are deflected off specimen and collected Disadvantage: Cells must be killed

26. Transmission Electron Microscopy • Up to approx. 1,000,000 X magnification • Transmitting electrons through very thin sections • (20 -100 nm) • Stained or coated with metals that increase contrast Cyanobacterium

27. Back to Bright Field Light Microscopy Methods to Enhance Contrast • Simple stains • One stain • Differential stains • Use a primary stain and a counterstain to distinguish cell types or parts • Gram stain, acid-fast stain and endospore stain • Special stains • Capsule and flagellar stains

29. Staining • Cationic dyes • Basic, with positive charges on the chromophore • Surfaces of microbes are negatively charged and attract basic dyes – positive staining • Anionic dyes • Acidic, with negative charges on the chromophore • negative staining – microbe repels dye & it stains the background

30. Culture microbes on media

31. Media – providing nutrients in the laboratory • Most commonly used: “complex” medium nutrient broth • liquid medium containing beef extract & peptone • nutrient agar • solid media containing beef extract, peptone & agar • Agar is a complex polysaccharide isolated from red algae • solid at room temp, liquifies at boiling (100oC), does not resolidify until it cools to 42oC • provides framework to hold moisture & nutrients • not digestible for most microbes Opposite of complex medium is simple medium

32. Culture microbes on media Inoculation – Introduction of a sample into a container of media – on agar plates, streaking or spreading Incubation – Under conditions that allow growth Isolation – Separating one species from another Inspection Identification

33. Isolation Technique Pure culture

34. Selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes

35. Differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

36. Physiological / Biochemical Tests • There are many type of media used to determine • Aerobic / anaerobic growth • Metabolism of carbohydrates • Production of degradative enzymes • Presence metabolic enzymes

37. There are many microbes that scientists do not know how to culture • We know they exist, but we just don’t know how to grow them in the lab • How do we know they exist? • Microscopic evidence • Isolate DNA & RNA • Polymerase chain reaction • Examine the nucleotide sequence • Detect products of respiration • CH4, CO2, H2S • Many species have been described & named, but never grown in the lab

38. rRNA Sequence • One of the greatest advancements in classifying organisms • Differences in the nucleotide sequence are used to classify prokaryotes • 16S rRNA sequences • 23S rRNA sequences Actually look at the DNA that codes for the rRNA

40. How is this accomplished? Extract DNA from a colony, or from an environmental sample without growing the organism PCR with primers for rRNA sequences Automated DNA sequencer Compare with known samples in a database