1. How we study microbes Chapter 3

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

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

4. 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. E E Virtual Image Reaches the eye

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

6. 6 Magnification Defined as amount of enlargement of specimen 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 Picture of Anton van Leeuwenhoek

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

8. 8 2  sin  Resolution = = wavelength of light  = refractive index  = angle between most divergent light ray gathered by lens and the center of the lens 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. 9 of Light Used Different colors of light have different 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 2  Resolution = = wavelength of light 500 nm ---------- = 200 nm 2 x 1.25

10. 10 Refractive Index of Medium Between Specimen and Lens Light path without oil Light path with oil 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 2  sin  Resolution =  = refractive index

11. 11 Oil Immersion Lens  for air  = 1.5 for glass  = 1.52 immersion oil

12. 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  = angle between most divergent light ray gathered by lens and the center of the lens Lens close to specimen Lens far from specimen 

13. 13 Numerical Aperture Website micro.magnet.fsu.edu/primer/java/nuaperture/ Visit this website 2  sin  Resolution =

14. 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. 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. 16 Types of Light Microscopy Bright-field Dark-field Phase contrast Fluorescence Differential Interference Contrast (DIC) (Also known as Nemarski Optics) Confocal

17. 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. 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. 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. 20 3 Views of a Cell Bright-field Dark-field Phase-contrast

21. 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. 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. 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. 24 Electron Microscopy page 76 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. 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. 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. 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

28. 28

29. 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. 30 Culture microbes on media

31. 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 (100 o C), does not resolidify until it cools to 42 o C –provides framework to hold moisture & nutrients –not digestible for most microbes Opposite of complex medium is simple medium

32. 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. 33 Isolation Technique Pure culture

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

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

36. 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. 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 CH 4, CO 2, H 2 S Many species have been described & named, but never grown in the lab

38. 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. 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