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Microscopy

Microscopy. Most microorganisms are in the micrometer size range. m = 1 meter cm = centimeter = 1/100m = 10 -2 meters mm = millimeter = 10 -3 meters μ m = micrometer = 10 -6 meters nm = nanometer = 10 -9 meters 1 Angstrom = 10 -10 meters pm = picometer = 10 -12 meters .

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Microscopy

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  1. Microscopy

  2. Most microorganisms are in the micrometer size range • m = 1 meter • cm = centimeter = 1/100m = 10-2 meters • mm = millimeter = 10-3 meters • μm = micrometer = 10-6 meters • nm = nanometer = 10-9 meters • 1 Angstrom = 10-10 meters • pm = picometer = 10-12 meters

  3. Size Comparisons Among Atoms, Molecules, and Microorganisms

  4. Light microscopy

  5. ElectronMicroscopeImages

  6. Magnificationvs. resolution • Magnification = increase in apparent size of an object • Resolution = ability to distinguish two objects as separate from each other

  7. Bacteria: Staining Techniques • Positive Stain (basic) • Negative Stain (acidic) • Gram Stain • Acid-Fast Stain • Capsule Stain • Spore Stain • Flagella Stain

  8. Why Stain ??? • A) Achieve Contrast • B) View Size, Shape, + Cellular Structures (cell wall, flagella, glycocalyx, spores, etc.) • C) Classify/Partially identify organisms

  9. Staining: Smear Preparation • Smear = a slide with microbes on it, ready to be stained 1) Label slide 2) Add water drop to the slide 3) Add the microbe to the water drop 4) Air-dry 5-10 minutes 5) Heat-fix (basic stains only, not acidic stains or the capsule stain)

  10. Simple Staining Reactions in Microbiology Positive Stain

  11. Gram Stain Procedure

  12. Acid-fast Stain Designed to identify Mycobacteria -- Mycobacterium tuberculosis -- Mycobacterium leprae Mycobacteria have a special wax layer in their cell wall (made of mycolic acid) Wax helps these bacteria to resist acid-alcohol de-staining step (“acid-fast” = have ability to retain the primary stain in spite of acid-alcohol treatment) Can be used on sputum Mycobacterium (acid-fast positive)

  13. Capsule Stain Capsule = Glycocalyx -- sticky layer around some bacteria -- helps them to retain water, attach to tissues, and avoid the immune system Klebsiella Pneumonia COMBINATION STAIN: two stains on top of each other; one is acidic (stains background), other is basic (stains the cell); capsule resists both stains and appears as a white “halo” around cells.

  14. Spore Stain Resistant structures formed by some bacterial species Examples: bacteria that cause anthrax, botulism, tetanus, gangrene, diarrhea (“C. diff.”) Difficult to stain, need to use steam and lots of stain to visualize them Can have “endospores” or “free spores”

  15. Flagella Stain • Provide motility (movement) • Long, thin proteins that are fragile, break easily • Difficult to stain and visualize • Other methods exist to look at motility (wet mount technique) Also go to wet mount video at http://www-micro.msb.le.ac.uk/video/motility.html

  16. Bacteria: Culturing and Counting Techniques • How to grow microbes: Types of media • How to isolate microbes: Throat swab / “Streak” plate • How to count microbes: Serial dilution / “Spread” plate

  17. Culture media PEA Agar for Gram-Positive bacteria Mannitol Salt agar for pathogenic staphylococci

  18. Selective Media Phenylethanol Agar, selective for Gram-positive organisms.

  19. Differential Media Example Used when Trying to examine “Staph” bacteria S. Aureus – potential pathogen S. Epidermidis – harmless resident of skin Plate contains a dye that turns yellow at low pH (if the bacteria are producing acid) S. Aureus can eat the sugars in the media (mannitol) and produces acid as a “waste” product S. Epidermidis cannot eat the sugar at all Mannitol Salt Agar

  20. Enriched Media NeisseriaGonorrhea on Chocolate Agar

  21. Streak Plate Technique GOAL: separate different bacterial species from each other when they are in a mixture ISOLATION of colonies: a colony represents a single bacterium and its overnight descendants Streak Isolation on Nutrient Agar

  22. Hemolysis Alpha = partial breakdown of the red blood cells (greening) Beta – total destruction of RBCs (white/clear zone) Gamma – no destruction of RBCs Alpha, Beta, and Gamma hemolysis

  23. Serial dilution of cultures

  24. Biochemical Tests • Bacteria and other microbes can be classified/identified according to the types of enzymes they possess {and thus the types of biochemical reactions they can perform}.

  25. Catalase Test Staphylococcus aureus Enterococcus faecalis

  26. COAGULASE TEST An enzyme produced by some, but not all, bacteria Positive reaction = clump or clot formation in the media within 2-6 hours Negative reaction – no clot Media is rabbit plasma broth Makes bacs more dangerous because unwanted clots are produced and the clot itself shields them from phagocytes

  27. UREASE TEST An enzyme produced by some, but not all, bacteria Urea – a toxic compound, kills bacteria (in stomach, in bladder, kidneys, etc.) Some bacteria can break down urea to carbon dioxide and ammonia (basic, can neutralize stomach acid) H. pylori is urease + Dye is pink when pH is basic

  28. Dichotomous key •  a map for the identification of organisms based on a series of choices between alternative characters •  can be stains, biochemical tests, antibiotic susceptibility, or other

  29. Dichotomous Key -- a simple example ------------------------------------------------------------------------------------------------------------------

  30. API-20E kit example (A) Culture ID #8101 {Patient 1 symptoms: severe abdominal cramps and watery diarrhea. There is little or no fever, and no vomiting.

  31. Parasitology • Parasitology = study of protozoa and multicellular parasites such as worms, ticks, lice, and fleas • Today - Examine microscope slide sets + Live “wet mounts”

  32. Coccidiosis Eimeria necatrix http://www.anri.barc.usda.gov/pbel/images/bigchicklittlechick.jpg

  33. Trypanosomes: African Sleeping Sickness LIFE CYCLE OF TSE-TSE FLY = VECTOR http://www.med.uni-marburg.de/stpg/ukm/lt/hygiene/schwarz/Trypanosoma.jpg

  34. Balantidiasis Balantidium coli

  35. Entamoeba histolytica http://www.weizmann.ac.il/Biological_Chemistry/images/mirelman.jpg http://www.microscope-microscope.org/applications/pond-critters/protozoans/sarcodina/entamoeba.htm

  36. Malaria Plasmodium spp. Female anopheles mosquito http://bepast.org/docs/photos/malaria/Malaria.jpg

  37. Eye-worm (loa loa) http://maven.smith.edu/~sawlab/fgn/pnb/loaloa.html

  38. PUBIC LICE (“CRABS”) ON LOCATION !! www.visualdxhealth.com

  39. Scabies Mites http://www.stanford.edu/class/humbio103/ParaSites2004/Scabies/scabies1.jpg

  40. Wet Mount Technique • Method to visualize living microbes • Uses a cover glass and “depression” slide • Also known as the “hanging drop” technique Planaria (flatworm)Trichomonas vaginalis

  41. Control of Microbes

  42. Measuring Zones of Inhibition Pour 25mL agar plates Grow bacteria in liquid culture to 100,000,000/ml Spread 150 microliters on plate Add antibiotic discs to plate Let bacteria grow overnight Measure ZOI and compare to standard table Antibiotic Disc Diffusion Assay

  43. Using the Spectrophotometer to count bacteria Absorbance is proportional to number of bacteria GO TO  http://www.physics.csbsju.edu/stats/chi_fit.html

  44. Salt • Used to preserve foods (meats/fish/etc.) • Works by dehydrating microbes -- (lose water, shrivel) • Creates hypertonic environment (re: osmotic stress) • Exception: Halophiles prefer 3% NaCl or ↑

  45. pH • Measures H+ ion concentration • ↑ H+ means more acidic (lower pH), • ↓ H+ means more basic (higher pH) • Most microbes are neutralphiles (5.5-8.5) • Some are acidophiles (<5.5) • A few are basophiles (>8.5) • Examples: “pickling” with vinegar (acid) or basify shampoos

  46. Filtration • Method to physically trap microbes • Used to purify liquids/air • Has tiny holes called “pores” • (anything larger than the pore size gets trapped on the filter itself) AcetatePlus VP vacuum filtration units with cellulose acetate

  47. Biotechnology and Genetic Engineering

  48. Examples of products made by recombinant DNA technology • HUMALOG – Human insulin made by E. coli bacteria • PROCRIT – Human erythropoietin made by mouse cells (helps anemics/cancer patients) • NEUPOGEN – helps humans grow more neutrophils, made by inserting human DNA into E. coli • RECOMBIVAX – Hepatitis B vaccine made by inserting viral DNA into yeast cells and growing up viral proteins !

  49. Construction of a Recombinant DNA Molecule

  50. Can we move DNA between these two organisms ??? Aequorea victoria (Sea Jelly ) E. coli (bacteria)

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