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3/20/2015 Microscopy 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 1 3/20/2015 Size Comparisons Among Atoms, Molecules, and Microorganisms FISH TAPEWORM 2 3/20/2015 Light microscopy Magnification vs. resolution • Magnification = increase in apparent size of an object • Resolution = ability to distinguish two objects as separate from each other 3 3/20/2015 Light microscopy has other optical configurations • Dark-field microscopy • Phase-contrast microscopy • Fluorescence microscopy Flourescence Microscopy Image Fluorescence microscopy of endothelial cells using three labels. Red labels the mitochondria, green labels the F-actin cytoskeleton and blue labels the nucleus. Image by Steve Karl 4 3/20/2015 Electron Microscopy Electron Microscope 5 3/20/2015 Electron Microscope Images Streptococcus pneumoniae Gram-positive diplococci with capsule (haloe) formation, located outside neutrophils. http://www.fujita-hu.ac.jp/~tsutsumi/case/case071.htm 6 3/20/2015 Legionella pneumophila As seen in the cytoplasm of macrophages Anthrax bacterium (Bacillus anthracis) with human blood cells. 7 3/20/2015 Pseudomonas aeruginosa Gram-negative rods floating within mucoid matrices. Bordetella pertussis Mainly seen here outside neutrophils 8 3/20/2015 Bacteria: Staining Techniques • • • • • • • Positive Stain (basic) Negative Stain (acidic) Gram Stain Acid-Fast Stain Capsule Stain Spore Stain Flagella Stain Why Stain ??? • A) Achieve Contrast • B) View Size, Shape, + Cellular Structures (cell wall, flagella, glycocalyx, spores, etc.) • C) Classify/Partially identify organisms 9 3/20/2015 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) Simple Staining Reactions in Microbiology Positive Stain 10 3/20/2015 Positive Stain Typical Bacillus stained with Crystal Violet Negative Stains Bacillus stained with negrosin Cocci stained with Negrosin 11 3/20/2015 Gram Stain Procedure Streptococcus mutans 12 3/20/2015 E. coli gram stain Gram Stain • 2 slides/group: • 1 bacterium (tube) • 1 gum-line sample • • • • • Procedure: Make Smear Heat-Fix Gram-Stain View with Microscope • Materials • • • • • • • • • • • • • Gloves Slides Pen Loop Toothpick Sparker/Bunsen Burner Bacteria Test Tube Rack Clothespin Staining Kit Transfer Pipette Drying/Bibulous Paper Microscope/Oil/Lens Paper 13 3/20/2015 Bacillus cereus with neutrophils 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 acidalcohol treatment) Can be used on sputum Mycobacterium (acid-fast positive) 14 3/20/2015 Capsule Stain Capsule = Glycocalyx -- sticky layer around some bacteria Klebsiella Pneumonia -- helps them to retain water, attach to tissues, and avoid the immune system 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. Bacteria from a dirty dish; 1600x, capsule stain (negrosin then safranin) http://picasaweb.google.com/marc.murison/BestMicro/photo#5114441791829109154 15 3/20/2015 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” Anthrax spore stain 16 3/20/2015 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 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 3/20/2015 Culture media PEA Agar for GramPositive bacteria Mannitol Salt agar for pathogenic staphylococci Selective Media Phenylethanol Agar, selective for Gram-positive organisms. 18 3/20/2015 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 Selective and Differential: McConkey Agar 19 3/20/2015 Enriched Media Neisseria Gonorrhea on Chocolate Agar CHECK YOUR UNDERSTANDING THIS IS EMB AGAR (Eosin-Methylene Blue) IT CONTAINS DYES THAT INHIBIT GRAM POSITIVE BACTERIA. IT CONTAINS LACTOSE THAT ALLOWS LACTOSE-FERMENTERS TO HAVE A GREEN/METALLIC COLOR THIS IS AN EXAMPLE OF which type of media: A. B. C. D. E. F. G. SELECTIVE DIFFERENTIAL ENRICHED ALL OF THE ABOVE A AND B B AND C I DON’T KNOW 20 3/20/2015 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 Materials needed: Gloves/swab/loop/tongue depressor/plate/sparker/alcohol/tape/pen 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 21 3/20/2015 Hemolysis http://gold.aecom.yu.edu/id/micro/hemolysisabg-72.jpg Serial dilution of cultures 22 3/20/2015 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}. Catalase Test Staphylococcus aureus Enterococcus faecalis 23 3/20/2015 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 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) Dye is pink when pH is basic H. pylori is urease + 24 3/20/2015 OXIDASE TEST CITRATE TEST 25 3/20/2015 INDOLE TEST BILE ESCULIN AGAR 26 3/20/2015 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 Dichotomous Key -- a simple example ---------------------------------------------------------------------------------------------------------------- 27 3/20/2015 Partially complete example….. ALL 13 Organisms Gram - Gram + Cocci, clusters Cocci, Chains Large Rods ML, SE EF BC, BM F F - LP, BS, BP FG F - Rods Short Rods PF, CV EC, KP, SM F Lactose SE Normal Rods + Glucose Lactose Glucose - Indole ML EC BC BM BP LP BS CV KP SM PF + motility SM KP 28 3/20/2015 29 3/20/2015 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. Example Data Table O A L O C H U T I G G M I S R S M A A culture N V D D D I 2 R D N E L A N O H A E M R no. P P H C C T S E A D L U N O R A C L Y A G 8101 + – + + – – – – + – – + + – + + + + – + Identification: Escherichia coli 5144572 30 3/20/2015 API-20E kit example (B) Culture ID: 8P14 Patient Symptoms: PAIN, fever, diarrhea and abdominal cramps Example Data Table O culture N no. P G A L O C H U T I G G M I S R S M A A V D D D I 2 R D N E L A N O H A E M R P C C T S E A D L U N O R A C L Y A H 8P14 – – + + – + – – – – – 7-digit ID code = 4501552 + + – + + – + – + Identification: Salmonella sp. 31 3/20/2015 Parasitology • Parasitology = study of protozoa and multicellular parasites such as worms, ticks, lice, and fleas • Today Examine microscope slide sets Live “wet mounts” and worm dissection Introduction: Coccidiosis Eimeria necatrix http://www.anri.barc.usda.gov/pbel/images/bigchicklittlechick.jpg 32 3/20/2015 Trypanosomes: African Sleeping Sickness LIFE CYCLE OF TSE-TSE FLY = VECTOR http://www.med.uni-marburg.de/stpg/ukm/lt/hygiene/schwarz/Trypanosoma.jpg Balantidiasis Balantidium coli 33 3/20/2015 Entamoeba histolytica http://www.weizmann.ac.il/Biological_Chemistry/images/mirelman.jpg http://www.microscope-microscope.org/applications/pond-critters/protozoans/sarcodina/entamoeba.htm Malaria Plasmodium spp. Female anopheles mosquito http://bepast.org/docs/photos/malaria/Malaria.jpg 34 3/20/2015 Eye-worm (loa loa) http://maven.smith.edu/~sawlab/fgn/pnb/loaloa.html PUBIC LICE (“CRABS”) www.visualdxhealth.com 35 3/20/2015 Scabies Mites http://www.stanford.edu/class/humbio103/ParaSites2004/Scabies/scabies1.jpg Parasite Lab Materials 1. Black Box – Protozoa Slides 2. White Box – Multicellular Parasite Slides 3. Green Jar – Preserved worms (for inspection) 4. White Jar – Preserved worms (for DISSECTION) Precautions/Safety – -- HANDLE SPECIMENS WITH FORCEPS AND GLOVES (+USE GOGGLES DURING DISSECTION) -- DISPOSE DISSECTED WORMS IN BIOHAZARD BIN 36 3/20/2015 Parasite Lab – Week 5 Microbiology Dissection -- Use your dissecting tools to make a longitudinal cut in the roundworm provided (Ascaris). Try to distinguish if your worm is male or female (see photo below). Note that the major structures you see will be used for sexual reproduction and digestion. Case Study: Chagas Disease Trypanosoma cruzi (causative agent) Reduviid “Kissing” Bug (Vector) Gross anatomy of a heart that has been damaged by chronic Chagas disease 37 3/20/2015 PORK TAPEWORM CYSTS IN THE BRAIN Top: A pork tapeworm (Taenia solium) cysticercus, the form in which the tapeworm is found in an infected brain. (Colorized image by P. W. Pappas and S. M. Wardrop, courtesy of P. W. Pappas, Ohio State University.) Bottom: T. solium cysticerci in the brain of a nine-year-old girl who died during cerebrospinal fluid extraction to diagnose her headaches. This was in the 1970s—if it had happened 10 years later, noninvasive computerized tomography would have given an accurate diagnosis, and the parasites could have been killed with drugs. (Image courtesy of Dr. Ana Flisser, National Autonomous University of Mexico.) 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 38 3/20/2015 Control of Microbes 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 39 3/20/2015 Using the Spectrophotometer to count bacteria Absorbance is proportional to number of bacteria GO TO http://www.physics.csbsju.edu/stats/chi_fit.html 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 ↑ 40 3/20/2015 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 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 41 3/20/2015 Biotechnology and Genetic Engineering Genetic engineering 42 3/20/2015 Restriction Enzymes and Recombinant DNA Construction of a Recombinant DNA Molecule 43 3/20/2015 Human genes can be cloned in bacteria “Artificial” Transformation 44 3/20/2015 Examples of products made by recombinant DNA technology 1. HUMALOG – Human insulin made by E. coli bacteria 2. PROCRIT – Human erythropoietin made by mouse cells 3. NEUPOGEN – helps humans grow more neutrophils, made by inserting human DNA into E. coli 4. RECOMBIVAX – Hepatitis B vaccine made by inserting viral DNA into yeast cells and growing up viral proteins ! Applications of Genetic engineering 45 3/20/2015 The Genetic Code Bt crop concerns http://www.biology-blog.com/images/blogs/10-2007/genetically-engineered-corn.jpg 46 3/20/2015 Golden Rice “THE GENE GUN” 47 3/20/2015 Can we move DNA between these two organisms ??? Aequorea victoria (Sea Jelly ) E. coli (bacteria) The pGLO plasmid 48 3/20/2015 THE PROCEDURE Expected Results LB/AMP/pGLO LB/AMP/ARA/pGLO LB/AMP LB 49 3/20/2015 Transformed Bacteria GFP Fly 50 3/20/2015 GFP mice and RFP cat Oinky Oinky…. 51 3/20/2015 Fun on a plate Living bacteria expressing 8 different colors of fluorescent proteins. BIOTERRORISM – “THE BIG 6” • ANTHRAX • SMALLPOX • TULAREMIA (“rabbit fever”) • HEMMORHAGIC FEVER VIRUSES (EBOLA/MARBURG) • PLAGUE • BOTULISM 52 3/20/2015 Epidemiology • Study of disease “determinants” in populations (infectious, environmental, genetic, and lifestyle) • Includes measurements of incidence, prevalence, distribution, and control of diseases • Usually involves collecting and analyzing data (heavy statistics!). Epidemiological terms • Epidemic: An outbreak of disease that attacks a large percentage of the population simultaneously and may spread through one or several communities. • Pandemic: When an epidemic spreads throughout the world. • Endemic: a disease that exists permanently in a particular region or population. Usually a small percentage of persons are affected. • Outbreak: a short epidemic (contained) 53 3/20/2015 Smoking vs. lung cancer per capita per year (country) Hungary 2515 Japan 2510 USA 2020 South Africa 1950 UK 1700 France 1690 USSR 1650 Brazil 1200 Philipines 1150 Venezuela 950 Zaire 150 India 100 The Oxford Atlas of the World, ISBN 0-19-520955-9, published in 1992 From Parkin, D. M. et al. CA Cancer J Clin 2005;55:74-108. Extras 54 3/20/2015 Genetic Vaccine Questions • What are some of the disadvantages of inactivated, subunit, and attenuated viral vaccines? • What are the potential advantages of genetic vaccines? • In what genetic form and how are genetic vaccines delivered to the body cells? • How can the positive effects of the vaccines be amplified/increased ? • What human genetic vaccine tests are currently being performed/attempted? RNA interference questions 1. How is RNA interference more precise than the interferon response? 2. What are some of the ultimate goals of “directed” RNA interference ? 3. What type of RNA proved most useful in RNAi, single or double stranded RNA ?? 4. What are siRNAs? MicroRNAs? 5. How does RNA interference help in learning about the functions of genes ? 6. What is the most difficult challenge facing human RNAi therapies ? 55 3/20/2015 Making Yogurt • Heat milk (450ml) to 83°C while stirring • Allow to cool to 43°C degrees • Put starter culture (1/2 cup) in separate mixing bowl • Slowly add milk to starter culture w/ stirring • Cover with foil and punch holes in foil • Incubate 2-6 hours at 30°C • Add fruit (optional) • Try it (if you are brave………) Questions for Food Poisoning Film • Who is getting sick, and why ?? • Where did physical control of microbes break down ??? • Why do some victims recover quickly, while others take 10+ years ??? 56