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Chapter 3 tools of the laboratory e mail


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Micro 1 lecture for chapter 3 tools of the laboratory for LATTC

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Chapter 3 tools of the laboratory e mail

  1. 1. Chapter 3 Tools of the Laboratory: The methods for studying microorganisms
  2. 2. Media <ul><li>Media provides nutrients for bacterial growth in the lab. Media can be solid ( agar ), semi-solid or liquid ( broth ). </li></ul><ul><li>General-purpose media: For microbes that only require basic nutrients for growth. </li></ul><ul><li>Enriched media others need a complex mix of specific nutrients. Enriched media are tailored to fit these difficult growers. Bacteria that are difficult to grow because they require specific media are called fastidious. </li></ul><ul><li>Selective Media : only allows certain bacteria to grow and stops other bacteria from growing. Selective media is important in isolating a specific bacteria from a mixed culture. Eg) feces- looking for Salmonella. </li></ul><ul><li>Differential Media: grows different microbes but brings out differences in them when they grow (different color colonies, media changes color, etc) - Used to test for certain things eg) fermentation of sugars </li></ul>
  3. 3. Examples of media
  4. 4. 5 I’s of microbiology <ul><li>To study microorganisms, we need to get them </li></ul><ul><li>from their natural environment, grow them </li></ul><ul><li>in a laboratory, isolate each species & study them. </li></ul><ul><li>How do microbiologists do this? </li></ul><ul><li>Inoculation </li></ul><ul><li>To culture bacteria, you introduce a small sample ( inoculum ) into a container of sterile nutrient medium . The microbial growth in that medium is called a culture. </li></ul><ul><li>The type of media depends on what you are culturing. </li></ul><ul><li>Samples can come from anywhere you are studying: body fluids, tissues, soil, water, inanimate objects etc. </li></ul>
  5. 5. 5 I’s of microbiology <ul><li>2) Incubation </li></ul><ul><li>Microorganisms have a specific temperature range & humidity that is best for its growth. </li></ul><ul><li>You would put the inoculated media in an incubator . </li></ul><ul><li>Most pathogenic organisms like temperatures from 20-40 o C (room temp. to body temp) </li></ul><ul><li>Usually incubate bacteria for 24-48 hours for good growth. </li></ul>
  6. 6. 5 I’s of microbiology <ul><li>3) Isolation </li></ul><ul><li>In order to separate one type of bacteria from another, you must isolate each cell from one another . </li></ul><ul><li>One isolated bacterial cell will multiply and grow into a mound of cells called a colony . A colony consists of all the same type of bacterial cells because they all are descendents of the first cell. </li></ul><ul><li>Technique: inoculate a large area with few bacteria. Spread out the cells so they are far apart. </li></ul><ul><li>* you will do this in lab </li></ul>
  7. 7. 5 I’s of microbiology <ul><li>4) Inspection </li></ul><ul><li>Examine growth. </li></ul><ul><li>Colony morphology </li></ul><ul><li>Appearance of a colony. </li></ul><ul><li>Size, shape, texture & color are characteristics of the bacteria. </li></ul><ul><li>Microscopic morphology : </li></ul><ul><li>Take a sample of colony & look at it under the microscope. With staining techniques, you can make out the shape, size and motility of the bacterial cell. </li></ul>
  8. 8. 5 I’s of microbiology <ul><li>5) Identification </li></ul><ul><li>How can you tell what kind of microorganism you have? </li></ul><ul><li>A lot of bacteria have the same shape & appearance. </li></ul><ul><li>You need to test their biochemistry or cell metabolism: </li></ul><ul><li>The kind of sugars do they digest </li></ul><ul><li>Products given off during growth </li></ul><ul><li>Enzymes they have </li></ul><ul><li>Mechanisms for deriving energy </li></ul><ul><li>Use or no use of oxygen </li></ul><ul><li>These are called biochemical tests. </li></ul><ul><li>Used to figure out exactly what species </li></ul><ul><li>of bacteria you have. </li></ul><ul><li>This is where differential media are useful! </li></ul>
  9. 9. Metric Units 0.0000000001 = 10 -10 Angstrom Å 0.000000001
  10. 10. Scale sizes End light microscope viewing power - Eukaryotic cells are measured in µm (100-10µm) - Bacteria are measured in µm (1-10µm) - Viruses are measured in nm
  11. 11. Try to visualize… <ul><li>A parvovirus is about 18nm across </li></ul><ul><li>E. coli (small bacteria) is about 2000nm long and 500nm wide </li></ul><ul><li>Lymphocyte 7000 nm in diameter. </li></ul><ul><li>Human hair has a diameter of 17,000nm </li></ul><ul><li>to 180,000nm depending on the hair type. </li></ul><ul><li>Change nm to inches to get a better idea of size: </li></ul><ul><li>Parvovirus is 18 inches (1.5 feet) </li></ul><ul><li>E. Coli would be 166.6 feet long x 41.6 feet wide </li></ul><ul><li>Lymphocyte is 583 feet in diameter </li></ul><ul><li>Human hair would be anywhere from ¼ mile to 2.8 miles in diameter!! </li></ul>How big is a…. Utah Cells’ alive How big?
  12. 12. <ul><li>Uses visible light to observe specimens </li></ul><ul><li>“ Compound” refers to the fact that there are 2 magnifying lenses that work together: </li></ul><ul><ul><li>Ocular lens (10x) </li></ul></ul><ul><ul><li>Objective lenses </li></ul></ul><ul><ul><li>(4x, 10x,40x,100x) </li></ul></ul><ul><li>How do you figure the total magnification of the image? </li></ul>Compound Light Microscope
  13. 13. Parts of compound microscope (for lab) <ul><li>Illuminator (light source) bulb </li></ul><ul><li>Condenser: directs light rays through specimen </li></ul><ul><li>Objective lens : lens closest to specimen (4x-100x) </li></ul><ul><ul><li>-Oil immersion objective (100x) Use oil with ONLY this objective. </li></ul></ul><ul><li>Ocular lens : lens you look through (10x) </li></ul><ul><li>Diaphragm: controls amount of light focused on object </li></ul><ul><li>Stage: area where specimen is placed </li></ul><ul><li>Coarse adjustment knob: get image into approximate focus under low power. </li></ul><ul><li>Fine adjustment knob: finely adjust focus of image under all powers. </li></ul><ul><li>Nose piece : holds objective lenses </li></ul><ul><li>Arm: area to hold microscope </li></ul>
  14. 14. Magnification & Resolution <ul><li>Magnification: how much an image is enlarged. </li></ul><ul><li>Resolution : ability to distinguish between 2 points a specified distance apart. </li></ul><ul><li>How close together can two objects be and still be seen as two distinct objects, rather than one large object? </li></ul><ul><li>Each microscope will have different limits of resolution. </li></ul><ul><li>Eg) compound scope has resolution of 0.2 µm </li></ul><ul><li>So how can we achieve good resolution with a high magnification? </li></ul>
  15. 15. Oil immersion <ul><li>The 40x objective lens is very small and does not allow much light to enter. The more light that hits our eye, the clearer the image. </li></ul><ul><li>Immersion oil is used b/c it has same refractive index (light bending ability) as glass so oil becomes “part of glass” and funnels more light into the objective lens. </li></ul><ul><li>Without immersion oil, </li></ul><ul><li>light will bend away when </li></ul><ul><li>it hits air & be lost. </li></ul>
  16. 16. Images with a compound light microscope Bacteria 1000x Amoeba 1000x Note the enormous size difference between one bacterial cell & the amoeba! Bacteria are prokaryotic & these cells are usually much smaller than eukaryotic cells (amoeba)
  17. 17. Finding bacteria with a microscope 100x 1000x 40x 400x
  18. 18. Electron microscope (EM) <ul><li>EM’s are used to visualize very small structures (viruses, parts of cells, large molecules etc.) </li></ul><ul><li>A beam of electrons are used instead of light. </li></ul><ul><li>Electrons are beamed into microscope traveling in waves too, but wavelength is much smaller (100,000 times smaller) than visible light waves= resolving power much better! </li></ul><ul><li>Uses computer to piece together image. </li></ul>
  19. 19. 2 types of Electron Microscopes <ul><li>Transmission EM (TEM) </li></ul><ul><li>Requires very thin sections </li></ul><ul><li>See internal structures </li></ul><ul><li>2D image </li></ul><ul><li>Scanning EM (SEM) </li></ul><ul><li>Sweeps across surface of specimen </li></ul><ul><li>See surface anatomy </li></ul><ul><li>3D image </li></ul>
  20. 20. Virus budding from cell Which is TEM? SEM?
  21. 21. Other types of microscopy <ul><li>Dark Field Microscopy: only allows peripheral light to hit specimen. Dark background & light specimen. </li></ul><ul><li>Phase-Contrast Microscopy: Differences in how light passes through the different structures are intensified & used to see internal structures well. </li></ul><ul><li>Confocal microscopy: uses a laser beam of light to scan specimen- sometimes the specimen is colorized with fluorescence. Can see fine detail, inside & out. </li></ul>
  22. 22. Staining <ul><li>Stains: colored substances with a positive or negative ion that attaches to the cells. </li></ul><ul><li>You must stain organisms to see shape, and other structures. </li></ul><ul><li>Unstained bacteria are mostly made of water and are nearly transparent when viewed through a light microscope (pictured on the left). </li></ul><ul><li>Note that most of the microbes are not visible, but a dust spec in the center of the field of view is visible. </li></ul>Unstained Stained 40x Stained 100x dust
  23. 23. Staining <ul><li>Stains are dyes that cling to the positive and negative charges of bacteria, but do not bind as readily to the background of a slide. </li></ul><ul><li>They therefore differentiate microbes from their surroundings. </li></ul><ul><li>types of dyes: </li></ul><ul><li>1) Basic dyes: positive (+) ion </li></ul><ul><li>2) Acidic dyes: negative (-) ion </li></ul><ul><li>*Most bacteria are slightly negatively charged…what kind of dye would work best for staining bacteria? </li></ul><ul><li>Examples of some stains </li></ul><ul><li>Crystal violet </li></ul><ul><li>Methylene blue </li></ul><ul><li>Malachite green </li></ul><ul><li>Safranin </li></ul>
  24. 24. Simple Stain vs Differential Stain <ul><li>Simple stain is used to highlight & stain the entire organism. </li></ul><ul><li>A simple stain only uses one color dye. </li></ul><ul><li>You can see the </li></ul><ul><li>1) shape </li></ul><ul><li>2) some basic structures (flagella) </li></ul><ul><li>3) relative size </li></ul><ul><li>Differential stain : uses 2 colored dyes. </li></ul><ul><li>The second dye is called a counterstain . Usually it’s a contrasting color to the principal stain. </li></ul><ul><li>A differential stain is used to </li></ul><ul><li>1) contrast two cell types 2) indicate cell parts. </li></ul><ul><li>Example: </li></ul><ul><li>Gram stain </li></ul><ul><li>Endospore stain </li></ul>
  25. 25. Gram stain <ul><li>The gram stain is a procedure using 2 colors to tell you what kind of cell wall a bacterial cell has. </li></ul><ul><li>2 basic types of cell walls: </li></ul><ul><ul><li>Gram positive </li></ul></ul><ul><ul><li>Gram negative </li></ul></ul><ul><li>Important to medical microbiology </li></ul><ul><li>Provides valuable information for treatment of disease caused by bacteria </li></ul>
  26. 26. Endospore stain <ul><li>Endospores are special resistant, dormant structures in bacterial cells </li></ul><ul><li>Can be formed by a few genera of bacteria. </li></ul><ul><li>Endospores cannot be stained by other techniques b/c wall of endospore is very thick. </li></ul><ul><ul><li>Malachite green added with heat (to penetrate endospore wall) </li></ul></ul><ul><ul><li>Safranin counterstain applied </li></ul></ul>
  27. 27. Test yourself! <ul><li>A fastidious organism must be grown in: </li></ul><ul><li>a) general purpose media c) solid media </li></ul><ul><li>b) differential media d) enriched media </li></ul><ul><li>2. Which of the following is not one of the Five I’s? </li></ul><ul><li>Inspection b) identification c) incubation d) induction </li></ul><ul><li>3. In order to identify bacteria, you must test to see what kind of chemical reactions are occurring inside the cells. Which choice would you use to do this? </li></ul><ul><li>a) look at colony morphology c) grow cells in enriched media </li></ul><ul><li>b) use biochemical tests d) look at cell morphology </li></ul><ul><li>4. The term ‘culture’ refers to_____________ </li></ul><ul><li>Growing bacteria in the body b) putting bacteria into a cell </li></ul><ul><li>Growing bacteria in media d) biochemical tests on bacteria </li></ul><ul><li>5. Resolution is _________ with a longer wavelength of light. </li></ul><ul><li>Improved b) worsened c) not changed d) not possible </li></ul>
  28. 28. <ul><li>6. What is the total magnification of the bacteria you are viewing if you are using the 4x objective?_________ </li></ul><ul><li>What is a colony? </li></ul><ul><li>Why are counterstains important? </li></ul><ul><li>Why is immersion oil important when using 100x objective? </li></ul><ul><li>What does the Gram stain tell you about bacteria? </li></ul><ul><li>Why must you stain bacteria before viewing under a micrscope? </li></ul><ul><li>Why does an electron microscope give you better resolution than a light microscope? </li></ul>