2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 4 Microscopy, Staining, and Classification Table 4.1 Metric Units of Length Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Convex lens Inverted, reversed, and enlarged image Focal point Specimen Glass Light Air Chicken egg Human red blood cell Large protozoan (Euglena) Chloroplasts Flea Typical bacteria and archaea Diameter of DNA VirusesProteins Ribosomes Amino acids Atoms Scanning tunneling microscope (STM) 0.01 nm10 nm Scanning electron microscope (SEM) 0.4 nm1 mm Transmission electron microscope (TEM) nm100 m Atomic force microscope (AFM) 1 nm10 nm Compound light microscope (LM) 200 nm10 mm Unaided human eye 200 m Mitochondrion Figure 4.3 The limits of resolution of the human eye and of various types of microscopes Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects, or between an object and background Important in determining resolution Staining increases contrast 2012 Pearson Education Inc. Microscopy Light Microscopy Bright-field microscopes Simple Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms 2012 Pearson Education Inc. Microscopy Light Microscopy Bright-field microscopes Compound Series of lenses for magnification Light passes through specimen into objective lens Oil immersion lens increases resolution Have one or two ocular lenses Total magnification (objective lens X ocular lens) Most have condenser lens (direct light through specimen) 2012 Pearson Education Inc. Figure 4.4 A bright-field, compound light microscope-overview Line of vision Ocular lens Path of light Prism Body Specimen Objective lenses Condenser lenses Illuminator Ocular lens Body Objective lenses Condenser Illuminator Remagnifies the image formed by the objective lens Base Fine focusing knob Coarse focusing knob Diaphragm Stage Arm Transmits the image from the objective lens to the ocular lens using prisms Primary lenses that magnify the specimen Controls the amount of light entering the condenser Focuses light through specimen Holds the microscope slide in position Light source Moves the stage up and down to focus the image Microscopy The microscopes we will be using are parfocal, which means that once you have it focused at one magnification using the coarse adjustment, you should only have to use the fine adjustment for the other objectives. Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Refracted light rays lost to lens Glass cover slip Light source Specimen Slide Without immersion oil Glass cover slip Light source Slide Microscope objective More light enters lens Lenses With immersion oil Immersion oil Microscopy Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm Greater resolving power and magnification Magnifies objects 10,000X to 100,000X Detailed view of bacteria, viruses, and large atoms Two types Transmission electron microscopes Scanning electron microscopes 2012 Pearson Education Inc. Figure 4.13 SEM images-overview Microscopy Rotate nosepiece to 4x objective Raise stage completely using coarse adjustment. Find area of specimen on slide with naked eye Place specimen over circle of light (adjust to area of interest using stage dials) Lower stage until specimen comes into focus. Switch to the 10x objective. Once focused here, switch to 40x objective. Once focused at 40x, move 40x objective out of the way. Place oil directly on slide and rotate objective labeled 100x (oil). Use ONLY fine focus (small knob) only to bring into view through ocular. Required for all microbiology preparations to assure that contaminants are not introduced. On a personal note, aseptic technique assures that infectious agents are not spread to you, fellow students, or the laboratory surfaces. Disinfection: The use of a physical or chemical procedure to virtually eliminate all recognized pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects. Disinfection Decontamination Sterilization: The use of physical or chemical procedures that destroy all microbial life forms, including highly resistant bacterial endospores. Autoclave: Pressurized steam at 15 psi and 121 o C for an average of 20 min (10 40 min depending on bulk and load) Microscope (with accessories) Inoculation loops Source of flame (Bunsen burner) Microscope slides and Cover slips Gram staining kits Petri dishes and proper growth media Incubators Autoclave (pressure cooker) Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant provided in lab. Growth media Agar plates Agar slants Brothliquid media General purpose Selective Differential General purpose: Supports growth of most non fastidious organisms Tryptic Soy Agar Selective: Favors the growth of one type of microorganisms and inhibits the growth of others Saboraud Dextrose Agar (SDA)grows fungi Differential Media: Distinguishes between different groups of bacteria on the basis of biochemical characteristics Required for all microbiology preparations to assure that contaminants are not introduced. On a personal note, aseptic technique assures that infectious agents are not spread to you, fellow students, or the laboratory surfaces. General rules of microbiology laboratory The inoculating loop is usually used for making transfers of bacterial cultures (see next few slides for technique). Allow the loop to cool sufficiently so that any organisms to be tested will not be killed by the hot wire, but do not allow the loop to contact anything during the cooling period or contamination will result. Learn to remove and replace the caps or lids efficiently without setting them on the countertop or leaving the cover off too long. After the transfer is completed the loop must be sterilized again. Follow the procedure outlined on the following slides to prevent splattering of infectious materials. It may be easier to work while sitting down. Attention to details and practice will allow you to work both rapidly and accurately. FLAMING A LOOP Flaming tubes Transferring Microorganisms to Slant Test Tubes Streaking a slant Procedure for Transferring Microorganisms To/From Test Tubes Hold loop with dominant hand Flame the loop Hold culture tube in other hand Remove cap with pinkie of dominant hand Flame mouth of culture tube Place loop into tube Flame mouth of culture tube and close Put the tube back in the rack. Open new culture tube or plate. If tube, flame the mouth. Inoculate Flame mouth of tube and close or close plate Flame loop Procedure for transferring microorganisms to/from plate Flame loop Pick up bottom of plate in non-dominant hand. Touch the loop to a bacterial colony. Replace the bottom of the plate on the top. Pick up the bottom of a new plate. Touch loop to plate. Replace the bottom of the plate on the top. Flame the loop. 2012 Pearson Education Inc. Staining Principles of Staining Staining increases contrast and resolution by coloring specimens with stains/dyes Smear of microorganisms (thin film) made prior to staining Spread culture in thin film over slide Pass slide through flame to fix it Air dry Figure 4.15 Preparing a specimen for staining Simple Stains Differential Stains Gram stain Acid-fast stain Endospore stain Special Stains Negative (capsule) stain Flagellar stain 2012 Pearson Education Inc. Staining Figure 4.16 Simple stains-overview Color of Gram-Positive Cells Color of Gram-Negative Cells Primary Stain: Crystal Violet Purple Mordant: Iodine Purple Decolorizing Agent: Alcohol-Acetone PurpleColorless Counterstain: Safranin PurpleRed Gram Stain Figure 4.17 The Gram staining procedure-overview Slide is flooded with crystal violet for 1 min, then rinsed with water. Result: All cells are stained purple. Slide is flooded with solution of ethanol and acetone for 1030 sec, then rinsed with water. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. Slide is flooded with iodine for 1 min, then rinsed with water. Result: Iodine acts as a mordant; all cells remain purple. Figure 4.18 The Ziehl-Neelsen acid-fast stain Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae Background stain Bacterium Capsule Figure 4.21 Flagellar stain of Proteus vulgaris Flagella