1. Lab Exercises Week 2: #2 Pure Culture #7 Defined and Undefined #22 Normal Skin Biota #6 Differential and Special Stains (Gram- stain) Pre lab due: 1/17/15 Post lab due: 1/31/15

2.  Pure culture defined as population of cells derived from a single cell Allows study of single species  Pure culture obtained using aseptic technique Minimizes potential contamination  Cells grown on culture medium Contains nutrients dissolved in water Can be broth (liquid) or solid gel 4.3. Obtaining a Pure Culture

3.  Need culture medium, container, aseptic conditions, method to separate individual cells With correct conditions, single cell will multiply Form visible colony (~1 million cells easily visible) Agar used to solidify Not destroyed by high temperatures Liquifies above 95°C Solidifies below 45°C Few microbes can degrade Growth in Petri dish Excludes contaminants Agar plate Growing Microorganisms on a Solid Medium

4.  Streak-plate method Simplest, most commonly used method for isolating Spreads out cells to separate Obtain single cells so that individual colonies can form Growing Microorganisms on a Solid Medium Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 2 3 4 6 5 Sterilize loop. Dip loop Into culture. Streak first area. Agar containing nutrients Starting point Streak second area. Sterilize loop. Sterilize loop.

5.  General categories of culture media Complex media contains variety of ingredients Exact composition highly variable Often a digest of proteins Chemically defined media composed of exact amounts of pure chemicals Used for specific research experiments Usually buffered 4.7. Cultivating Prokaryotes in the Laboratory

6.  Hundreds of types of media available Regardless, some medically important microbes, and most environmental ones, have not yet been grown in laboratory 4.7. Cultivating Prokaryotes in the Laboratory

7.  Special types of culture media Useful for isolating and identifying a specific species Selective media inhibits growth of certain species 4.7. Cultivating Prokaryotes in the Laboratory Differential media contains substance that microbes change in identifiable way Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (a)(b) Zone of clearing Colony a: © Christine Case/Visuals Unlimited; b: © L. M. Pope and D. R. Grote/Biological Photo Service

8.  Simple staining involves one dye  Differential staining used to distinguish different types of bacteria 3.2. Microscopic Techniques: Dyes and Staining

9.  Acid-fast staining used to detect Mycobacterium Includes causative agents of tuberculosis and Hansen’s disease (leprosy) Cell wall contains high concentrations of mycolic acid Waxy fatty acid that prevents uptake of dyes Harsher methods needed Used to presumptively identify clinical specimens 3.2. Microscopic Techniques: Dyes and Staining

10.  Capsule stain  Some microbes surrounded by gel-like layer Stains poorly, so negative stain often used India ink added to wet mount is common method 3.2. Microscopic Techniques: Dyes and Staining © Dr Gladden Willis/Visuals Unlimited/Getty 10  m Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

11.  Flagella stain  Flagella commonly used for prokaryotic motility Too thin to be seen with light microscope Flagella stain coats flagella to thicken and make visible Presence and distribution can help in identification 3.2. Microscopic Techniques: Dyes and Staining 1  m Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © E. Chan/Visuals Unlimited

12.  Endospore stain  Members of genera including Bacillus, Clostridium form resistant, dormant endospore Resists Gram stain, often appears as clear object Endospore stain uses heat to facilitate uptake of primary dye (usually malachite green) by endospore Counterstain (usually safranin) used to visualize other cells 3.2. Microscopic Techniques: Dyes and Staining © Jack M. Bostrack/Visuals Unlimited 10  m Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

13.  Gram stain most common for bacteria  Two groups: Gram-positive, Gram-negative Reflects fundamental difference in cell wall structure 3.2. Microscopic Techniques: Dyes and Staining Crystal violet (primary stain) 3 4 1 2 Cells stain purple. Cells remain purple. Iodine (mordant) Safranin (counterstain) Gram-positive cells remain purple; Gram-negative cells appear pink. Alcohol (decolorizer) Gram-positive cells remain purple; Gram-negative cells become colorless. State of BacteriaAppearanceSteps in Staining (a)(b) 10 µm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. b: © Leon J. Le Beau/Biological Photo Service

14.  Gram-positive cell wall has thick peptidoglycan layer The Gram-Positive Cell Wall (a) (c) Cytoplasmic membrane Peptidoglycan Gram-positive (b) Gel-like material Peptidoglycan and teichoic acids Cytoplasmic membrane Cytoplasmic membrane Peptidoglycan (cell wall) Gel-like material N-acetylglucosamineN-acetylmuramic acidTeichoic acid 0.15 µm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (c): © Terry Beveridge, University of Guelph

15.  Gram-negative cell wall has thin peptido- glycan layer  Outside is unique outer membrane  Periplasm  LPS The Gram-Negative Cell Wall Lipoprotein Peptidoglycan (a) Peptidoglycan Cytoplasmic membrane (d) Lipopolysaccharide (LPS) Porin protein Outer membrane (lipid bilayer) Periplasm Cytoplasmic membrane (inner membrane; lipid bilayer) Peptidoglycan Outer membrane Periplasm Cytoplasmic membrane (c) Periplasm Outer membrane Lipid A Core polysaccharide O antigen (varies in length and composition) 0.15 µm (b) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (d): © Terry Beveridge, University of Guelph

16.  Crystal violet stains inside of cell, not cell wall Gram-positive cell wall prevents crystal violet–iodine complex from being washed out Decolorizing agent thought to dehydrate thick layer of peptidoglycan; desiccated state acts as barrier Solvent action of decolorizing agent damages outer membrane of Gram-negatives Thin layer of peptidoglycan cannot retain dye complex Cell Wall Type and the Gram Stain