Micro 3 New.ppt.pdf tools the laboratory the method
- 1. Chapter 3 Tools of the Laboratory: The Methods for Studying Microorganisms Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 2. * Subculture INOCULATION INCUBATION ISOLATION INSPECTION INFORMATION GATHERING The sample is placed into a container of medium that will support its growth. The medium may be in solid or liquid form, and held in tubes, plates, ļ¬asks, and even eggs. The delivery tool is usually a loop, needle, swap or syringe. Inoculated media are placed in a controlled environment (incubator) to promote growth. During the hours or days of this process, a culture develops as the visible growth of the microbes in the container of medium. Some inoculation techniques can separate microbes and spread them apart to create isolated colonies that each contain a single type of microbe This is invaluable for identifying the exact species of microbes in the sample, and it paves the way for making pure cultures. Cultures are observed for the macroscopic appearance of growth characteristics. Cultures are examined under the microscope for basic details such as cell type and shape. This may be enhanced through staining and use of special microscopes. Additional tests for microbial function and characteristics are usually required. This may include inoculations into specialized media that determine biochemical traits, immunological testing, and genetic typing. Such tests will provide speciļ¬c information unique to a certain microbe. SPECIMEN COLLECTION One goal of these procedures is to attach a name or identity to the microbe, usually to the level of species. Any information gathered from inspection and investigation can be useful. Identiļ¬cation is accomplished through the use of keys, charts, and computer programs that analyze the data and arrive at a ļ¬nal conclusion. IDENTIFICATION Staining Streak plate Keys Blood bottle Incubator Bird embryo Biochemical tests Drug sensitivity Immunologic tests DNA analysis Pure culture of bacteria Microbiologists begin by sampling the object of their interest. It could be nearly any thing or place on earth (or even Mars). Very common sources are body ļ¬uids, foods, water, soil, plants, and animals, but even places like icebergs, volcanoes, and rocks can be sampled. Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 3. * The Microscope Key characteristics of a reliable microscope are: ā¢ Magnification ā ability to enlarge objects ā¢ Resolving power ā ability to show detail
- 4. * Magnification in most microscopes results from an interaction between visible light waves and the curvature of a lens. ā The extent of enlargement is the magnification. Magnification
- 5. * Parts of the Microscope Ocular (eyepiece) Interpupillary adjustment Body Arm Coarse adjustment knob Fine focus adjustment knob Stage adjustment knobs Nosepiece Objective lens (4) Mechanical stage Substage condenser Aperture diaphragm control Base with light source Field diaphragm lever Light intensity control Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Leica Microsystems Inc.
- 6. * ā The objective lens forms the magnified real image ā The real image is projected to the ocular where it is magnified again to form the virtual image Magnification in Two Phases Retina Brain Objective lens Specimen Ocular lens Eye Condenser lens Light source Virtual image Formed by ocular lens Real image formed by objective lens Light rays strike specimen Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 7. * ā¢ Total magnification of the final image is a product of the separate magnifying powers of the two lenses objective power ocular power total magnification x = Total Magnification Retina Brain Objective lens Specimen Ocular lens Eye Condenser lens Light source Virtual image Formed by ocular lens Real image formed by objective lens Light rays strike specimen Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 8. * The capacity to distinguish or separate two adjacent objects and depends on ā The wavelength of light that forms the image along with characteristics of the objectives Resolution (a) (b) Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 9. * Quantifying Resolution ā¢Visible light wavelength is 400 nmā750 nm ā¢Numerical aperture of lens ranges from 0.1 to 1.25 ā¢Shorter wavelength and larger numerical aperture will provide better resolution ā¢Oil immersion objectives resolution is 0.2 Ī¼m ā¢Magnification between 40X and 2000X Resolving Power (RP) Wavelength of light in nm 2 X Numerical aperture of objective lens =
- 10. * The Purpose of Oil Air Oil Objective lens Slide Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 11. Magnification & Resolution 0.2 Āµm 1.0 Āµm Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 12. Concept Check: In order to get the best resolution possible with a light microscope, you would want: A. Long wavelengths of light B. Short wavelengths of light C. The wavelength doesnāt affect resolution
- 13. * Variations on the Optical Microscope ā¢ Bright-field ā most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens
- 14. * Variations on the Optical Microscope ā¢ Dark-field ā brightly illuminated specimens surrounded by dark field; used for live and unstained specimens
- 15. * Variations on the Optical Microscope ā¢ Phase-contrast ā transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures
- 16. * Fluorescence Microscope ā¢ Modified microscope with an ultraviolet radiation source and filter. ā¢ Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence ā¢ Useful in diagnosing infections Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Invitrogen Corporation
- 17. * Scanning Confocal Microscope ā¢ Uses a laser beam of light to scan the specimen. ā¢ Integrates images to allow focus on multiple depths or planes.
- 18. * Electron Microscopy ā¢ Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds ā¢ Electron waves are 100,000 times shorter than the waves of visible light ā¢ Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength ā¢ Magnification between 5,000X and 1,000,000X
- 19. Comparing Microscopes: * Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Light Microscope Lamp Electron gun Electron beam Ocular lens Viewing screen Eye Image Transmission Electron Microscope (a) (b) Condenser lens Light rays Specimen Objective lens
- 20. cilia MA C macro- nucleus pre cv chambe r cv fv13 pa k mic pa k myoneme pi Courtesy Dr. Richard Allen * 2 Types of Electron Microscopes ā¢ Transmission electron microscopes (TEM) ā transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. Viruses (a) Hazelton, PR and Gelderblom, HR. 2003. Electron microscopy for Rapid diagnosis of Emerging Infectious Agents. Emerging Infectious Diseases 9:294-303. 0.1 mm Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (b)
- 21. * 2 Types of Electron Microscopes ā¢ Scanning electron microscopes (SEM) ā provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.
- 22. Concept Check: What kind of microscope was used to get this image of Rotavirus? A. Brightfield B. Phase Contrast C. Scanning Electron D. Transmission Electron Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Colony with zone Of beta hemolysis Ā© Kathy Park Talaro
- 23. * Specimen Preparation for Optical Microscopes ā¢ Wet mounts and hanging drop mounts ā allow examination of characteristics of live cells: size, motility, shape, and arrangement ā¢ Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.
- 24. * Staining ā¢ Dyes are used to create contrast by imparting color ā¢ Basic dyes ā cationic, positively charged chromophore ā¢ Positive staining ā surfaces of microbes are negatively charged and attract basic dyes (a) Basic Dye Cell envelope Positive-Type Staining (a and b) Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 25. * Staining ā¢ Acidic dyes ā anionic, negatively charged chromophore ā¢ Negative staining ā microbe repels dye, the dye stains the background Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cell envelope Cell envelope Acidic Dye (b) (c) Acidic Negative Staining (c)
- 26. * Staining ā¢ Simple stains ā one dye is used; reveals shape, size, and arrangement ā¢ Differential stains ā use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain) ā¢ Structural stains ā reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains
- 27. Staining Examples * Methylene blue stain of Corynebacterium (1,0003) (a) Simple Stains (b) Diļ¬erential Stains Gram stain Purple cells are gram-positive. Red cells are gram-negative (1,0003). Acid-fast stain Red cells are acid-fast. Blue cells are non-acid-fast (7503). Spore stain, showing spores (green) and vegetative cells (red) (1,0003) (c) Structural Stains India ink capsule stain of Cryptococcus neoformans (5003) Flagellar stain of Proteus vulgaris. Note the ļ¬ne fringe of ļ¬agella (1,5003). Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Harold J. Benson CD C Ā© Jack Bostrack/Visuals Unlimited Ā© Steven R. Spilatro, Department of Biology, Marietta College, Marietta, OH Ā© A.M. Siegelman/Visuals Unlimited Ā© Dr. Rita Colwell & Dr. Munirul Alam
- 28. * The 6 Iās of Culturing Microbes Inoculation ā introduction of a sample into a container of media to produce a culture of observable growth Isolation ā separating one species from another Incubation ā under conditions that allow growth Inspection Information gathering Identification
- 29. * Isolation ā¢ If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cellsā a colony. A colony consists of one species. Mixture of cells in sample Microscopic view Cellular level Macroscopic view Colony level Incubatio n Parent cells Separation of cells by spreading or dilution on agar medium Microbes become visible as isolated colonies containing millions of cells. Growth increases the number of cells. Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 30. * ā Streak plate technique ā Pour plate technique ā Spread plate technique Isolation Techniques 1 Steps in a Streak Plate; this one is a four-part or quadrant streak. 2 3 4 5 (a) (c) Steps in Loop Dilution; also called a pour plate or serial dilution 2 1 "Hockey stick" (e) Steps in a Spread Plate 2 1 3 2 1 3 Note: This method only works if the spreading tool (usually an inoculating loop) is resterilized (ļ¬amed) after each of steps 1ā4. Loop containing sample Loop containing sample Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Kathy Park Talaro Ā© Kathy Park Talaro Ā© Kathy Park Talaro (b) (d) (f)
- 31. * Inspection ā¢ If a single species is growing in the container, you have a pure culture but if there are multiple species than you have a mixed culture. ā¢ Check for contaminants (unknown or unwanted microbes) in the culture.
- 32. * Ways to Identify a Microbe: ā¢ Cell and colony morphology or staining characteristics ā¢ DNA sequence ā¢ Biochemical tests to determine an organismās chemical and metabolic characteristics ā¢ Immunological tests Oxidase ā Acinetobacter spp.* Moraxella spp. Oxidase + Ferments maltose Grows on nutrient agar Reduces nitrite Does not reduce nitrite Does not grow on nutrient agar Does not ferment sucrose or lactose Ferments sucrose; does not ferment lactose Ferments lactose; does not ferment sucrose Gram-negative cocci and coccobacilli Neisseria meningitidis Neisseria lactamica** Neisseria sicca N. gonorrhoeae Branhamella catarrhalis Does not ferment maltose Scheme for Diļ¬erentating Gram-Negative Cocci and Coccobacilli (a) (b)
- 33. * Media: Providing Nutrients in the Laboratory Media can be classified according to three properties: 1. Physical state ā liquid, semisolid, and solid 2. Chemical composition ā synthetic (chemically defined) and complex 3. Functional type ā general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration
- 34. * Physical States of Media Liquid ā broth; does not solidify Semisolid ā contains solidifying agent Solid ā firm surface for colony formation ā Contains solidifying agent ā Liquefiable and nonliquefiable Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Kathy Park Talaro
- 35. * ā The most commonly used solidifying agent ā Solid at room temperature, liquefies at boiling (100o C), does not re-solidify until it cools to 42o C ā Provides framework to hold moisture and nutrients ā Not digestible for most microbes Agar
- 36. * ā Nutrient broth ā liquid medium containing beef extract and peptone ā Nutrient agar ā solid media containing beef extract, peptone, and agar Most Commonly Used Media
- 37. * ā¢ Synthetic ā contains pure organic and inorganic compounds in an exact chemical formula ā¢ Complex or nonsynthetic ā contains at least one ingredient that is not chemically definable ā¢ General purpose media ā grows a broad range of microbes, usually nonsynthetic ā¢ Enriched media ā contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes Chemical Content of Media
- 38. Examples of Enriched Media * Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Colony with zone Of beta hemolysis Ā© Kathy Park Talaro Ā© Kathy Park Talaro
- 39. * Selective media: contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes Differential media: allows growth of several types of microbes and displays visible differences among those microbes Selective & Differential Media Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (a) General-purpose nonselective medium (All species grow.) Selective medium (One species grows.) Mixed sample (b) General-purpose nondiļ¬erential medium (All species have a similar appearance.) Diļ¬erential medium (All three species grow but may show diļ¬erent reactions.) Mixed sample
- 40. Concept Check: CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patientās sample is inoculated and based on the color of the colonies you can identify the pathogen. CHROMagar is best described as: A. Enriched B. Selective C. Differential D. Complex Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ā© Kathy Park Talaro
- 41. * Some media can be both Selective & Differential
- 42. * Miscellaneous Media ā¢ Reducing medium ā contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria
- 43. * Miscellaneous Media ā¢ Carbohydrate fermentation medium ā contains sugars that can be fermented, converted to acids, and a pH indicator to show this reaction Gas bubble Outline of Durham tube Cloudines s indicating growth Ā© Harold J. Benson Copyright Ā© The McGraw-Hill Companies, Inc. Permission required for reproduction or display.