Microbiology BIO210 Observing Microbial Cell (PDF)

Microbiology – BIO210 Observing the Microbial cell CH2 Outline: ❖Understand the key characteristics of Microscope ❖Culturing & Identifying microorganisms  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display....

Microbiology – BIO210 Observing the Microbial cell CH2 Outline: ❖Understand the key characteristics of Microscope ❖Culturing & Identifying microorganisms  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. IDENTIFICATION INOCULATION One goal of these procedures is to The sample is placed into a container of medium attach a name or identity to the microbe, that will support its growth. The medium may be in usually to the level of species. Any solid or liquid form, and held in tubes, plates, information gathered from inspection flasks, and even eggs. The delivery tool is usually a and investigation can be useful. loop, needle, swap or syringe. Identification is accomplished through the use of keys, charts, and computer programs that analyze the data and arrive at a final conclusion. Bird Streak plate embryo Keys INFORMATION SPECIMEN Blood bottle GATHERING COLLECTION INCUBATION Additional tests for microbial function and Microbiologists begin by sampling the Inoculated media are placed in a characteristics are usually required. This may object of their interest. It could be nearly controlled environment (incubator) include inoculations into specialized media that any thing or place on earth (or even Mars). to promote growth. During the hours or days determine biochemical traits, immunological Very common sources are body fluids, of this process, a culture develops as the testing, and genetic typing. Such tests will foods, water, soil, plants, and animals, visible growth of the microbes in the provide specific information unique to a certain but even places like icebergs, volcanoes, container of medium. microbe. and rocks can be sampled. DNA Biochemical Drug analysis tests sensitivity Incubator INSPECTION ISOLATION Immunologic tests Cultures are observed for the macroscopic Some inoculation techniques can separate appearance of growth characteristics. Cultures microbes and spread them apart to create isolated are examined under the microscope for basic colonies that each contain a single type of microbe details such as cell type and shape. This may This is invaluable for identifying the exact species be enhanced through staining and use of of microbes in the sample, and it paves the way for special microscopes. making pure cultures. Pure culture of bacteria Staining Subculture  The Microscope Key characteristics of a reliable microscope are: Magnification – ability to enlarge objects Resolving power – ability to show detail *  Magnification Magnification in most microscopes results from an interaction between visible light waves and the curvature of a lens. The extent of enlargement is the magnification  Parts of the Microscope Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Interpupillary adjustment Ocular (eyepiece) Body Nosepiece Arm Objective lens (4) Coarse Mechanical stage adjustment knob Substage condenser Fine focus Aperture diaphragm control adjustment knob Base with light source Stage adjustment knobs Field diaphragm lever Light intensity control © Leica Microsystems Inc.  Magnification in Two Phases Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Brain Retina Eye – The objective lens forms the Ocular lens magnified real image Virtual image Formed by ocular lens – The real image is projected Objective lens Specimen to the ocular where it is Light rays strike Real image formed magnified again to form the specimen by objective lens virtual image Condenser lens Light source  Total Magnification Brain Retina Eye Ocular lens Total magnification of the final image is a product of the separate Virtual image magnifying powers of the two lenses Objective lens Formed by ocular lens Light rays Specimen strike Real image objective ocular total specimen formed power x = by objective power magnification Condenser lens lens Light source  Resolution ▪ The capacity to distinguish or separate two adjacent objects and depends on -The wavelength of light that forms the image along with characteristics of the objectives (a) (b) * Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  Quantifying Resolution Wavelength of light in nm Resolving Power (RP) = 2 X Numerical aperture of objective lens Visible light wavelength is 400 nm–750 nm Numerical aperture of lens ranges from 0.1 to 1.25 Shorter wavelength and larger numerical aperture will provide better resolution Oil immersion objectives resolution is 0.2 μm *  The Purpose of Oil A special type of oil to fill the gap between the microscope objective lens and the microscope slide. This process improves the light gathering ability of the microscope, allowing for sharper images and more accurate measurements.  Variations on the Optical Microscope Bright-field – most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens *  Variations on the Optical Microscope Dark-field – brightly illuminated specimens surrounded by dark field; used for live and unstained specimens *  Variations on the Optical Microscope Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures  Fluorescence Microscope Modified microscope with an Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ultraviolet radiation source and filter. Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence Useful in diagnosing infections © Invitrogen Corporation  Scanning Confocal Microscope Uses a laser beam of light to scan the specimen. Integrates images to allow focus on multiple depths or planes.  Electron Microscopy Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds Electron waves are 100,000 times shorter than the waves of visible light Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength Magnification between 5,000X and 1,000,000X  Comparing Microscopes Light Microscope Transmission Electron Microscope Lamp Electron gun Electron beam Condenser lens Light rays Specimen Objective lens Image Ocular lens (a) Eye (b) Viewing screen Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  2 Types of Electron Microscopes Viruses Transmission electron microscopes (TEM) 0.1 mm Hazelton, PR and Gelderblom, HR. 2003. Electron microscopy for Rapid diagnosis of Emerging Infectious Agents. Emerging Infectious Diseases 9:294-303. (a) cili – transmit electrons through the specimen. a myonem e Darker areas represent thicker, denser parts pa and lighter areas indicate more transparent, p k i mi c less dense parts. pre cv chamb er c pa k MA C v macro - nucleu s fv1 3 * (b) Courtesy Dr. Richard Allen Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 Types of Electron Microscopes Scanning electron microscopes (SEM) – provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.  Specimen Preparation for Optical Microscopes Wet mounts and hanging drop mounts – allow examination of characteristics of live cells: size, motility, shape, and arrangement 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. *  Staining Dyes are used to create contrast by imparting color Basic dyes – cationic, positively charged chromophore - Positive staining – surfaces of microbes are negatively charged and attract basic dyes  Staining Acidic dyes – anionic, negatively charged chromophore - Negative staining – microbe repels dye, the dye stains the background  Staining Simple stains – one dye is used; reveals shape, size, and arrangement (e.g., Methylene blue stain) Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain) Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains  The 6 I’s of Culturing Microbes Inoculation – introduction of a sample into a container of media to produce a culture of observable growth Isolation – separating one species from another Incubation – under conditions that allow growth Inspection Information gathering Identification  Isolation If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells— a colony. A colony consists of one species. Mixture of cells in sample Separation of cells by spreading or Microscopic view Parent Cellular level cells dilution on agar medium Incubation Growth increases the number of cells. Microbes become visible as isolated Macroscopic view colonies containing Colony level millions of cells. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  Isolation Techniques Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Note: This method only works if the spreading tool (usually an inoculating loop) is resterilized (flamed) after each of steps 1–4. – Streak plate Loop containing sample technique 1 2 3 4 5 (a) Steps in a Streak Plate; this one is a four-part or quadrant streak. © Kathy Park Talaro Loop containing sample (b) – Pour plate technique 1 2 3 1 2 3 © Kathy Park Talaro – Spread plate (d) (c) Steps in Loop Dilution; also called a pour plate or serial dilution 1 technique (f) "Hockey stick" (e) Steps in a Spread Plate 2  Inspection 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. Check for contaminants (unknown or unwanted microbes) in the culture.  Ways to identify a microbe: Cell and colony morphology or staining characteristics DNA sequence Biochemical tests to determine an organism’s chemical and metabolic characteristics Immunological tests  Media: Providing Nutrients in the Laboratory Media can be classified according to three properties: 1. Physical state – liquid, semisolid, and solid 2. Chemical composition – synthetic (chemically defined) and complex 3. Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration  Physical States of Media Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Liquid – broth; does not solidify Semisolid – contains solidifying agent Solid – firm surface for colony formation © Kathy Park Talaro – Contains solidifying agent – Liquefiable and nonliquefiable  Agar – The most commonly used solidifying agent – Solid at room temperature, liquefies at boiling (100oC), does not re-solidify until it cools to 42oC – Provides framework to hold moisture and nutrients – Not digestible for most microbes  Most Commonly Used Media – Nutrient broth – liquid medium containing beef extract and peptone – Nutrient agar – solid media containing beef extract, peptone, and agar  Chemical Content of Media Synthetic – contains pure organic and inorganic compounds in an exact chemical formula Complex or nonsynthetic – contains at least one ingredient that is not chemically definable General purpose media – grows a broad range of microbes, usually nonsynthetic Enriched media – contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes  Examples of Enriched Media Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Colony with zone of beta hemolysis © Kathy Park Talaro © Kathy Park Talaro  Selective & Differential Media Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mixed sample Selective media: Differential media: General-purpose Selective medium allows growth of contains one or more (a) nonselective medium (One species grows.) several types of (All species grow.) agents that inhibit growth Mixed sample microbes and of some microbes and displays visible encourage growth of the differences among desired microbes those microbes General-purpose Differential medium nondifferential medium (All three species grow but may (All species have a similar show different reactions.) appearance.) (b) Some media can be both Selective & Differential  Miscellaneous Media Reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria *  Miscellaneous Media Carbohydrate fermentation medium – contains sugars that can be fermented, Gas bubble converted to acids, and a pH indicator to show this reaction Outline of Durham tube Cloudiness indicating growth © Harold J. Benson Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.