Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Chapter 3 Tools of the Laboratory: The Methods for Studying Microorganisms

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 2

3 The Microscope Key characteristics of a reliable microscope are: Magnification – ability to enlarge objects Resolving power – ability to show detail

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 4 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. Magnification

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Parts of the Microscope 5

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. –The objective lens forms the magnified real image –The real image is projected to the ocular where it is magnified again to form the virtual image Magnification in Two Phases 6

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 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 7

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 8 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 Resolution

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 9 Quantifying Resolution 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 Magnification between 40X and 2000X Resolving Power (RP) Wavelength of light in nm 2 X Numerical aperture of objective lens =

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 10 The Purpose of Oil

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Magnification & Resolution 11

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 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 doesn’t affect resolution 12

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 13 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 doesn’t affect resolution

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 14 Variations on the Optical Microscope Bright-field – most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 15 Variations on the Optical Microscope Dark-field – brightly illuminated specimens surrounded by dark field; used for live and unstained specimens

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 16 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 17 Fluorescence Microscope Modified microscope with an ultraviolet radiation source and filter. Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence Useful in diagnosing infections

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 18 Scanning Confocal Microscope Uses a laser beam of light to scan the specimen. Integrates images to allow focus on multiple depths or planes.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 19 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Comparing Microscopes: 20 Copyright © McGraw-Hill Education. 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 2 Types of Electron Microscopes Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. 21

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education 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.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 23 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.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 24 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 (a) Basic Dye Cell envelope Positive-Type Staining (a and b) Copyright © McGraw-Hill Education. Permission required for reproduction or display.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 25 Staining Acidic dyes – anionic, negatively charged chromophore Negative staining – microbe repels dye, the dye stains the background Copyright © McGraw-Hill Education. Permission required for reproduction or display. Cell envelope Acidic Dye (b) (c) Acidic Dye Negative Staining (c)

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 26 Staining Simple stains – one dye is used; reveals shape, size, and arrangement 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Staining Examples 27

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 28 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 29 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.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 30 –Streak plate technique –Pour plate technique –Spread plate technique Isolation Techniques

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 31 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.

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 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 32

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 33 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

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Physical States of Media Liquid – broth; does not solidify Semisolid – contains solidifying agent Solid – firm surface for colony formation –Contains solidifying agent –Liquefiable and nonliquefiable 34

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 35 –The most commonly used solidifying agent –Solid at room temperature, liquefies at boiling (100 o C), does not re-solidify until it cools to 42 o C –Provides framework to hold moisture and nutrients –Not digestible for most microbes Agar

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 36 –Nutrient broth – liquid medium containing beef extract and peptone –Nutrient agar – solid media containing beef extract, peptone, and agar Most Commonly Used Media

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 37 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 Chemical Content of Media

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Examples of Enriched Media 38

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 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 39

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patient’s 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 © McGraw-Hill Education. Permission required for reproduction or display. © Kathy Park Talaro 40

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patient’s 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 © McGraw-Hill Education. Permission required for reproduction or display. © Kathy Park Talaro 41

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 42 Some media can be both Selective & Differential

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 43

Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 44 Miscellaneous Media Carbohydrate fermentation medium – contains sugars that can be fermented, converted to acids, and a pH indicator to show this reaction