Microbiology: Basic and Clinical Principles Chapter 1 PDF

Microbiology: Basic and Clinical Principles Second Edition Chapter 1 Introduction to Microbiology Presented by Janet Dowding, Ph.D....

Microbiology: Basic and Clinical Principles Second Edition Chapter 1 Introduction to Microbiology Presented by Janet Dowding, Ph.D. St. Petersburg College Copyright © 2023 Pearson Education, Inc. All Rights Reserved Clinical Case Copyright © 2023 Pearson Education, Inc. All Rights Reserved A Brief History of Microbiology (1 of 2) After this section, you should be able to: Define the term microorganism and give examples of microbes studied in microbiology. Explain the distinction between a pathogen and an opportunistic pathogen. Compare the theories of biogenesis and spontaneous generation, and summarize Louis Pasteur’s role in proving biogenesis. Describe how Robert Koch helped shape the germ theory of disease and list his postulates of disease. Identify the goals of aseptic technique, and explain and why it is important in healthcare facilities and laboratories. Copyright © 2023 Pearson Education, Inc. All Rights Reserved A Brief History of Microbiology (2 of 2) After this section, you should be able to: Discuss how Semmelweis, Lister, and Nightingale contributed to healthcare. Outline the basic steps of the scientific method, distinguish an observation from a conclusion, and compare a scientific law to a theory. Copyright © 2023 Pearson Education, Inc. All Rights Reserved What Is Microbiology? (1 of 6) Microbiology is the study of microorganisms or microbes, which are often invisible to the naked eye. Copyright © 2023 Pearson Education, Inc. All Rights Reserved  What Is Microbiology? (2 of 6) The term microbe encompasses… – Cellular, living microorganisms such as bacteria, archaea, fungi, protists, and helminths – Nonliving/noncellular entities such as viruses and prions (infectious proteins) – Microorganisms that are not microscopic such as some fungi, helminths, and protists (however, part of their life cycle is microscopic) Copyright © 2023 Pearson Education, Inc. All Rights Reserved What Is Microbiology? (3 of 6) Table 1.1 Living and Nonliving Agents Studied in Microbiology Microbe Cell Type Notes Bacteria Prokaryotic Unicellular;* pathogenic and nonpathogenic Archaea Prokaryotic Unicellular; nonpathogenic; most live in extreme environments Protists Eukaryotic Unicellular and multicellular; pathogenic and nonpathogenic (unicellular example: amoebae; multicellular example: algae) Fungi Eukaryotic Unicellular and multicellular; pathogenic and nonpathogenic (unicellular example: yeast; multicellular example: mushrooms) Helminths Eukaryotic Multicellular;* parasitic roundworms and flatworms Viruses Not cells; nonliving Infect animal, plant, or bacterial cells; can have a DNA or RNA genome Prions Not cells; nonliving; Not discovered until the 1980s; transmitted by transplant or infectious proteins ingestion; some prion diseases are inherited *Unicellular = one-celled organism; multicellular = organism made of many cells Copyright © 2023 Pearson Education, Inc. All Rights Reserved What Is Microbiology? (4 of 6) At least half of Earth’s life is microbial Microbes inhabit almost every region of our planet – Deep-sea trenches to glaciers Copyright © 2023 Pearson Education, Inc. All Rights Reserved What Is Microbiology? (5 of 6) Prokaryotic cells – Evolved about 3.5 billion years ago – Earliest life forms – Include unicellular bacteria and archaea Eukaryotic cells – All multicellular organisms and a number of unicellular microorganisms (e.g., amoebae and yeast) – Endosymbiotic theory Copyright © 2023 Pearson Education, Inc. All Rights Reserved What Is Microbiology? (6 of 6) Microbiology spans a wide variety of fields: – Healthcare – Agriculture – Industry – Environmental sciences Humans rely on microbes for many things: – Food production – Making medications – Breaking down certain environmental hazards Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes and Disease Pathogens are microbes that cause disease – About 1,400 pathogens are known to infect humans – 1 characteristically different colonies Copyright © 2023 Pearson Education, Inc. All Rights Reserved Specimens Are Often Stained Before Viewing With a Microscope (1 of 5) Stains, or dyes, increase contrast so the sample is easier to see Most bacterial staining techniques involve: – Making a smear of the specimen – Fixing the specimen by exposing it to heat (or chemical reagent) – Staining of the specimen Copyright © 2023 Pearson Education, Inc. All Rights Reserved Specimens Are Often Stained Before Viewing With a Microscope (2 of 5) Basic dyes are some of the most commonly used stains – Dye is positively charged – Attracted to the negatively charged cell surface – Result: cell appears the color of the dye Examples: – Methylene blue – Crystal violet – Safranin – Malachite green Copyright © 2023 Pearson Education, Inc. All Rights Reserved Specimens Are Often Stained Before Viewing With a Microscope (3 of 5) Acidic dyes are used in negative staining – Dye is negatively charged – Repelled from negatively charged cell surface – Result: stain the background of a specimen Examples: – Nigrosin – India ink Copyright © 2023 Pearson Education, Inc. All Rights Reserved Specimens Are Often Stained Before Viewing With a Microscope (4 of 5) Mordants are chemicals that may be required in certain staining procedures to interact with a dye and fix, or trap, it on or inside a treated specimen Examples: – Iodine – Alum – Tannic acid Copyright © 2023 Pearson Education, Inc. All Rights Reserved Specimens Are Often Stained Before Viewing With a Microscope (5 of 5) Most microbiological staining techniques are classified as: – Simple – Structural – Differential Copyright © 2023 Pearson Education, Inc. All Rights Reserved Simple Stains Simple staining techniques use one dye – Used to determine size, shape, and/or cellular arrangement Copyright © 2023 Pearson Education, Inc. All Rights Reserved Structural Stains (1 of 4) Flagella Staining – Prokaryotes can have single or multiple flagella with diverse arrangements – Mordants are added to coat the thin flagella and then a basic dye is applied Copyright © 2023 Pearson Education, Inc. All Rights Reserved Structural Stains (2 of 4) Capsule Staining – Capsules are sticky carbohydrate-based structures that some bacteria produce – Both a basic dye (stains the cell) and acidic dye (stains the background) are used – Capsule appears as a clear halo Copyright © 2023 Pearson Education, Inc. All Rights Reserved Structural Stains (3 of 4) Bacterial Endospore Staining – Endospores are specialized dormant structures that certain bacteria form in harsh conditions – Specimen is heated to drive the dye (malachite green) into the spores – Nonsporulating cells are stained with safranin Copyright © 2023 Pearson Education, Inc. All Rights Reserved Structural Stains (4 of 4) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Differential Stains: Gram and Acid-Fast Differential staining highlights differences in bacterial cell walls in order to discriminate between classes of cells Examples: – Gram stain – Acid-fast stain Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (1 of 6) Gram stain classifies bacteria as either Gram-positive or Gram-negative Gram-positive cells will appear purple and Gram- negative cells will appear pink Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (2 of 6) The Gram stain technique is as follows: – Crystal violet (primary stain) is added to a heat- fixed bacterial smear – Iodine (mordant) is added forming an insoluble crystal violet-iodine complex (CV-I complex) – Acetone-alcohol (decolorizing step) is used to rinse the sample – Safranin (counterstain) is added to the sample Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (3 of 6) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (4 of 6) To understand how it works we must first discuss cell wall composition… – Gram-positive cell walls ▪ Contain a thick layer of peptidoglycan ▪ No outer membrane – Gram-negative cell walls ▪ Contain a thin layer of peptidoglycan ▪ Contain an outer membrane rich in lipids Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (5 of 6) Results of the acetone-alcohol treatment on… Gram negative: – Dissolves the outer membrane – Damages the thin peptidoglycan layer – CV-I washes out Gram positive: – Slightly damages the thick peptidoglycan – Dehydration makes it less permeable – CV-I is retained Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gram Stain (6 of 6) Experimental errors can happen… If the sample is decolorized too long – Thick peptidoglycan layer of Gram-positive cell walls is damaged – CV-I complex is rinsed out of the cells – Gram-positive cells appear Gram-negative To minimize Gram property errors, fresh cultures between 24 and 48 hours old should be used Interpreting results can be difficult... – Variations in cell walls Copyright © 2023 Pearson Education, Inc. All Rights Reserved Acid-Fast Staining (1 of 4) Acid-fast stain distinguishes between cells with and without waxy cell walls Acid-fast bacteria – Contain waxy cell walls rich in mycolic acid – Retain red-colored primary dye after exposure to an acid wash Non–acid-fast cells – Red primary stain is washed away after exposure to an acid wash Copyright © 2023 Pearson Education, Inc. All Rights Reserved Acid-Fast Staining (2 of 4) Ziehl-Neelsen method – Carbol-fuchsin (primary dye) is added to a heat- fixed smear – Sample is steamed for several minutes to drive the red dye into the bacteria – Acid-alcohol (decolorizing agent) is used to rinse the sample – Methylene blue (counterstain) is added to the sample Copyright © 2023 Pearson Education, Inc. All Rights Reserved Acid-Fast Staining (3 of 4) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Acid-Fast Staining (4 of 4) Important diagnostic tool for detecting: – Mycobacterium species – Nocardia species Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microscopy Is Central to Microbiology Van Leeuwenhoek’s microscopes reached about 300× magnification Today’s microscopes allow us to see samples 20 million times smaller than the visibility of the eye Micrographs, or pictures taken through a microscope, allow us to document and share microscopy observations Copyright © 2023 Pearson Education, Inc. All Rights Reserved Light Microscopy Light microscopy uses visible light to illuminate the specimen Photons in a light wave interact with the specimen and are then channeled up to the viewer’s eyes through a series of lenses The compound light microscope is the most common type of optical microscope Copyright © 2023 Pearson Education, Inc. All Rights Reserved Figure 1.16 Parts of a Compound Light Microscope Copyright © 2023 Pearson Education, Inc. All Rights Reserved Parts of the Compound Light Microscope (1 of 2) Objective lens is near the specimen – Come in varieties that usually include 4×, 10×, 40×, and 100 × Ocular lens sits at the top of the microscope near the viewer’s eyes Final magnification is determined by multiplying the magnification of the ocular and objective lenses Copyright © 2023 Pearson Education, Inc. All Rights Reserved Parts of the Compound Light Microscope (2 of 2) Condenser lenses sharpen light into a precise cone to illuminate the specimen Iris diaphragm controls amount of light aimed at the specimen to improve contrast Coarse focus knob allows the viewer to roughly focus the image by adjusting the distance between the objective lens and specimen Fine focus knob allows for precision focusing Copyright © 2023 Pearson Education, Inc. All Rights Reserved Resolution Resolution is the ability to distinguish two distinct points as separate – The naked eye has a resolution of about 0.1 mm (100,000 nm) – Most compound light microscopes magnify up to 1,500 × with resolution of about 200 nm Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oil Immersion (1 of 2) Refractive index is the degree to which a substance bends light – Air has a lower refractive index than glass – Light passes through a slide then into the air above the slide where it scatters – Light is not channeled through the objective lens – To get a sharp image at 100 × objective lens, immersion oil is used Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oil immersion (2 of 2) Immersion oil is formulated to have the same refractive index as glass Copyright © 2023 Pearson Education, Inc. All Rights Reserved Types of Light Microscopy Bright Field Dark Field Phase Contrast Differential Interference Contrast Copyright © 2023 Pearson Education, Inc. All Rights Reserved Table 1.3 (1 of 2) Table 1.3 Comparing Light Microscopy Techniques: Amoeba Proteus Viewed with Different Light Microscopy Techniques Microscopy Technique Image Equipment Notes Bright Field Darker Compound Illuminates sample with solid cone of contrasting light light; image formed based on how light image on a microscope is absorbed; sample must be stained or A micrograph illustrates a pink amoeba with a dark pink nucleus against a light background. bright have natural coloration background Dark Field Negative Modified Illuminates sample with hollow cone of image, where condenser in light; image formed based on how light the sample a compound is scattered as opposed to how light is A micrograph illustrates a bright blue amoeba with distinct organelles against a dark background. appears light on light absorbed, so staining is not necessary; a darker microscope negative image made by dark field background microscopy should not be confused with negative staining; visualizes unstained specimens (live or dead) and stained specimens Copyright © 2023 Pearson Education, Inc. All Rights Reserved Table 1.3 (2 of 2) Table 1.3 [continued] Microscopy Technique Image Equipment Notes Phase Contrast Negative image, Modified Illuminates sample with hollow cone of A micrograph illustrates an amoeba whose cell membrane appears and organelles appear bright against a dark background. where the sample condenser light; a device in the microscope (i.e., a appears light on a in a phase plate) interacts with light that darker compound passes through the viewed sample— background light thereby enhancing image brightness, microscope shading, and contrast; visualizes unstained specimens (live or dead) and stained specimens Differential One side of Modified Illuminates specimen with polarized light Interference Contrast specimen compound (uniformly oriented light) as opposed to a (or Nomarsky) appears brighter light hollow cone of unorganized nonpolarized A micrograph illustrates an amoeba that appears three-dimensional against a darker background. than the other microscope light used in phase contrast microscopes side, providing a false three- dimensional appearance Copyright © 2023 Pearson Education, Inc. All Rights Reserved Electron Microscopy (1 of 4) Resolution improves with smaller wavelengths – Smallest wavelength of visible light is 400 nm – Smallest wavelength of electron beams is 1 nm Table 1.4 Comparison of Electron Microscopy to Light Microscopy Light Microscopes Electron Microscopes Use light waves to image the specimen Use an electron beam to image the specimen Small, portable, and affordable Large, requires special designated space, expensive Simple, cheap, and easy sample preparation that Lengthy and complex sample preparation requires requires minimal training substantial training Color images possible Only black-and-white images (though color may be added later, as an aftereffect) 1,000 × 500,000 × Most microscopes provide a maximum of 1,000 times Can magnify over 500,000 times Resolution of 200 nm 0.2 nm or about 1,000 times better than the best compound light microscopes Specimens can be living or dead Specimens are all dead Stains often used, but certain forms can be done Specimens often must be stained with an electron-dense without staining and can visualize live cells substance like osmium or gold Copyright © 2023 Pearson Education, Inc. All Rights Reserved Electron Microscopy (2 of 4) How it works… – Shoots electrons at a specimen – Electrons interact with the specimen and an image is generated – Provides high-magnification and high-resolution images Very expensive Requires considerable training to use Copyright © 2023 Pearson Education, Inc. All Rights Reserved Electron Microscopy (3 of 4) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Electron Microscopy (4 of 4) Two main classes of electron microscopes: – Transmission electron microscopes (TEM) – Scanning electron microscopes (SEM) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Transmission Electron Microscopy (T EM) (1 of 2) Most common form of electron microscopy 1 million times magnification and 1,000 times better resolution Samples must be extensively pretreated Specimens cannot be thicker than 1/285th of a human hair Copyright © 2023 Pearson Education, Inc. All Rights Reserved Transmission Electron Microscopy (TEM) (2 of 2) Electron beam passes through the specimen Hits a detector Generates 2D images of internal structures Copyright © 2023 Pearson Education, Inc. All Rights Reserved Scanning Electron Microscopy (SEM) Electron beam scans over the specimen Detectors sense how the electrons interact with the surface of the specimen Generates a 3D image of the surface Copyright © 2023 Pearson Education, Inc. All Rights Reserved Using Fluorescence in Microscopy (1 of 3) Fluorescence occurs when a substance absorbs energy (ultraviolet [UV] light) and then emits that energy as visible light Fluorochromes are fluorescent dyes that can be used to stain samples so they will fluoresce when illuminated by a UV light microscope Copyright © 2023 Pearson Education, Inc. All Rights Reserved Using Fluorescence in Microscopy (2 of 3) Examples of fluorochromes – Hoechst: binds to DNA and emits a blue glow – Auramine-rhodamine: binds acid-fast bacteria and emits a reddish-yellow glow – Calcofluor-white: binds cellulose and chitin Copyright © 2023 Pearson Education, Inc. All Rights Reserved Using Fluorescence in Microscopy (3 of 3) Immunofluorescence – Uses fluorescent dyes linked to antibodies that can recognize a specific target – Can be used for identification of bacteria in blood cultures, virus identification in patient samples, and screening for bacteria in food-processing plants Copyright © 2023 Pearson Education, Inc. All Rights Reserved Table 1.5 UV Light Microscopy and Probe Microscopy Techniques (1 of 2) Blank Microscopy Image Type of Notes Technique Sample Fluorescence General Flat image Live or fixed The UV waves cause visible light to be Imaging Fluorescence with coloration released from fluorochromes; advantage A micrograph illustrates a curved rod-shaped microbe with green fluorescence against a dark background. based on over light microscopy is not improved fluorochrome resolution or magnification, but easy and used sensitive detection; allows for the detection of even a single molecule in a sample Fluorescence Confocal 3D image Live or fixed Eliminates blurriness associated with Imaging A micrograph illustrates green thread-like structures running across the cell and concentrated at the membrane, a solid orange nucleus in the center, and an accumulation of blue dots next to the nucleus. standard fluorescence microscopes; images are taken at different planes of focus, and then these photo “slices” are compiled to generate a 3D image Copyright © 2023 Pearson Education, Inc. All Rights Reserved Table 1.5 UV Light Microscopy and Probe Microscopy Techniques (2 of 2) Table 1.5 [Continued] Blank Microscopy Image Type of Sample Notes Technique Probe Scanning Tunneling 3D image Atoms can be A probe sharpened to a single atom Techniques A micrograph illustrates a fluorescent green colored double helix structure of D N A on a three-dimensional elevation. visualized; sample at the tip shoots electrons at the must conduct sample surface; elevations or dips in electricity, which sample surface are registered to limits what can be make the image; not as good as visualized scanning electron microscopy for detecting steep rises or deep valleys in sample Probe Atomic Force 3D image Atoms can be Probe is dragged or tapped along Techniques A micrograph illustrates an elevated, fluorescent pink tear-drop structure with white tips connected by purple thread-like structures. visualized; live specimen surface; not as good as samples under scanning electron microscopy for Physiological detecting steep rises or deep valleys conditions or fixed in sample samples Copyright © 2023 Pearson Education, Inc. All Rights Reserved Visual Summary: Introduction to Microbiology Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (1 of 4) Summary of the case: – Cholera is an acute, infectious diarrheal illness – Originally thought it was caused by “miasma” (harmful air) – Officials addressed sewage issues in poor areas – Improved sanitation led to decreased cholera cases – Koch isolated comma-shaped bacteria (Vibrio cholerae) from cholera patients, but couldn’t infect healthy hosts – Pettenkofer drank a culture of V. cholerae but didn’t develop full- blown cholera – V. cholerae is a naturally occurring waterborne bacterium that invades humans through contaminated drinking water and foods – Hundreds of V. cholerae strains (O1 and O139 are responsible for most outbreaks) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (2 of 4) Summary of the case: – V. cholerae colonize copepods; adheres to the surface around the oral region and egg sacs – V. cholerae benefit copepod by secreting a substance that helps the egg sac rupture – Bacteria benefit by having an environment rich in food and safety from protozoans – During starvation V. cholerae colonies on agar plates change from smooth to wrinkled colonies Copyright © 2023 Pearson Education, Inc. All Rights Reserved  Think Clinically: Be S.M.A.R.T. About Cases (3 of 4) 1. Provide a possible explanation for why Koch experienced difficulties in establishing an animal model for cholera. 2. What type of electron microscopy was most likely used to obtain the data about V. cholerae’s colonization of copepod external surfaces? Explain your reasoning. 3. We now know the miasma theory is wrong, so how did proper sewage management help reduce cholera cases, if not by reducing the stench? 4. How would you define the symbiotic relationship between copepods and V. cholerae? Explain your definition. 5. How would you describe the biota classification or status of V. cholerae in humans? How about in copepods? Copyright © 2023 Pearson Education, Inc. All Rights Reserved  Think Clinically: Be S.M.A.R.T. About Cases (4 of 4) 1. What is the most likely explanation for why Max von Pettenkofer did not develop a classic case of cholera after drinking the V. cholerae bacteria? 2. Why might V. cholerae cells from rugose colonies be better able to survive harsh conditions than cells from smooth colonies? Explain your reasoning. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Copyright This work is protected by United States copyright laws and is provided solely for the use of instructors in teaching their courses and assessing student learning. Dissemination or sale of any part of this work (including on the World Wide Web) will destroy the integrity of the work and is not permitted. The work and materials from it should never be made available to students except by instructors using the accompanying text in their classes. All recipients of this work are expected to abide by these restrictions and to honor the intended pedagogical purposes and the needs of other instructors who rely on these materials. Copyright © 2023 Pearson Education, Inc. All Rights Reserved