1 Applied Microbiology 2 nd lecture: Chapter

2 Applied Microbiology 1 st part Introduction into Microbiology Book: Foundations in Microbiology, Basic Principles, Book: Foundations in Microbiology, Basic Principles, 6 th edition, by Talaro, McGraw-Hill 6 th edition, by Talaro, McGraw-Hill 1 st lecture: Chapter (Main Themes of Microbiology + Chemistry of Biology) 2 nd lecture: Chapter 3 + part of 11 (Methods studying microorganisms + Agents for microbial control) 3 rd Lecture: Chapter 4 (Prokaryotic cells) 4 th Lecture: Chapter 5 (Eukaryotic cells) 5 th Lecture: Chapter 6 (Viruses) 6 th Lecture: Chapter 7 (Microbial growth) 7 th Lecture: Chapter 8 (microbial metabolism) 8 th Lecture: Chapter 13 (microbe-human interaction)

3 Tools of the Laboratory Sources of Microorganisms

4 Tools of the Laboratory The 5 I’s of Culturing Microbes 1. Inoculation – introduction of a sample into a container of media to produce a culture of observable growth 2. Incubation – under conditions that allow growth 3. Isolation –separating one species from another 4. Inspection 5. Identification

5 Tools of the Laboratory Inoculation - Producing a culture

6 Tools of the Laboratory Incubation

7 Isolation – Separating one species from another

8 Tools of the Laboratory Inspection + Identification

9 Tools of the Laboratory Isolation – Separating one species from another 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. 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. A colony consists of one species.

10 Tools of the Laboratory Methods for Isolation of Bacteria Isolation techniques include: Isolation techniques include: streak plate techniquestreak plate technique pour plate techniquepour plate technique spread plate techniquespread plate technique

11 Tools of the Laboratory Media -> Providing nutrients -> Media can be classified according to three properties: -> Media can be classified according to three properties: Physical state – liquid, semisolid and solid Physical state – liquid, semisolid and solid Chemical composition – synthetic (chemically defined) and nonsynthetic (complex) Chemical composition – synthetic (chemically defined) and nonsynthetic (complex) Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay

12 Tools of the Laboratory Media -> Physical states Liquid – broth; does not solidify -> nutrient broth Liquid – broth; does not solidify -> nutrient broth Semisolid – clot-like consistency; contains solidifying agent (agar or gelatin) Semisolid – clot-like consistency; contains solidifying agent (agar or gelatin) Solid – firm surface for colony formation -> nutrient agar Solid – firm surface for colony formation -> nutrient agar contains solidifying agentcontains solidifying agent liquefiable and nonliquefiableliquefiable and nonliquefiable

13 Tools of the Laboratory Media -> Physical states -> Most commonly used solidifying agent is agar -> Most commonly used solidifying agent is agar A complex polysaccharide isolated from red algae A complex polysaccharide isolated from red algae solid at room temp, liquefies at boiling (100 o C), does not resolidify until it cools to 42 o Csolid at room temp, liquefies at boiling (100 o C), does not resolidify until it cools to 42 o C provides framework to hold moisture and nutrientsprovides framework to hold moisture and nutrients not digestible for most microbesnot digestible for most microbes

14 Tools of the Laboratory Media -> Physical states Liquid media semisolid media

15 Tools of the Laboratory Media -> Chemical composition Synthetic – contains pure organic and inorganic compounds in an exact chemical formula -> defined media Synthetic – contains pure organic and inorganic compounds in an exact chemical formula -> defined media Complex or nonsynthetic – contains at least one ingredient that is not chemically definable Complex or nonsynthetic – contains at least one ingredient that is not chemically definable General purpose media- grows a broad range of microbes, usually nonsynthetic 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 Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

16 Tools of the Laboratory Media -> Chemical composition Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

17 Tools of the Laboratory Media -> Chemical composition 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 desired and undesired microbes

18 Tools of the Laboratory Media -> Chemical composition Differential media

19 Tools of the Laboratory Incubation, Inspection, and Identification 1. Incubation – temperature-controlled chamber at appropriate temperature and atmosphere microbe multiplies and produces macroscopically observable growthmicrobe multiplies and produces macroscopically observable growth 2. Inspection – observation; macroscopic and microscopic pure culture – grows only single known species of microorganismspure culture – grows only single known species of microorganisms mixed cultures – hold two or more identified species or microorganismsmixed cultures – hold two or more identified species or microorganisms contaminated culture – once pure or mixed culture that has unwanted microbes growingcontaminated culture – once pure or mixed culture that has unwanted microbes growing

20 Tools of the Laboratory Incubation, Inspection, and Identification 3. Identification – macroscopic and microscopic appearance, biochemical tests, genetic characteristics, immunological testing

21 Tools of the Laboratory Maintenance and disposal of cultures -> Potentially hazardous cultures and specimens are usually disposed of in two ways: steam sterilizationsteam sterilization Incineration (burning)Incineration (burning) -> Culture collections : American Type Culture Collection (ATCC) German Collection of Microorganisms and Cell Cultures (DSMZ)

22 Tools of the Laboratory The Microscope Key characteristics of a reliable microscope are: Magnification – ability to enlarge objects Magnification – ability to enlarge objects Resolving power – ability to show detail Resolving power – ability to show detail Magnification in most microscopes results from interaction between visible light waves and curvature of the lens. angle of light passing through convex surface of glass changes – refractionangle of light passing through convex surface of glass changes – refraction Depending on the size and curvature of the lens, the image appears enlarged.Depending on the size and curvature of the lens, the image appears enlarged. extent of enlargement - magnificationextent of enlargement - magnification

23 Tools of the Laboratory The Microscope

24 Tools of the Laboratory Resolution Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power function of wavelength of light that forms the image along with characteristics of objectivesfunction of wavelength of light that forms the image along with characteristics of objectives Visible light wavelength is 400 nm – 750 nm. Visible light wavelength is 400 nm – 750 nm. Numerical aperture of lens ranges from 0.1 to Numerical aperture of lens ranges from 0.1 to Oil immersion lens requires the use of oil to prevent refractive loss of light. Oil immersion lens requires the use of oil to prevent refractive loss of light. Shorter wavelength and larger numerical aperture will provide better resolution. Shorter wavelength and larger numerical aperture will provide better resolution. Oil immersion objectives resolution is 0.2 μm. Oil immersion objectives resolution is 0.2 μm. Magnification between 40X and 2000X Magnification between 40X and 2000X

25 Tools of the Laboratory Types of Light Microscopes Bright-field – most widely used; specimen is darker than surrounding field; live and preserved stained specimens Bright-field – most widely used; specimen is darker than surrounding field; live and preserved stained specimens Dark-field – brightly illuminated specimens surrounded by dark field; live and unstained specimens Dark-field – brightly illuminated specimens surrounded by dark field; live and unstained specimens Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

26 Tools of the Laboratory Fluorescence Microscope Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence Useful in diagnosing infections Useful in diagnosing infections Confocal

27 Tools of the Laboratory Electron Microscopy Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds 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. 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. Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. Magnification between 5,000X and 1,000,000X Magnification between 5,000X and 1,000,000X Transmission electron microscopes (TEM) Transmission electron microscopes (TEM) Scanning electron microscopes (SEM) Scanning electron microscopes (SEM)

28 Tools of the Laboratory Electron Microscopy Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. 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. 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. TEM SEM

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30 Tools of the Laboratory Probing Microscopes -> Scanning tunneling microscope (STM) -> Atomic force microscope (AFM) Carbon monoxide man -> STM

31 Tools of the Laboratory Staining Simple stains – one dye is used; reveals shape, size, and arrangement 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) Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: gram stain, acid-fast stain and endospore stain) Special stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains Special stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains

32 Microbial Control Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area Primary targets are microorganisms capable of causing infection or spoilage: Primary targets are microorganisms capable of causing infection or spoilage: vegetative bacterial cells and endosporesvegetative bacterial cells and endospores fungal hyphae and spores, yeastfungal hyphae and spores, yeast protozoan trophozoites and cystsprotozoan trophozoites and cysts wormsworms virusesviruses prionsprions

33 Microbial Control

34 Microbial Control Relative Resistance of Microbes Highest resistance Highest resistance bacterial endospores, prionsbacterial endospores, prions Moderate resistance Moderate resistance Pseudomonas sp.Pseudomonas sp. Mycobacterium tuberculosisMycobacterium tuberculosis Staphylococcus aureusStaphylococcus aureus protozoan cystsprotozoan cysts Least resistance Least resistance most bacterial vegetative cellsmost bacterial vegetative cells fungal spores and hyphae, yeastfungal spores and hyphae, yeast enveloped virusesenveloped viruses protozoan trophozoitesprotozoan trophozoites

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36 Microbial Control Terminology and Methods of Control Sterilization – a process that destroys all viable microbes, including viruses and endospores; microbicidal Sterilization – a process that destroys all viable microbes, including viruses and endospores; microbicidal Disinfection – a process to destroy vegetative pathogens, not endospores; inanimate objects Disinfection – a process to destroy vegetative pathogens, not endospores; inanimate objects Antiseptic – disinfectants applied directly to exposed body surfaces Antiseptic – disinfectants applied directly to exposed body surfaces Sanitization – any cleansing technique that mechanically removes microbes Sanitization – any cleansing technique that mechanically removes microbes Degermation – reduces the number of microbes Degermation – reduces the number of microbes

37 Microbial Control Microbial death Permanent loss of reproductive capability, even under optimum growth conditions Permanent loss of reproductive capability, even under optimum growth conditions

38 Microbial Control Factors that affect death rate The effectiveness of a particular agent is governed by several factors: Number of microbes Number of microbes Nature of microbes in the population Nature of microbes in the population Temperature and pH of environment Temperature and pH of environment Concentration or dosage of agent Concentration or dosage of agent Mode of action of the agent Mode of action of the agent Presence of solvents, organic matter, or inhibitors Presence of solvents, organic matter, or inhibitors

39 Microbial Control Factors that affect death rate

40 Microbial Control Methods of Physical Control 1. Heat – moist and dry 2. Cold temperatures 3. Desiccation (dry) 4. Radiation 5. Filtration

41 Microbial Control Methods of Physical Control Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins Dry heat – moderate to high temperatures; dehydration, alters protein structure; incineration Dry heat – moderate to high temperatures; dehydration, alters protein structure; incineration

42 Microbial Control Heat Resistance and Thermal Death Bacterial endospores most resistant – usually require temperatures above boiling Bacterial endospores most resistant – usually require temperatures above boiling

43 Microbial Control

44 Microbial Control Steam under pressure – sterilization Steam under pressure – sterilization Autoclave 15 psi/121 o C/10-40min Autoclave 15 psi/121 o C/10-40min Steam must reach surface of item being sterilized Steam must reach surface of item being sterilized Item must not be heat or moisture sensitive Item must not be heat or moisture sensitive Mode of action – denaturation of proteins, destruction of membranes and DNA Mode of action – denaturation of proteins, destruction of membranes and DNA

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46 Microbial Control Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value 63°C - 66°C for 30 minutes (batch method) 63°C - 66°C for 30 minutes (batch method) 71.6°C for 15 seconds (flash method) 71.6°C for 15 seconds (flash method) Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes

47 Microbial Control Dry heat using higher temperatures than moist heat Incineration – flame or electric heating coil Incineration – flame or electric heating coil ignites and reduces microbes and other substancesignites and reduces microbes and other substances Dry ovens – o C- coagulate proteins Dry ovens – o C- coagulate proteins

48 Microbial Control Ionizing radiation – deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA, Ionizing radiation – deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA, gamma rays, X-rays, cathode raysgamma rays, X-rays, cathode rays used to sterilize medical supplies and food productsused to sterilize medical supplies and food products

49 Microbial Control Filtration Physical removal of microbes by passing a gas or liquid through filter Physical removal of microbes by passing a gas or liquid through filter Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms

50 Microbial Control Microbes on a normal unwashed hand