Microbiology Workshop

1. Microbiology Workshop 1st lecture: Chapter 3 + 11

2. Microbiology Workshop 1st lecture: Chapter 3 + part of 11 (Methods studying microorganisms+ Agents for microbial control) 2nd Lecture: Chapter 7 (Microbial growth) 3rd Lecture: Chapter 8 (microbial metabolism) Book: Foundations in Microbiology, Basic Principles, 6th (7th) edition, by Talaro, McGraw-Hill

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. Tools of the Laboratory 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. • A colony consists of one species.

10. Tools of the Laboratory Methods for Isolation of Bacteria • Isolation techniques include: • streak plate technique • pour plate technique • spread plate technique

11. Tools of the Laboratory Media -> Providing nutrients -> Media can be classified according to three properties: • Physical state – liquid, semisolid and solid • Chemical composition – synthetic (chemically defined) and nonsynthetic (complex) • 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 • Semisolid – clot-like consistency; contains solidifying agent (agar or gelatin) • Solid – firm surface for colony formation -> nutrient agar • contains solidifying agent • liquefiable and nonliquefiable

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

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

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 growth 2. Inspection – observation; macroscopic and microscopic • pure culture – grows only single known species of microorganisms • mixed cultures – hold two or more identified species or microorganisms • contaminated 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 sterilization • 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 • 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 – refraction • Depending on the size and curvature of the lens, the image appears enlarged. • extent 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 • function of wavelength of light that forms the image along with characteristics of objectives • Visible light wavelength is 400 nm – 750 nm. • Numerical aperture of lens ranges from 0.1 to 1.25. • Oil immersion lens requires the use of oil to prevent refractive loss of light. • Shorter wavelength and larger numerical aperture will provide better resolution. • Oil immersion objectives resolution is 0.2 μm. • 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 • 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

26. Tools of the Laboratory Fluorescence Microscope • 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 • 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 • 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 • Transmission electron microscopes (TEM) • 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. • 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

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 • 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

32. Microbial Control • 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: • vegetative bacterial cells and endospores • fungal hyphae and spores, yeast • protozoan trophozoites and cysts • worms • viruses • prions

33. Microbial Control

34. Microbial Control Relative Resistance of Microbes • Highest resistance • bacterial endospores, prions • Moderate resistance • Pseudomonas sp. • Mycobacterium tuberculosis • Staphylococcus aureus • protozoan cysts • Least resistance • most bacterial vegetative cells • fungal spores and hyphae, yeast • enveloped viruses • protozoan trophozoites

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

37. Microbial Control Microbial death • 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 • Nature of microbes in the population • Temperature and pH of environment • Concentration or dosage of agent • Mode of action of the agent • Presence of solvents, organic matter, or inhibitors

39. Microbial Control Factors that affect death rate

40. Microbial Control Methods of Physical Control • Heat – moist and dry • Cold temperatures • Desiccation (dry) • Radiation • Filtration

41. Microbial Control Methods of Physical Control • Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins • 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

43. Microbial Control

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

46. Microbial Control • 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) • 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

47. Microbial Control Dry heat using higher temperatures than moist heat • Incineration – flame or electric heating coil • ignites and reduces microbes and other substances • Dry ovens – 150-180oC- coagulate proteins

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

49. Microbial Control Filtration • 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

50. Microbial Control Microbes on a normal unwashed hand