A221 Microbiology Lecture Notes - AY 2024 Sem 2 - PDF

A221 MICROBIOLOGY LESSON 01 Spontaneity LECTURE NOTES Module Chair: Won Choong Yun Last Updated: 23 September 2024 Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Interactive Seminar Time...

A221 MICROBIOLOGY LESSON 01 Spontaneity LECTURE NOTES Module Chair: Won Choong Yun Last Updated: 23 September 2024 Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Interactive Seminar Time Activity 8:30am – 9:45am Module story and ice breaker 9:45am – 10:30am Break 10:30am – 12:00pm Lecture Part I and Worksheet Activity Part I 12:00pm – 1:30pm Lunch break 1:30pm – 3:00pm Lecture Part II and Worksheet Activity Part II 3:00pm – 3:30pm PQ and discussion 3:30pm Class ends Copyright © 2024 by Republic Polytechnic, Singapore  LECTURE PART I Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE A 17th century recipe for spontaneous production of mice… Sweaty, Soiled clothes art_brownpot 21 days later Wheat grains https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Another 17th century recipe for spontaneous production of maggots… Days later For thousands of years, people believed that certain living things arose from “vital forces” present in non-living or decomposing matter. This ancient idea is known as “spontaneous generation” (i.e. abiogenesis). This theory was also used to explain phenomena such as food spoilage → https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Even after single-celled microorganisms were discovered during the late-1600s by Antonie van Leeuwenhoek (see video below), the idea of spontaneous generation persisted. Through van Leeuwenhoek’s Eyes: Microbiology in a Nutshell (2:02): https://www.youtube.com/watch?v=lU9duPteXcg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Two Hypotheses on the Origin of Life Over the subsequent 200 years, scientists battled over the two hypotheses for the origin of simple life forms: kair2.JPG Abiogenesis Biogenesis: (i.e. spontaneous generation): Living things arise from Living things arise from living things of their non-living things same kind Here are some of the key experiments documented from this period… https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Francesco Redi 1668 experiment Experimental setup: Open jar Sealed jar Gauze-covered jar Observations: Flies entered the jar, No flies, eggs or No flies entered the jar, but they laid laid eggs that hatched maggots on the eggs on the gauze that hatched maggots on the meat meat maggots, or eggs fell through the gauze and hatched maggots on the meat https://www.britannica.com/science/spontaneous-generation Conclusion: The presence of maggots on the meat did NOT result from spontaneous generation but from _the eggs laid by the files._________. https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE John Needham 1745 experiment Experimental setup: Sealed Days later Microbial growth Chicken and corn Broth turns broth cloudy Briefly boiled broth https://www.timetoast.com/timelines/history-of-the-spontaneous-generation Conclusion: The presence of microbial growth in the broth resulted from spontaneous generation https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Louis Pasteur 1859 experiment Experimental setup: Broth in the flasks was boiled for 1hr and allowed to cool. Open S-shaped Open flask Cotton plugged flask neck flask Beef broth Observations: Broth turns Broth remains Broth remains cloudy clear clear https://www.britannica.com/science/spontaneous-generation Conclusion: Spoilage of the broth did NOT result from spontaneous generation but from the growth of microorganisms (present in the surrounding air) https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Louis Pasteur 1859 experiment (continued) Experimental setup: Broth turns cloudy Conclusion: Spoilage of the broth did NOT result from spontaneous generation but from the growth of microorganisms (from the surrounding air) that had settled in the bend of the S-shaped neck. https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How did Louis Pasteur prove that spontaneous generation did not occur? ▪ Boiling of broth rendered solution “sterile” (i.e. completely free of all living microbial life forms) and evacuated the air in the flask, hence the broth stays clear. ▪ Tipping of flask to allow broth to come into contact with contaminants (i.e. undesired microorganisms) and resulted in a cloudy broth (evidence of microbial growth). ▪ Thus, a source of live microbes is required for the proliferation of the microbes in broth. spontaneous generation https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE John Tyndall 1877 experiment Experimental setup: Hay infusion in the flask was boiled for 1hr and allowed to cool Does this mean that Pasteur was wrong to debunk spontaneous generation? Hay infusion https://sciencelessonsthatrock.c om/setting-up-a-hay-infusion- for-your-microscope-unit-html/ Observations: Hay infusion is made by steeping hay (which provides sugars for microbial growth) in water collected from a pond, lake, stream, or Hay infusion remains clear Hay infusion turns cloudy puddle. (absence of microbial growth) (presence of microbial growth) Conclusion: Microorganisms exists in two forms - one that is sensitive to heat and one that is resistant to boiling https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Discovery of bacterial endospores Tyndallisation was developed to eliminate the heat-resistant microbes. Day 1 Day 2 Day 3 Hay infusion with heat-resistant microbes 37C for 24hrs 37C for 24hrs Sterilised hay infusion Boil/ steam Boil/ steam Boil/ steam for 30-60mins for 30-60mins for 30-60mins In the same year, Ferdinand Cohn endospores discovered that Bacillus subtilis forms endospore (i.e. the heat-resistant form of bacteria) by detailed microscopy studies. What do we know about microorganisms today? https://doi.org/10.1111/j.1574-6976.2000.tb00540.x https://commons.wikimedia.org/wiki/File:Jan_Antoon_Garemijn_-_The_Pandreitje_in_Bruges_-_WGA08468.jpg Copyright © 2024 by Republic Polytechnic, Singapore  WORKSHEET ACTIVITY PART I Copyright © 2024 by Republic Polytechnic, Singapore  LECTURE PART II Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What could have caused food spoilage? Primarily microorganisms (i.e. microbes) ▪ Organisms of microscopic size (i.e. too small to be seen by naked eye) or submicroscopic size (i.e. too small to be seen under light microscope) ▪ Mostly unicellular such as ▪ Bacteria (Prokaryote, e.g. Escherichia coli) ▪ Yeasts (Eukaryote, e.g. Saccharomyces cerevisiae) ▪ Some are multicellular such as To cover in L08 ▪ Moulds (Eukaryote, e.g. Rhizopus stolonifer) Bacteria exist in various shapes Budding yeasts Common bread mould Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How are microorganisms classified? – 3-domain classification system Focus for Prokaryotic cells ▪ Living organisms are classified Lesson 1-7 in a three-domain classification system based on nucleotide sequences in rRNA. ▪ Members of the Domain of Eukarya are eukaryotic cells. ▪ Members of the Domain of Bacteria and Archaea are prokaryotic cells. ▪ Bacteria differ from Archaea in numerous ways (e.g. rRNA sequences, cell wall and cell membrane composition). Berry Chess. Talaro's Foundations in Microbiology. (Edition 12) Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How are microorganisms named? ▪ Binomial nomenclature is a formal way of naming species of living things by giving each a scientific name composed of two parts. ▪ It is a universal language for scientists to use when classifying organisms. ▪ The scientific name of each organism is a combination of: ˃ Generic (genus)  First letter of the genus is always capitalised ˃ Specific (species)  Species name begins with a lower-case letter ▪ When typed, the scientific names are always italicized. Escherichia coli or E. coli Homo sapiens ▪ When written, the scientific names are always underlined separately. Escherichia coli or E. coli Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What are the structure and functions of bacterial cells? ▪ Bacteria are prokaryotic cells that have no nucleus Prokaryotic cell structure and function (3.12): https://www.youtube.com/watch?v=jpGMbcSbb0E Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What is the size range of bacteria? ▪ Most bacterial cells fall between ___1µ-10µm_______ in size. Microorganisms Size Comparison (2.20): https://www.youtube.com/watch?v=h0xTKxbIElU&t=13s Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How do bacteria reproduce? ▪ Bacteria reproduce via a process called binary fission and can cause food spoilage. Binary Fission (2.19): https://www.youtube.com/watch?v=u5UyBrgVsvs Copyright © 2024 by Republic Polytechnic, Singapore https://doi.org/10.1002/adma.202300875  OFFICIAL (CLOSED) \ NON-SENSITIVE Why did bacterial growth still occur after boiling? – Recall John Tyndall’s 1877 experiment Experimental setup: Hay Experimental setup: Hay infusion infusion in in the the flask flask was was boiled boiled for for 1hr 1hr and and allowed allowed to to cool cool Hay infusion that Hay infusion does NOT contain that contains endospores endospores Hay infusion Observations: Observations: ▪ The natural habitat for spore-forming bacteria is found in the soil Hay infusion Hay infusion remains clear turns cloudy ▪ Since hay is grown in soil and the sugars (absence of (presence of in hay provide an excellent medium for microbial Hay growth) infusion remains clear microbial Hay growth) infusion turns cloudy bacterial growth, endospores are (absence of microbial growth) (presence of microbial growth) commonly found in hay infusions. Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What are endospores? ▪ Some bacterial species (e.g. Bacillus anthracis and Clostridium tetani) are able to form endospores during harsh environmental conditions such as lack of nutrients ▪ Endospores are extremely resilient to harsh conditions e.g. boiling temperatures, radiation, disinfectants and desiccation, that would easily kill vegetative cells Released endospore (green) Endospores (green) Endospore Vegetative cell Vegetative cell (red) https://doi.org/10.1016/j.idcr.2022.e01416 Clostridium sp. Bacillus subtilis (after endospore staining was performed) Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How are endospores formed? Endospore formation (1:30): https://www.youtube.com/watch?v=NAcowliknPs Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE Sporulation versus germination The most common Why don’t bacteria trigger of sporulation remain in their is nutrient deprivation “resistant form” Sporulation indefinitely? (6-12hrs in most spore-forming Vegetative cell species) (1.5hr in most spore- forming species) Endospore Germination Resistant to harsh Susceptible to harsh environmental conditions environmental conditions Metabolically Favorable Metabolically active inactive/dormant growth Able to multiply Unable to multiply condition Berry Chess. Talaro's Foundations in Microbiology. (Edition 12) Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What makes endospores so resilient? ▪ Endospores are resistant to heat, freezing, desiccation, radiation and many chemicals (i.e. acids, bases, alcohol). ▪ Exosporium (forms the outermost layer of some endospores) play roles in protection, adhesion and spore virulence. ▪ The thick cortex and spore coat surrounding Berry Chess. Talaro's Foundations in the spore provide protection against radiation Microbiology. (Edition 12) and chemicals. ▪ The deposition of calcium dipicolinate in the endospore removes water and dehydrates the endospore, which makes it resistant to high temperature. ▪ There are also production of special proteins in the core of the endospores that protect its DNA from chemicals. How then do we ▪ Boiling for 5 hours is insufficient to kill endospores. achieve sterility? Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE How does Tyndallisation kill endospores and achieve sterility? Day 1 Day 2 Day 3 Hay infusion with endospores 37C for 24hrs 37C for 24hrs Sterilised hay infusion Boil/ steam Boil/ steam Boil/ steam for 30-60mins for 30-60mins for 30-60mins ▪ Heat treatment (i.e. boiling or exposure to steam) for 30-60 mins kills heat-sensitive vegetative cells. ▪ However, heat treatment is repeated for a total of 3 times with about 24hr incubations at 37C between the heat treatments. ▪ The incubations allow remaining endospores to germinate into vegetative cells, which will be killed upon subsequent heat treatment. Is there a faster way to achieve sterility? Copyright © 2024 by Republic Polytechnic, Singapore  WORKSHEET ACTIVITY PART II Copyright © 2024 by Republic Polytechnic, Singapore  OFFICIAL (CLOSED) \ NON-SENSITIVE What have you learnt today? ▪ Differentiate between biogenesis and abiogenesis ▪ Define spontaneous generation, biogenesis, and abiogenesis ▪ Explain the differences between biogenesis and abiogenesis ▪ Identify the contributions to microbiology made by Pasteur and his experiment to debunk the theory of spontaneous generation. ▪ Explain the rationale behind why each experiment was conducted, and what each experimental set up was trying to prove. ▪ Explain how Pasteur’s experiments supported the theory of biogenesis ▪ Justify why not all bacteria are killed when heated to high temperatures ▪ State what an endospore is, and explain how it helps some bacteria to adapt to harsh conditions ▪ Explain that bacteria exist in various shapes ▪ List and identify the different shapes that bacteria can be found in ▪ Give examples of bacteria (such as Bacillus) that may exist in the form of endospores ▪ Explain how binomial nomenclature is used in the naming of microorganisms ▪ Describe how the system of binomial nomenclature uses genus and specific epithet names ▪ Differentiate between genus and species Copyright © 2024 by Republic Polytechnic, Singapore