MICR20010 Lecture 2 2023.pptx

MICR20010 Lecture 2 Culturing Microbes Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science Any questions? • Email module coordinator • Dr. Tadhg O Croinin • [email protected] • View intro lecture video on brightspace for all module info Lecture 1 • • • • Define...

MICR20010 Lecture 2 Culturing Microbes Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science Any questions? • Email module coordinator • Dr. Tadhg O Croinin • [email protected] • View intro lecture video on brightspace for all module info Lecture 1 • • • • Define Microbiology Different types of microbes Role and application of microbes Importance of microbiology in: – Agriculture – Food industry – Animal and plant health • Role of Microbiologists • History of Microbiology Learning Outcomes • • • • How to culture microbes Difficulties working with microbes Sterile growth media Handling microorganisms – – – – – Inoculation Incubation Isolation Inspection Identification • Disposal of cultures • Disinfectants and Antiseptics How to culture microbes? • During 1880s Scientists realised that the study of microorganisms would require ways to visualise and handle them. 1. 2. 3. 4. Preparation of sterile growth media Separating microbes from each other Growing microbes under controlled conditions Preparing specimens for microscopic examination Difficulties working with microbes • Most microbes exist in complex communities e.g. soil or human mouth – Individual bacterial species need to be isolated before they can be studied • Microbes need to be grown under artificial conditions • Microbes are invisible to the human eye – problems with contamination • Aseptic technique to prevent contamination Sterile Growth Media • Before handling and growing microbes sterile growth media is required • Bacteria are grown in a medium (latin for middle) containing nutrients • Medium can be liquid, water based (broth) or solid (agar Petri plates) Common methods of sterilising bacterial growth media • Boiling – 100OC for 30 mins - Kills cells • Autoclave – 121OC for 30 mins - Kills all cells and spores • Dry heat – 150OC for 120 mins - Kills all cells and spores Handling Microorganisms: • The five I’s ➊ Innoculation ➋ Incubation ➌ Isolation ➍ Inspection ➎ Identification Innoculation or producing a culture • To grow/cultivate/culture microbes, a tiny sample (the inoculum) is introduced or inoculated into nutrient medium, which provides an environment in which the organisms multiply. • Growth in nutrient broth can be observed as a cloudy suspension which is termed a culture. Nutrient agar provides a surface for colonies to develop. • The inoculum may be a clinical specimen e.g. blood; a soil sample, a water sample, a sewage sample, a food sample etc. Growth of bacteria on agar nutrient medium • AGAR: MELTS AT 100OC, SOLIDIFIES AT 40OC • STERILIZED BY AUTOCLAVING • BACTERIA GROW AS COLONIES • SINGLE COLONY PURIFICATION Single colony purification Each bacterial colony is derived from a single cell Single colony purification = Single bacterial cell purification Obtaining Pure Cultures from an Isolation Plate Growth of bacteria in liquid nutrient medium Bacterial growth Sterile Incubation • Microorganisms are grown in an incubator, which provides optimal temperature and gas content. An incubator speeds up the process of multiplication and production of a culture Isolation • Concept of separating cell from other cells and providing it with adequate nutrients and space to grow • The ability to grow microbes in pure form in essential in the study of their biology. • The nutrients required to grow bacteria in medium vary depending on the bacterial species. – Each microorganism has its own nutritional requirements. – Can be exploited in identifying microbes. – > 500 different media for growing bacteria – reflects bacterial diversity COLONIES OF BACTERIA Inspection • Examination of colonies to determine if culture is pure – Each colony forms from a single cell – therefore the colony is an isolated population of an individual bacterial species • The appearance of the colony is useful in identifying the bacterial species – Therefore in isolating a bacterial species, it is important that all the colonies appear the same • Different colony type means that the culture is not pure or is mixed. – If culture is contaminated or mixed, a single colony of the desired species is subcultured • In broth culture, not possible to determine if growth of more than one bacterial species has occurred. Identification • Macroscopic or colony morphology • Microscopic morphology • Biochemical characteristics • Genetic characteristics Disposal of cultures Sterilisation: Removal and destruction of all microbes in or on an object Physical Methods: •Heat - Moist Heat. Boiling water, flowing steam. - Cells & most viruses. Not spores • (Tyndallisation –intermittent boiling) – Steam (Autoclaving) 121oC - 15-30min. • All spores/viruses/cells. Media & equipment - Dry Heat (Hot air), 1 hour at 171oC, – Incineration (burning) 1 sec or more at 1000oC) How an autoclave works Disposal of cultures • Radiation – Ionising e.g. X rays, gamma rays, secs-hrs. OHradicals, damage to DNA. – Sterilize pharmaceuticals, medical supplies – Nonionising e.g. UV light. DNA damage. Operating theatres, kitchens Radiation sterilisation Westport Co 60, Cs 135 gamma rays Medial Products: sutures, gloves, gowns, face masks, dressing, syringes, surgical staplers Radura symbol to label irradiated food Chemical sterilisations methods Gaseous agents e.g. ethylene oxide Sterilisation service provider, Tullamore Ethylene oxide sterilisation EtO alkylates nucleic acids, proteins and enzymes causing cell death 12-24hour cycles Sterilizes a wide variety of delicate items that heat and moisture can damage Commonly used for surgical equipment in hospitals Gas chamber Safety: EtO is toxic and flammable Disinfection • Related process • Reduction in bioload including the removal of pathogens • Methods – Chemical – heat, e.g. pasteurisation – filtration • Often easier to achieve than sterilisation and adequate for instruments in contact with mucous membranes, e.g. endoscopes Pasteurisation: Use of heat (e.g. 75°C, 15 seconds) to kill pathogens and reduce the number of spoilage micro-organisms in food and beverages (milk, fruit juice, wine, beer). Balance between removing microbes and affecting taste or quality of product Antiseptics and disinfectants • Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane – should not be taken internally. • Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents. Antiseptics and disinfectants • Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. • Examples: chlorine, hypochlorites, chlorine compounds, copper sulfate, quaternary ammonium compounds. Antiseptics and disinfectants • Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink. Appropriate handwashing facilities Antiseptic hand wash Further Reading • Microbiology an Introduction, Tortora, Funke and Case 12th Ed. • Chapter 6 “Microbial Growth” • Chapter 7 “The control of Microbial Growth”