M1L4 - Growth, Isolation and Cultivation and Deactivation of Microorganisms.pdf

GROWTH, ISOLATION, CULTIVATION, & CONTROL/DESTRUCTION OF MICROORGANISMS https://th.bing.com/th/id/R.627fc7f486931eedf7fd6a262cd4d6bd?rik=G1ROIdR0SWKzFw&riu=http%3a%2f%2fwww.imperial.ac.uk%2fImageCropToolT4%2fimageTool%2fuploaded-images%2fMicrobiology--tojpeg_1493806871478_x2.jpg&e...

GROWTH, ISOLATION, CULTIVATION, & CONTROL/DESTRUCTION OF MICROORGANISMS https://th.bing.com/th/id/R.627fc7f486931eedf7fd6a262cd4d6bd?rik=G1ROIdR0SWKzFw&riu=http%3a%2f%2fwww.imperial.ac.uk%2fImageCropToolT4%2fimageTool%2fuploaded-images%2fMicrobiology--tojpeg_1493806871478_x2.jpg&ehk=ZnVYYeEMWKzfUZfATHLo5zA7Z39ns55ZTe%2fzZG7eQyU%3d&risl=&pid=ImgRaw&r=0 Bacterial Growth Growth of Microorganisms (Bacteria) BACTERIAL GROWTH ▪ In multicellular organisms: growth is manifested by an increase in size and mass. ▪ In unicellular microorganisms (e.g. bacteria): growth is usually defined as the increase in number and mass. ▪ Most bacteria and archaea are haploid and reproduce via binary fission. Phases of Bacterial Growth Growth of Microorganisms (Bacteria) Binary Fission in Prokaryotes 1. In the initial step of binary fission, the DNA replicates or copies its chromosome. 2. Next, each copy of the DNA is attached to a separate portion in the cell. 3. In the next step, the cell elongates to approximately twice its average cell length and since the DNA is attached to different locations of the cell, they separate from one another. 4. At this point, a pinch is formed at the center, creating a septum that serves as a separation wall between the 2 compartments of the cell. 5. Finally, the cell splits into new daughter cells, each having a copy of the DNA  Factors Affecting Growth Growth of Microorganisms (Bacteria) FACTORS AFFECTING GROWTH a) Nutrients – carbon, nitrogen + other macro- and micronutrients b) Temperature – psychrophiles, mesophiles, thermophiles, hyperthermophiles c) Moisture – availability of water (water activity, Aw) d) Aeration – different oxygen requirements e) pH - expression of acidity or alkalinity https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.04%3A_Temperature_and_Microbial_Growth Phases of Bacterial Growth Growth of Microorganisms (Bacteria) Phases of Bacterial Growth: The Growth Curve During cell division, a cell reproduces to form 2 cells so that for each generation, both the cell number and cell mass doubles. https://microbiologynotes.org/bacterial-growth-curve-definition-phases-and-measurement/ Measurement of Growth Growth of Microorganisms (Bacteria) MEASUREMENT OF GROWTH: Standard Plate Count (SPC) ✓ In this method, viable cells are counted, or those cells that can divide and reproduce. ✓ It is called standard plate count because agar plates are needed for this method. ✓ Assumption: one viable cell will give rise to one colony that will grow on an agar plate = no. of colonies reflects the cell no. Advantage: can determine # of living cells Disadvantage: underestimation of cell population https://pharmacyscope.com/methods-of-isolation-of-pure-culture/ Serial Dilution Growth of Microorganisms (Bacteria) SERIAL DILUTION A turbid bacterial broth contains millions of bacteria thus, when it is plated it will only produce a lawn of bacterial growth. Serial dilution is done initially to “thin out” or decrease the concentration of cells so that A dilution is prepared by adding a specific volume or weight of distinct colonies can be sample to a volume of sterile diluent such as water, saline water or phosphate buffer to dilute or make the bacterial counted after plating. suspension less concentrated than the original sample. Series of tubes become less concentrated. https://chemistnotes.com/biochemistry/isolation-of-bacteria/ Plating Methods Growth of Microorganisms (Bacteria) PLATING METHODS After serial dilution, plating technique can be done either by pour-plate or spread-plate method: a. In pour-plate method, 0.1 to 1 milliliter of culture is pipetted to a sterile petri plate. Molten agar medium at around 50 degrees Celsius is poured onto the plate and is mixed gently before the agar solidifies. b. In spread-plate method, 0.1 milliliter of culture is pipetted on the surface of an agar plate. The aliquot will be spread on the surface of the agar plate using a sterile glass spreader or L-rod.  Plating Methods Growth of Microorganisms (Bacteria) For both methods, the number of colonies that grow on the plates should not be too many or too few. If colonies are too many, colonies may fuse with one another leading to erroneous counts. If colonies are too few, the statistical significance will be low. An example of agar plates with colonies Valid no. of colonies in the for a standard plate count. plate count method: 25 to 250 colonies https://microbenotes.com/plate-count-agar-pca/ Computation Growth of Microorganisms (Bacteria) To compute for the colony-forming units or CFU per ml, the product of the average number of colonies and dilution factor is divided by the volume plated in ml. DF or dilution factor is equal to 1 divided by the dilution. Sample Computation Growth of Microorganisms (Bacteria) SAMPLE COMPUTATION Steps Isolation & Cultivation of Microorganisms Definition of Terms Isolation & Cultivation of Microorganisms PURE CULTURE (AXENIC CULTURE) a culture which contains a single species of microorganism a population of cells arising from a single cell to study a single type of microorganism to characterize an individual species Isolation Techniques Isolation & Cultivation of Microorganisms STEPS IN PREPARING PURE CULTURES Isolation Transfer desired colony to a slant or stab Verify the purity (Gram staining) Microscopy (morphological characteristics) Re-streak on agar medium Physiological and biochemical tests Make stock cultures (preservation Definition of Terms Isolation & Cultivation of Microorganisms CULTIVATION NUTRIENTS -increasing the population of - extracellular substances which microorganisms by providing provide the cell with materials for their nutritional and physical building protoplasm and for energy requirements generation CULTURE MEDIUM - any nutrient material for growth and cultivation of microorganisms in the laboratory  Culture Media Isolation & Cultivation of Microorganisms USES OF CULTURE MEDIUM ✓ for growth and maintenance of microbial cultures ✓ to favor the production of particular compounds ✓ to study microbial action on some https://microbeonline.com/types-of-bacteriological-culture-medium/ constituents of the medium Culture Media Isolation & Cultivation of Microorganisms Nutrient materials for growing and cultivating microorganisms Must contain essential elements needed by the microorganisms C, H, O, N, P, S – basic elements in the cell for biosynthesis and energy production Ingredients vary from one type of media to another. pH is properly adjusted since different microorganisms have different pH requirements Culture Media Isolation & Cultivation of Microorganisms Almost all solid media has agar as its component Solidifying agent with no nutritive value Very few microorganisms can degrade agar Melts at 100°C, solidifies at 40°C Image from: https://www.indiamart.com/proddetail/agar-agar- powder-bacteriological-grade-18746755488.html Types of Culture Media Isolation & Cultivation of Microorganisms 1. Based on composition Synthetic – all components + their conc. are known Complex – made from digests of microbial, animal, or plant products; nutritional composition is not known precisely Types of Culture Media Isolation & Cultivation of Microorganisms Types of Culture Media Isolation & Cultivation of Microorganisms 2. Based on consistency Image from: https://www.jlindquist.com/generalmicro/dfnewthiopage.html Liquid (Broth) – no solidifying agent Semi-solid – 0.5% - 0.7% solidifying agent Solid – 1.5% - 2.0% solidifying agent (agar/gelatin) Types of Culture Media Isolation & Cultivation of Microorganisms 3. Based on purpose/use a. General Purpose - can support most or almost all types of species e.g. Nutrient Agar (NA) , Potato Dextrose Agar (PDA) b. Differential - distinguishes one type of bacteria from another; with special reagents like pH indicators or dyes e.g. Eosin Methylene Blue Agar (EMBA) c. Selective - allows the growth of a specific type of microorganism only; with selective agents (ex. salts, dyes, antibiotics, etc.) e.g. Bacillus Cereus Agar (BCA) d. Enrichment - used to increase the number of microorganisms with unusual physiological characteristics; with special nutrients (ex. blood) e.g. Blood Agar e. Assay - of prescribed composition used for assay of vitamins, amino acids and antibiotics; used to determine qualitative/quantitative production of such a compound by an organism Types of Culture Media Isolation & Cultivation of Microorganisms 3. Based on purpose/use Image from: https://orbitbiotech.com/mannitol-salt-agar/ Image from: https://orbitbiotech.com/blood-agar-%CE%B1-hemolysis-%CE%B2-hemolysis-%CE%B3- Image from: https://microbiologyinfo.com/citrate- Image from: https://microbiologie-clinique.com/nutrient-broth.html hemolysis-alpha-prime-wide-zone-alpha-hemolysis-composition-procedure-usus/ utilization-test-principle-media-procedure-and-result/ Media Preparation Isolation & Cultivation of Microorganisms Guide for the Determination of Total Volume Media Preparation Isolation & Cultivation of Microorganisms Example: Prepare 10 plates (maximum vol.), 10 big stabs, and 10 small slants of Nutrient Agar (NA). How much NA (in ml) should be prepared? 10 plates x 20ml/plate = 200ml 10 big stabs x 10ml/big stab = 100ml 10 small slants x 3ml/small slant = 30ml TOTAL VOL. TO BE PREPARED: 330ml NA Media Preparation Isolation & Cultivation of Microorganisms How much of each component (in ml) should be prepared to make 330ml NA? Isolation Techniques Isolation & Cultivation of Microorganisms ASEPTIC TRANSFER/TECHNIQUE Isolation Techniques Isolation & Cultivation of Microorganisms ASEPTIC TRANSFER/TECHNIQUE Isolation Techniques Isolation & Cultivation of Microorganisms Isolation Techniques Isolation & Cultivation of Microorganisms STREAK PLATING - The inoculum is streaked over the agar goal: to “thin out” the population - Usually, by the third or fourth quadrant, only a few organisms are transferred which will give discrete colony forming units (CFUs). - Surface colonies formed Isolation Techniques Isolation & Cultivation of Microorganisms https://www.youtube.com/watch?v=hHk0dRuNr_U Isolation Techniques Isolation & Cultivation of Microorganisms SPREAD AND POUR PLATING Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Objective to retain the viability of the stock culture for a long period of time while maintaining its purity Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Advantages: 1. Simple; some growth can be removed under the oil 2. Still preserved even after the removal Disadvantages: 1. Viability of mcgs varies with species 2. Viable for 1-3 years only Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Advantages: 1. Long term survival 2. Characteristics of cultures are less likely to change 3. Smallness of storage containers Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION: LYOPHILIZATION Lyophilization (youtube.com) Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION Culture Preservation Isolation & Cultivation of Microorganisms CULTURE PRESERVATION: IN LIQUID NITROGEN A Look inside the Lab: Liquid Nitrogen Freezer - Vaccine Makers Project (youtube.com) Importance Control and Destruction of Microorganisms Why control growth of microorganisms? Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Steam Sterilizer Operation - YouTube Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Agents Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms https://www.fishersci.com/shop/products/emd-millipore- millex-sterile-syringe-filters-pes-membrane-green-4/p-114965 https://www.ilabot.com/cleanroom-equipment/biosafety-cabinet-class-2.html Methods Control and Destruction of Microorganisms Working principle of Laminar Air Flow & Biosafety Cabinets - YouTube Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Introduction to Spray Drying - YouTube Methods Control and Destruction of Microorganisms 3W Drying single drum dryer (youtube.com) Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms https://www.worldhealth.net/news/one-step-towards- daily-use-deep-uv-light-source-sterilization-and- disinfection/  Methods Control and Destruction of Microorganisms https://www.worldhealth.net/news/one-step-towards-daily-use- deep-uv-light-source-sterilization-and-disinfection/ https://thatsinteresting.scienceblog.com/2013/08/09/ive-got-uv-under-my-skin/ Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms WHY? Bactericidal but not sporicidal Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Methods Control and Destruction of Microorganisms Is it recommended to apply hydrogen peroxide to open wounds? - NO. Rather, it is detrimental and prevents the healing process!! - It also kills normal cells within the wound.  Methods Control and Destruction of Microorganisms Some Factors Affecting the Antimicrobial Activity of Biocides Biocide = A chemical or physical agent, usually broad spectrum, that inactivates microorganisms Factors inherent to the biocide: Factors inherent to the cell: a. Concentration a. Presence of biofilm b. Contact time b. Type of microorganisms c. Formulation c. Number of microorganisms d. Temperature  Methods Control and Destruction of Microorganisms ANSWERS TO QUESTIONS: 1. What is the proper usage of hydrogen peroxide? a. Cleaning the dishwasher, kitchen sink, counters and cutting boards b. Washing vegetables c. Deep cleaning the toilet d. Shining mirrors and glass surfaces e. Whitening old porcelain f. Removing stains from clothes g. Treat plants with fungal infection h. Cleaning out the litter box i. Cleaning makeup brushes j. Whitening the teeth https://www.healthline.com/health/hydrogen-peroxide-uses  Methods Control and Destruction of Microorganisms ANSWERS TO QUESTIONS: 2. What are the proper brushing techniques? How To Properly Brush Your Teeth (The RIGHT Way!) (youtube.com)  Methods Control and Destruction of Microorganisms ANSWERS TO QUESTIONS: 3. How do soaps work? How Soap Works - 3D Animation (youtube.com)  Methods Control and Destruction of Microorganisms ANSWERS TO QUESTIONS: 4. What happens when fluoride is ingested? Fluoride in Water - The Good, The Bad & The Ugly (youtube.com)