Isolation and Cultivation of Microorganisms PDF

CHAPTER 3: Isolation & Cultivation of Microorganisms Prepared by: Domini Theresa M. Pecundo, MSc Expected Learning Outcomes At the end of this chapter, you should be able to: 1. Enumerate and describe the types and applications of culture media; 2. Compare varying isolation techniques and explai...

CHAPTER 3: Isolation & Cultivation of Microorganisms Prepared by: Domini Theresa M. Pecundo, MSc Expected Learning Outcomes At the end of this chapter, you should be able to: 1. Enumerate and describe the types and applications of culture media; 2. Compare varying isolation techniques and explain the principles behind each; 3. Describe basic culture preservation techniques; and 4. Explain the importance of culture collections and list examples.  Topics 1 Culture media 2 Isolation techniques 3 Preservation techniques 4 Culture Collections Definition of terms https://www.asmscience.org/content/education/imagegallery/image.3069 Pure Culture (Axenic Culture) a culture which contains a single species of microorganism a population of cells arising from a single cell  Definition of terms https://www.atcc.org/~/media/PDFs/webinars/Presen tations/2017/Best%20Practices%20Microbiology.ashx Cultivation increasing the population of microorganisms by providing their nutritional and physical requirements Definition of terms Nutrients extracellular substances which provide the cell with materials for building protoplasm and for energy generation https://www.atcc.org/en/Documents/Marketing_Literature/Bacterial_Culture_Guide/Growth_Media.aspx Definition of terms Culture Medium any nutrient material for growth and cultivation of microorganisms in the laboratory https://microbiologyinfo.com/list-of-culture-media-used-in-microbiology-with-their-uses/ 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://www.sciencephoto.com/media/723037/view/bacterial-growth-on-culture-media constituents of the medium Types of culture media According to physical state: Liquid (Broth) – no solidifying agent Solidifying agent: Semi-solid – 0.1 - 0.5% solidifying agent agar or gelatin Solid – 1.5 – 2.0% solidifying agent https://www.labtestsguide.com/culture Types of culture media https://www.researchgate.net/figure/Conformation-of- http://www.biomerieux-culturemedia.com/product/36-gardnerella-agar Helicobacter-pylori-through-urease-test_fig3_318021754 LIQUID SOLID Types of culture media https://quizlet.com/340178083/motility-test-flash-cards/ SEMI SOLID Types of culture media According to chemical composition: Synthetic – all components are chemically defined Complex – not all components are chemically defined e.g. potato infusion (plant origin) beef extract (animal origin) yeast extract (microbial origin) https://www.thermofisher.com/blog/food/a-colorful-future-for-culture-media/  Types of culture media https://www.amazon.com/Laboratory-Grade- https://alphabiosciences.com/beef-extract-b02-104/ https://www.ddbiolab.com/frontoffice/article/670929 Bacteriological-Agar-Powder-100g/dp/B0731X58Y3 Types of culture media According to principal function, purpose or application: General Purpose can support most or almost all types of species e.g. Nutrient Agar (NA), Potato Dextrose Agar (PDA) https://www.thomassci.com/scientific- https://www.3m.com/3M/en_US/company-us/all-3m-products/~/3M- supplies/Nutrient-Agar-Plates Potato-Dextrose-Agar/?N=5002385+3292766879&rt=rud Types of culture media Potato Dextrose Agar for enumeration of molds http://www.moldbacteriaconsulting.com/fungi/mould-aspergillus-how- https://www.rrnursingschool.biz/microbiology/info-xqv.html does-it-affect.html Aspergillus flavus Penicillium chrysogenum  Types of culture media According to principal function, purpose or application: Differential distinguishes one type of bacteria from another with special reagents like pH indicators or dyes e.g. Eosin Methylene Blue Agar (EMBA) https://www.dreamstime.com/stock-photo-black-bacterial-colonies-salmonella- species-salmonella-shi-shigella-agar-ss-agar-selective-differential-medium-plate- image81636361 Types of culture media https://microbeonline.com/nutrient-agar-composition-preparation-uses/ https://www.pinterest.ph/pin/37436240625365577/ E. coli on NA E.coli on EMBA Types of culture media http://www.medical-labs.net/thiosulphate-citrate-bile- https://quizlet.com/126981642/media-flash-cards/ / salts-sucrose-agar-tcbs-agar-2422 Spirit blue agar TCBS agar (left - lipase, right + lipase) Types of culture media https://jcm.asm.org/content/43/11/5768/figures-only  Types of culture media According to principal function, purpose or application: 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) https://microbeonline.com/types-of-bacteriological-culture-medium/ Types of culture media Bacillus Cereus Agar https://www.thermofisher.com/blog/food/bacillus-cereus-selective-media-for-unpasteurized-food-samples/ Types of culture media According to principal function, purpose or https://www.fishersci.co.uk/shop/products/chocolate-agar-vitox/13245359 application: Enrichment used to increase the number of microorganisms with unusual physiological characteristics with special nutrients (ex. blood) e.g. Blood Agar Types of culture media https://microbenotes.com/hemolysis-of-streptococci/ Blood Agar Types of culture media According to principal function, purpose or application: 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 https://www.mdpi.com/2073-4344/9/7/608/htm Types of culture media https://microdok.com/methyl-red-test/ https://slideplayer.com/slide/4691873/ Fermentation media Types of culture media Triple Sugar Iron Medium Types of culture media Antibiotic sensitivity testing http://www.shreechamundeswariceramic.com/uosjh/disk-diffusion-method.html Using Disk Diffusion Assay Isolation Techniques 1. Plating Colony - a macroscopically visible (surface or subsurface) growth or cluster of microorganisms on a solid medium https://www.allposters.com/-sp/Escherichia-Coli-Bacteria-Colonies-on- Macconkey-Agar-Culture-Dish-Lactose-Fermenters-are-Red- Posters_i6016226_.htm Isolation Techniques Types of Plating https://present5.com/bacterial-culture-methods-by-konrad-t-juszkiewicz-md/ Streak plating https://homepages.wmich.edu/~rossbach/bios312/L https://paramedicsworld.com/microbiology-practicals/streak- abProcedures/Streak%20plate%20procedure.html plate-culture-method-isolation-bacteria-microorganism-pure- culture/medical-paramedical-studynotes Isolation Techniques https://www.quora.com/What-is-the-difference-between-the-pour-plate-method-and-the-spread-plate-method-in-isolation-of-bacterial-colonies  Isolation Techniques 2. Enrichment Culture isolation of specific types of microorganisms by a combination of nutrient and physical conditions used for the isolation of unusual physiological types of microorganisms which are https://www.medixcorp.com/catalog/detail.asp?Item_id=6783 present in small numbers and which grow slowly Isolation Techniques Enrichment Culture https://www.reddit.com/r/dankmemes/comments/8odhig/cultural_enrichment_creates_colonies_its_just/  Isolation Techniques 3. Serial Dilution used if the desired microorganism is present at a higher level than any other microorganism https://www.researchgate.net/figure/Schematic-diagram-representing-the-serial-dilution-and-plating-process-adapted-from_fig9_304901960 Isolation Techniques https://www.researchgate.net/figure/Schematic-diagram-representing-the-serial-dilution-and-plating-process-adapted-from_fig9_304901960 Serial dilution and plating  Isolation Techniques Serial dilution and plating http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/lab6enumeration.html  Isolation Techniques Isolation Techniques 4. Single-Cell Isolation Technique uses a micropipette or a microprobe to physically pick a single cell and transfer it on an agar medium https://v3.pubpub.org/pub/fiber-microprobe-for-selective-electroporation Isolation Techniques Isolation Techniques 5. Membrane Filter Technique for samples with low population uses a sterile membrane filter having a pore size that retains microorganism https://www.slideshare.net/HamaNabaz/water-microbiology-21923074 Isolation Techniques https://www.vtei.cz/en/tag/total-coliforms/ https://www.it4ip.be/product-portfolio/ipblack/ Steps in Preparing Pure Cultures Isolation Transfer desired colony to a slant or stab Verify the purity microscope re-streak on agar medium physiological and biochemical test Make stock cultures Culture Preservation https://www.atcc.org/~/media/PDFs/webinars/Presentations/2017/Best% 20Practices%20Microbiology.ashx Objective to retain the viability of the stock culture for a long period of time while maintaining its purity Culture Preservation Methods 1. Periodic transfer to fresh media Considerations time interval of transfers proper medium proper storage temperature https://www.dreamstime.com/photos-images/slant-agar.html Culture Preservation Methods 2. Overlaying cultures with mineral oil http://www.mut.unito.it/en/Collezione https://www.wikiwand.com/en/Mineral_oil Aim: limit the availability of O2 reduced metabolic rate Culture Preservation Methods 2. Overlaying cultures with mineral oil Advantages Simple enables one to remove some growth under the oil and inoculate it in a fresh medium and still preserve the initial culture https://www.mcguffmedical.com/mineral-oil-heavy-16-ounce-bottle Culture Preservation Methods 2. Overlaying cultures with mineral oil Disadvantage viability of microorganisms varies with species https://catalog.hardydiagnostics.com/cp_prod/content/hugo/ofbasalmedium.htm  Culture Preservation Methods Culture Preservation Methods 3. Freeze-drying (lyophilization) temperature = -20oC Employs: rapid drying in frozen state https://cleanroomtechnology.com/news/article_page/Preserv ing_reference_cultures_for_QC_and_compliance/88703 https://www.spectrumchemical.com/OA_HTML/Equipment_Appliances_Freeze-Dryers.jsp Culture Preservation Methods 3. Freeze-drying (lyophilization) Advantages long-term survival less opportunity for changes in the characteristics of culture small storage containers https://plantpath.ifas.ufl.edu/rsol/CulturePreservation.html Culture Preservation Methods Culture Preservation Methods https://www.vekamaf.com/equipment/liquid-nitrogen-freezer-for- safe-and-dry-storage-of-your-samples/ 4. Freezing with liquid nitrogen temperature = -196oC Considerations: cryoprotective agent (glycerol) liquid-nitrogen refs https://microbeonline.com/maintenance-and-preservation-of-pure-cultures-of-bacteria/ Culture Preservation Methods 5. Drying Samples are grown on sterile paper disk saturated with nutrient, then the disks are allowed to air dry and stored aseptically drying temperature = 45oC Limitation: for spore- and cyst-formers https://slideplayer.com/slide/6427649/ BANKING MICROBES https://www.atcc.org/~/media/PDFs/webinars/Presentations/2017/Best% 20Practices%20Microbiology.ashx Culture Collections organizations which maintain authentic pure cultures of microorganisms provide ‘type’ strains to microbiologists throughout the world  Examples https://www.glassdoor.com/Photos/American-Type-Culture-Collection-Office-Photos-IMG433203.htm https://www.liofilchem.com/products/liofilchem-products.html American Type Culture Collection (ATCC) National Collection of Type Cultures (Maryland) (NCTC) (London) Examples https://jcm.brc.riken.jp/en/catalogue_e https://www.facebook.com/pncm.uplb/ Philippine National Collection of Microorganisms References 1. Madigan M, Bender K, Buckley D, Sattley W, Stahl D. 2019. Brock Biology of Microorganisms. 15th edition. NY: Pearson Education Limited. 2. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless) /6%3A_Culturing_Microorganisms/6.3%3A_Culturing_Bacteria/6.3A%3A_Culture_Media 3. https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Micr obiology_Labs/Microbiology_Labs_II/Lab_03%3A_Obtaining_Pure_Cultures_from_a_Mixe d_Population 4. https://www.biologydiscussion.com/organism/culture-organism/obtaining-pure-culture- of-microorganisms-6-methods/55042 5. https://www.atcc.org/~/media/PDFs/webinars/Presentations/2017/Best%20Practices%20 Microbiology.ashx 6. http://www.biologydiscussion.com/micro-biology/preserving-microbial-cultures-top-5- methods/17821  Copyright © 2020. Domini Theresa M. Pecundo. All Rights Reserved. No part of this file may be published without the creators’ consent.