2Tues__Pure culture techniques
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2Tues__Pure culture techniques

Course Number: BIO 205, Fall 2009

College/University: Miramar College

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Lab Exercise 5: Pure culture techniques OBJECTIVES 1. Perform a streak-plate to separate the cells of a mixed culture so that discrete colonies can be isolated. 2. Perform a pour-plate (loop) dilution to separate cells of a mixed culture and compare growth characteristics beneath and on the surface of the agar. INTRODUCTION As you learned in Lab 4, microbes exist everywhere, and very rarely do they occur as a...

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Exercise Lab 5: Pure culture techniques OBJECTIVES 1. Perform a streak-plate to separate the cells of a mixed culture so that discrete colonies can be isolated. 2. Perform a pour-plate (loop) dilution to separate cells of a mixed culture and compare growth characteristics beneath and on the surface of the agar. INTRODUCTION As you learned in Lab 4, microbes exist everywhere, and very rarely do they occur as a single species. Robert Koch, known as the father of medical microbiology, was one of the first to recognize that isolating a microbe (in his case, bacterium) away from other microbes was crucial for his own argument that microbes cause disease, as well as understanding characteristics of the microbe itself. His studies on Bacillus anthracis contributed to many of the laboratory techniques we still use today, including the method for isolating pure cultures of bacteria. The most commonly used method in the laboratory for isolating microbes is the streak plate, and to a lesser extent, the pour plate. Both methods rely on dilution of bacterial cells in a sample to the point at which a single cell can divide giving rise to a single pure colony. The pure colony is essentially a clone of cells that all are identical copies of the original cell and can be used for further study. Lab Exercises I. Streak plate method Table supplies 1 mixed culture of Serratia marcescens, Escherichia coli, and Chromobacterium Individual supplies 1 environmental body broth (from Lab Exercise 4) 2 nutrient agar plates Inoculating loop violaceum Protocol: 1. Prepare lab bench by removing extraneous items and cleaning surface with table disinfectant. 2. Label the bottom surface of your sterile agar plates. 3. Using the T-method (illustrated below) or quadrant method, outline the sections on the bottom of the agar plate. 4. Obtain mix culture and shake gently to suspend organisms. 5. Flame the loop until it is red-hot and let cool. 6. Remove cap of mix culture and flame the mouth of the tube (do NOT place cap down on the table). 7. Insert the loop into the tube and remove a loopful of broth with bacterial cultures. 8. Lightly flame the mouth of the culture tube again. 9. Return the cap to the tube and set in your tube rack. 10. Streak the plate, following either the T-method or quadrant streak shown below. Do not gouge into the medium with the loop and keep the lid over the plate as much as possible. 11. Flame the loop before setting it down. 12. Repeat the above protocol (# 4-11) using your body broth from Lab 4 and a new agar plate. Step 1: Use the sterile cooled loop, get inoculum and and draw over the agar surface in the first section. Flame and then cool the loop. Step 2: Use the sterile cooled loop and draw of the agar surface in the second section. Note that you do NOT go back to the broth and you overlap with the first section a few times. Flame the loop and allow to cool. Step 3: Use the sterile cooled loop and draw of the agar surface in the third section. Again do NOT go back to the broth overlap and with the second section only. Flame the loop. Now place the plate in incubator. 13. Incubate the plates inverted at 30 C for 2 days. (NOTE: we will have to set up a separate 30 C incubator for this experiment. Follow instructions on where to put these plates). See animation at: http://www.sumanasinc.com/webcontent/animations/content/streakplate.html II. Pour plate method Table supplies Mixed culture of S. marcescens, E. coli, and C. violaceum Team supplies 3 nutrient agar pours 3 empty Petri dishes Inoculating loop 1. Label the bottom of three sterile Petri plates I, II, and III. 2. Remove 3 tubes of nutrient agar from the 50 C water bath in the back of the room. (NOTE: you must work quickly at this point or the nutrient agar will solidify in the tube). 3. Using aseptic technique, take one loopful from the mixed culture and add to the nutrient agar in tube I. 4. Mix the tube by rolling vigorously between the palms of both hands. DO NOT shake the tubes as the caps are not secure and medium will splash out. 5. Immediately, and using aseptic technique, take a loopful of the agar from tube I and add it to tube II. 6. As soon as this is done, you may pour the contents of tube I into the plate labeled plate I, being sure to pour it into the bottom of the plate. 7. Repeat steps 4-6, transferring a loopful of tube II to tube III, and then pouring the contents into plates II and III. 8 . Allow media to solidify and then incubate the plates inverted at 30 C for 2 days. (NOTE: we will have to set up a separate 30 C incubator for this experiment. Follow instructions on where to put these plates). DATA AND OBSERVATIONS 1. Evaluation of streak plates: Show within the circle the distribution of the colonies on your streak plate. Streak plate Did you get clear isolation of purple Chromobacterium violaceum, red Serratia marcescens, and white E. coli? If not, you maybe need to practice the technique again by subculturing the colonies (your instructor might want you to do this regardless). To subculture, use your wire loop to pick an isolated colony that you will use as inoculum for another streak plate. 2. Evaluation of pour plates: Show the distribution of colonies on the pour plate II or III. Indicate which colonies are growing on the medium and which are growing within it. Plate dilution ________ DISCUSSION 1. Compare your results from the streak-plate and pour-plate methods. Which method achieved the best separation of species? 2. Which specie(s) are able to grow both on the surface and within the medium? In which location are the colonies larger? Why? 3. In regards to bacterial growth on solid media, define the term colony. 4. Explain how dilution is a common approach employed by any pure culture technique. 5. What advantage does the streak-plate method have over the pour-plate method? 6. Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50 C? 7. Provide two reasons why plates should be inverted during incubation

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Miramar College - BIO - 205
Lab Exercise 6: The smear and simple stainingOBJECTIVES1. Prepare bacterial smears for the microscopic visualization of bacteria. 2. Perform a simple staining procedure. 3. Compare the shapes and arrangements of bacterial cells.INTRODUCTIONSmears Much
Miramar College - BIO - 205
Lab Exercise 9: Acid-fast stainOBJECTIVES1. Understand the chemical basis for the acid-fast stain 2. Perform a successful acid-fast stain to differentiate between acid-fast and nonacid fast organisms.INTRODUCTIONBacteria belonging to such genera as My
Miramar College - BIO - 205
Lab Exercise 7: The negative stainOBJECTIVES1. Perform a negative staining procedure 2. Understand the benefits of using negative stains.INTRODUCTIONAs mentioned in lab exercise 6, not all dyes stain the bacterial cell. However, acidic dyes can be use
Miramar College - BIO - 205
Lab Exercise 9: Special stains for cell structures: spore and capsule stainsOBJECTIVES1. Understand the chemical basis for the spore and capsule stains. 2. Perform a successful spore stain to differentiate between bacterial spores and vegetative cells.
Miramar College - BIO - 205
Lab Exercise: Lethal effects of temperatureOBJECTIVES1. To predict and determine the thermal death point (TDP) and thermal death time (TDT) for various organisms based on physiological and ecological characteristics.INTRODUCTIONMany factors affect how
Miramar College - BIO - 205
Lab Exercise: Lethal effects of ultraviolet lightOBJECTIVES1. To understand the mechanism by which UV light causes damage and how this can lead to cell death. 2. To perform experiments to test the varying resistance to UV light of different microbesINT
Miramar College - BIO - 205
Lab Exercise: Effects of temperature on growthOBJECTIVES1. To determine optimum growth temperature and observe effects of temperature on such processes as pigment production.INTRODUCTIONMicrobes grow over a broad temperature range that extends from be
Miramar College - BIO - 205
Lab Exercise 12: Effects of oxygen on growthOBJECTIVES1. To differentiate between oxygen requirements using anaerobic culture media.INTRODUCTIONMicrobes can be classified into 5 groups based on their requirement for air that contains 20% oxygen: 1. Ob
Miramar College - BIO - 205
Lab Exercise: Effects of pH on growthOBJECTIVES1. To determine optimum pH for growth for organisms with different characteristicsINTRODUCTIONpH is an important environmental condition to consider, as an excess of either hydrogen ions (H+), as exists i
Miramar College - BIO - 205
Lab Exercise: Antibiotics- Evaluation using Kirby Bauer method.OBJECTIVES1. Compare the antimicrobial capabilities of different antibiotics. 2. Compare effectiveness of with different types of bacteria. 3. Utilize aseptic techniques.INTRODUCTIONYou ma
Miramar College - BIO - 205
Lab Exercise: Antiseptics and Disinfectants- Evaluation using filter paper methodOBJECTIVES1. Compare the antimicrobial capabilities of different antiseptic and disinfectant chemicals. 2. Compare effectiveness of antiseptics with different type of bacte
Miramar College - BIO - 205
Lab Exercise: TransformationOBJECTIVES1. Understand the process of transformation and how it is used in a laboratory setting for expression of genes (i.e. production of proteins). 2. Perform a successful transformation using the pTOM plasmid.INTRODUCTI
Miramar College - BIO - 205
Lab Exercise: DNA fingerprinting or Restriction Fragment Length Polymorphism (RFLP) analysisOBJECTIVES1. Understand the basis of DNA fingerprinting technology, including restriction enzyme digestion, polymerase chain reaction, and agarose gel electropho
Miramar College - BIO - 205
Lab Exercise: Dental Microbiology- Snyder agarOBJECTIVES1. To become familiar with organisms responsible for dental carriers in the mouth. 2. To perform experiments that demonstrate susceptibility to caries formation.INTRODUCTIONMany microbes are know
Miramar College - BIO - 205
Lab Exercise: A Physiological & Cultural Examination of the Minor UnknownOBJECTIVES1. Become familiar with media used in identification of microbes, and how these media work to test for different physiological characteristics. 2. Perform physiological t
Miramar College - BIO - 205
Lab Exercise: Diversity of MicrobesBacteriophagesOBJECTIVES1. To understand the lytic cycle of bacteriophages 2. To be able to make serial dilutions. 3. To be able to determine concentrations of viral samples from PFU counts on plates.INTRODUCTIONViru
Miramar College - BIO - 205
Lab Exercise: Diversity of Eukaryotic MicrobesOBJECTIVES1. To observe representatives of major types of microbes. 2. To cultivate select representatives of major types of microbes. 3. Understand key characteristics of the different eukaryotic microbes a
Miramar College - BIO - 205
Lab Exercise: Simulating an Influenza EpidemicOBJECTIVES1. Describe how immunizations work to cause immunity. 2. Define herd immunity. 3. Define antigenic shift and antigenic drift and their relationship to immunization efficacy. 4. Describe the various
Miramar College - BIO - 205
Biology 205 Microbiology Staph, Strep & Enteric Unknown Lab ReportThis assignment will be worth a total of 70 points toward your total course grade. These lab reports will loosely follow a journal article format as seen in scientific journals. They can b
Miramar College - BIO - 205
Lab Exercise: Staphylococcal, Streptococcal & Enteric UnknownsOBJECTIVES1. Identify each of the organisms worked with in this lab exercise. 2. Understand the use of differential and selective media in the identification of bacteria and the enrichment of
Miramar College - BIO - 205
Microbiology Major Unknown Fall 2009Lab dynamicsYou will be isolating and identifying two unknown bacteria from a mixed culture using the morphological and biochemical methods with which you are already familiar. For any determinative work, it is absolu
Miramar College - BIO - 205
Lab Exercise: Examining Water Quality: Most Probable Number & Colilert Test Kit Lab OBJECTIVES 1. Understand the use of MPN to determine likely fecal water contamination. 2. Understand the use of MUG, ONPG and -galactosidase in the Colilert test kit. 3. U
Miramar College - BIO - 205
GamePlanLecture Returnexam Microbialsymposiumreminder Taxonomy Identificationandclassification ofmicrobes Cladogramsanddichotomouskeys APO3:BergeysManualanddichotomous keys Lab Transformationresults DNAfingerprintingScientificnamesandmeaningsScientific
Miramar College - BIO - 205
Game PlanLecture Eukaryotes Fungi Algae Protozoa Helminths Viruses,Viroids&PrionsLabReviewoftransformationlab & DNAfingerprintlab BiotechnologyData Discussionchapters12&13 eukaryotes,viruses&prionseukaryoticmicrobesTable 12.1FungalDiseases(mycoses)
Miramar College - BIO - 205
G a m e P la nLecture Ep id e m io lo g y Dis e a s e te rm ino lo g y C la s s ific a tio no finfe c tio us d is e a s e De ve lo p m e nto fd is e a s e C a s e s tud y :th e b ird fluLab C o m p le te e xe rc is e s 3 9 4 0 S y nth e tic e p id e m i
Miramar College - BIO - 205
C h a p te r1 6 /1 7 :Im m une s y s te mLab SSE UnknownsLecture Chapter 16: Nonspecific defenses Firstlineofdefense Formedelements Secondlineofdefense Complementsystem Chapter 17: Specific defenses Antibodies Humoralresponse Cellularresponse ContinueAP
Miramar College - BIO - 205
G a m e p la nLe c tureAntib o d y a ntig e nb ind ing Hum o ra lim m unity C e llula rim m unity C lo na ls e le c tio na nd im m uno lo g ic a lm e m o ryLabS ta p h ,S tre p a nd Ente ric Unkno wnsO ve rvie wo fim m unityO ve rvie wo fim m unity
Miramar College - BIO - 205
G a m e p la nLecture Va c c ina tio ns ELIS As Lab C o ntinue S ta p h ,S tre p a nd Ente ric Unkno wnsIm m uno lo g ic a la p p lic a tio ns :va c c ine s1 7 9 6 Ed wa rd Je nne rTable 18.3Im m uno lo g ic a la p p lic a tio ns :va c c ine sAttenu
Miramar College - BIO - 205
C ha pter 19: I mmunol ogi ca l di sor der sLectur e H yper sensi ti vi ti es Autoi mmune di seasesLa b M ajor unknown Water sampl es for next cl ass (get col l ecti on bottl es)H yper sensi ti vi ti esT ype I : anaphyl acti c r eacti onsSensi ti zat
Miramar College - BIO - 205
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Miramar College - BIO - 205
G a m e p la nLe c ture S kina nd e y e d is e a s e s S tud y G uid e fo rExa m 4 is p o s te d to We b C TLa b Ma jo rUnkno wn Wa te rMic ro b io lo g y Dental Micro next classS kinfirs tline o fd e fe ns eS kinle s io nsS ta p h y lo c o c c a lin
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C h a p te r2 2 :Dis e a s e s o fth e ne rvo us s ys te mLecture NS o ve rvie w Ba c te ria ld is e a s e s Vira ld is e a s e s Fung a ld is e a s e sLab Ma jo runkno wn C o m p le te d e n ta lm ic ro Wa te rm ic ro b io lo g yT h e ne rvo us s ys t
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Miramar College - BIO - 205
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Miramar College - BIO - 205
C ha pter 25: D i sea se of the di gesti ve tr a ctLectur e Str uctur e Upper di gesti ve system di seases Food poi soni ng Bacter i al enter i ti s Vi r al enter i ti s Pr otozoan di seases H el mi nthi c di seasesD i gesti ve systemD ental car r i es
Miramar College - BIO - 205
Chapter 26: Disease of the urinary and reproductive tractsLecture R e p ro d uc tive s truc ture s S T Ds Urina ry tra c tinfe c tio nsLab La b Exa mFe m a le urina rys ys te mFe m a le re p ro d uc tive s ys te mMa le urina rya nd re p ro d uc tive
Miramar College - BIO - 205
StudentPresentationQuestions(listedinnoparticularorder)15/17students. UpdatedOctober15th3:30pm Maribel 1. TreponemapallidumhasbeentermedtheStealthpathogen.Why? 2. WhataretwomaindifferencesbetweenChlamydiaeandTreponemamicrobes?Robin 1) Whatare2waysviruses
Miramar College - BIO - 205
BIOL 205 General Microbiology- BIOL 205 Fall 2009Dr. Laura W. Murphy Office: S5-101G Phone: 619-388-7539 E-mail: lmurphy@sdccd.edu Dr. Tobey Tam Email: ttam@sdccd.eduLocation/ Times-Tuesdays and Thursdays 8:00 am 12:20 pm, Room S5-109 Dr. Murphy: Dr. T
Miramar College - BIO - 205
Biology 205 General Microbiology Miramar CollegeTentative Lecture ScheduleChapters should be read prior to the scheduled lecture. This will help with your comprehension and retention of material and will assist you in completing preparatory quizzes and
Miramar College - BIO - 205
name date organism media name date organism media name date organism media name date organism media name date organism media name date organism media name date organism media name date organism media name date organism medianame date organism media name
Miramar College - BIO - 205
Microbial diversity symposium Fall 2009You will now have th e o pportunity to r esearch a small subset of microorganisms and get the chance to teach the r est of us abo ut th em! Th e for mat of o ur symposium will be r elaxed and interactive, so th at a
Miramar College - BIO - 205
APO 3: Identification and Classification- Bergeys Manual Group members _ _ _ _This exercise is desi gned to help you n avigate Bergeys Manual and become famili ar with the types of char acteristics an d tests used to i denti fy microor ganisms. You will
Miramar College - BIO - 205
microbesscientific namesthe golden age of microbiologyGreat Microbiologistsbiogenesis: refuting spontaneous generation1668 Francesco Redi1837 Schwann (Schultze)1768 Spallanzani1864 Pasteurhuman bacteriamicrobesmicrobes
Miramar College - BIO - 205
review material chapters two & fiveunit I review materialacids & bases acid dissociates into H+Figure2.6 HCl H+ + Cl base dissociates into OH NaOH Na+ + OHacid-base balance H+ in solution expressed as pH pH = -log [H+] increasing [H+] increases
Miramar College - BIO - 205
resolutionFigure 3.2staining increases resolutionmicroscopySEM & TEMFigure 3.8differential stains: Gram reactionsdifferential stains: acid-fast (AFB) stainFigure 3.11special stains: capsulespecial stains: endosporespecial stains: flagella
Miramar College - BIO - 205
cellular morphologycellular arrangementFigure 4.2 & 4.3the glycocalyxFigure 4.6flagellaflagellaaxial filaments/endoflagellafimbriae & piliFigure 4.11the cell wallcell wall: peptidoglycanFigure 4.13athe cell wallcomparing the cell wallGram p
Miramar College - BIO - 205
nutritional classificationnutritional classificationenergy sourcesunlight preformed molecules organic compound inorganic compound organic compound inorganicphotochemoorganolithoheteroauto--trophelectron donorcarbon sourceoxidation-reductionFigure
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microbial population growthcompare figure 6.14direct methods: dilution & platingfigure 6.15direct methods: microscopic countsdirect methods: filtrationdirect methods: most probable numberfigure 6.18bindirect methods: spectrophotometry1 0.9 0.8 0.
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the control of microbial growth disinfectants/antiseptics surface/skin sterilization no microbial life commercial sterilization killing C. botulinum spores sanitization microbe level too low to cause illness decimal reduction timedefining microb
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chaptereight: reviewinformationterminology genetics studyofgenes& inheritedfactors DNAsegmentthat encodesafunctional product,usuallyaprotein allofthegeneticmaterial inacell molecularstudyof genomes gene/visibleproductof gene gene genome genomics ge
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chapter nine: biotechnologyrecombinant DNA technologyDNA synthesissynthesis: reverse transcriptionFigure 9.9multiplying DNA: polymerase chain reaction (PCR)Figure 9.4transfection: gene gunsprotoplast fusiontransfection : electroporationtransfect
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chapters 10 & 11 taxonomy, phylogenetics and prokaryotic diversitythe 16S rDNA family treeorganism classificationidentification:identification: serologyFigure 10.10identification: immunoblotsidentification:genetic analysisgenetic analysisphyloge
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chapters 12 & 13 eukaryotes, viruses & prionsEukaryaeukaryotic microbesTable 12.1eukaryotes: sexual reproductionprotozoamolds & yeastsalgaeFigure 12.11aphytoplanktonhelminthschapter 13 viruses & prionsviral characteristicsFigure 13.2viral ta
Miramar College - BIO - 205
chapter 14 principles of disease & epidemiologynormal microbiota10 the number of human cells!epidemiology descriptive retrospective collection & analysis of data frequency & pattern: who, what, when, where analytical (experimental) study of a disea
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chapter 15 microbial mechanisms of pathogenicitypathogenesispathogenesisportals of entry & exitentryexitinoculation vs. diseasebacterial toxinsexotoxin source metabolic function chemical make-up fever? neutralized by antitoxin? Gram positivea meta
Miramar College - BIO - 205
chapter 16: nonspecific defenses of the hosthost defensesFigure 16.11st defense: physical barriers2nd defense: white blood cellsphagocytosisphagocytosisinflammationrubor, tumor, calor, dolor functio laesafeveradapted from Figure 15.6interferons
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chapters 18 & 19: practical applications of immunology immune system disordersantibody response & productioncompare Figure 17.10immunizationacquiring immunityisolation & identification methodsagglutination reactionsFigure 18.4precipitation reactio
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chapter 20 antimicrobial drugschemotherapeutic agentsantimicrobial drug actionFigure 20.2antiviralsFigure 20.16penicillin & cell wall synthesisGFA: sulfanilamidessynergism: SXTFigure 20.13modes of bacterial resistancebacterial resistancedisk/b
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chapters 27 & 28 environmental microbiology & applied & industrial microbiologythe carbon cyclecoccolithophoresFigure 27.3the nitrogen cycleFigure 27.4the sulfur cycleFigure 27.7life without sunshinemicrobial decompositionFigure 27.9municipal w
Miramar College - BIO - 205
Miramar College Biology 205 Microbiology Midterm Exam Study Guide These learning objectives are intended as a study guide. This is not necessarily a complete guide and as such is not intended to be the sole source of your studies. You should use your note
Miramar College - BIO - 205
Miramar College Biology 205 Microbiology Midterm Exam II Study Guide These learning objectives are intended as a study guide. This is not necessarily a complete guide and as such is not intended to be the sole source of your studies. You should use your n