chapter 6: measuring microbial growth microbial growth requirements binary fission & biotic potential microbial population growth direct methods: dilution & plating The spread plate method Inoculate plate containing solid medium. 100μl Bacterial dilution Spread inoculum over surface evenly. Colonies grow only on surface of medium. direct methods: microscopic counts indirect methods: most probable number - control + control pH 11 pH 10 1 pH 8 pH 7 pH 6 growth relative to control indirect methods: spectrophotometry pH optimum 0.8 0.6 0.4 0.2 0 growth requirements: temperature growth requirements: temperature food spoilage growth requirements: osmotic pressure osmoprotectants/compatible solutes growth requirements: pH All environments • DNA/RNA PO4 buffer • low pH causes tumbling • membrane gathers H+/OH• ion circuits: Na+/H+ antiport • surface proteins pH stable Alkaline environments • Na+/H+ antiport • H+ motive force changes Acidic environments • not permeable to H+ living with oxygen: anti-oxygen enzymes oxygen requirements chemical growth requirements biological macromolecule synthesis media: meeting physical & chemical needs MLS-1 growth optima 2.2 DSIC-II (NH4Cl) - 85 hours 2 DSIC-II (glutamine) - 30 hours 1.8 1.6 OD (600nm) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 time (minutes) media DSIC-II Chemically Defined Medium EM-II Chemically Defined Medium 80g NaCl (sodium chloride) 70g NaCl 0.5g NH4Cl (ammonium chloride) OR 14mM glutamine 0.8g NH4Cl 0.6g KH2PO4 (monopotassium phosphate) 0.8g KH2PO4 2.5g K2SO4 (potassium sulfate) 10g Na2SO4 0.1g Na2S2O35H2O (sodium thiosulfate) 0.8g Na2S9H2O (Sodium sulfide) dissolved in 10ml H2O, filter sterilized & added to cooled media 1ml vitamin solution (10mg biotin, 35mg nicotinamide, 30mg thiamine dichloride, 10mg pyridoxyl chloride, 10mg Ca-panthenoate, 5mg vitamin B12, in 100ml dH2O) No vitamin solution 1ml trace elements (5.2g EDTA, 190mg CoCl22H2O, 0.1g MnCl24H2O, 1.5g FeCl24H2O, 6mg H3BO3, 17mg CuCl22H2O, 188mg Na2MoO42H2O, 25mg NiCl26H2O, 70mg ZnCl2, 30mg VOSO42H2O, 2mg Na2WO42H2O, 2mg NaHSeO3, in 1l dH2O) 1ml SLA trace elements (1.8g FeCl24H2O,; 250mg CoCl26H2O,; 10mg NiCl26H2O,; 10mg CuCl25H20,; 70mg MnCl24H2O,; 100mg ZnCl2,; 500mg H3BO3,; 30mg Na2MoO42H2O; 10mg Na2SeO35H2O; 1l dH2O) 0.2g MgSO47H2O (autoclaved separately) 0.1g MgCl26H2O (autoclaved separately) 0.2g CaCl22H2O (autoclaved separately) 50mg CaCl22H2O (autoclaved separately) 20g NaHCO3 (autoclaved separately) 20g NaHCO3 (autoclaved separately) dH2O up to 1L dH2O up to 1L, store anaerobically special media types differential & selective media Chapter Six Learning Objectives 1. How do most bacterial cells reproduce? Why do bacterial cells have tremendous biotic potential? 2. Discuss what is happening to a bacterial culture during the fourth phases of population growth. Are they growing, dividing, what is driving the change in population number? http://professorcrista.com/files/animations/posted_animations/bacterial_growth_curve.html http://professorcrista.com/files/animations/posted_animations/binary_fission.html https://www.youtube.com/watch?v=lpI4WCM_9pM&feature=youtu.be (Go to 4:00) 3. Categorize, discuss the pros and cons and understand the mechanism of action for each of the following methods of counting the number of bacterial cells in culture: serial dilution and spread plating, microscopic counts, MPN, and spectrophotometry. http://education.wichita.edu/saltymicro/ecology_interactives/serial_dilution.html 4. How do temperature, osmotic pressure, pH and oxygen changes affect the growth of a bacterial culture? How are organisms classified according to their needs in regard to these physical aspects of the environment? What adaptations might various bacterial species have in order to combat less than desirable environments? 5. Six chemical growth requirements were discussed in lecture. What are these used for in the bacterial cell? How does the availability of these affect the growth of a bacterial culture? 6. How are differential and selective media useful in isolating, identifying and enriching for particular bacterial species? 7. How do complex and chemically defined media differ? When is each useful for the routine culture of bacterial cultures? chapter seven: control of microbial growth the control of microbial growth treatment effectivity • • • • • number of microbes/length of exposure microbial characteristics environment: moisture & temperature organic matter vegetations/biofilms 1. cells populate substrate 2. extracellular polymeric substances (EPS) produced & attaches 3 & 4. biofilm architecture develops and matures 5. cells are released from the biofilm the benefits of a biofilm protection from Abx, toxins and immune cells tobramycin ciprofloxacin planktonic cells biofilm cells biofilm colony open symbols are untreated (control) terminology • decimal reduction time (DRT)- opposite of decimate • thermal death point (TDP) – lowest temperature when all cells killed in 10 min. • thermal death time (TDT) – time to kill all cells at given T heat •dry heat- oxidation – flaming – incineration – hot-air sterilization •moist heat- denaturization – autoclave – pasteurization • not sterile • 63°C for 30 min • flash (UHST): 72°C for 15 sec • ultra heat treatment (UHT): >135°C for <1 sec •moist vs. dry – hot air: 170˚C, 2 hr – autoclave: 121˚C, 15 min filtration • physical removal of organisms • protects heat-labile components ionizing & nonionizing radiation ionizing radiation ionizes H2O OH OH damages DNA nonionizing radiation damages DNA chemical control: use-dilution test useful for testing bactericidal properties dip metal ring in bacteria dry at 37°C place ring in disinfectant 10 minutes @ 20°C culture in broth check survivability chemical control: disk-diffusion method filter paper is soaked with disinfectant paper placed on “seeded” agar plate incubated examined for zone of inhibition chemical microbial control * for study Chapter Seven Learning Objectives 1. Define sterilization, commercial sterilization, disinfection, antisepsis, degerming and sanitization. Understand what is meant by “-stat,” “-lytic” and “-cide”. http://professorcrista.com/files/animations/posted_animations/chemotherapeutic_agents.html 2. How do the following affect the effectivity of a given microbial control agent: microbial load, exposure length, microbial characteristics, moisture, temperature, organic matter and vegetations/biofilms? 3. How is a biofilm produced? How are the microbes within it so protected from the environment? 4. Why is moist heat better than dry heat at killing microbes? 5. Define thermal death point, thermal death time, and decimal reduction time. 6. When is filtration a useful method of controlling microbial growth? 7. How do ionizing and non-ionizing radiation work to control microbial growth? 8. What experimental method discussed in class is useful to determine the bactericidal properties of a given chemical? Which would you use if you were only concerned with the bacteriostatic properties? 9. How would you rate the various kinds of microorganisms in terms of their resistance to the chemical control of microbial growth? chapter eight: microbial genetics the hereditary material Griffith 1927 & Avery, et al. 1944 the “transforming principle” coined by Griffith, identified by Avery the hereditary material Hershey Chase, 1952 the bacterial chromosome plasmids • F factor (conjugative plasmid) – genes for sex pili and plasmid transfer • dissimilation plasmids – enzymes to catabolize unusual compounds • R factors – antibiotic resistance