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BSC242 week of 2/8

by: Alexandra

BSC242 week of 2/8 BSC 242

GPA 4.01

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lecture notes 2/9 and 2/11
Microbiology and Man
Daryl W. Lam
Class Notes
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This 16 page Class Notes was uploaded by Alexandra on Thursday February 11, 2016. The Class Notes belongs to BSC 242 at University of Alabama - Tuscaloosa taught by Daryl W. Lam in Winter 2016. Since its upload, it has received 36 views. For similar materials see Microbiology and Man in Biology at University of Alabama - Tuscaloosa.


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Date Created: 02/11/16
Microbial Growth 02/09/2016 ▯ Growth= increase in number of cells NOT cell size  Physical requirements for growth o Temp  (In C) 0°C=32°F  examples  psychrophiles: -10° to 20°  psychrotrophs: -10° to 30°  mesophiles: 10° to 50° (meso think middle)  thermophiles: 40° to 70°  hyperthermophiles: 65° to 110°  Min growth temp  Optimum growth temp  Maximum growth temp o  Improper cooling  It is major reason for food-borne illnesses  Leaving cooked foods at room temp too long  Refrigerating large quantities of food in deep containers o pH  most bacteria grow between pH 6.5 and 7.5  molds and yeasts grow between pH 5 and 6  acidophiles grow in acidic environments (below pH 7) o osmotic pressure  microbes obtain nutrients from their environment  Hypertonic environments (increased salt/sugar) cause plasmolysis  Plasmolysis: cell loses water  Extreme or obligate halophiles require high osmotic pressure (dead sea bacteria)  Facultative halophiles tolerate high osmotic pressure (staphylococci)  Chemical requirements for Growth o Carbon  Used for structural organic molecules, energy source  Heterotrophs use organic carbon sources  (hetero=different, troph=feeder)  Autotrophs (photosynthetic organisms) use CO to mak2 sugars o Nitrogen  In amino acids, proteins, nucleic acids  Most bacteria decompose proteins  Some bacteria use NH 4+ or NO 3-  A few bacteria use N in2nitrogen fixation o Sulfur  In amino acids (cys, met), thiamine and biotin  Most bacteria decompose proteins 2-  Some bacteria use SO 4 or H2S o Phosphorus  In DNA, RNA, ATP and membranes (phospholipids)  PO 43-is a source of phosphorus o Trace elements  Inorganic elements required in small amounts  Usually as enzyme cofactors  Ex  Zinc, iron, magnesium, iodine o Oxygen (O ) 2  Obligate aerobe  Requires molecular oxygen to live  Facultative anaerobe  Can grow w/ or w/o molecular oxygen  Obligate anaerobe  Does not use molecular oxygen & is killed in its presence  Aerotolerant anaerobes  Does not use molecular oxygen but is not affected by its presence  Microaerophiles  Grows best in an environment with less molecular oxygen than is normally found in air  Toxic forms of Oxygen (pgs 155-156) -  Superoxide free radicals: O 2  O 2 2 H —superoxide dismutase—> H O 2 2 + O 2 2-  Peroxide anion: O 2 (toxic)  2 H O —catalase—> 2 H O + O 2 2 + 2 2  H 2 2 2 O —peroxidase—> 2 H O 2  Singlet oxygen: O -1 2  Very reactive, causes damage to cells  Hydroxyl radical (OH)  Very reactive o Organic growth factors (“essential” in diet)  Organic compounds obtained from environment  Vitamins, amino acids, purines, and pyrimidines ▯ Culture Media  Culture medium o Nutrients prepared for microbial growth o Must contain proper  Nutrients (C, N, energy sources)  pH  salt conc  Temp  Oxygen requirements o Types  Broth  Used for large # of cells in small place  Agar slant  Used for transport & sustaining of cultures  Agar deep  Used for motility & O 2equirement  Agar plate  Used for isolated colonies  Agar  Complex polysaccharide  Used as solidifying agent for culture media in Petri plates, slants, and deeps  Generally not metabolized by microbes  Liquefies at 100°C  Solidifies ~40°C o Chemically defined media exact chemical composition is known o Complex media extracts and digests of yeasts, meat, or plats (nutrient broth, nutrient agar)  Sterile o No living microbes (including endospores)  Inoculum o voluntary introduction of microbes into medium  Contamination o Involuntary introduction of microbes into medium  Culture o Microbes growing in/on culture medium ▯ Anaerobic Culture Methods  Reducing media o Contain chemicals (thioglycolate or oxyrase) that combine O 2 and deplete it o Heated to drive off O 2  Anaerobic jar o Chemicals produce H and CO2which rem2ve O by combining 2 it in presence of a catalyst with H to 2orm water  Anaerobic chamber  Oxyrase: combines with O with H and removes O as water is 2 2 2 formed ▯ Capnophiles require High CO (Low O 2 2  Candle jar (candle generates CO 2  CO –2acket ▯ Selective (S) Media  Clinical specimens are often contaminated by other microbes  Suppresses unwanted microbes and encourages growth of desired microbes  Examples o Media containing antibiotics (only resistant ones grow) o Sabouraud’s Dextrose Agar (low pH suppresses bacteria and favors yeasts) ▯ Differential (D) Media  Makes it easy to distinguish colonies of different microbes from each other  Examples o Blood agar (clear zones around Streptococcus pyogenes from throat swabs) o X-gal plates (lactose positive colonies appear blue; lactose negative colonies appear white) [pg 248 Figure 9.11] ▯ Mannitol salts Agar plates (MSA)  S: high NaCl inhibits many microbes  D: pH indicator (phenol) turns yellow to indicate acid from fermentation ▯ Eosin-Methylene Blue Agar (EMB)  S: dyes inhibit Gram-positives  D: lactose fermenters turn dark; E. coli gets a green shade also ▯ Enrichment Media  Encourages growth of desired microbe ▯ Type Purpose Chemically defined Growth of chemoautotrophs and photoautotrophs; microbiological assays Complex Growth of most chemoheterotropic organisms Reducing Growth of obligate anaerobes Selective Suppression of unwanted microbes; encouraging desired microbes Differential Differentiation of colonies of desired microbes from others Enrichment Similar to selective media but designed to increase numbers of desired microbes to detectable levels Definitions Pure culture (axenic) Contains only 1 species or strain Colony (CFU: colony-forming unit) Population of cells arising from a single cell or spore ▯ ▯ Preserving Bacteria Cultures  Deep-freezing: -50° to -95°C  Lyophilization (freeze-drying): frozen (-54° to -72°C) and dehydration in a vacuum ▯ ▯ Reproduction in Prokaryotes (asexual)  Binary fission o Bacteria o Organism separate to form 2 new organisms o Cytoplasm is split evenly o Steps  Cell elongates and DNA is replicated  Cell wall and plasma membrane begin to grow inward  Cross-wall forms completely around divided DNA  Cell separate  (a) a diagram of the sequence of cell divison  (b) a thin section of a cell of Bacillus licheniformis starting to divide  Budding o Yeast (only a few bacteria) o New organism is from old organisms and cytoplasm splits unevenly  Conidiospores (actinomycetes)  Fragments of filaments ▯ ▯ Generation time: time it takes a cell to divide (or population to double)  Ex: began with 5 cells, ended with 160 cells. How many generations?  5*2=10 *2=20 *2=40 *2=80 *2= 160 (5 generations) ▯ Important graph ▯ ▯ ▯ Direct Measurements of Microbial Growth  Plate counts: perform serial dilutions of a sample o inoculate Petri plates from serial dilutions o spread and pour plates o after incubation, count colonies on plates that have 25-250 colonies ▯  Filtration  Direct microscopic count ▯ Estimating Bacterial Numbers by Indirect Methods  Turbidity (spectrophotometry) o Measuring light passing through  Metabolic activity  Dry weight ▯ ▯ Chapter 7 ▯ The control of Microbial Growth Sephis Refers to microbial contamination Asepsis The absence of contamination Aseptic technique Used in lab to prevent contamination of solutions and cultures (aseptic surgery techniques prevent microbial contamination of patients) Sterilization Removal of all microbial life (including endospores) Commercial sterilization Enough heat to kill Clostridium botulinum endospores Disinfection Removal of pathogens from surfaces Antisepsis Removal of pathogens from living tissue Degerming Removal of microbes from a limited area Sanitization Lower microbial counts on eating utensils Biocide/Germicide Kills microbes Bacteriostasis Inhibiting, not killing, microbes Bacterial populations die at a constant logarithmic rate ▯ Effectiveness of antimicrobial treatment  Depends on o Number of microbes o Environment (organic matter, temp, biofilms) o Time of exposure o Microbial characteristics  Biofilms o Matrix of polysaccharides, DNA and protein=slime o Bacteria that are grouped together in communities that provide great benefits from that grouping o Biological systems: functional communities o Usually attached to rocks, teeth or mucous membranes (single species or diverse group) o Share nutrients and are protected from harmful factors o Conjugation of genetic material between organisms o Super resistant to microbicides ▯ Actions of Microbial Control Agents  Alteration of membrane permeability  Damage to proteins  Damage to nucleic acids ▯ Physical methods of microbial control  Heat o Thermal death point (TDP)  Lowest temp. at which all cells in a culture are killed in 10 min. o Thermal death time (TDT)  Time to kill all cells in a culture o Moist heat denatures proteins  Autoclave: steam under pressure  Pressure and temperature in autoclave  Standard autoclave run= 121C (15psi) for 15 min  Steam must directly contact all surfaces of solid objects  Steam Sterilization  Steam must contact item’s surface  Pasteurization reduces spoilage organisms and pathogens  Equivalent treatments o 63°C for 30min (classic pasteurization) o high temp, short time (HTST): 72°C for 15sec o ultra high temp (UHT): 140°C for <1sec (sterilizes)  Dry heat sterilization kills by oxidation o Flaming o Incineration o Hot-air sterilization Filtration Removes microbes High pressure Denatures proteins Low temp Inhibits microbial growth Refrigeration Deep freezing Lyophilization Desiccation (absence of water) Prevents metabolism Osmotic pressure Causes plasmolysis Dried microbes are a problem in hospitals  Radiation damages DNA o Ionizing radiation  Kills microbes on certain meats, vegetables, spices, plasticware, mail, some medical supplies (x-rays, gamma rays, etc.)  High degree of penetration o Nonionizing radiation (UV) light  Germicidal lamps found in hospitals, some vaccines are treated with UV  Low degree of penetration ▯ Chemical Methods of Microbial Control  Principles of effective disinfection o Conc. of disinfectant o Organic matter present o pH o time o temp  evaluating a disinfectant o use-dilution test  metal rings dipped in test bacteria are dried  dried cultures are placed in disinfectant for 10 min at 20°C  rings are transferred to culture media to determine whether bacteria survived treatment o disk-diffusion method ▯ Types of Disinfectants  Phenol and Phenolics (Lysol and Amphyl) o Disrupt membranes and proteins o Active in organic matter and effective against mycobacteria  Bisphenols (hexacholorphene, Triclosan) o Disrupt plasma membranes  Biguanides (Chlorhexidine) o Disrupt plasma membranes o Combined with detergent or alcohol in surgical scrubs  Halogens o Often effective against mycobacteria and endospores o Affect enzyme activity o Iodine  Binds to proteins  Available as a Tincture of Iodine (iodine in alcohol) or as the Iodophor (e.g. Betadine) o Chlorine  Biocidal activity is based on formation of hypochlorous acid (HOCl, a strong oxidizer) when in water (e.g. Clorox bleach)  Used in water treatment  Chlorine dioxide gas is used to treat large areas  Alcohols (ethanol, isopropanol) o Denature proteins, dissolve lipids o Can enhance effectiveness of other agents (e.g. tinctures) o Used to degerm skin  Heavy Metal (Ag and Cu) o Denature proteins (combines with sulfhydryl groups) o 1% silver nitrate used in eyes of newborns (before antibiotics available) o silver impregnated wound dressings and catheters o copper sulfate is used to kill green algae in ponds, pools, and fish tanks  Surface-active agents or surfactants o Decrease surface tension of molecules Soap Degerming Acid-anionic detergents Sanitizing Quaternary ammonium compounds Bactericidal, denature proteins, Cationic detergents disrupt plasma membrane o Soaps emulsify the oily film on skin together with any debris and this mix gets washed away with water (scrubbing) o QUATS  are derivatives of the ammonium ion;  probably affect the membrane.  Some pseudomonades can grow in quat solutions.  Quats are not useful against mycobacteria or endospores.  Bactericidal against gram-positive bacteria and less active against gram-negative bacteria  Chemical food preservatives: retard spoilage o Organic acids  Inhibit metabolism  Sorbic acid, benzoic acid, and calcium propionate  Control molds and bacteria in foods and cosmetics o Nitrite  Prevents endospore germination: meat products o Antibiotics  Nisin and natamycin prevent spoilage of cheese  Safe for human consumption  Aldehydes: very effective antimicrobial o Inactivate proteins by cross-linking with functional groups (-NH2, -OH, -COOH, -SH) o Glutaraldehyde, formaldehyde, and ortho-phthalaldehyde (OPA) o Effective on endospores and mycobacteria  Gaseous Sterilants o Denature proteins o Ethylene oxide (used in a chamber) o Penetrates well o Very effective on all microbes and endospores o Cross-linking of DNA and proteins (stops vital cellular functions) o Toxic to humans and a suspected carcinogen  Peroxygens o Oxidizing agents o O (3zone), H O 2pe2oxide), benzoyl peroxide (acne treatment), peracetic acid (a sporocide) o Not good for open wounds o Catalase in human tissues readily inactivates hydrogen peroxide producing oxygen gas ▯ ▯


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