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Hunter College Microbiology Exam 2 Notes

Transcript of Exam2Lecture5-1

3/28/2011 5:22:00 PMChapter 6: Microbial Growth * Refer to pictures The Requirements for Growth y Physical requirements o Temperature o o y pH osmotic pressure Minimum growth temperature Optimum growth temperature Maximum growth temperatue

Chemical Requirements o C,N, S, P, trace elements, oxygen, organic growth factor

y

Plasmolysis (Fig. 6.4)

Psychropiles/psychotrophes y y pH y y y Most bacteria grow bw pH 6.5-7.5 Molds and yeasts grow bw pH 5-6 Acidophiles grow in acidic environment Grow between 0-15C and 10-25C Cause food spoilage

Osmotic Pressure -Hypertonic environments or an increase in slat or sugar cause plasmolysis -Extreme or obligate halophiles require high osmotic pressure (ie 30% salt Great Sea, Red Sea) -Facultative halophiles can tolerate high osmotic pressure (2%-15% salt) What would happen if cell put into distilled water? Plasmolysis*

Chemical Requirements for Bacterial Growth Carbon y y y Nitrogen y y y Amino acids and proteins Most bacteria decompose proteins (recycle) Some bacteria use ammonium ions NH4 or nitrate ions Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2

y Sulfur y y y Phosporus y y

A few bacteria(cyanobacteria) use N2 in nitrogen fixation

In amino acids, thiamine, and biotin Most bacteria decompose proteins to get sulfur Some bacteria use SO4-2 or H2S

In DNA, RNA, ATP and membranes PO43- is a source of phosporus

Trace Elements y y Inorganic elements required in small amounts Usually as enzyme cofactors

The Effect of Oxygen on Microbial Growth (Table 6.1) A. obligate aerobe- needs oxygen to survive B. facultative anaerobe- retain the ability to ferment to undergo anaerobic metabolism. Organisms not on top are facultative anaerobe. eg. E.Coli C. obligate anaerobe- cannot use molecule oxygen, but they can use oxygen from water eg. Plastrinium- causes tetanus and botulism D. Aerotolerant anaerobe-they dont use oxygen but can still grow in the presence of it. E. Microaerophile-can only grow in a very SPECIFIC level of oxygen

Toxic Oxygen Singlet oxygen: O2- boosted to a higher energy state Superoxide radicals O2y y y When you use oxygen as an electron acceptor Try to grab electrons from nearest molecule SOD (enzyme)* o O2-2+ O2-2 +2H+ Peroxide anion O2 y y y-2

* H2O2 +O2

catalase or peroxidase neutralize H2O2 catalase- produce H2O and O2 peroxidase- H2O

Hydroxyl radical (OH-) y y y Happens in cytoplasm Through ionizing radiation All of these could be toxic to humans or bacteria

Organic Growth Factors

-Organic compounds obtained from the environment. Varies from organism to organism -Vitamins, amino acids, purines, and pyramidines

Biofilms (Fig 6.5) -Microbial communities -form slime or hydrogels y bacteria attracted by chemicals via quorum sensing: communication between one cell and another. Drugs to block the quorum sensing receptors. y y y place gets too crowded, people have to leave. Important in digestive systems of cows. Need to digest cellulose. Grow upwards to increase surface area. Causes bacterial infections.

-share nutrients -sheltered from harmful factors - patients with indwelling catheters received contaminated heparin. -bacterial numbers in contaminated heparin were too low to cause infection -84-421 after exposure, patients developed pseudomonas infection -Pseudomonas fluorescens were cultured from catheters. -What happened? y y y They grew and multiplied by themselves. They are antibiotic resistance. Heparin might signal biofilm growth

Table 6.2: A chemically defined medium for growing a typical chemoheterotroph, such as E.Coli Table 6.4: Composition of Nutrient Agar, a Complex medium for the growth of heterotrophic bacteria Culture Media -Culture Media: nutrients prepared for microbial growth. -Sterile: no living microbes -Inoculum: introductions of microbes intro mediums -Culture: microbes growing in/on culture medium Growing a Culture -nutrients -pH -moisture -oxygen -initially sterile Agar -Complex polysaccharide

-Used as solidifying agent for culture media in Petri plates, slants, and deeps -Generally not metabolized by microbes -Liquefies at 100C -Solidifies at ~40C Culture Media (Table 6.2, 6.4) -Chemically defined media: exact chemical composition is known -complex media: you dont know the exact compositions. Include extracts and digests of yeasts, meats or plants y y Nutrient Broth Nutrient Agar

Anaerobic Culture Methods y y Anaerobic jar (fig. 6.6) Anaerobic chamber (fig. 6.8)

Reducing media -Contains chemicals (thioglycolate or oxyrase) that combine O2. -Heated to drive off O2. y Capnophiles -microbes that require high CO2 conditions -CO2 packet -Candle jar -multiple methods present Biosafety Level 1:No special precautions 2: Lab coats, gloves, eye protection 3: Biosafety cabinets to prevent airborne transmission 4: Sealed, negative pressure Selective Media -suppress unwanted microbes and encourage desired microbes. -chemically defined: salt media is preventing some bacterial species while allowing others to grow -Staphylococcus aureus- metabolizes manatose to make acidic environment (yellow) - Staphylococcus epidermidis- growing but basic Differential Media -make it easy to distinguish colonies of different microbes Enrichment Culture -encourages growth of desired microbe -assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Figure 6.6, 6.7

y y y y

inoculate phenol-containing culture medium with soil and incubate transfer 1 mL to another flask of the phenol medium, and incubate transfer 1 mL to another flask of the phenol medium and incubate only phenol-metabolizing bacteria will grow after much dilutions later.

Binary Fission (Fig 6.12A) Cell Division Bacterial Growth Curve

Obtaining Pure Cultures -A pure culture contains only one species or strain -A colony is a population of cells arising from a single cell or spore or from a group of attached cells. -A colony is often called a colony-forming unit (CFU) -The streak plate method is used to isolate pure cultures Preserving Bacterial Cultures -deep freezing: -50C to -95C -Lyophilization (freeze-drying): frozen (-54 to 72C) and dehydrated in a vacuum. The Growth of Bacterial Culture Reproduction in Prokaryotes y y y y y Binary fission Budding Conidiospores (actinomycetes)-chains when they reproduce Fragmentation of filaments Bacterial growth happens exponentially- based on generation number and binary fission can figure out how many cells are being made. Generation Time y If 100 cells growing for 5 hours produced 1,720,320 cells: o o Number of generation= [log #of cells (end) log #cells (beg)]/(0.301) log of 2 Generation time= (60min x 5hours)/ number of generations= 21 min/generation.

Bacterial Growth Curve Phases of Growth (log graph, not linear) 1. Lag phase -not much bacterial growth -no exponential growth 2. Log phase -exponential growth -assuming optimal conditions

-reaches point of capacity 3. Stationary phase -# cells growing = # cells dying -no exponential growth 4. Death phase -nutrients have been used up -exponential decrease Direct Measurement of Microbial Growth 1. Serial Dilutions 1:10 9 mL broth in each tube 1:100 1:1000 1:10,000 1:100,000 2. Plate Counts a) pour plate method b) the spread plate method 3. Plate Counts y 4. After incubation, count colonies on plates that have 25-250 colonies (CFUs)

Counting Bacteria by Membrane Filtration Fig 6.18

5.

Direct Microscopic Count -Serial dilution must be performed -number of bacteria/mL= #of cells counted/ volume of area counted

6.

Turbidity Indirect Methods -turbidity -metabolic activity -dry weight

Direct Methods -plate counts -filtration -MPN -direct microscopic count

3/28/2011 5:22:00 PMThe terminology of microbial control -Sepsis-microbial contamination -Asepsis- the absence of significant contamination -Aseptic surgery-prevent microbial contamination of wounds Sterilization- removing all microbial life Commercial sterilization- killing C. botulinum endospores Disinfection- removing pathogens Antisepsis- removing pathogens from living tissue Degerming- removing microbes from a limited area Sanitization- lowering microbial counts on eating utensils Biocide/germicide- kills microbes Bacteriostasis- inhibiting growth, NOT killing Population Death Rate is Constant A Microbial Death Curve Actions of Microbial Control Agents y y y Heat y y y y y y y y y Thermal death point (TDP)-lowest temperature at which all cells in a culture are killed in 10 min. Thermal death time (TDT)- time during which all cells in a culture are killed. Minutes to kill 90% of a population at a given temperature (fig 7.2) Moist heat denatures proteins Autoclave: steam under pressure Steam must contact items surface Reduces spoilage organisms and pathogens Equivalent treatments 63 C for 30 min Decimal Reduction Time (DRT) Moist Heat Sterilization alternation of membrane permeability damage to proteins damage to nucleic acids (fig 7.1 A)

Steam Sterilization Pasteurization

y y y y

High temperature short time- 72C for 15 sec Ultra high temp- 140 C for 0.3 uM Membrane filtration removes microbes > 0.22 uM Low temp inhibit microbial growth o o o y y y Radiation y Ionizing radiation (X rays, gamma rays, electron beams) o o y y Ionizes water to release OH Damages DNA Damages DNA Kill by heat; not especially antimicrobial Refrigeration Deep freezing Lyophilization

Physical Methods of Microbial Control

High pressure denatures proteins Dessication prevents metabolism Osmotic Pressure causes plasmolysis

Nonionizing radiation (UV, 260 nm) o Microwaves o

Principles of Effective Disinfection y y y y Concentration of disinfectant O