physical requirements for growth prefixes and suffixes: bacteria are highly diverse in the types of...
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Physical requirements for growth
• Prefixes and suffixes:• Bacteria are highly diverse in the types of
conditions they can grow in. – Optimal or required conditions implied by “-
phile” meaning “love”
• Some bacteria prefer other conditions, but can tolerate extremes– Suffix “-tolerant”
• Note the difference!
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http://www.kodak.com/global/images/en/health/filmImaging/thermometer.gif
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When growing microbes..
• The physical/chemical conditions that are most important – Presence or absence of oxygen– Temperature range– pH range– Water activity (how “wet”)
• Note that by changing the conditions to make them unfavorable we can prevent bacterial growth.
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Oxygen: friend or foe?• Early atmosphere of Earth had none
– First created by cyanobacteria using photosynthesis– Iron everywhere rusted, then collected in atmosphere
• Strong oxidizing agent• Reacts with certain organic molecules, produces
free radicals and strong oxidizers :– Singlet oxygen, H2O2(peroxide), O3
- (superoxide), and hydroxyl (OH-) radical.
– Free radicals are highly reactive chemicals that damage proteins, nucleic acids, and other cell molecules.
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Protections of bacteria against oxygen
– Bacteria possess protective enzymes, catalase and superoxide dismutase.
– Catalase breaks down hydrogen peroxide into water and oxygen gas.
– Superoxide dismutase breaks superoxide down into peroxide and oxygen gas.
– Anaerobes missing one or both; slow or no growth in the presence of oxygen.
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Fe3+ -SOD + O2- → Fe2+ -SOD + O2
Fe2+ -SOD + O2- + 2H+ → Fe 3+ -SOD + H2O2
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5Relation to Oxygen
• Aerobes: use oxygen in metabolism; obligate.
• Microaerophiles: require oxygen (also obligate), but in small amounts.
• Anaerobes: grow without oxygen; SEE NEXT
•Capnophiles: require larger amounts of carbon dioxide than are found normally in air.
A: aerobeB: microaerophile
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6Anaerobes grow without O2
• Classifications vary, but our definitions: – Obligate (strict) anaerobes:
killed or inhibited by oxygen.– Aerotolerant anaerobes: do
not use oxygen, but not killed by it.
– Facultative anaerobes: can grow with or without oxygen
C: could be facultative or aerotolerant.D: strict anaerobe
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7Effect of temperature
• Low temperature– Enzymatic reactions too slow; enzymes too stiff– Lipid membranes no longer fluid
• High temperature– Enzymes denature, lose shape and stop functioning– Lipid membranes get too fluid, leak– DNA denatures
• As temperature increases, reactions and growth rate speed up; at max, critical enzymes denature.
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8Bacteria and temperature
• Bacteria have temperature ranges (grow between 2 temperature extremes), and an optimal growth temperature. Both are used to classify bacteria.
• As temperature increases, so do metabolic rates.• At high end of range, critical enzymes begin to
denature, work slower. Growth rate drops off rapidly with small increase in temperature.
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9Classification of bacteria based on temperature
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10Terms related to temperature
• Special cases:– Psychrotrophs: bacteria that grow at “normal”
(mesophilic) temperatures (e.g. room temperature” but can also grow in the refrigerator; responsible for food spoilage.
– Thermoduric: more to do with survival than growth; bacteria that can withstand brief heat treatments.
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11pH Effects
• pH = -log[H+]
• Lowest = 0 (very acid); highest = 14 (very basic) Neutral is pH 7.
• Acidophiles/acidotolerant grow at low pH
• Alkalophiles/alkalotolerant grow at high pH
• Most bacteria prefer a neutral pH– Many grow well from pH 6 to 8
• Some bacteria create their preferred conditions– Lactobacillus creates low pH environment in vagina
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12Low water activity:halophiles, osmophiles, and xerotolerant
• Water is critical for life; remove some, and things can’t grow. (food preservation: jerky, etc.)
• Halophiles/halotolerant: relationship to high salt.– Marine bacteria; archaea and really high salt.
• Osmophiles: can stand hypertonic environments whether salt, sugar, or other dissolved solutes– Fungi very good at this; grandma’s wax over jelly.
• Xerotolerant: dry. Subject to desiccation. Fungi best– Bread, dry rot of wood– Survival of bacterial endospores.
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13Bacterial growth defined
• Since individual cells double in size, then divide into two, the meaningful increase is in the population size.
• Binary fission: cell divides into two cells. No nucleus, so no mitosis.
• Cells do not always fully detach; produce pairs, clusters, chains, tetrads, sarcina, etc.
• “GROWTH” = increase in number of bacteria
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14Mathematics of bacterial growth
• Because bacteria double in number at regular intervals, they grow exponentially:
• N = N0 x 2n where N is the number of cells after n number of doublings and N0
is the starting number of cells.
• Thus, a graph of the Log of the number of bacteria vs. time is a straight line.
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15
The Bacterial Growth Curve
• Bacteria provided with an abundant supply of nutrients will increase in number exponentially, but eventually run out of nutrients or poison themselves with waste products.
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1. Lag phase2. Exponential or
Log phase3. Stationary
phase4. Decline or
Deathphase.
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16Growth curve (continued)
• Lag phase: growth lags; cells are acclimating to the medium, creating ribosomes prior to rapid growth.
• Log phase: cells doubling at regular intervals; linear graph when x-axis is logarithmic.
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•Stationary phase: no net increase in cell numbers, some divide, some die. Cells preparing for survival.•Decline phase: highly variable, depends on type of bacteria and conditions. Death may be slow and exponential.
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17More about Growth
• The Growth curve is true under ideal conditions; in reality, bacteria are subject to starvation, competition, and rapidly changing conditions.
• Generation time: the length of time it takes for the population to double.
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•Growth of bacteria is nonsynchronous, not every bacterium is dividing at the same time.
•Instead of stepwise curve, smooth curve
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Exponential growth
• “Balanced growth”– Numbers of bacteria are doubling at regular
intervals.– All components of bacteria are increasing in amount
at the same rate• 2x as many bacteria = 2x as much protein, 2x as
much peptidgolycan, 2x as much LPS, etc.– During exponential growth, bacteria are not limited
for any nutrients, i.e. they are not short of anything.
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19Measurement of cell numbers
• Direct methods: cells actually counted.– Petroff-Hausser
counting chamber (right), 3D grid. Count the cells, multiply by a conversion factor.
– Dry a drop of cells of known volume, stain, then count.
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Coulter Counter20
Coulter-counter: single-file cells detected by change in electric current.
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21Counting cells with plates
• Viable plate count– Relies on bacteria being alive,
multiplying and forming colonies.
– Spread plate: sample is spread on surface of agar.
– Pour plate: sample is mixed with melted agar; colonies form on surface and within agar.
– Colonies counted with a colony counter.
biology.clc.uc.edu/.../Meat_Milk/ Pour_Plate.htm
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22Filtration and plate counting
http://dl.clackamas.cc.or.us/wqt111/coliform-8.jpg
http://www.who.int/docstore/water_sanitation_health/labmanual/p25bs.jpg
•Membrane filters are very thin with a defined pore size, e.g. 0.45 µm.
•Bacteria from a dilute sample are collected on a filter; filter placed on agar plate, colonies counted.
•Used when concentration of bacteria is low.
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23Spectrophotometry
• Bacteria scatter light, making a turbid (cloudy) suspension.
• Turbidity is usually read on the Absorbance scale– Not really absorbance, but Optical Density (OD)
• More bacteria, greater the turbidity (measured as OD)
Based on www.umr.edu/~gbert/ color/spec/Aspec.html
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24More about Spectrophotometry
– Does NOT provide an actual number unless a calibration curve (# of bacteria vs. O.D.) is created.• Indirect counting method
– Quick and convenient, shows relative change in the number of bacteria, useful for determining growth (increase in numbers).
– Does NOT distinguish between live and dead cells. To create a calibration curve, best to plot OD vs. number of cells determined with microscope (not plate count).
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25Biomass: • Measure the total mass of cells or amount of any
component such as protein, PS, DNA, KDO.• Especially when cells are doubling,
the amounts of all the components of a cell are increasing at the same rate, so any could be measured.– Not so in stationary phase.
In this example, total biomass increases exponentially over time.
http://www.pubmedcentral.nih.gov/pagerender.fcgi?artid=242188&pageindex=10#page