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! http://www.kodak.com/global/images/en/health/filmImaging/thermometer.gif

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.

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.

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. Fe3+ -SOD + O2- → Fe2+ -SOD + O2 Fe2+ -SOD + O2- + 2H+ → Fe 3+ -SOD + H2O2

Relation to Oxygen Aerobes: use oxygen in metabolism; obligate. Microaerophiles: require oxygen (also obligate), but in small amounts. Anaerobes: grow without oxygen; SEE NEXT A: aerobe B: microaerophile Capnophiles: require larger amounts of carbon dioxide than are found normally in air.

Anaerobes 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

Effect of temperature Low temperature High 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.

Bacteria 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.

Classification of bacteria based on temperature

Terms 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.

pH 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

Low 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.

Bacterial 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

Mathematics 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.

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. Lag phase Exponential or Log phase Stationary phase Decline or Death phase. 4 3 2 1

Growth 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. 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.

More 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. Growth of bacteria is nonsynchronous, not every bacterium is dividing at the same time. Instead of stepwise curve, smooth curve

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.

Measurement 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.

Coulter Counter Coulter-counter: single-file cells detected by change in electric current.

Counting 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

Filtration and plate counting 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. http://dl.clackamas.cc.or.us/wqt111/coliform-8.jpg http://www.who.int/docstore/water_sanitation_health/labmanual/p25bs.jpg

Spectrophotometry 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

More 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).

Biomass: 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