1 Effect 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.

2 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.

3 Classification of bacteria based on temperature

4 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.

5 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 –What is pH of human blood? Some bacteria create their preferred conditions –Lactobacillus creates low pH environment in vagina

6 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.

7 Miscellaneous conditions Radiation (solar, UV, gamma) –Can all damage cells; bacteria have pigments to absorb energy and protect themselves. –Endospores are radiation resistant. –Deinococcus radiodurans: extremely radiation resistant Extremely efficient DNA repair, protection against dessication damage to DNA. Barophiles/barotolerant: microbes from deep sea –Baro- means pressure. Actually require high pressure as found in their environment.

8 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

9 Mathematics of bacterial growth Because bacteria double in number at regular intervals, they grow exponentially: N = N 0 x 2 n where N is the number of cells after n number of doublings and N 0 is the starting number of cells. Thus, a graph of the Log of the number of bacteria vs. time is a straight line.

10 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 2.Exponential or Log phase 3.Stationary phase 4.Decline or Death phase.

11 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.

12 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

13 Measurement of growth 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: single-file cells detected by change in electric current.

14 Measurement of growth -2 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. biology.clc.uc.edu/.../Meat_Milk/ Pour_Plate.htm

Coulter Counter 15 Coulter-counter: single- file cells detected by change in electric current.

16 Filtration: ual/p25bs.jpg Membrane filters are very thin with a defined pore size, e.g µm. Bacteria from a dilute sample are collected on a filter; filter placed on agar plate, colonies counted.

17 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 color/spec/Aspec.htmlwww.umr.edu/~gbert/ color/spec/Aspec.html

18 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).

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. 19

20 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.