Most bacteria reproduce by BINARY FISSION. One cell roughly doubles in volume, contents, and divides into 2 cells. There are exceptions: bacteria that divide by unequal fission (budding), but this does not affect cell number, only mass parameters.

Batch Growth Curve reflects behavior of a population, not that of an individual cell. See Fig. 20.

LAG PHASE is a period of adaptation, if required. Lag may be quite long when stationary phase cells are transferred to fresh medium or when cells in a rich medium are transferred to a nutrient-poor medium.

EXPONENTIAL PHASE: Cells more uniform in properties--balanced growth and balanced increase in all components occurs.

STATIONARY PHASE: Growth slows as wastes accumulate, nutrients are depleted. No net increase or decrease in cell numbers occurs (death balances growth). Cells are significantly different in properties in stationary phase (it is a differentiated state).

DEATH PHASE: Death rate is greater than growth rate. Cell lysis may occur. Note: death is also exponential

Bacterial populations increase EXPONENTIALLY, not arithmetically. Plot of cell number as a function of time is an exponential curve, not a straight-line.

N_t = Number of cells at time t

N₀ = Initial number of cells

n = number of generations that have occured

k = average (mean) growth rate = 1/g

k is a rate & has units of reciprocal time.

g = mean doubling time (generation time) = 1/k

g is a time, and has the units of time.

Dt = time interval

Textbook describes calculations to determine the specific growth rate constant (m), also known as the "instantaneous growth-rate constant." m = 0.693k or m = 0.693/g

k is an average value over a population of cells, while m is more related to rate of individual activities.

Average growth rate is easily calculated during the exponential phase of growth. In mid-exponential phase, growth is BALANCED--probably rarely attained in the wild, but can be produced in laboratory. Relative rate of synthesis is same as growth rate. An important concept in physiological studies is that cells should be in reproducible conditions--achieved by manipulating cells under standard conditions and working with cells in mid- to late-exponential phase of growth. Text makes the statement that a physiological experiment done with a poorly characterized culture is "all but useless." This is not so. It is completely useless--it can not be repeated, and if it can't be repeated, it is completely useless.

1. Cell Number

A. Viable cell count--plating under standard conditions. Counts colony forming units (cfus)

B. Petroff-Hauser Counter Chamber (hemocytometer)--not very sensitive or accurate; counts all cells (alive or dead)

C. Coulter Counter--electronic counting device. Conductivity changes when cell passes a small orifice, causing a voltage pulse

2. Cell Mass

A. Dry Weight--remove H₂O by drying and simply weigh the dried cell mass. Insensitive and not very accurate.

B. Light scattering is proportional to cell volume, not cell numbers. Not good for low numbers, but very easy to measure with spectrophotometer or nephelometer. Scattering increases with decreasing wavelength of light, so blue light is more sensitive than red or far red light.

C. Colorimetric assay for protein, DNA, RNA, Chlorophyll, etc. Usually simple and sensitive.

A linear relationship exists between the biomass produced and the amount of the limiting nutrient for a given organism under a given set of conditions when all other nutrients are in excess.

Example: tryptophan auxotroph of E. coli

The amount of biomass will be linearly related to amount of tryptophan added to the growth medium, if all other nutrients are in excess. Establish a standard curve by adding known amounts of tryptophan to a fixed amount of cells and media. Add a test material, and quantitate growth. This BIOASSAY allows quantitation of the amount of tryptophan in the test material.

Growth yields have been used to make guesses concerning fermentative metabolism mechanism. Since ATP consumption in biosynthesis, polymerization and assembly is virtually constant, yield of cells per mole of input carbon/energy source can be used to comparatively. Fermentations typically yield constant, integral values (1 ATP per mole, or 2 ATP per mole), and given the uncertainties, there is a large and meaningful difference. Difference between 24 and 30 would be less meaningful and harder to determine. In respirations there are too many variable in general, although exceptions exist where YATP is still useful.

Radiochemical labeling has advantages over purely chemical methods: allows smaller samples and greater sensitivity.

Choice of label is important, and requires careful thought and knowledge of biochemistry. Common labels include: ³H, ¹⁴C, ³²P, ³⁵S

General labels will label many types of molecules

[¹⁴C]-glucose would label all organic compounds in a chemoheterotroph (¹⁴CO₂ for an autotroph). Adding unlabeled (cold) compounds may suppress labeling of specific molecules

[³²P]-phosphate would label primarily RNA, DNA, phospholipids

More specific labels:

[³H]-thymidine DNA

[³H]-uracil RNA

[³⁵S]-methionine Proteins

[¹⁴C]-diaminopimelic acid Murein

[¹⁴C]-heptose LPS

Using mutants dependent upon exogenous compounds (e.g., auxotrophs) can cause much greater specificity of labeling.

Heavy isotopes (²H, ¹³C, ¹⁵N) can also be used in separations based on density or may be important for enzyme mechanism studies

Isotopic labeling may also include inhibitors of synthesis of certain (macro)molecules.

Penicillins, Cephalosporins: Murein

Cerulenin" Fatty acids

Chloramphenicol Proteins

Fusidic Acid Proteins

Rifampicin RNA

Streptolydigin RNA

Nalidixic Acid DNA

Mitomycin C DNA

Addition of precursor or metabolic building block will first lead to labeling of the pool, and knowledge of pool size and use is helpful. Most amino acid pools are small, and completely turn over in 10-20 seconds. Thus, after a minute or so, the specific activity of the pool will be equal to the added material. Specific activity of macromolecules will depend upon the growth rate and the length of labelling. Would require longer-term labeling, and depends upon the stability of the compound being considered (proteins and DNA differ from RNA).

Small molecules often separated from macromolecules by precipitation with 5% Trichloroacetic Acid (TCA). Precipitates proteins, RNA, DNA but not small molecules.

Measuring incorporation as a function of time allows an estimate of growth rate during BALANCED GROWTH.

During unbalanced growth, rates of synthesis of components are distinct from growth rate and are variable as a function of time. PULSE LABELING, short-term labeling, is used to estimate the instanteous rate of synthesis. To extract information concerning rate of biosynthesis requires that pool size be small and that macromolecules are stable.

PULSE-CHASE LABELING: the pulse is followed by large amount of unlabeled compound. Allows behavior of pool to be monitored (see Fig. 11 of text). Difference between total uptake and pool rise and fall will equal incorporation into macromolecule.

RNA labeling presents a special challenge, since there are two classes of RNA: stable (tRNA, rRNA) and unstable (mRNA). RUN-OUT labeling can distinguish the two classes. Add label and inhibit initiation of new RNA synthesis (rifamycin addition). Incorporation occurs into all RNA classes. Monitor loss of label macromolecular pool as mRNA is degraded back to nucleotides. The ratio of peak labeling to steady-state will give proportion of RNA synthesis that is stable vs. unstable. (See Fig. 13, p. 223).