MB 351 - Lecture 18 Flashcards
Microbial Growth is defined as…
an increase in the number of cells, not cell size -populations
Bacterial growth occurs through
Binary fission
Stages of binary fission 1.
cells elongate to approximately twice their original length and DNA is replicated
Stages of binary fission 2.
a partition is formed that constricts the cell into two daughter cells -cell wall and plasma membrane begin to constrict
Stage 3.
cross-wall forms, completely separating the two copies of DNA -nascent septum formation continues until the two daughter cells are…
Stage 4.
Cells separated. Two daughter cells are pinched off.
Generation time or Doubling Time
when one cell divides to form two – the time required for this process to occur (the rate of exponential growth)
Bacterial growth curve phases (batch culture – closed system) Phase 1
Lag Phase (length of time is highly variable): immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged -dependent on “history” of inoculum -change in cell composition but no cell division -adaptation to new medium conditions -resynthesis of damaged cell constituents
Phase 2
Log (exponential) phase: A pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. Phase is relatively short. Cells in this phase are most active metabolically (good for experiments) -# of cells doubles in fixed time period?
Phase 3
Stationary phase. The growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes. Secondary metabolites are produced during this phase. Spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation to prepare for dormant period
Phase 4
Death phase. Viable cell population declines – decreases geometrically (exponentially
Population growth is limited by:
- exhaustion of available nutrients 2. accumulation of inhibitory metabolites or end products 3. exhaustion of “biological” space
Bacterial Growth Curve looks like…
You can plot the growth curve for exponentially increasing population as plotted logarithmically (dashed line) and arithmetically (solid line). Plotting problems can be avoided by graphing Log10 of the population numbers.
Calculating Generation Time
Two basic way to measure growth:
Direct measurement and indirect measurement
Direct measurement
such as by measuring cell numbers (counting), or by total mass which is often directly proportional to cell number
Indirect means of measurement
measuring turbidity, metabolic activity, or dry weight
Direct microscopic counts using counting _________.
counting chambers or Petroff-Hausser cell counters. These are glass slides with precisely machined chambers and coverslips, so that cells in very small volumes can be counted using a microscope.
-dead cells can not be distinguished from living ones, it is hard to see small cells and unstained cells, and labor intensive
Viable Cell Counts and CFUs
Viable cell counts, also called colony/plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be serially diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (CFU) and the number of CFU’s is related to the viable number of bacteria in the sample. A viable cell is one that is able to divide and form offspring, and in most situations of cell counting, these are the ones we are most interested in. However, note that a Viable count NOT a total cell count, only those that are viable will be observed. Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are (1) only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth, for most microbes we don’t know how to culture them in the lab. There are other experimental problems associated: it is common for pipetting or dilution errors to occurs, resulting in poor erroneous cell and colony numbers and or distribution of cells. For accurate info., it is critical that each colony comes from only one cell, so chains and clumps of cells must be broken apart. Only those plates between 30-300 CFUs can be used to calculate the CFU/ml, as that is statistically significant.
Use spectrophotometers to measure the amount of light transmitted by an organism
Turbidity measurements. Measures light that is not scattered by the sample. More cells means more scattering, which results in less light transmission.
- dead cells are counted, sensitivty is limited
- Advantage: fast, doesn’t destroy cells
