MB 351 - Lecture 18

Question 1

Microbial Growth is defined as…

Answer 1

an increase in the number of cells, not cell size -populations

Question 2

Bacterial growth occurs through

Answer 2

Binary fission

Question 3

Stages of binary fission 1.

Answer 3

cells elongate to approximately twice their original length and DNA is replicated

Question 4

Stages of binary fission 2.

Answer 4

a partition is formed that constricts the cell into two daughter cells -cell wall and plasma membrane begin to constrict

Question 5

Stage 3.

Answer 5

cross-wall forms, completely separating the two copies of DNA -nascent septum formation continues until the two daughter cells are…

Question 6

Stage 4.

Answer 6

Cells separated. Two daughter cells are pinched off.

Question 7

Generation time or Doubling Time

Answer 7

when one cell divides to form two – the time required for this process to occur (the rate of exponential growth)

Question 8

Bacterial growth curve phases (batch culture – closed system) Phase 1

Answer 8

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

Question 9

Phase 2

Answer 9

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?

Question 10

Phase 3

Answer 10

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

Question 11

Phase 4

Answer 11

Death phase. Viable cell population declines – decreases geometrically (exponentially

Question 12

Population growth is limited by:

Answer 12

1. exhaustion of available nutrients 2. accumulation of inhibitory metabolites or end products 3. exhaustion of “biological” space

Question 13

Bacterial Growth Curve looks like…

Answer 13

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.

Question 14

Calculating Generation Time

Answer 14

Question 15

Two basic way to measure growth:

Answer 15

Direct measurement and indirect measurement

Question 16

Direct measurement

Answer 16

such as by measuring cell numbers (counting), or by total mass which is often directly proportional to cell number

Question 17

Indirect means of measurement

Answer 17

measuring turbidity, metabolic activity, or dry weight

Question 18

Direct microscopic counts using counting _________.

Answer 18

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

Question 19

Viable Cell Counts and CFUs

Answer 19

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.

Question 20

Use spectrophotometers to measure the amount of light transmitted by an organism

Answer 20

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