Week 3 enumeration methods

Question 1

describe the bacterial growth curve

Answer 1

• seen in batch culture/ closed system
• population increase is exponential, usually by binary fission sometimes budding

lag phase: bac getting used to environment (may need to repair or turn on genes before growth)

Log phase: period of balanced growth, exponential inc in number of living bac cells

stationary phase: plateau in number of livng bacterial cells: rate of cell division and death roughly equal

Death or decline phase: exponential decrease in # of living bacterial cells

Question 2

describe the formation of endospores to compensate for deteriorating growth conditions

Question 3

describe the steps to the changes in gene expression for the formation of an endospore

Answer 3

1. DNA replicates
2. Membranes form around the DNA
3. Forespore forms additional membranes
4. Protective cortex forms around the spore
5. Protein coat forms around the cortex
6. spore is released

Question 4

what are chemostats

Answer 4

• used to maintain constant exponential growth in lab
• nutrients are refreshed and wastes removed
• maintains log phase
• can be used for pure culture (ex physiological studies, or when growing a particular strain in industry)

Question 5

plotting generation time

Answer 5

generation time (g) is the time it takes for cells to double in number

• plot it as a log phase growth (Semilog scale)

Question 6

explain biphasic log phase curve

Answer 6

• due to change in growth conditions
  ex: if you had glucose and lactose, glucose is used preferentially but when that runs out then needs to turn on lac operon and then will sue lactose

^does not grow at same rate bc no longer optimum conditions

Question 7

what are the 3 enumeration methods

Answer 7

1. Viable plate counting
2. Optical density measurements
3. Hemocytometer

Question 8

describe viable plate counting

Answer 8

indirect method

• must fall in 30-300 range
• problems is organism is filamentoud (lots of cells give rise to one colony)
• sometimes need to conc cells to get the on plate
• consider spread vs pour plates, pour can inocualte larger volume but not goo for heat sensitive or obligate aerobes bc need 55 deg molten agar and organisms are obmerged

Question 9

describe optical density measurements

Answer 9

• indirect method
• looking at cloudiness of broth culture
• light is scattered to determine NOT absorbed
• uses a spectrophotometer - one scan with blank I0 = 100% then with suspension which scatters light giving reading of I_T

OD = log (I₀/ I_T)

• once above 0. ratio, proportionality is lose and results dont mean much, will tell you there is less than there actually is bc of backscattering and blocking
• to compensate can make a dilution -> note never an exact measurement “quick and dirty method

Question 10

describe a hemosytometer

Answer 10

direct method

• can do instantly - look through a microscope and count on slides
• slide has depression that hold 50 microliters
• count cells on grid and see how many are in 5 squares then extrapolate

*get cells/mL

drawbacks: for dilute concentrations wont get enough cells in 50 microliters to get a statistically significant amount