L15: Bacterial Growth And Measurement Of Growth

Question 1

Bacterial binary fission

Answer 1

Bacterial reproduction

-> pop increase in number (logarithmic)

Question 2

Generation time (g)

Answer 2

The time for cell to produce 2 cells or time for bacterial pop to double in no.

Question 3

As no. of bacteria increases in environment..

Answer 3

Nutrients used up

Metabolic wastes that may be toxic accumulate

Living space may become limited

Aerobes may suffer from oxygen depletion

-> bacterial growth stops

Question 4

Significance of bacterial growth

Answer 4

Has human health implications: bacterial meningitis, cholera, food poising from staphylococci or salmonella, plague, tetanus etc

Has animal health implications: anthrax in ruminants, mastitis in dairy herds etc

Has environmental impacts: eutrophication (enrichment of aquatic environment with nutrients e.g. from sewage pollution), blooms of toxic cyanobacteria, acid sulphate soils, composting and sewage treatment

Provides for industrial applications: producing products (e.g. cheese, antibiotics, insulin), industrial processes (e.g. leaching metals from low grade ores)

Question 5

Stationary cells

Answer 5

Non-dividing cells inoculated into fresh medium and begin to grow and multiply

Question 6

Phases in bacterial growth in culture

Answer 6

Lag phase -> exponential or log phase -> stationary phase -> death phase

Y axis: log number of viable cells

X axis: time

Question 7

1. Lag phase

Answer 7

Inoculate stationary cells

Cells have to adapt to new physiological conditions

Synthesis of cellular constituents (ribosomes & enzymes) begins

Energy (ATP) become available for growth

Cell volume increases but no cell division-> growth rate (k) and generation time (g)= 0

Rates of physiological process, initially unbalanced -> different genes are upregulated at different times

DNA replication and cell division initiated (cell division and hence pop growth begins)

Question 8

2. Exponential (or Log) Phase

Answer 8

Bacteria are growing and dividing at max rate possible for particular strain , medium, and environment

Growth: balanced - relative synthesis rates are constant (DNA, ribosomes, enzymes, cell walls etc)

Growth rate (or growth rate constant). k=no. of generations per unit time, often per hour

k and g are constant

Graph of log [no.] vs. time produces straight line (slope = k)

Rate of growth depends in conditions (temp, nutrients). Different (stable) conditions -> exponential growth occurs but at different rate

Exponential growth continues until some factor becomes limiting. Limiting factors-> drive culture into stationary phase

Question 9

3. Stationary phase of growth

Answer 9

Total viable cell numbers remain constants (k=0): cells are not dividing (nutrients or O2 have been depleted, but remain metabolically active) or metabolic waste/toxin may have built up and cell death or inactivation balances cell division

Question 10

Properties of cultures in stationary phase

Answer 10

Cells tend to be small

Cells have different composition of cellular constituents compared to growing cells

Growth again unbalanced

Available energy used for maintenance (cell survival mechanisms e.g. endospore formation may be initiated)

Question 11

4. Cell death phase

Answer 11

Cells begin to die (nutrient deprivation or toxic waste build up -> irreplaceable damage to cells)

K becomes -ve

Pop death rate usually logarithmic (constant proportion of cell dies per unit time)

Question 12

Hypotheses of cells in death phase

Answer 12

‘Classical view’: even when cells transferred to fresh medium, did not resume growth. Assumed cells died but did not lyse

Other hypotheses:

1. Do bacteria in death phase actually become ‘viable but not nonculturable’ cells? These are dormant cells, could possibly resume growth
2. Does a fraction of pop of cells die due to activation of programmed cell death genes? Dead cells then release nutrients which support growth of other cells. Dying cells sacrifice themselves for rest

Question 13

5. Long term stationary phase

Answer 13

Some exhibit this phase

Can last months

Pop. continually evolves, actively growing cells use nutrients released by dying cells

Successive waves of genetically distinct variants

Question 14

What about bacterial pop growth in natural world?

Answer 14

Bacteria in soils, waters, surfaces, intestinal tracts: subject to stimulants and inhibitors of growth

Natural systems: complex. Variable in physical, chemical and bio factors affecting pop.

Many microbes form biofilms: aggregations of microbes in complex communities, growing on surfaces and held together by extracellular polymers

Growth cycles fluctuate to reflect environmental variability and complexity

Question 15

How is bacterial growth measured?

Answer 15

Measure cell biomass

Measure cell no.

Question 16

Measure cell biomass

Answer 16

Dry weight determination: cells in broth collected by centrifugation, washed and dried in oven -> weighed. Insensitive (>10^9 cells/ml required), very slow (1 or 2 days in oven). Used for studies on fungi & filamentous bacteria

Wet weight determination: weigh pellet after centrifugation -> result usually less consistent than dry weight, insensitive (varies) but rapid

Question 17

Direct (or total) cell counts

Answer 17

Example: microscope counts

Not very sensitive (> 10^7 cells/ml required)

Rapid method (completed in ~30 mins)

Uses counting chamber: side view of chamber showing coverglass and space beneath holds bacterial suspension. Top view: grid in centre of slide. In grid, bacteria in several squares are counted -> used to calculate conc of cells in original sample

Question 18

Viable cell counts: serial dilution and plating (direct)

Answer 18

Plates with 30-300 colonies are counted

0.1ml aliquots are taken and spread onto surface of agar plates using sterile glass or plastic spreader

Question 19

Viable cell counts: conc/filtration (direct)

Answer 19

Membrane filter on filter support-> water sample filtered through membrane filter (0.45um pore size will trap most bacteria) -> membrane filter removed and placed in plate containing appropriate medium -> incubation for 24 hrs -> typical colonies

Membranes with different pore sizes trap different microorganisms. Incubation times for membranes also vary with medium and microorganism

Question 20

Most probable number (MPN) (direct)

Answer 20

Statistical estimate of probable pop in liquid by diluting and determining endpoints for microbial growth

One cell would give rise to turbid broth after O/N incubation

Used in water analysis

Question 21

Turbidity (indirect growth measures)

Answer 21

Bacterial cells affect passage of light through a solution

Measured by light absorbance or its inverse, light transmittance

Very rapid method and often used

Inaccurate when cell densities are high

Question 22

Measuring turbidity

Answer 22

Spectrophotometer has 2 scales: bottom scale shows absorbance and top scale: percent transmittance

Absorbance increases as % transmittance decreases so absorbance=1 when %transmittance=0

Determination of microbial number by measurement of light absorption. As pop and turbidity increase, more light is scattered and so absorbance reading increases

Question 23

Desirable to maintain cells in exponential growth: applications of knowledge of bacterial growth

Answer 23

Useful in genetic and physiological experiments: cells in identical growth state, relative synthetic rates constant for all cell constituents thus reproducibility in repeated experiments

Useful in industrial fermentations to make primary metabolites (produced during active growth)

Question 24

Applications of knowledge of bacterial growth: stationary phase is needed for other purposes

Answer 24

Secondary metabolites: products of metabolism synthesised after growth has been completed

E.g. antibiotics produced in stationary and death phases

Question 25

Applications of knowledge of bacterial growth

Answer 25

Chemostat enables cells to be kept in exponential phase, at specific growth rate (provide constant supply of one essential nutrient at limiting conc = continuous culture system)