5- Microbial growth

Question 1

Sources of nutrients

Answer 1

Macronutrients: required in large amounts: Carbon, Nitrogen, Hydrogen, etc.

Micronutrients:required in trace amounts: Iron (ETC), trace metals

Growth factors: molecules that the microorganism needs for growth but cannot synthesize by itself. Some growth factors are the by-product or waste of another microorganism (vitamin, aa, purine, etc.)

Question 2

What is the growth of the population?

Answer 2

Increase number of cells or biomass

Question 3

How do most prokaryotes multiply?

Explain

Answer 3

By binary fission

The cell grows in size until it forms a partition (septum) that
constricts the cells into 2 daughter
cells.
Each daughter cell receives one copy
of the chromosome, sufficient ribosomes, macromolecules, monomers and other molecules to exist as an independent cell.

Question 4

Cell division and peptidoglycan synthesis- what does it require?
What are wall bands?

Answer 4

Cell division requires synthesis of new cell wall material, but also requires its destruction by autolysins.
At the division ring (FtsZ ring),
autolysins create some gaps in
the peptidoglycan. This allows
rearrangement of the
peptidoglycan and synthesis of a
new cell wall.

Wall bands: scar between old and
new peptidoglycan.

Question 5

What is Bactoprenol?

Answer 5

Bactoprenol allows peptidoglycan subunit to be

exported across the cytoplasmic membrane.

Question 6

What is MacConkey?

Answer 6

A selective medium.
Bile salts inhibit growth of Gram+,
permissive for Gram-, enteric pathogens.

Differentiate between Lactose
fermenters (Pink) and Lactose nonfermenters
(colorless).
• Lactose à glucose + galactose.
Glucose à glycolytic pathway à
fermentation: lactate (lactic acid,
reduce pH).
• E. coli forms dark pink colonies with
bile precipitate.

Question 7

What is Mannitol-salt?

Answer 7

Selective medium, high NaCl
concentration: inhibits most Gram-and many Gram+.

• Used for isolation or detection of
Staphylococcus.

• Mannitol fermenters: +, yellow; -, pink.
• Staphylococcus aureus is mannitol +.

Question 8

How do you measure microbial presence/growth?

Answer 8

Evaluate contamination of food, water, etc.

• Ensure enough microorganisms are inoculated during process requiring them:
beer, wine, yogurt, cheese, etc.

• Evaluate the efficiency of antimicrobial agents.
• Study microbial populations from different ecosystems.

• Measure effect of mutation of genes involved in metabolic pathway, survival,
protection, virulence, etc.

Question 9

What are the 2 methods for viable counts?

Answer 9

Spread-plate method and pour-plate method

Question 10

What is serial dilution?

Answer 10

Bacterial cultures can reach
high number of cells (billions).

• To get a viable count of such
cultures, serial dilutions have to
be made.

• The results, and reproducibility,
are strongly affected by the skills of the technician.

CFU =
number of colonies/
dilution plated × volume plated

Question 11

How do you do microscopic counts?

Answer 11

Petroff-Hausser chamber

Count all cells: dead, alive, and cells that cannot be
grown in lab. Viability staining can be used to differentiate dead (red) and live (green) cells.

• Fast, no need to wait until bacteria has grown.
• Small cells can be missed, motile cells are hard to
count and must be immobilized.

Question 12

What is the flow cytometry?

Answer 12

Beher at counting big cells: protozoan, yeast, mammalian
cells, etc.

• Detection of fluorescent dyes
allows labeling of specific cell
types or species.

• Can be used to sort cells according to size, shape, labeling, etc.

Question 13

What is the turbidimetric method? And by what is it affected?

Answer 13

Measure the contribution of alive
and dead cells to turbidity.

• Affected by the behavior of cells:
– Clumping
– Attachment to surfaces

OD (Optica density) is affected by properties of cells: size, shape, composition, cell inclusion, etc.
• A standard curve must be made and the relationship between OD and cell number must be established empirically. Some mutations may affect this relationship.

Question 14

Population growth; generation time, formula

Answer 14

Generation time: time needed for the
population to double.

g = t/n
g = generation time
t = time
n = number of generation
• Generation time depends on the
growth medium and the conditions.

• When the conditions are right,
microorganisms can grow
exponentially; the population
doubles at a constant rate.

N = N02’n
N, number of cells
N0, number of cells, initially
n, number of generation

Question 15

Growth cycle in bath culture; lag phase; exponential phase; stationary phase; death phase

Answer 15

Lag phase: time needed by the bacteria to adjust to the new condition, slow growth.

• Exponential phase: doubling of the population at a constant rate.

• Stationary phase: limiting nutrients are depleted or accumulation of a waste product that inhibits growth; growth is stopped. No net increase in cell number,
cells are still metabolically active, induction of “survival” systems.

• Death phase: cells start to die, metabolism has stopped, in some cases cell death
occurs with cell lysis. Death phase is also an exponential function.

• Batch cultures are continually being affected by the metabolic activities of the growing microorganism: depletion of nutrients, genera@on of toxic waste.

Question 16

What is the equilibrium in natural environment?

What is the main limiting factor?

Answer 16

Division rate = death rate

concentration of a limiting growth factor

Question 17

What is chemostat? fresh medium; overflow

What are the parameters?

Answer 17

Fresh medium: supply of limiting nutrients.
Overflow: death of microorganism

Parameters: concentration of a limiting nutrient (fresh medium and aeration) and dilution rate (addition of fresh medium and washout)
Once equilibrium is reached, nbr of cell is constant, the growth rate = death rate (washout)

Question 18

What factors affect growth?

Answer 18

- Nutrients
– Temperature
– pH
– Osmolarity
– Oxygen
– Pressure
– Radiation (visible light, UV light)

Question 19

What are extremophiles?

Answer 19

microorganisms
that grow preferentially under
extreme conditions.

Question 20

Temperature; minimum, maximum and optimum growth rate

Answer 20

Minimum: membrane gelling; transport processes so slow that growth cannot occur

Maximum: protein denaturation; collapse of the cytoplasmic membrane; thermal lysis

Optimum: enzymatic reactions occuring at max possible rate

Question 21

Temperature for: Psychrophile; Mesophile; Thermophile; Hyperthermophile;

Answer 21

Psychrophile; 4 Mesophile; 39 Thermophile; 60 Hyperthermophile; 88 or 106

Question 22

Adaptation of microbial life in cold temperature; cryoprotectants
At what T microbial culture can be preserved?

Answer 22

Adaptation to cold temperature:
– Changes in protein structure and sequence so the enzymes are active at low
temperature.

– Transport across the membrane functions optimally at low temperature.

– Requires modification of the cytoplasmic membrane so it stays fluid at low
temperatures.

– Cold-shock proteins which help keep proteins active.

– Cryoprotectants: antifreeze proteins, glycerol. They help prevent the
formation of ice crystals that can puncture the cytoplasmic membrane.

• Cold temperature does not kill microorganisms, ice crystals do!

• Microbial cultures are preserved at -80°C or -196°C in liquid nitrogen.
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Question 23

What is Barophilic (piezophilic)?

Answer 23

growth best at high pressure

Question 24

Adaptation to high T

Answer 24

• Adaptation to high temperature:
– Changes in protein sequence so the enzymes are not denatured by the high
temperature and can stay active. These enzymes are heat-stable.

– Transport across the membrane functions optimally at high temperature.

– Requires modification of the cytoplasmic membrane so it remains stable at
high temperatures, for example the lipid monolayer of Archaea.

– Heat-shock proteins which help keep proteins in the active conformation.

– Protection mechanisms to ensure stability of DNA (GC rich).

• Hot temperatures kill microorganisms (thermal lysis).
• Endospores help resist high temperatures (but they are not metabolically active).

Question 25

pH- Adaptation to: low pH; high pH; internal pH

Answer 25

• Adaptation to low pH:
– Changes of the cytoplasmic membrane to resist high concentration of protons. Usually the membrane
requires high concentration of protons for stability. Bacteria lyse at higher pH, because the membrane
becomes unstable.

• Adaptation to high pH:
– Changes of the cytoplasmic membrane to resist low concentration of protons.
– Use of Na+ gradient for transport and motility (low concentration of protons outside, pmf is hard to
maintain).
– Keep the electron transport chain close to the ATPase, so protons that are pumped out do not diffuse away.

• Internal pH: ideally close to the pH of the environment but:
– DNA is acid-labile
– RNA is alkali-labile
– Internal pH limits: 4.6 – 9.5 (with protection system, not fully understood yet).

• Buffers are usually used in growth medium to keep the pH steady because bacterial waste tends to affect pH (Ex.: MacConkey Agar).

Question 26

Osmotic effect; halophile

Answer 26

Microorganisms that can grow at high
salt concentrations are called
halophiles.
• Usually requires NaCl for growth.
• Seawater: 3%

Question 27

What are the toxic forms of oxygen? How does it happen?

Answer 27

O2- (superoxide); H2O2 (hydrogen peroxide); OH (hydroxyl radical)

During aerobic respiration O2 is reduced to H2O.

During oxygenic
photosynthesis H2O is oxidized to O2.

During these processes, toxic forms of oxygen are produced.

• Flavoproteins, quinone and iron-sulfur proteins, present in virtually all cells, can also reduced O2 to O2-

• Toxic forms of oxygen oxidize cell components, stopping key metabolic
pathways and destroying key structures.

Question 28

Resistance to toxic forms of oxygen?

Answer 28

Aerobes and facultative aerobes usually have catalase and superoxide dismutase.

• Anaerobes may or may not contain such enzymes, but if they do, the activity of
these enzymes is clearly not sufficient to allow the organism to grow under oxic
conditions; however it maybe sufficient for the cell to survive.