5. Microbial growth

Question 1

What are Growth factors?

Answer 1

• substances that microorganism needs for growth but cannot synthesize by itself.
• can be by-product or waste of another microorganism.
  e. g.Vitamins, amino acids, purines, pyrimidines or other organic molecules

Question 2

What is the process of binary fission?

Answer 2

• the cell grows in size until it forms a partition (septum) that constricts the cells into 2 daughter cells.
  cell elongation–> Septum formation–> Completion of septum formation of walls; cell separation

Question 3

Each daughter cell receives how many copies of the chromosome from the binary fission?

Answer 3

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

Answer 4

Cell division requires both the synthesis of new cell wall material, and destruction by autolysins.

Question 5

Bactoprenol

Answer 5

• a lipid acts as a membrane carrier

- allows peptidoglycan subunit to be exported across the cytoplasmic membrane.

Question 6

Autolysins

Wall bands

Answer 6

• create some gaps in the peptidoglycan at the division ring
• allows rearrangement of the peptidoglycan and synthesis of a new cell wall.
• Wall bands: scar between old and new peptidoglycan.

Question 7

Selective medium: MacConkey

Answer 7

• Bile salts inhibit growth of Gram+ ; permissive for Gram- , enteric pathogens.

Question 8

MacConkey plate can be used to differentiate___?

Answer 8

Lactose fermenters (Pink) lactose positive
Lactose non- fermenters (colorless)
Lactose–> glucose + galactose.
Glucose–> glycolytic pathway->fermentation: lactate (lactic acid, reduce pH).
e.g. E.coli forms dark pink colonies with bile precipitate–> lactose fermenters

Question 9

Mannitol-salt plate

Answer 9

• 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 10

1. Viable counts

Answer 10

1) spread-plate method: sample is pipetted onto agar plate
2) pour-plate method: sterile plate
» Need a permissive growth medium.
» Results are reliable when there are between 30-300 colonies.

Question 11

• Serial dilution

Answer 11

choose 1 plates which has between 30-300 colonies
do NOT use average value between 2 plates.
Because there should be only 1 plate around 30-300
CFU = (number of colonies)/ (dilution plated) × (volume plated)

Question 12

2. Microscopic counts

- Viability staining

Answer 12

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

Question 13

• Petroff-Hauser chamber

Answer 13

sample between coverslip and slide is 0.02mm. Whole grid has 25 large squares, total area 1 mm and total volume 0.02 mm^3

Question 14

3. Flow cytometry

Answer 14

• put all cells in a straight line, use a laser to count the cells
  • Better 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 15

4. Turbidimetric method

Answer 15

• use spectrophotometer, shoot beam of light in the culture and measure the turbidity of the culture
• can NOT differentiate live and dead cells.
  • Measure the contribution of alive and dead cells to turbidity.
  • Affected by the behavior of cells:
  – Clumping
  – Attachment to surfaces
  • Optical 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 16

Population growth:

• generation time?
• factors affecting generation time?

Answer 16

• Generation time: time needed for the population to double.
• 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=N0 (2^n)
n–>number of generation

Question 17

how to calculate generation time?

Answer 17

g= t/n

time/ number of generation

Question 18

Growth cycle in batch culture

Answer 18

• 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, sometimes cell death occurs with cell lysis. Death phase is also an exponential function.
• Depletion of nutrients, generation of toxic waste. can continuously affect growth

Question 19

Continuous culture

- Chemostat

Answer 19

• can be used to keep the microorganisms in a constant growth rate over a long period of time.
• vessels with broth, inoculate with cells
  1. fresh medium: input of fresh broth
  2. overflow: remove some liquid if you add some
  e.g.as the waste is removed, a fraction of the population are removed as well
  so the population remains constant

Question 20

Open system in natural environment

Answer 20

• Most natural environments are open systems:
– constant supply of nutrients and diffusion of waste.
– competition with other microorganisms.
– Predation (protozoan, worms, etc.).
– changing environmental conditions.

Question 21

most environmental systems can reach an equilibrium: division rate equals death rate.
because…..?

Answer 21

concentration of a limiting growth factor

Question 22

As the concentration of a limiting nutrient increases,

Answer 22

• at the beginning, cells growth rate and yield are both increased
• When the growth rate reaches the maximum, only cells yield increases along with the nutrient concentration

Question 23

As the dilution rate increases,

Answer 23

• addition of fresh medium makes the cell’s growth rate increases and reaches the maximum, and growth rate decreases to 0 during washout
• doubling time (also generation time) reduces

Question 24

Washout

Answer 24

removing fresh medium too fast so the cells are losing and can NOT match the growth rate

Question 25

the growth rate equals the death rate (washout) when….?

Answer 25

Once equilibrium is reached, number of cells is constant; the growth rate equals the death rate (washout).

Question 26

Factors affecting microbial growth:

Answer 26

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

Question 27

Extremophiles:

Answer 27

microorganisms that prefers to grow under extreme conditions.

Question 28

The temperature range for most microorganisms is __degrees around the optimal temperature.??

Answer 28

is typically 25-40 °C around the optimal temperature.

Question 29

psychrotolerant

Answer 29

Organisms that can grow at 0°C but have optimal temperature around 20 to 40 °C

Question 30

1. Temperature
   - Adaptation to cold temperature
   e. g. Cryprotectants?

Answer 30

1) Cryoprotectants: antifreeze proteins, glycerol. –> prevent the
formation of ice crystals that can puncture the cytoplasmic membrane
»ice crystals will kill microorganisms but cold temp won’t
2) changes in protein structure and sequences–>enzymes are active at low temp
3) membrane transport functions optimally at low temp
4) modification of plasma membrane –> maintain fluidity
5) cold-shock proteins –> keep proteins active

Question 31

• Adaptation to high temperature:

Answer 31

1) Endospores help resist high temp
» hot temp kill microorganisms
2) changes in protein sequences–> heat-stable enzymes won’t denature at high temp and stay active
3) Modification of cytoplasmic membrane–>remains stable at high temp
e.g. lipid monolayer of Archea
4) heat-shock proteins
5)protection mechanism: ensure stability of DNA (GC rich bc GC(3 bonds) is stronger than AT(2 bonds)

Question 32

2. pH

- Adaptation to low pH & high pH ?

Answer 32

• 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.

Question 33

Internal pH

Answer 33

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).

Question 34

Buffers are usually used in growth medium to keep the pH steady because …….?

Answer 34

bacterial waste tends to affect pH (Ex.: MacConkey Agar).

Question 35

3. Osmotic effect

- halophiles.?

Answer 35

Halophiles: Microorganisms that can grow at high salt concentrations
- Usually requires NaCl for growth.

Question 36

Obligate and facultative aerobes are usually grown in liquid with constant shaking of the culture to ensure…?

Answer 36

Obligate and facultative aerobes are usually grown in liquid with constant shaking of the culture to ensure sufficient O2 concentration in the medium.

Question 37

Growth and oxygen concentration

• Thioglycolate?
• reazurin?

Answer 37

• Thioglycolate is added to the medium. It reduces O2 to H2O, which creates a gradient of oxygen concentra@on.
• Redox indicator reazurin is used to differentiate oxic and anoxic zones (pink when oxidized).

Question 38

Toxic forms of oxygen

Answer 38

During aerobic respiration O2 is reduced to H2O. During oxygenic photosynthesis H2O is oxidized to O2. Along with the production of toxic forms of oxygen
Toxic forms of oxygen oxidize cell components, stopping key metabolic pathways and destroying key structures.

Question 39

O2 can be reduced to to O2- by…?

Answer 39

Flavoproteins, quinone and iron-sulfur proteins, can reduced O2 to O2-.

Question 40

resistance to toxic forms of oxygenic aerobes&facultative aerobes vs. Anaerobes ?

Answer 40

• 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.