Topic 6: Cultivating Microorganisms

Question 1

Anabolism

Answer 1

Energy-consuming process.
introduces compounds into macromolecules like DNA, RNA, lipids, and proteins

Question 2

Catabolism

Answer 2

Releases energy by breaking down chemicals or by harnessing light

Question 3

Macronutrients

Answer 3

Required by ALL cells to build macromolecules
- C, N, P, S, O

Question 4

Micronutrients

Answer 4

Required by only some cells
- Fe, Cu, Na, Mg, Mn, and others
—- Fe is sometimes considered to be a macronutrient because it is essential to almost all organisms. It is often a population-limiting micronutrient

Question 5

Assigning metabolic categories (naming)

Answer 5

1. Energy Source
   - Chemicals (chemotrophs)
   - Light (phototrophs)
2. Electron Source
   - Organic matter (organotrophs)
   - Inorganic chemicals, like water (lithotrophs)
   *only archaea and bacteria can be litho
3. Carbon source
   - Fixed organic C-C bonds (heterotrophs)
   - Gaseous inorganic CO2 (autotrophs)

Question 6

Which of the following nutritional categories is unlikely to exist? In other words, which category would be energetically unfavourable?

Chemoorganoautotroph
Chemolithoheterotroph
Photolithoautotroph
Photolithoheterotroph

Answer 6

Chemoorganoautotroph

If a microorganism can oxidize organic carbon for energy and electrons (requires a lot of organic carbon), it can also assimilate that carbon source and would not need to use energy to fix inorganic carbon into biomass.

Question 7

Oxygen Tolerance

Answer 7

Aerobic growth: Uses oxygen
- Obligate aerobes: Require O2
- Microaerophiles: Grow best in low levels of O2

Anaerobic growth: Occurs without oxygen
- Aerotolerant anaerobes: Are not harmed by O2, but don’t use it
- Obligate anaerobes: Cannot grow when O2 is present
- Facultative anaerobes: Can grow in the absence of O2, but grow better when it is present

Question 8

Toxic Oxygen Species

Answer 8

Reactive oxygen species (ROS): responsible for harmful effects of oxygen

• Pigments, enzymes (e.g., superoxide dismutase, catalase, peroxidase), and antioxidants used for protection

Question 9

pH Tolerance

Answer 9

• pH affects macromolecule structures and transmembrane electrochemical gradients

Each microbe has an optimal pH range:
- Acidophiles (low pH, 0-5.5)
- Neutrophiles (neutral pH, 5.5-8.5)
- Alkalophiles (high pH, 8.5-13.5)

Regardless of pH preference, intracellular pH stays relatively neutral, internal pH found as low as 4.6 or as high as 9.5

Question 10

Moisture Content

Answer 10

• Different solute concentrations can result in influx or efflux of water. This can cause stress to the cell, causing it to either swell or shrink
• Water must also be available for biochemical reactions
• measure in terms of water activity (aw)
• interactions with solutes can decrease aw values
• pure water aw=1.0
• seawater aw= 0.98
• most bacteria require aw > 0.9

-Cytoplasm typically has a higher solute concentration than the external environment
– Water tends to move into the cell
– Positive water balance
- Water will flow out in a hypertonic environment.

Water loss is prevented by increasing internal solute concentration by pumping in inorganic ions from environment or synthesis/concentration of compatible solutes

Question 11

Temperature

Answer 11

• Growth max, min, and optima are organism-specific. They can be modified by factors such as growth medium composition.
• can affect molecular structure, membrane fluidity and enzyme function

Question 12

Psychrotolerant organisms

Answer 12

• Able to grow ~0°C
• Optimal growth between 20–40°C
• “mesophiles capable of low-temperature growth”

Question 13

Psychrophiles

Answer 13

• Minimum <0°C, Optimum ~10–15°C, Maximum ~20°C
• Higher proportion of unsaturated fatty acids in the membrane phospholipids

Question 14

Mesophiles

Answer 14

• Optimal growth between 10–55°C
• Most common

Question 15

Thermophiles and Hyperthermophiles

Answer 15

• Hyperthermophiles: Optimal growth between 80–130°C
• Thermophiles: Optimal growth between 55–80°C
• Critical amino acid substitutions in key locations to produce heat-tolerant folds
• Increases in ionic bonds between acidic and basic residues to resist protein unfolding
• Certain solutes stabilize proteins

Question 16

Explain why microorganisms are cultivated on both solid and liquid media

Answer 16

• Solid media are essential for isolating and maintaining pure cultures
• Liquid media are crucial for large-scale growth, experimental control, and biotechnological applications.

Question 17

Complex and Defined media

Answer 17

Complex media:
- Unknown composition
- Fast and easy to do
- Contains peptone and yeast extract

Defined media:
- Known chemical composition
- Long process

Question 18

Selective media

Answer 18

Allows for the isolation of microorganisms by inhibiting the growth of other microorganisms

Question 19

Differential media

Answer 19

Allows certain microbes to be recognized based on visual reactions in the medium

Question 20

Enriched media

Answer 20

Used to increase a population of microbes with a specific property by providing nutrients needed for target population and not providing nutrients needed for another

Question 21

Direct count method

Answer 21

Known volume is loaded onto a slide with a grid, and cells are counted under a light microscope.

Advantages: Cheap; fast; easy
Disadvantages: Can’t differentiate between viable and dead cells
*unless use stain that binds to DNA + RNA and tells you by colour if alive or dead

Question 22

Viable Cell Count

Answer 22

• Serial dilutions of a culture are prepared, and then the spread plate or pour plate method is used to count the number of colonies.
• Colony forming units (CFUs) per milliliter of initial culture can be calculated by multiplying the number of colonies by the inverse of the dilution factor

Question 23

Turbidity Measurements

Answer 23

A spectrophotometer is used to measure the absorbance of a culture at various time points, and a growth curve can be constructed.

Question 24

Bacterial Growth Curve

Answer 24

A graphical representation of the growth of a population of bacteria over time

• Lag phase: Microorganisms are preparing for steady growth while adjusting to new culture conditions
• Exponential phase: Microorganisms are replicating at a constant and steady exponential rate
• Stationary phase: Replication has either halted or it is now equal to the death rate
• Death phase: Nutrients are depleted and waste levels are high; cells are dying at a steady exponential rate

Analysis of a growth curve
- Generation time: The time to double the population in the exponential phase
- Growth rate: The number of generations per unit of time (inverse of the generation time)
- Growth yield: The maximum population density under given incubations and/or amount of cellular material produced by the culture

Question 25

Why are continuous cultures useful?

Answer 25

- Keeps microbes in exponential growth to harvest cells or products of cellular metabolism - Provides limited but continuous flow of nutrients - Chemostat: A system that brings in fresh medium and removes old medium/microbes

Question 26

Types of Filters

Answer 26

Pre-filter: - Removes large particles prior to other forms of filtration; can be depth filter, cheesecloth, or Whatman filter paper Polymer membrane filter: - Conventional filter made of cellulose acetate or cellulose nitrate; pore diameter is variable during production; “sieve-like action”; used for routine sterilization Nucleopore filters: - Thin, polycarbonate film ~10 μm thick; consistent pore size; useful for microscopy

Question 27

Describe methods for controlling microbial growth (4)

Answer 27

1. Filtration: physical removal of microbes - For sterilization (rapid, effective, non-destructive) - To separate microorganisms by size - Nylon/Teflon filters with pore size of 0.2 or 0.45 μm remove bacteria and archaea - Viruses can be removed using ultrafiltration methods with a pore size of 10–100 nm --- Problems: - Large particles clog filters; viscous liquids don’t filter well; ultrafiltration requires high pressure 2. Temperature Manipulation: - Heat denatures proteins and nucleic acids (100°C kills most microbes quickly) --- Potential problems with using heat alone to kill microorganisms: - Hyperthermophiles and endospores might not be destroyed - Some materials can’t be heated 3. Radiation: Electromagnetic or Ionizing radiation 4. Chemical Control: - Chemicals can target specific groups (e.g., microbicidal, bacteriocidal, fungicidal, algicidal, viricidal) -- Nonselective: not used internally; can affect sulfhydryl groups for example -- Selective: useful for treating diseases (e.g., antibiotics)

Question 28

Methods of Temperature Manipulation

Answer 28

1. Autoclaves: use heat and pressure to sterilize; standard settings: 121°C and 15 psi 2. Pasteurization: reduces microbial load; destroys pathogens; increases shelf-life; does NOT sterilize - HTST: 72°C for 15 s (most common process) - UHT: 135°C for <1 s - ESL: Filtration, then lower-temp treatment 3. Refrigeration and freezing: reduces microbial growth and spoilage

Question 29

Radiation Types

Answer 29

Electromagnetic Radiation: - UV radiation (260-280 nm wavelengths) --- Used to reduce microbial burden; does not sterilize --- Damages DNA by forming thymine dimers --- Exploited to control microbial growth on non-living surfaces and in water; not used for tissues Ionizing Radiation: - Cobalt-60 or cesium --- Used for sterilization --- Extensive protein and DNA damage --- Higher energy for better penetration of materials --- Limited to large industrial operations due to cost and hazards

Question 30

Antimicrobial Agents

Answer 30

Bacteriostatic: inhibits cell growth to give immune system time to work - Example, tetracycline Bacteriocidal: causes cell death (still there but not viable) - Example, kanamycin Bacteriolytic: causes cell lysis (visible cell count decrease) - Example, penicillin

Question 31

Disinfectants vs Antiseptics

Answer 31

D: substance used on inanimate surfaces, tables, floors, windows, anything that's not tissue A: used on living issue, like teeth, mouth hands Many compounds can be either an antiseptic or a disinfectant, depending on the concentration. ***For example, you might put hydrogen peroxide on a cut at a 3% concentration. Hydrogen peroxide at 100% is only ever a disinfectant; it would be harmful on your skin

Question 32

Commonly used disinfectants

Answer 32

Alcohols: Example: Ethanol - Routinely used in laboratory settings; also present in most hand sanitizers Effect: membranes Phenolic compounds: Example: Triclosan - Added to numerous products, including some soaps, deodorants, and cosmetics Effect: membranes Oxidizing agents: Example: Sodium hypochlorite - Commonly added to swimming pools and hot tubs to inhibit microbial growth Effect: remove electrons Others: Example: Benzalkonium chloride - Major ingredient in Lysol Effect: membranes Example: Glutaraldehyde - Often used to prepare biological specimens Effect: crosslinks proteins

Question 33

Broad spectrum and Narrow Spectrum antibiotics

Answer 33

- Broad-spectrum (e.g., tetracycline) - narrow spectrum (e.g., polymixin B, penicillin) Effects include: - inhibition of cell wall synthesis (e.g., penicillin) - protein synthesis (e.g., tetracycline) - disruption of cell outer membrane (e.g., polymyxin B)