Bacterial Growth and Physiology

Question 1

Bacterial Growth (multiply) Why? Because that’s how they cause disease. Some release toxins, but most problems arise from bacterial growth. Static antibiotics stop multiplication so immune system. Cytic kill bacteria.

Answer 1

General features.
Bacterial replication is the generation of 2 complete daughter cells from one cell.
Bacteria multiply by binary fission by these steps:
Replication of DNA.
Polar separation of daughter chromosomes.
Generation of the cross-wall.
Separation.

One bacteria becomes two, not four.

Question 2

Generation time and Double time

Answer 2

The time for one cell to become two is the generation time. This is also the amount of time required for a number of cells in culture to double.
DOUBLING TIME = GENERATION TIME
All descendants of a single cell are clones (copies).
Most replication is asynchronous.
Cell numbers are generally measured by cell concentration or biomass.

Question 3

longer antibiotic regimen req’d for baceria in abcess or blood?

Answer 3

abcess, b/c it has less of the “tools” necessary to grow. Abcess bacteria have longer generation time

Question 4

The Growth Curve

Answer 4

A saturated broth culture is used to inoculate fresh media.
Bacterial counts are taken at different time points and plotted as cell number versus time.

Have steeper line in blood than abcess

Lag phase.
The bacteria adapt to the new nutrient-rich environment.
Log phase (logarithmic or exponential growth).
The bacteria are doubling every generation time (~20 minutes for E. coli) New cell material is synthesized at a constant rate.

Stationary Phase
Nutrients are exhausted; toxic products begin to build up. The bacteria remain at a relatively constant number
Death phase
Bacteria begin to die because of toxicity. Not all bacteria have a death phase.

Question 5

Why colonize bacteria?

Answer 5

Determine type and number

A bacterial colony is tens of millions of individual bacteria from a single organism (clone).
In order to determine the concentration of bacteria in liquid culture, the culture is diluted and plated onto media. Each colony represents one bacterium from the original culture (but it’s much easier to see).

For a given culture, at each time point:
Make 10-fold dilutions of a culture.
Spread a known volume on an agar plate.
Allow colonies to grow.
Count the number of colonies.
Calculate original concentration (at time of sampling.
NOTE: This only represents VIABLE bacteria

Question 6

Determination of bacterial concentration by serial dilution and colony forming units (CFU).

Answer 6

BAsed on dilution concentrations. Sample put on is .1mL

Only count ones that are ALIVE!!!

Question 7

Bacterial Cultivation

Growth Requirements

Answer 7

Depends on bacterial genetic information

*All of the elements for organic matter (including and especially a carbon source).
*Ions for energy generation, catalysis, and osmotic maintenance.

Gene products aren’t all expressed at the same time. Can induce expression by crx right environment (carbon source).

Question 8

Bacterial Cultivation Energy Sources

Answer 8

Energy sources:

Fermentation. - Anaerobes
The formation of ATP NOT coupled toelectron transfer.
Respiration. - Aerobic bacteria
The formation of ATP via oxidative phosphorylation (chemical reduction of an oxidant) wherein ATP is formed during electron transfer.
Photosynthesis. (no medically relevant bacteria here)
ATP is formed via the reduction of an oxidant via light energy. Similar to respiration.

Question 9

Can use fermentation products to determine type of microbes

Answer 9

Test question will come from this:

Question 10

Nutritional Requirements

Answer 10

Nutritional requirements.
The growth media must contain: carbon, nitrogen, sulfur, phosphorus, calcium, magnesium, potassium, iron, and other minerals in small amounts.
Iron is NOT required for the growth of Borrelia species (in other words, there are often exceptions).
Anything that cannot be synthesized by the bacterium, but is required for growth, must be provided (e.g. vitamins) or obtained from the host (eg. siderophores:iron chelators)

Question 11

Classification based on nutritional requirements:

Answer 11

Heterotroph.
Requires pre-formed organic compounds like sugars, amino acids, and vitamins.
Autotroph.
Can synthesize everything it needs from inorganic compounds like CO2.
Hypotroph.
Is an obligate intracellular pathogen requiring the host to provide organic compounds (in this way, it may also be called an intracellular heterotroph).

Question 12

Bacterial Uptake of Nutrients

Answer 12

• *Diffusion through the cytoplasmic membrane**
• Uptake of nutrients from the media through the cytoplasmic membrane occurs with the help of specific carrier proteins called permeases. This happens in three basic ways:

Question 13

Diffusion through the cytoplasmic membrane

Answer 13

1) Carrier mediated diffusion (facilitated diffusion).
- Nutrients travel across a concentration gradient, from highest to lowest concentration. E.g., if the concentration of glycerol inside of the organism drops, the cell will take up more glycerol.
- Concentration dependant
- NOT energy dependant

• *Phosphorylation-linked transport (group translocation).**
  
  • This is an E-dependent process, also known as group translocation.
  • Certain sugars are taken up this way.
  • The sugars are co-transported with a carrier in the membrane of the cells and chemically modified (phosphorylated) in the process.
• *Active transport.**
  
  • Also an E-dependent process.
  • Requires the generation of a proton-motive force.
  • Protons are pumped out of the cell, creating a flux of protons in and out of the organism.
  • Molecules are coupled to the uptake of protons, making the overall energetics favorable.
  • Requires a symport for the uptake of protons and the nutrient.

Question 14

There are 5 basic types of bacteria classified by their oxygen requirements:

Answer 14

• *Obligate aerobes:** Must have O2 for growth.
• *Obligate anaerobes:** No requirements for O2, these organisms are actually killed by oxygen radicals generated during the metabolism of O2.
• *Facultative anaerobes:** Can grow with or without O2.
• *Microaerophilic**: Must grow at low concentrations of O2 (less than 20%).
• *Aerotolerant anaerobes:** Similar to facultative anaerobes, but prefer anaerobic (fermentative) growth.

Question 15

Superoxide dismutase

Answer 15

Aerobic Bacteria need these enzymes to catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide. Simply stated, SOD out-competes damaging reactions of superoxide, thus protecting the aerobic pk’s from superoxide toxicity.

Question 16

Temperature Requirements

Answer 16

There are 3 different classifications of bacteria based on optimal growth temperature:
Psychrophiles: Grow best in the range of 0–20ºC.
Mesophiles: Grow best in the range of 20–45ºC.
Thermophiles: Grow best in the range of 45–90ºC.

Question 17

Bacterial Growth Media

Answer 17

The basic requirements for bacterial growth in the laboratory must be supplied in the media. There are hundreds of different media recipes.

Liquid media is a broth solution. Solid media is usually the broth recipe plus ~15% agar to make agar plates.

NOTE: Defined media is made with chemicals at known concentrations.

Question 18

Differential and Selective media.

Answer 18

Differential media: Supplies nutrients and indicators (e.g. pH or RBC) for visual determination of which organisms are present.

• Selective media:**Selects AGAINST the growth of particular bacteria by the addition of dyes, acid/base, salts, or antibiotics.*
• NOTE: Some media are both differential AND selective. An example is MacConkey agar, which has* Bile salts and crystal violet inhibit the growth of gram positives (SELECTIVE) and has lactose plus a pH indicator to indicate the fermentation of lactose (DIFFERENTIAL). Do NOT memorize the r**ecipe.