Bacterial growth week 3

Question 1

Describe 3 methods for measuring bacterial growth in culture and the drawbacks of each.

Answer 1

I. Bacterial Growth is required to cause diseases

(except for a few diseases caused by ingestion of bacterial exotoxins)

A. Measurement of bacterial growth in broth culture

1. turbidity (light scattering) - measured in spectrophotometer (alive plus dead)
2. direct particle count (alive plus dead) - microscope, or electronic particle analyzer

Problems with above methods:

1) Only accurate within a range.
2) Can’t discriminate between Live & Dead bacteria

3. viable cell count - colony forming units (CFU) on agar, one colony from one live bacterium

Problems:

1) Can only measure live bacteria.
2) Assumes complete dispersion which may not occur in bacteria such as Staph aureus which grow in clusters

Question 2

State the 4 phases of a bacterial growth plot.

Answer 2

lag, exponential growth, stationary, declin

Question 3

State the physiological processes that occur in the lag phase of bacterial growth.

What occurs during the logarithmic (exponential) growth phase? What determines how long this phase lasts?

Answer 3

Lag phase: bacteria adapt to culture medium

• generation of proper redox potential, siderophore (iron chelator) production,
• synthesis of needed enzymes
• cell elongation, mass increase
• chromosome replication
• septum formation, preparation for division

Logarithmic phase: rapid cell division – Maximum % viable

• cells divide by binary fission
• exponential increase in cell numbers
• lasts as long as nutrients are available or until toxic products inhibit growth
• responsible for rapid onset of disease, like meningitis

Question 4

What changes to bacterial cell growth occur in the stationary phase? What physiological changes occur in this phase? Explain the ability for the host to repond during the stationary phase.

What changes to bacterial cell growth occur in the decline phase? What is the rate of this phase dependent upon?

Answer 4

Stationary phase: rate of cell division = rate of cell death

• adaptive changes (synthesis of inclusion bodies, or sporulation in some
• viable cell number (CFU) remains constant
• genera)
• still induces host response - sometimes detrimental (endotoxin released when gram negative cells die)
• exotoxin production accelerates

Decline phase: cells die

• rate of death depends on exhaustion of nutrients, temperature, pH, O2, moisture
• bacteria survive for long periods - moist soiled bandages and bedding are infectious until sterilized

Question 5

What temperature and pH ranges are ideal for bacterial growth?

Define the cardinal temperatures and state what defines each.

Answer 5

temperature: usually 37 degrees C

pH - most pathogens require pH near 7.2-7.6

Minimum growth temperature is the lowest temperature at which the organism will be able to grow, though sluggishly.
-­‐
Optimum growth temperature is the temperature at which the organism grows the fastest kinetically.
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Maximum growth temperature at which growth is possible

Question 6

What is the optimum temperature range for human pathogens?

Answer 6

Btwn 20 and 45 degrees C

Question 7

What nutrional factors are needed for bacterial growth? Do the needs vary btwn organisms?

What kind of bacteria (aerobes, anaerobes) require Fe? Why? How do they obtain iron? Be specific about the process.

Explain how the requirement for Fe in these bacteria may serve as a virulence factor.

Answer 7

Nutritional factors needed for growth

• water
• preformed organic compounds for energy and as carbon source
• requirement for amino acids, purines, pyrimidines, and vitamins vary among organisms
• inorganic ions (i.e. K+, Mg2+, Fe2+, Na+, PO4-)
  
  • iron required by aerobes as special components of cytochrome system
  • siderophores are secreted, solubilize ferric iron, and return with the iron to the siderophore receptor. Transport system is in the plasma membrane
  • the ability of an organism’s siderophores to compete for iron with transferrin and lactoferrin in human body may serve as a virulence factor

Question 8

# Define the following terms: obligate aerobes, facultative anaerobes, obligate anaerobes, microaerophiles, aerotolerant
.

Give examples of bacteria in each category.

Answer 8

Respiration: final electron acceptor is O2

Obligate aerobes: Require O2 to live: Mycobaterium thrives in the lungs

Facultative anaerobes: most pathogens, E.coli for example. Use aerobic oxidation in the presence of O2 but can switch to anaerobic metabolism when O2 not available.

• a. glucose pyruvate (glycolysis or variations on glycolysis)
• b. pyruvate enters the TCA cycle. Generates ATP and intermediates for
• c. Electron transport and oxidative phosphorylation source of most ATP biosynthesis.
  
  • In one turn of the TCA cycle, 3 NAD and 1 FAD act as electron acceptors
  • NADH-> flavoproteins-> cyt b-> cyt c-> cyt a O2
  • Cytochromes contain iron and undergo reversible Fe(II)-Fe(III) valence changes to carry electrons towards O2

Fermentation: no O2 available

Obligate anaerobes: Clostridia that cause tetanus, botulism. These organisms are poisoned by O2

Facultative anaerobes (when no O2 is present)

• Organic compounds serve as both electron donors and acceptors
• Substrate level phosphorylation is source of ATP (no electron transport)
• Glycolysis: glucose pyruvate lactate, or other compounds (industrial value, useful in identification)

Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube but not the very top (H. pylori)

Aerotolerant: organisms do not require oxygen as they metabolize energy anaerobically. Unlike obligate anaerobes however, they are not poisoned by oxygen. They can be found evenly spread throughout the test tube. (S. pyogenes)

Question 9

What is pasteurization? When is it used and why?

What is disinfection?

Answer 9

Pasteurization: (62 C for 30 minutes) destroys pathogens but doesn’t affect flavor of product (i.e. milk)

Disinfection: chemical substances able to kill microorganisms on surfaces but too toxic to apply internally

Question 10

How do alkylating agents work as disinfectants? Name two alkylating agents that are used.

Name 3 oxidizing disinfectants and explain how they work.

Answer 10

Alkylating agents (alkylates terminal hydroxyl, amino, carboxyl, and sulfhydryl groups on proteins)

• Ethylene oxide: gas used to sterilize objects that are heat sensitive
• Formaldehyde: gas dissolved in water

Oxidizing agents

• Hydrogen peroxide (oxidant form is hydroxyl radical)
  
  • kills bacteria, but not spores at 3%-6% (topical)
  • kills spores, too, at 10%-25% (instruments)
• Iodine (best at low pH) oxidizes essential enzymes; used to disinfect skin (Betadine)
  
  • kills organisms and spores
  • activity reduced by organic compounds; must clean skin surface first
• Chlorine compounds oxidize sulfhydryl groups
  
  • hypochlorous acid (HOCl, Chlorox); 5%-10% solution used to sterilize infectious
  • laboratory materials before discarding
  • hypochlorite ion

Question 11

Name 3 membrane disrupting agents.

Answer 11

Membrane disrupting agents

• Quartenary ammonium compounds
• Alcohols
  
  • ethanol or isopropanol
  • used to clean skin surface before Betadine
  • disinfection of anal thermometers
  • 70% is more effective than 95%
• Soaps/Detergents

Question 12

What is the difference btwn bactericidal and bacteriostatic antibiotics?

State the mechanism of each of the drugs below and whether they are bactericidal or bacteriostatic.

Answer 12

Bactericidal (kills bacteria). Bacteriostatic (stops bacterial growth, allows immune system to control)

Bactericidal (kills bacteria)

• Penicillins and cephalosporins inhibit bacterial cell wall (murein) formation.
• Nalidixic acid and the quinolones bind to bacterial DNA gyrase (enzyme that reintroduces supercoils after replication), preventing DNA replication.
• Aminoglycosides (streptomycin, kanamycin and neomycin) bind to bacterial 30S ribosomal subunit and inhibit elongation. Subunits held together more tightly.

Bacteriostatic (stops bacterial growth, allows immune system to control)

• Rifampin binds bacterial RNA polymerase and prevents transcription.
• Chloramphenicol, lincomycin and erythromycin bind to bacterial ribosomes and inhibit peptide bond formation.

Question 13

What is the MOA of sulfafonimide drugs?

Answer 13

Inhibit bacterial enzyme required for folic acid synthesis: dihydropteroate synthetase. Bacteria cannot take up folic acid from the environment.