Medical Microbiology Chapter 6 Microbial Nutrition and Growth

Medical Microbiology Chapter 6 – Microbial Nutrition and Growth

I. Growth requirements

A. Microbial Growth

– increase in a population of microbes

B. Result of microbial growth

1. Discrete colony

-aggregation of cells ring from single parent cell

2. Biofilm

– collection of microbes living on a surface in a complex community

C. Reproduction results in growth

D. variety of nutrients used for energy needs/build molecules/build cellular structures

E. Most common nutrients contain necessary elements

1. C2

2. O2

3. N2

4. H2

F. nutrients obtained from variety of sources

II. Nutrients: Chemical and Energy Requirements

A. 3 things all cells need for metabolism – C2, energy, and electrons

1. 2 groups based on source

a. autotrophs – use inorganic C2 from CO2

b. heterotrophs – use C2 from catabolism of organic molecules they get from other organisms

2. 2 groups of organisms based on source of energy

a. chemotrophs – from inorganic substances

b. phototrophs – from light

B. oxygen requirements

1. O2 essential for obligate aerobes

2. O2deadly for obligate anaerobes

a. toxic forms of O2highly reactive

b. toxic forms excellent oxidizing agents

-causes irreparable damage to cells

3. 4 toxic forms of O2

a. singlet oxygen – produced during metabolism

b. superoxide radicals – formed from incomplete reduction of O2

c. peroxide anion – in H2O2 formed from breakdown of superoxide radicals

d. hydroxyl radical – incomplete reduction of H2O2

4. different categories according to tolerance/intolerance of O2

a. aerobes

-need O2

-anthrax; TB

b. anaerobes

– O2 toxic

-tetanus; botulism

c. facultative anaerobes

– can live with both but prefers no O2

-E. coli

d. aerotolerant anaerobes

– insensitive to presence of O2 - use fermentation

-lactobacilli (used to make pickles and cheese)

e. microaerophiles – require 2-10% O2 (like in the stomach)

-syphilis; Lyme disease

C. N2 requirements – growth limiting nutrient

1. anabolism often ceases because of insufficient N2

2. N2 acquired from organic and inorganic nutrients

3. all cells recycle N2 from amino acids and nucleotides

4. N2 fixation by certain bacteria essential to life on Earth

a. 79% of atmosphere N2

b. can’t be used but need it

c. bacteria convert it to usable form

D. other chemical requirements

1. phosphorus

2. sulfur

3. trace elements – required in small amts.

4. growth factors

-necessary organic chemicals that can’t be made by certain organisms

III. Physical requirements

A. temperature

1. effect of temperature on proteins

a. too high – bonds break

b. at lower temperature – bonds more likely to form

2. effect of temperature on cell membranes and organelles

a. too low – membranes become rigid and fragile

b. too high – membranes become too fluid

3. minimum and maximum growth temperature

a. lowest and highest at which metabolism continues

b. optimum growth temperature

produces highest growth rate

4. different classifications according to temperature requirements

a. psychrophiles – 00C - 200C (320F – 680F)

b. mesophiles – 100C – 450C (500F – 1130F)

c. thermophiles – 400C – 700C (1040F – 1580F)

d. hyperthermophiles – 800C and up (1760F and up)

e. bacteria in body at optimal temperature – 370C (98.60F)

B. pH

1. organisms sensitive to changes in acidity

- H+ and OH- interfere with H2 bonding

2. neutrophiles grow best in narrow range around neutral pH

3. acidophiles grow best in acidic habitats

4. alkalinophiles live in alkaline soils and H2O

C. physical effects of H2O

1. microbes require H2O

-dissolve enzymes/nutrients

2. H2O important reactant in many metabolic reactions

3. most cells dies in absence of H2O

a. some have cell walls that retain H2O

b. endospores and cysts cease most metabolic activity

4. 2 physical effects of H20

a. osmotic pressure

b. hydrostatic pressure

5. osmotic pressure

a. pressure exerted on semipermeable membrane by solution containing solutes that can’t freely cross

membrane

b. hypotonic solutions

-have lower solute concentrations

-cell bursts

c. hypertonic solutions

-have greater solute concentrations

-cell crenates

d. isotonic

– solute concentrations = on both sides of membrane
-cell maintains shape

e. restricts organisms to certain environments

-obligate halophiles

– need high osmotic pressure

– Great Salt Lakes

-facultative halophiles

-can tolerate – do not need – high osmotic pressure

-Staph aureus

f. most bacteria live in hypotonic or isotonic solutions

6. hydrostatic pressure

a. water exerts pressure in proportion to its depth

b. barophiles live under extreme pressure

-membranes and enzymes depend on pressure to maintain their shape

IV. Associations and Biofilms

A. Organisms live in association with different species

1. antagonistic relationships – one species benefits and one is harmed

2. synergistic relationships - both species benefit

3. symbiotic relationships – live in close nutritional or physical contact – become interdependent

B. Biofilms

1. complex relationships among numerous microorganisms

2. develop an extracellular matrix

a. adheres cells to one another

b. allows attachment to a substrate

c. sequesters nutrients

d. may protect individuals in biofilm

3. form on surfaces often as result of quorum sensing

-microorganisms respond to density of nearby microorganisms

-secrete molecules into environment

-act as signal molecules

-increase density, increase in signal molecules

-signal molecules bind to receptors on cells

-binding to receptors exceeds threshold amount -

- triggers suppressed gene

4. many microorganisms more harmful as part of biofilm

-Dental plaque – biofilm

– bacteria digest nutrients and secrete acid as byproduct

-aid eats holes in teeth – cavities

V. Culturing microorganisms

A. Inoculum (sample) introduced into medium (collection of nutrients)

1. environmental specimens – soil, water, etc.

2. clinical specimens – blood, tissue, feces, sputum, etc.

3. stored specimens – above specimens stored in a lab

B. Culture

-microorganisms grown from an inoculum

C. obtaining pure cultures

1. cultures composed of cells rising from single progenitor

2. progenitor is termed a CFU – colony forming unit

3. aseptic technique prevents contamination of sterile substances or objects

4. 2 common isolation techniques

-streak plates

– inoculating loop used to spread inoculum across surface of solid medium in petri dish

-incubation colonies different

-pour plates

– gradually dilutes sample

–done by series of dilutions

-1 mL sample introduced into 9 mL of liquid medium 3 times

-pour into warm agar in petri dish

-colonies form at surface and in medium

-CFU’s become isolated/separate

D. Culture media

1. nutrient broth (liquid) and nutrient agar (solid)

1. majority of prokaryotes have not been grown in culture medium

2. 6 types of general culture media

a. defined media – exact chemical composition known

b. complex media – exact chemical composition unknown plus nutrients and growth factors

c. selective media – favor/inhibit growth of specific microorganisms

d. differential media – causes differences to be exhibited in bacteria or media

e. anaerobic media – cells protected from O2

f. transport media – used to carry clinical specimens; speed in transporting important

3. can make petri dishes with solid media

4. can make slant tube with solid media

-large surface area for aerobic growth

-butt of slant tube – for anaerobic bacteria

E. special culture techniques

1. used to monitor/control CO2 levels

2. animal and cell cultures

a. used where artificial media are inadequate

b. for obligate intracellular parasites – viruses, rickettsia, chlamydias

3. low O2 culture – used to mimic intestinal tract/respiratory tract/other body tissues

4. enrichment culture – used to enhance growth of less abundant organisms to observable level

F. Preserving cultures

1. refrigeration – for short period of time

2. deep-freezing – for years

3. lyophilzation

a. for decades

b. freeze drying

-removes H2O from cells

-revived by adding cells to liquid culture media

VI. Growth of Microbial populations

A. Generation time

1. time required for bacterial cell to grow/divide (1-3 hours)

2. time required for population of cells to double in number

3. binary fission

a. results in exponential (logarithmic) growth

b. 1 cell -7 hours – 2 million cells

c. use scientific notation to deal with huge numbers involved

d. steps

-DNA replicates

-chromosomes separate

-new cell membrane/cell wall formed

-cells separate

4. dependent on chemical and physical conditions

B. four phases of growth

1. lag phase

a. cells adjusting to new environment

b. not much reproduction

c. synthesizing enzymes

2. log (exponential) phase

a. rapid growth

b. more cells produced than die

c. metabolic rate at maximum

d. when symptoms of disease show up

e. vulnerability to antibiotics highest at this stage

3. stationary phase

a. # of cells produced = # of cells dying

b. metabolic rate down

4. death (decline) phase

-cells dying faster than produced

5. chemostat

a. controls amount of limiting nutrients entering/leaving culture

b. maintains culture in particular phase – usually log phase

VII. Measuring Microbial Reproduction
A. Labs must determine size of microbial population.

B. Direct/Indirect methods used

C. Direct methods

1. serial dilution and viable plate counts

a. plates with 25-250 colonies

b. multiply number of colonies by the reciprocal of the dilution

c. gives estimate of # of bacteria per mL

2. membrane filtration

a. for small population density

b. pour large amt. of sample through membrane filter

c. cells trapped in membrane

d. transfer membrane to solid medium

e. incubate

f. count colonies

3. most probable number

a. used to count microorganism that can’t grow on solid media

b. used when regular counts required

c. samples of waste, water, and food

d. samples with too few organisms for plate count

4. microscopic counts

a. use microscope

b. microscope slide has grid on it

c good for speedy count

5. electronic counts

-used for bigger cells

D. Indirect methods

1. metabolic activity – rate at which population utilizes nutrients and produces waste

2. dry weight – filter cells from medium dried, and weighed; organisms killed in process

3. turbidity – cloudiness

-can use spectrophotometer

-measures amt. of light transmitted through culture – speedy

F. measuring microbial reproduction

-genetic methods

-isolate DNA sequences of unculturable prokaryotes

-used to estimate # of these microbes