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L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
LECTURE 4. MICROBIAL GROWTH
1. Microbial survival strategies
2. Microbial growth
3. Effects of the environment
4. Microbial cultures
5. Growth measurements
6. Antimicrobial agents
7. Antibiotics
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.8. EXTREMOPHILISM AND EXTREMOPHILES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
CULTURE: a system used to allow the multiplication of a microbial population and reach a high microbial density
Culture components:Nutrients (medium)Inoculum(absence of contamination)
Culture types:Pure (or axenic)
Mixed
(According to the metabolic categories):C sourceE sourceMacronutrients (N, O, P, S, salts, vitamins, etc.)Micronutrients (normally, present as salt contaminants)H2OpH (buffers)WARNING! Auxotrophs vs prototrophs Inoculation
Preparation
Sterilization
Incubation
Types of culture media:Liquid / solidDefined, syntheticComplexSelective“Test”…
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
Closed system (only energy, and sometimes gases, are interchanged with the external environment; no cells or disolved products).
Growth curve with 4 phases.
4.1. BATCH (DISCONTINUOUS) CULTURE
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
N = N02n
N= Number of cells after n generationsN0 = Number of cells at the beginning
tg = Generation time(time needed to double cell number)m= specific growth rate (time units -1)(number of generations per time unit)tg = ln 2 / (hours)
Exponential growth
4. MICROBIAL CULTURES
4.1. BATCH CULTURE
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.1. BATCH CULTURE
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
Y = (X – Xo) / S
Y = yieldX, Xo = cells/ ml
S = nutrient concentration at to
Escherichia coli Tg= 20 min 4000 X Earth weight
LIMITING SUBSTRATE
Net growth: final biomass – initial biomass (inoculum)
Yield: unit of biomass produced per unit of nutrient consumed
4.1. BATCH CUTURE
µ depends on nutrient
concentration
µ = µ max S
Ks + S
After 48 hours
4. MICROBIAL CULTURES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
Cultures can be kept for long periods of time. Medium is added and culture removed, keepin V constant
4.2. CONTINUOUS CULTURE (“steady state”)
V entrance constant, [nutrient] constant, [cell] constant. V changes, µ changes and a new [cell] is reached
4. MICROBIAL CULTURES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4.3. CULTURES ON SOLID MEDIA
4. MICROBIAL CULTURES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
5. GROWTH MEASUREMENTS
Only balanced growth (ordered increase of all cell components) can be measured properly…
5.1. BIOMASS
Dry weight
Absorbance (cell density)*
5.2. CELL COMPONENTS
Nucleic acids, proteins, enzymatic activities
5.3. CELL NUMBRES
Total cells
Viable cells
(culturable)***
Counting chamber
Flow cytometer
Plate counts
Most probable number (MPN)
***VBNC: Viable but not culturable
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
Description of natural microbial communities
How many microbes are present in a natural sample?“Who” are they?
What do they do? (niche) How do they relate to each other and to other organisms?
(competition, antagonisms, symbioses, etc.)
Direct counts
Plate count (“viables”)
SAMPLE
5.3. CELL NUMBERS
5. GROWTH MEASUREMENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
Problems encountered when counting “viables” (i.e. when culturing)
Are they dead?Are they viable but not culturable (VBNC)*?
They do not grow on standard culture media
*Important in public health
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
ANTIMICROBIAL AGENTS: either (i) limit or inhibit microbial growth or (ii) destroy microorganisms
• Sterilization: a process that destroys all living organisms and their viruses from an object or habitat.
• Disinfection: partial elimination or inhibition of microbes, normally pathogens Disinfectant: (chemical) agents used to disinfect; used on inanimate objects.
• Antisepsis: prevention of sepsis or infection (antiseptic agents are used over tissues to prevent infections, normally less toxic than disinfectant agents).
• Germicide: destroy germs (pathogens) and non-pathogens, but not necessarily spores (bactericide, algaecide, fungicide, virocide...)
IMPORTANT CONCEPTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
ANTIMICROBIAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH6.1. EFFECTS OF ANTIMICROBIAL AGENTS
BACTERIOSTATIC BACTERICIDE
BACTERIOLYTIC
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.2. FACTORS THAT AFFECT THE EFFICIENCY OF ANTIMICROBIAL AGENTS
• Population size: the same fraction of the microbial population is destroyed in each time interval; a larger population needs more time to be completely eliminated than a smaller one.
• Population composition: different microbes have different sensitivity to antimicrobial agents
• Concentration and performance of the antimicrobial agent• Exposure time• Temperature• Local environment: pH, organic matter, biofilms, etc.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
MOIST HEAT
Boiling in water for 10 minutes destroys
vegetative cells and eukaryotic spores but
NOT bacterial endospores
Autoclave: temperatures higher than 100oC (pressure) with water saturated
steam. Time: 10-15 minutes. Depends on the sample volume.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
PASTEURIZATION NO STERILIZATION
Food treatment (milk...)
Old method: 63oC for 30 minutes.
Fast pasteurization(HTST: high-temperature short-term): 72oC for 15 seconds.
Sterilization at ultrahigh temperature (UHT: ultra-high temperature): 140-150oC for 1-3 seconds.
DRY HEAT
Oven at 160-170 oC from 2 to 3 hours
Not suitable for thermosensitive materials
Used for glass, oil and other materials Suele utilizarse para material de vidrio, aceite y otros materiales
Clostridium botulinum endospores
Moist heat: 5 min at 121 oC
Dry heat: 2 hours at 160oC
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
LOW TEMPERATURES
Inhibit growth
Used to preserve (not to sterilize or disinfect)
FILTRATION
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
RADIATION
UV (ceiling, biological safety hoods)
Ionizing radiation: very good sterilizing agent.
Pharmaceutical companies
Disposable clinical materials
Meat and other foods (spices)
Comercial radiation of spices and
seasonings, world data
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
GermicidesDisinfectant and antispeticAntibiotics SELECTIVE TOXICITY
6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.5. MEASURING ANTIMICROBIAL ACTIVITY
MINIMUM INHIBITORY CONCENTRATION
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
BACTERIOSTATIC BACTERICIDE BACTERIOLYTIC
MAIN ANTIBIOTIC TARGETS
Cell wall synthesis
Protein synthesis
Cell membrane integrity
Nucleic acids synthesis
Essential cofactors synthesis
A chemical substance produced by a microorganism (fungi or bacteria) that kills or inhibits the growth of another microorganism.Normally, they have selective toxicity*: the ability of a compound to inhibit or kill pathogenic microorganisms without adversely affecting the host. Thus, they can be used as chemotherapeutical agents.Some antibiotics are semi-synthetic.
“*The magic bullet”
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
ANTIBIOTIC MECHANISMS
7. ANTIBIOTICS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014