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Microbiology
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Microbial GrowthLecture 5 Chapter 6
TODAY’S OBJECTIVES1. What environmental factor may influence the growth of microorganisms?
2. Characterize types of bacteria in regards to temperature, oxygen, and pressure requirements;
3. Why is pH important for bacterial growth and how can we control pH in culture media;
4. Understand the relationship between temperature and food preservation;
5. Describe the enzymes observed in microbes that protect them against toxic O2 products;
6. Explain why carbon, nitrogen, phosphorous and sulfur are important for bacterial growth;
7. Describe the formation of biofilms and summarize their importance in natural environments, industrial settings, and medicine;
8. Define quorum sensing and provide examples of cellular processes regulated by quorum sensing;
9. Differentiate between chemically defined and complex media;
10. Justify the use of the different media types and techniques;
11. Describe binary fission as observed in bacteria and archaea.
12. Compare the three reproductive strategies used by bacteria other than binary fission.
13. Summarize the two major events in a typical bacterial cell cycle.
14. Define generation time.
15. Describe the four phases of a microbial growth curve observed when microbes are grown in a batch culture
16. Describe three hypotheses proposed to account for the decline in cell numbers during the death phase of a growth curve.
17. Correlate changes in nutrient concentrations in natural environments with the four phases of a microbial growth curve
18. Evaluate direct cell counts, viable counting methods, and cell mass measurements for determining population size.
19. Explain why plate count results are expressed in terms of colony-forming units (CFUs).
The Influence of Environmental Factors on Growth
Bacteria are adapted to a specific environment;
Physical and chemical changes in the environment can influence growth;
Environmental limits varies among species
TemperaturepHOsmolarityOxygenPressure
The Requirements for Growth
• Physical requirements– Temperature– pH– Osmotic pressure
• Chemical requirements– Carbon– Nitrogen, sulfur, and phosphorous– Trace elements– Oxygen– Organic growth factors
Physical Requirements• Temperature:
– Microbes cannot regulate their internal temperature– Enzymes have optimal temperature at which they function
optimally– High temperatures may inhibit enzyme functioning and be
lethal
Temperature ranges for microbial growth
Adaptations of thermophiles• Protein structure stabilized by a variety of means
– more H bonds– more proline– chaperones
• Histone-like proteins stabilize DNA• Membrane stabilized by variety of means
– more saturated, more branched and higher molecular weight lipids
– ether linkages (archaeal membranes)
Thermus aquaticus
• Organisms exhibit distinct cardinal growth temperatures– Minimum growth temperature– Optimum growth temperature– Maximum growth temperature
Temperature
• Psychrotrophs – Grow between 0C and 20 to 30C– Cause food spoilage
• Thermophiles– Optimum growth temperature of 50 to 60C– Found in hot springs and organic compost
• Hyperthermophiles – Optimum growth temperature >80C
Microbiology & Food preservation
pH
• Most bacteria grow between pH 6.5 and 7.5• Molds and yeasts grow between pH 5 and 6• Acidophiles grow in acidic environments
pH
• Acidophiles– growth optimum between pH 0
and pH 5.5
• Neutrophiles– growth optimum between pH
5.5 and pH 7
• Alkaliphiles (alkalophiles)– growth optimum between pH
8.5 and pH 11.5
Sulfur Caldron, Yellowstone
National ParkAcidic condition
Lake Magadi,Kenya
Alkaline condition
pH homeostasis• Most microbes maintain an
internal pH near neutrality– the plasma membrane is
impermeable to proton– exchange potassium for
protons
• Acidic tolerance response – pump protons out of the
cell– some synthesize acid
and heat shock proteins that protect proteins
• Many microorganisms change the pH of their habitat by producing acidic or basic waste products
Solutes and Water Activity
• Changes in osmotic concentrations in the environment may affect microbial cells– hypotonic solution (lower osmotic concentration)
• water enters the cell/cell swells may burst– hypertonic (higher osmotic concentration)
• water leaves the cell/membrane shrinks from the cell wall (plasmolysis) may occur
Microbes Adapt to Changes in Osmotic Concentrations
• Reduce osmotic concentration of cytoplasm in hypotonic solutions– mechanosensitive (MS) channels in plasma membrane allow
solutes to leave• Increase internal solute concentration with compatible
solutes to increase their internal osmotic concentration in hypertonic solutions– solutes compatible with metabolism and growth.
Osmotic Pressure
• Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis due to high osmotic pressure
• Extreme or obligate halophiles require high osmotic pressure (high salt)
• Facultative halophiles tolerate high osmotic pressure
Extremely Adapted Microbes
♦Halophiles♦grow optimally in the presence of
NaCl or other salts at a concentration above or about 0.2M.
♦Extreme halophiles♦require salt concentrations of 2M and
6.2M♦extremely high concentrations of
potassium♦cell wall, proteins, and plasma
membrane require high salt to maintain stability and activity
Plasmolysis
Chemical Requirements
• Carbon– Structural backbone of organic molecules– Chemoheterotrophs use organic molecules as
energy– Autotrophs use CO2
Chemical Requirements
• Nitrogen– Component of proteins, DNA, and ATP– Most bacteria decompose protein material for the
nitrogen source– Some bacteria use NH4
+ or NO3– from organic
material– A few bacteria use N2 in nitrogen fixation
Chemical Requirements
• Sulfur– Used in amino acids, thiamine, and biotin– Most bacteria decompose protein for the sulfur
source– Some bacteria use SO4
2– or H2S
• Phosphorus – Used in DNA, RNA, and ATP– Found in membranes– PO4
3– is a source of phosphorus
Trace Elements
• Inorganic elements required in small amounts• Usually as enzyme cofactors• Include iron, copper, molybdenum, and zinc
Oxygen
• Obligate aerobes—require oxygen (20% O2)• Facultative anaerobes—grow via fermentation or
anaerobic respiration when oxygen is not available• Obligate anaerobes—unable to use oxygen and
are harmed by it• Aerotolerant anaerobes—tolerate but cannot use
oxygen• Microaerophiles—require oxygen concentration
lower than air (2-10% O2)
Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
Oxygen easily reduced to toxic reactive oxygen species (ROS) that can damage DNA, RNA, proteins and lipids:• Singlet oxygen: (1O2
−) boosted to a higher-energy state and is reactive
• Superoxide radicals: O2
• Peroxide anion: O22–
• Hydroxyl radical (OH•)
Aerobes produce protective enzymes– superoxide dismutase (SOD)– Catalase– peroxidase
Basis of Different Oxygen Sensitivities
Enzymes help neutralize these toxic reactive oxygen species and detect and repair macromolecules damaged by oxidation
Organic Growth Factors
• Organic compounds obtained from the environment
• Vitamins, amino acids, purines, and pyrimidines
Biofilms
• Most microbes grow attached to surfaces (sessile) rather than free floating (planktonic), forming microbial communities.
• These attached microbes are members of complex, slime enclosed communities called a biofilm.
• Biofilms are ubiquitous in nature in water.• Can be formed on any conditioned surface. • Share nutrients• Shelter bacteria from harmful environmental factors
Biofilm Formation• Microbes reversibly attach to conditioned surface and release
polysaccharides, proteins, and DNA to form the extracellular polymeric substance (EPS)
• Additional polymers are produced as microbes reproduce and biofilm matures
• A mature biofilm is a complex community of microorganisms
• Heterogeneity is differences in metabolic activity and locations of microbes
• Interactions occur among the attached organisms – exchanges take place metabolically,
DNA uptake and communication
Biofilms• Protects microbes from harmful agents
– UV light, antibiotics, antimicrobials – 1000x resistant to microbicides
• When formed on medical devices, such as implants, often lead to illness– Involved in 70% of infections– Catheters, heart valves, contact lenses, dental caries
• Found in digestive system and sewage treatment systems; can clog pipes– Sloughing off of organisms can result in contamination of water
phase above the biofilm such as in a drinking water system
Cell to Cell Communication Within the Microbial Populations
• Bacterial cells in biofilms communicate in a density-dependent manner called quorum sensing
• Produce small proteins that increase in concentration as microbes replicate and convert a microbe to a competent state
– DNA uptake occurs, bacteriocins are released.
Quorum Sensing
• Acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms– diffuses across plasma membrane– once inside the cell it induces expression of target genes
that regulate a variety of functions.
• Processes regulated by quorum sensing involve host-microbe interactions– symbiosis – Vibrio fischeri and bioluminescence in squid– pathogenicity and increased virulence factor production– DNA uptake for antibiotic resistance genes
LABORATORY CULTURE OF CELLULAR MICROBES
CULTURE MEDIA
• Need to grow, transport, and store microorganisms in the laboratory
• Culture media is solid or liquid preparation • Must contain all the nutrients required by the organism
for growth• Classification
• chemical constituents from which they are made• physical nature• function
Culture Media
• Culture medium: nutrients prepared for microbial growth
• Sterile: no living microbes• Inoculum: introduction of microbes into a
medium• Culture: microbes growing in or on a culture
medium
Culture Media
• Agar – Complex polysaccharide – Used as a solidifying agent for culture media in
Petri plates, slants, and deeps– Generally not metabolized by microbes– Liquefies at 100C– Solidifies at ~40C
Culture Media
• Chemically defined media: exact chemical composition is known– Fastidious organisms are those that require many
growth factors provided in chemically defined media• Complex media: extracts and digests of yeasts,
meat, or plants; chemical composition varies batch to batch– Nutrient broth– Nutrient agar
Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli
Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides
Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria
Anaerobic Growth Media and Methods
• Reducing media– Used for the cultivation of anaerobic bacteria– Contain chemicals (sodium thioglycolate) that
combine O2 to deplete it
– Heated to drive off O2
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Clamp withclamp screw
Lid withO-ring gasket
Envelope containinginorganic carbonate,activated carbon,ascorbic acid,and water
Anaerobic indicator(methylene blue)
Petri plates
CO2
H2
Figure 6.7 An anaerobic chamber.
Airlock
Armports
Special Culture Techniques
• Capnophiles – Microbes that require high CO2 conditions
– CO2 packet– Candle jar
Special Culture Techniques
• Biosafety levels– BSL-1: no special precautions; basic teaching labs– BSL-2: lab coat, gloves, eye protection– BSL-3: biosafety cabinets to prevent airborne
transmission– BSL-4: sealed, negative pressure; "hot zone"
• Exhaust air is filtered twice through HEPA filters
Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.
Selective and Differential Media• Selective media
– Suppress unwanted microbes and encourage desired microbes
– Contain inhibitors to suppress growth
• Differential media – Allow distinguishing of colonies of different microbes on
the same plate
• Some media have both selective and differential characteristics
Enrichment Culture
• Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels
• Usually a liquid
Table 6.5 Culture Media
Bacterial Division
• Bacteria and Archaea:– Haploid– Reproduce asexually
• Increase in number of cells, not cell size– Binary fission– Budding– Fragmentation of filaments
• Conidiospores (actinomycetes)
Figure 6.12a Binary fission in bacteria.
Plasma membraneCell wall
DNA (nucleoid)
Cell elongates andDNA is replicated.
Cell wall andplasma membranebegin to constrict.
Cross-wall forms,completelyseparating thetwo DNA copies.
Cellsseparate.
A diagram of the sequence of cell division
Figure 6.12b Binary fission in bacteria.
Partially formed cross-wall
Cell wall
DNA (nucleoid)
A thin section of a cell of Bacilluslicheniformis starting to divide
Bacterial Cell Cycle
• Cell cycle is sequence of events from formation of new cell through the next cell division
• Two pathways function during cycle– DNA replication and partition– cytokinesis
Single origin of replication
Proteins needed for DNA synthesis
Move in both directions
Terminus
Generation Time• Time required for a cell to divide
– 20 minutes to 24 hours
• Binary fission doubles the number of cells each generation
• Total number of cells = 2number of generations • Growth curves are represented logarithmically
GROWTH CURVE: WHEN ONE BECOMES TWO AND TWO BECOME FOUR…
• Increase in cellular constituents that may result in:• increase in cell number• increase in cell size
• Growth refers to population growth rather than growth of individual cells
• Observed when microorganisms are cultivated in batch culture
• Has four distinct phases
Phases of Growth• Lag phase• Log phase• Stationary phase• Death phase
Lag Phase• Cell synthesizing new components
– e.g., to replenish spent materials– e.g., to adapt to new medium or other
conditions• Varies in length
– in some cases can be very short or even absent
Exponential Phase• Also called log phase• Rate of growth and division is constant and
maximal• Population is most uniform in terms of
chemical and physical properties during this phase
Stationary Phase• Closed system population growth eventually ceases, total
number of viable cells remains constant – active cells stop reproducing or reproductive rate is balanced by
death rate
Possible Reasons for Stationary Phase• Nutrient limitation• Limited oxygen availability• Toxic waste accumulation• Critical population density reached
58
Senescence and Death Phase• Two alternative hypotheses
– cells are Viable But Not Culturable (VBNC)• cells alive, but dormant, capable of new growth when
conditions are right
• Programmed cell death– fraction of the population genetically programmed to
die (commit suicide)
59
Prolonged Decline in Growth• Bacterial population continually evolves• Process marked by successive waves of
genetically distinct variants• Natural selection occurs
Direct Measurement of Microbial Growth
• Direct measurements–count microbial cells– Plate count– Filtration– Most probable number (MPN) method– Direct microscopic count
Plate Counts
• Count colonies on plates that have 30 to 300 colonies (CFUs)
• To ensure the right number of colonies, the original inoculum must be diluted via serial dilution
• Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method)
Figure 6.16 Serial dilutions and plate counts.
The pour plate method The spread plate method
Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method The spread plate method
0.1 ml
Inoculate platecontainingsolid medium.
Spread inoculumover surfaceevenly.
Colonies growonly on surfaceof medium.
1.0 or 0.1 ml
Inoculateempty plate.
Add meltednutrient agar.
Swirl to mix.
Coloniesgrow on andin solidifiedmedium.
Bacterialdilution
Filtration• Solution passed through a filter that collects
bacteria• Filter is transferred to a Petri dish and grows
as colonies on the surface
The Most Probable Number (MPN) Method
• Multiple tube test• Count positive tubes• Compare with a statistical table
• Volume of a bacterial suspension placed on a slide
• Average number of bacteria per viewing field is calculated
• Uses a special Petroff-Hausser cell counter
Direct Microscopic Count
Number of bacteria/ml =Number of cells counted
Volume of area counted
Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added hereand fills the shallow volume over thesquares by capillary action.
Bacterialsuspension
Cover glass
Slide
Cross section of a cell counter.The depth under the cover glass and the areaof the squares are known, so the volume of thebacterial suspension over the squares can becalculated (depth × area).
Location of squares
Microscopic count: All cells inseveral large squares arecounted, and the numbers areaveraged. The large squareshown here has 14 bacterial cells.
The volume of fluid over thelarge square is 1/1,250,000of a milliliter. If it contains 14cells, as shown here, thenthere are 14 × 1,250,000 =17,500,000 cells in a milliliter.
Estimating Bacterial Numbers by Indirect Methods
• Turbidity—measurement of cloudiness with a spectrophotometer
• Metabolic activity—amount of metabolic product is proportional to the number of bacteria
• Dry weight—bacteria are filtered, dried, and weighed; used for filamentous organisms
Figure 6.21 Turbidity estimation of bacterial numbers.
Light source
LightSpectrophotometer
BlankScattered lightthat does notreach detector
Light-sensitivedetector
Bacterial suspension
Viable counting: Alive or dead?
• Whether or not a cell is alive or dead isn’t always clear cut in microbiology– Cells can exist in a
variety of states between ‘fully viable’ and ‘actually dead’