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Microbial Growth Lecture 5 Chapter 6

Lect 5 Ch 6

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Page 1: Lect 5 Ch 6

Microbial GrowthLecture 5 Chapter 6

Page 2: Lect 5 Ch 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).

Page 3: Lect 5 Ch 6

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

Page 4: Lect 5 Ch 6

The Requirements for Growth

• Physical requirements– Temperature– pH– Osmotic pressure

• Chemical requirements– Carbon– Nitrogen, sulfur, and phosphorous– Trace elements– Oxygen– Organic growth factors

Page 5: Lect 5 Ch 6

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

Page 6: Lect 5 Ch 6

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

Page 7: Lect 5 Ch 6

• Organisms exhibit distinct cardinal growth temperatures– Minimum growth temperature– Optimum growth temperature– Maximum growth temperature

Page 8: Lect 5 Ch 6

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

Page 9: Lect 5 Ch 6

Microbiology & Food preservation

Page 10: Lect 5 Ch 6

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

Page 11: Lect 5 Ch 6

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

Page 12: Lect 5 Ch 6

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

Page 13: Lect 5 Ch 6

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

Page 14: Lect 5 Ch 6

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.

Page 15: Lect 5 Ch 6

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

Page 16: Lect 5 Ch 6

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

Page 17: Lect 5 Ch 6

Plasmolysis

Page 18: Lect 5 Ch 6

Chemical Requirements

• Carbon– Structural backbone of organic molecules– Chemoheterotrophs use organic molecules as

energy– Autotrophs use CO2

Page 19: Lect 5 Ch 6

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

Page 20: Lect 5 Ch 6

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

Page 21: Lect 5 Ch 6

Trace Elements

• Inorganic elements required in small amounts• Usually as enzyme cofactors• Include iron, copper, molybdenum, and zinc

Page 22: Lect 5 Ch 6

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)

Page 23: Lect 5 Ch 6

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Page 24: Lect 5 Ch 6

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

Page 25: Lect 5 Ch 6

Organic Growth Factors

• Organic compounds obtained from the environment

• Vitamins, amino acids, purines, and pyrimidines

Page 26: Lect 5 Ch 6

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

Page 27: Lect 5 Ch 6

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

Page 28: Lect 5 Ch 6

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

Page 29: Lect 5 Ch 6

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.

Page 30: Lect 5 Ch 6

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

Page 31: Lect 5 Ch 6

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

Page 32: Lect 5 Ch 6

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

Page 33: Lect 5 Ch 6

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

Page 34: Lect 5 Ch 6

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

Page 35: Lect 5 Ch 6

Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

Page 36: Lect 5 Ch 6

Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

Page 37: Lect 5 Ch 6

Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

Page 38: Lect 5 Ch 6

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

Page 39: Lect 5 Ch 6

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

Page 40: Lect 5 Ch 6

Figure 6.7 An anaerobic chamber.

Airlock

Armports

Page 41: Lect 5 Ch 6

Special Culture Techniques

• Capnophiles – Microbes that require high CO2 conditions

– CO2 packet– Candle jar

Page 42: Lect 5 Ch 6

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

Page 43: Lect 5 Ch 6

Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

Page 44: Lect 5 Ch 6

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

Page 45: Lect 5 Ch 6

Enrichment Culture

• Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels

• Usually a liquid

Page 46: Lect 5 Ch 6

Table 6.5 Culture Media

Page 47: Lect 5 Ch 6

Bacterial Division

• Bacteria and Archaea:– Haploid– Reproduce asexually

• Increase in number of cells, not cell size– Binary fission– Budding– Fragmentation of filaments

• Conidiospores (actinomycetes)

Page 48: Lect 5 Ch 6

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

Page 49: Lect 5 Ch 6

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

Page 50: Lect 5 Ch 6
Page 51: Lect 5 Ch 6

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

Page 52: Lect 5 Ch 6

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

Page 53: Lect 5 Ch 6

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

Page 54: Lect 5 Ch 6

Phases of Growth• Lag phase• Log phase• Stationary phase• Death phase

Page 55: Lect 5 Ch 6

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

Page 56: Lect 5 Ch 6

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

Page 57: Lect 5 Ch 6

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

Page 58: Lect 5 Ch 6

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)

Page 59: Lect 5 Ch 6

59

Prolonged Decline in Growth• Bacterial population continually evolves• Process marked by successive waves of

genetically distinct variants• Natural selection occurs

Page 60: Lect 5 Ch 6

Direct Measurement of Microbial Growth

• Direct measurements–count microbial cells– Plate count– Filtration– Most probable number (MPN) method– Direct microscopic count

Page 61: Lect 5 Ch 6

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)

Page 62: Lect 5 Ch 6

Figure 6.16 Serial dilutions and plate counts.

Page 63: Lect 5 Ch 6

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

Page 64: Lect 5 Ch 6

Filtration• Solution passed through a filter that collects

bacteria• Filter is transferred to a Petri dish and grows

as colonies on the surface

Page 65: Lect 5 Ch 6

The Most Probable Number (MPN) Method

• Multiple tube test• Count positive tubes• Compare with a statistical table

Page 66: Lect 5 Ch 6

• 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

Page 67: Lect 5 Ch 6

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.

Page 68: Lect 5 Ch 6

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

Page 69: Lect 5 Ch 6

Figure 6.21 Turbidity estimation of bacterial numbers.

Light source

LightSpectrophotometer

BlankScattered lightthat does notreach detector

Light-sensitivedetector

Bacterial suspension

Page 70: Lect 5 Ch 6

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’