Microbial Growth - U of L Personal Web Sitespeople.uleth.ca/~selibl/Biol3200/CourseNotes/GrowthCh6.docx · Web viewMicrobial Growth Why study growth? Important to understanding biology

Biol 3400Tortora et al. Chap 6

Microbial Growth

Why study growth? Important to understanding biology of an organism – growth is essential to any organism's

existence Information on growth is required for control microoganisms

Definitions of Growth Steady increase in all the chemical components of an organism that may result in an increase

cell size, cell number or both

Increase in biomass as measured by changes in Dry weight increase Increase in absorbance Increase in cellular constituents Protein Nucleic acids other constituents e.g., peptidoglycan and chitin

Growth results in increased cell size and frequently cell division

Particularly relevant to unicellular organisms:o In unicellular organisms cell growth results in increase in numberso In multicellular organisms cell growth results in an increase in organism size

I. Factors that Affect Growth

A. Chemical factors Nutrients are substances used in biosynthesis and energy release and are therefore required

for growth One must define nutritional requirements in order to cultivate the microbe in the laboratory Chemical factors are supplied by i) the culture medium (pl. - media) that contains substrates

required for growth and ii) culture conditions (i.e., aerobic vs anaerobic conditions).

1. Macroelements (major elements - C, O, H, N, S, P, K, Ca, Mg, and Fe) required in large amounts by the cell – >95% of cells are composed of macroelements

(sometimes call macronutrients) C, O, H, N, S, P are components of macromolecules

Carbono Life on earth is carbon basedo Half of the dry weight of a typical cell is carbon

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Nitrogeno Nitrogen makes up approximately 14% of the dry weight of a typical cello Major constituent of protein and nucleic acids, some carbohydrates and lipidso NH3, NO3

-, N2 (nitrogen fixation) and organic N compounds (e.g., amino acids) from the environment. Some bacteria use atmospheric nitrogen (N2) as a nitrogen source

Phosphoruso component of phospholipids and nucleic acids, nucleotides such as ATP, some proteinso available as organic and inorganic forms in the environment

Sulfuro structural role in methionine and cysteine as well as a number of vitamins (thiamine,

biotin), coenzyme A and some carbohydrateso available usually from inorganic sources SO4

2- or H2S and organic sulfur compounds such as cysteine

K, Ca, Mg, and Fe are cations in cells and required for a variety of roles e.g., - cofactors (K+, Ca2+, Mg2+, and Fe2+ or Fe3+)

- stabilize membranes and ribosomes (Mg2+)- contribute to heat resistance of endospores (Ca2+) - components of biomolecules such as cytochromes (Fe2+ and Fe3+)

2. Trace elements or Micronutrients required in lesser or trace amounts. Critical to cell function Many are metals – structural role with many enzymes - cofactors often trace elements present in medium components or water provide all that is required for

growth Co, Cu, Mn, Mo, Ni, and Zn are needed by most cells. Some cells require Cr, Se, W, and V

3. Oxygen

a) Aerobic organisms growth at full atmospheric O2 tensions (21% O2 in the atmosphere) facultative organisms (under appropriate nutrient and culture conditions) can grow under

either aerobic or anaerobic condition obligate aerobes - require O2 for growth O2 is poorly soluble - forced aeration is often used in culture systems to provide O2

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b) Anaerobic organisms obligate (strict) anaerobes - grow only in the absence of O2; sensitive to O2 and brief exposure

will kill these organisms; perhaps because these organisms are unable to detoxify some of the products of O2 metabolism

lack a respiratory system and can’t use oxygen as a terminal electron acceptor These organisms do use oxygen found in cellular materials

Obligate anaerobiosis - prokaryotes, and a few groups of fungi and protozoa

Toxic forms of oxygen Oxygen itself is not toxic to anaerobic organisms – rather it is certain derivatives that are toxic reduction of O2 in respiration produces several toxic products singlet oxygen (1O2

-) – produced photochemically and biochemically (peroxidase activity). Outer shell electrons become highly reactive; carry out spontaneous and undesirable oxidations in the cell

hydrogen peroxide (H2O2) – Produced during aerobic respiration; damage cell components but not as toxic as O2

.-, or OH· superoxide (O2

.-) – Formed in small amounts during aerobic respiration; highly reactive and can oxidize any organic compound in the cell

hydroxyl radical (OH·) - most reactive, instantly oxidize any organic substance in the cell. All cells contain flavoproteins, quinines, thiols, and iron-sulfur proteins that can react with O2 and

produce superoxide Ionizing radiation is the major source of hydroxyl radicals. Small amounts of hydroxyl radicals

can be produced from H2O2. A number of enzymes have evolved to detoxify oxygen species

Catalaseo destroys H2O2 o H2O2 + H2O2 2 H2O + O2

o Catalase test - 30% H2O2 place on cells. Cells with catalase activity produces vigourous bubbling as O2 is released

Peroxidase o destroys H2O2 but does not produce O2. May require a reductant such as NADHo H2O2 + 2H+ 2 H2O

Superoxide dismutase (SOD)o Destroys superoxideo Indispensable to aerobic cellso O2

.- + O2.- + 2H+ H2O2 + O2

o Generally works in tandem with catalase: 4O2- + 4H+ 2H2O + 3O2

Superoxide reductaseo Found in some obligately anaerobic prokaryoteso O2

.- + 2H+ + cytochrome creduced H2O2 + cytochrome coxidized

o Avoids production of O2 as found with SODo H2O2 may then be removed by peroxidase activity

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Aerobes and facultative anaerobes usually produce superoxide dismutase and catalase

c) Aerotolerant anaerobes tolerate O2 and grow in its presence even though they can’t use oxygen. Aerotolerant organisms can tolerate oxygen because they produce SOD or equivalent system that

neutralizes toxic oxygen species. Usually lack catalase activity

d) Microaerophiles grow only at reduced O2 concentrations (2 to 10%) These organisms have limited capacity to respire or have some oxygen-labile molecules;

sensitivity to oxygen may also be due to the sensitivity superoxide radicals and peroxides

O2 usually excluded from culture systems by one or a combination of the following mechanisms Fill container to the top and seal Boil medium to drive out O2

Use reducing agents that react with O2; reduces it to H2O (e.g., thioglycolate, cysteine, H2S) Seal containers under O2 free gas Use redox indicators such as resazurin to indicate the presence of O2. Use O2 consuming devices (catalyst) Work under a stream of O2 free gas or in an anoxic glove box/anaerobic chamber

4. Other required elements Some microbes may have particular requirements that reflect their specific environment

(Halophiles require Na+) and morphology (Diatoms and Silicon dioxide based cell walls)

5. Growth Factors Some microbes have the enzymes and biochemical pathways needed to synthesize all cellular

components using minerals and sources of energy, carbon, nitrogen, phosphorus and sulfur. Other microbes lack one or more enzymes necessary to synthesize essential constituents – they get

these constituents or precursors from the environment Growth factors are organic compounds that are essential cellular components or precursors of these

components but cannot be synthesized by the organism Major Classes of Growth factors

1. amino acids2. purine and pyrimidines3. vitamins (e.g., thiamine, biotin, cobalamin, pyridoxine)

Other growth factors include heme (nonprotein component of many cytochromes) or cholesterol

Understanding growth factor requirements has practical implicationso Bioassays using microbes to detect the specific growth factor that they need. Growth-

response assay – uses this approach to detect the amount of a growth factor in solution. These assays can be specific, sensitive, simple and quantitative

o Manufacture of growth factors by specific microorganisms (e.g., Vitamin D by Saccharomyces) in industrial fermentations

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B. Physical (or environmental) Factors

1. The Effect of Temperature on Growth

Cardinal temperatures (Fig 6.1) Depend on environmental factors such as pH and available nutrients

a) Minimum temperature - below which cells are inactive reduced membrane fluidity – perhaps affects nutrient transport or proton gradient formation

b) Optimum temperature highest rate of growth and reproduction, always nearer maximum temperature

c) Maximum temperature - above which growth is not possible Growth stops because of inactivation of one or more key proteins, damages transport carriers

or other proteins, or thermal disruption of membrane

Cardinal temperatures vary for different organisms Medium composition can have a slight affect Temperature optima usually vary from 0C to 75C

Pyrolobus fumarii (archaeon) - maximum temperature = 113C

Growth temperature range for a particular organisms usually spans 30 to 40C

Distinguish five groups of microbes based on temperature optima

i) Psychrophiles Grow well at 0C and have an optimum temperature 15C and a maximum temperature

around 20C heat sensitive and unable to survive temperate climates

Adaptations to Psychrophilyo Enzymes, transport systems and protein synthetic apparatus work well at low

temperatures

enzymes with low temperature optima o greater amounts of -helix and lesser amounts of sheet secondary structureo greater amounts of polar amino acids and lesser amounts of hydrophobic amino acids

membranes contain higher amounts of unsaturated fatty acidso some psychrophiles have membranes higher in polyunsaturated fatty acids

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ii) Psychrotolerant (psychrotrophs, facultative psychrophiles) grow at 0C but have optima of 20 - 30C

iii) Mesophiles Optimum temperature between 25 and 40C Minimum temperature between 15 and 20C Maximum temperature 45C Most common type of microbe

e.g., E. coli Optimum temperature < 39CMaximum temperature < 48CMinimum temperature 8C

iv) Thermophiles Optimum temperature between 50 and 60C Minimum temperature around 45C Maximum temperature 45C

Only prokaryotes grow above 60C The most thermophilic organisms are Archaea Nonphototrophic organisms are able to grow at higher temperatures than phototrophic forms

v) HyperthermophilesOptimum temperature > 80C Extreme thermophiles are usually Archaea The highest growth temperatures for an archaeon is 113C (Pyrolobus fumarii)

Adaptations to Thermophily

i) Enzymes and other proteins are heat stable Subtle amino acid substitutions Increased number of salt bridges Densely packed hydrophobic interiors The presence of certain solutes such as di-inositol phosphate and diglycerol phosphate

ii) Macromolecules function optimally at high temperatures

iii) Membrane is heat stable Membrane lipids are more branched, rich in saturated fatty acids and of higher molecular

weight In some cases they have lipid monolayers (diglycerol tetraethers)

iv) DNA is stabilized by special histone – like proteins

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Review cell membrane structure – Chapter 4

Why don’t eukaryotes grow above 60C?

Applications of ThermophilyHigh temperature enzymese.g., feed pelleting process

PCR – Taq DNA polymerase from Thermus aquaticus

2. The Effect of pH on Growth All organisms have a characteristic pH range within which growth is possible. The range is

usually 2 – 3 pH units. In nature, environmental pH ranges from 5 to 9 Few organisms can growth at pH < 2 and > 10 pH is a great influence on growth rate pH is important because of its effect on proteins (charge is important to protein

conformation) as well as the plasma membrane

a) neutrophiles - pH optimum between 5.5 and 8 Most bacteria grow well within the pH range of 6 - 9

b) alkaliphiles - prefer growth under alkaline conditions (pH 8.0 to 11.5) many produce enzymes that work well at high pH – useful for the detergent industry

c) acidophiles - restricted to growth at low pH values – between 0 and 5.5 Fungi are generally more acid tolerant than bacteria – many grow at pH 4 to 6 Some Bacteria and Archaea are obligate acidophiles

e.g., Bacteria - Thiobacillus Archaea - Sulfolobus

pH has an important effect on stability of acidophile plasma membrane

Intracellular pHo Intracellular pH is usually between pH 6 to 8 but internal pH as low as 4.6 and as high as

9.5 have been measuredo Maintained by pumping H+ across the membrane, internal buffering and synthesizing new

proteins (e.g., acid shock proteins and heat shock proteins) that function by pumping protons or acting as chaperones

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3. Osmotic Effects on Growth Microbes require water to grow – their cells are 80 – 90% water Water availability depends not only on amount of water present in any environment but also

the concentration of solutes present (e.g., salts, sugars,…). Water activity (aw) - amount of water that is free to react = availability of water in a

substance aw = a ratio of the vapour pressure of the air in equilibrium with a substance or solution to the

vapour pressure of pure water (1/100 the relative humidity of a solution) aw ranges between 0 and 1 Most bacteria require an aw of 0.9 for active metabolism Most organisms are adversely affected by very low water activity (They suffer from

plasmolysis) In nature osmotic effects are of interest mainly in habitats with high salt concentration

a) Halophilic bacteria A organism requiring salt (NaCl) for growth microbes found in the sea (which is 3% NaCl) usually have a growth requirement for salt

Mild halophile – salt requirements between 1 and 6% Moderate halophile - salt requirements between 7 and 15% Extreme halophiles - salt requirements between 15 and 30% (e.g., Archaebacteria such as

Halobacterium species)

Halotolerant organisms can withstand some reduction in aw but generally grow best without added solute

Osmotolerant – grow over a wide range of water activity Osmophiles - require high solute (e.g., sugar) concentration for growth Xerophiles – able to grow in very dry environments (i.e., made dry by lack of water)

How does an organism grow under low aw?Increases internal solute concentration Pumps inorganic ions (e.g., K+) into the cell Synthesize or concentrate an organic solute (e.g., proline, glycine betaine, sucrose,

trehalose, mannitol) These substances must not inhibit biological processes; they are usually highly water

soluble

How does an organism grow under high aw?

II. Microbial Growth in Natural EnvironmentsMost natural ecosystems are complex and constantly changing Low concentrations of usable nutrients (Oligotrophic) Competition

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Growth in an environment depends on the nutrient supply and the microbes tolerance for the environment.

Liebig’s law - the total biomass of an organism will be determined by the nutrient present in the lowest concentration relative to the organism’s requirements

Shelford’s law – there are limits to environmental factors below and above which a microorganism cannot survive and grow regardless of the nutrient supply

Most bacteria are likely to experience starvation. How do they deal with nutrient limitation?

Reduction in cell size Change in morphology – increase surface area and ability to absorb nutrients Shutdown of metabolism except for housekeeping maintenance genes

Biofilms Most microbes are typically found in biofilms in nature Biofilms consist of cells embedded in EPS (Chapter 4) Microbes in biofilms share nutrients, communicate (e.g., quorum sensing), exchange genetic

information and are sheltered from adverse environmental factors (i.e., desiccation, antibiotics, host immune response)

Microbes in biofilms can be 1000X more resistant to antimicobial compounds Microbes in biofilms can carry out complex chemical processes (i.e., breakdown of plant cell

walls such as occurs in the rumen)

III Culture Media A culture medium (pl = media) is a nutrient solution used to grow microorganisms in the

laboratory. The growth medium is the most important factor when culturing microbes There are vast differences in the biosynthetic capacities of microorganisms and thus a need

for a variety of culture media. Knowledge of the microoganism’s normal habitat is useful in selecting an appropriate medium

Specialized media are used for a variety of purposes, including isolation and identification of microorganisms, testing antibiotic sensitivities, water and food analysis, industrial microbiology

Factors like temperature, pH, Oxygen and pressure must also be considered when culturing micoroganisms

Inoculum (pl. = inocula) = microbes introduced into a culture medium to initiate growth. These cells multiply and are referred to as the culture.

Fastidious microorganisms - have very rigorous or complex requirements (e.g., for vitamins, amino acids...)

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A. Chemical and Physical Types of Culture Media

1. Chemically defined (synthetic) media the exact chemical composition of the medium is known measured amounts of highly purified inorganic and organic chemicals are added to distilled

water

BM+G (chemically defined medium)Ingredient g/L in dH2OGlucose 2.0(NH4)2SO4 2.0K2HPO4 0.5Monosodium glutamate 5.0MgSO4.7H2O 0.3MnSO4.H2O 0.05CaCl2 0.08ZnSO4.7H2O 0.005CuSO4.5H2O 0.005FeSO4.7H2O 0.0005

2. Complex media certain components are of unknown composition and these components may change from

batch to batch. Use of this type of medium results in the loss of control of nutrient composition

Luria Burtani (LB; Chemically undefined or Complex medium)Ingredient g/L in dH2 O Yeast Extract 5.0Tryptone 10.0NaCl 5.0

Tryptic Soy Broth (TSB Chemically undefined or Complex medium)Ingredient g/L in dH2 O Tryptone 17.0Peptone 3.0Glucose 2.5NaCl 5.0Dipotassium phosphate 2.5

Refer to appendix 8 of lab manual for other examples of complex media

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3. Liquid or solidified media Both liquid and solidified media are routinely used in microbiology Solidified media is particularly important for the establishment of pure cultures as well as

determination of cell number. It is often desirable to have cells produce colonies (visible, isolated masses of cells) - Colonies come in different shapes, sizes, textures and colors, and colonial morphology may be useful in identifying a microorganism

Agar is the most commonly used solidifying agent. It is extracted from red algae and is a sulfated heteropolymer of D-galactose, 3,6-anhydro-L-galactose and D-glucuronic acid. Agar is added to a final concentration between 1 and 2% with 1.5% w/v being the most commonly used concentration.

Agar is particularly well suited for this application because it melts at a relatively high temperature (90C) but does not solidify until it reaches 45C. Moreover, very few microorganisms can hydrolyze agar.

Agar is melted during sterilization and the molten medium is poured into Petri dishes and allowed to solidify

B. Functional Types of Culture Media Complex media such as tryptic soy broth are called general purpose media or supportive

media because they sustain the growth of many microorganisms For some particularly fastidious organisms additional components such as whole blood or

serum must be added. These media are referred to as enriched media and designed to better mimic natural conditions (i.e., host for pathogens)

Selective medium A medium with a composition favoring growth of certain types of microorganisms while

inhibiting growth of any other microorganisms that may be present.

Examples

Differential medium A medium that contains substance(s) that permits for the differentiation of particular

metabolic activities during growth. Useful in distinguishing particular groups of microbes and may provide information useful in identification

Examples

Selective and differential characteristics may be combined in a single medium

Examples

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C. Enrichment technique Developed by Beijerinck The use of culture media or conditions that favour growth of one type or group of

physiologically related microorganisms over all other microorganisms present in the sample

D. Notes on culturing microbes not all microbes can be cultured in the laboratory General usage media generally permit the growth of a wide variety of microbes. At times it is desirable to use environmental or nutritional factors to selectively cultivate a

certain group or kind of microorganism.

Aseptic Technique Series of steps used to minimize contamination during the manipulations of cultures and

sterile culture media Sterilize all media and implements for handling materials of interest Clean working area Limit exposure to potential sources of contamination

Preparation of Pure Cultures

Streak plate technique Dilution Deposition of individual cells or clumps of cells (known as colony forming

units or CFU) on agar medium Cell growth multiplication resulting in the production of colonies (visible mass of

cells) each isolated colony on the streak plate is assumed to have originated from a

single CFU (It is unknown whether the cells in the colony came from a single cell or a clump of cells)

Preserving Bacterial Cultures1. Refrigeration at 4C short term solution - several weeks to several months duration depends on type of medium

2. Glycerol stocks Sterile glycerol is added to liquid cultures to a final concentration of 15 – 25% The stocks are placed in small plastic tubes with tight fitting lids (i.e., preferably screw cap

tubes with gaskets in the lids) The glycerol stocks are stored at -20C (1 to 2 years) or -80C (up to 10 years or more)

3. Lyophilization Freeze drying Culture is quick-frozen at temperatures ranging from -50 to -90C and then dried under

vacuum on a lyophilizer; freeze dried cultures are stored in sealed glass ampules for extended periods of time.

Microbial Culture Collections

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Sources of microbial cultures Cultures are distributed for a fee or free depending on the culture collection

ATCC American Type Culture CollectionDSMZ Deutsche Sammlung von Mikrooganismen und Zellkulturen

NCTC National Collections of Type Cultures and Pathogenic FungiNCIMB National Collections of Industrial and Marine BacteriaEGSC E. coli Genetic Stock CentreBGSC Bacillus Genetic Stock CentreFGSC Fungal Genetic Stock Centre

IV. Growth of Microbial Cultures

i) Eukaryotic Cell Cycle – review Biol 1010 notes

ii) Prokaryotic cell cycle most often is accomplished by Binary Fission but budding, fragmentation and other

processes may occur

Mother cell two daughter cells …Generation time (g)

Binary fission in E. coli takes 20 minutes under optimal conditions Required as many as 2000 chemical reactions Length of time depends on a number of factors, including nutrition, genetics and

environment

Rapidly Growing Cells In E. coli, the cell cycle takes 60 min to complete: 40 minutes for DNA replication and

partitioning and 20 min for septum formation and Cytokinesis But E. coli can complete this entire process in 20 min under optimal conditions This is possible because E. coli starts a second round of DNA replication (and sometimes a

third and a fourth round) before the first round of replication is completed.

A. Population Growth

Growth rate change in cell number or cell mass per unit time

Generation interval for the formation of two cells from one cell

Generation time (doubling time)

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time it takes for one cell to become two cells time it takes for the population to double depends on growth medium and conditions

1. The Mathematics of Growth (Growth Equations)

Growth by binary fission results in exponential growth of the population (Figure 6.13 & 6.14)

Geometric progression of the number 2

21222324

(1) Nt = N02n

Nt = final number of cells at time tN0 = initial number of cellsn = number of generations that have occurred during period of exponential growth

Solving for n (where all logarithms are to the base 10)

log Nt = log N0 + n log 2 and

(2) n = log N t - log N 0 = log N t - log N 0

log 2 0.301

Growth rate can also be expressed as the mean growth rate constant (k). The specific growth rate is a measure of the number of generations that occur per unit time

(3) k = n/t = log N t - log N 0

0.301t

Can now calculate the mean generation time (g) or mean doubling time.When the population doubles t = g and Nt = 2N0; substitute 2N0 into (3)

(4) k = log (2 N 0) - log N 0 = log 2 + log N0 – log N0 = 1/g 0.301g 0.301g

Therefore

(5) g = 1/k

Generation time can also be calculated from the slope of a line obtained in a semi-log plot of exponential growth

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(6) slope = 0.301/g ; g = 0.301/slope

How can we use growth rate information?

2. Culture Systems"Fermentation" - cultivation of microorganisms in a controlled, enclosed system

i. Batch CultureA fixed volume of liquid medium is inoculated and incubated for an appropriate period of time with no further addition of microorganisms or growth substrates

closed environment most common method of microbial cultivation nutrient concentration is a determinant of growth rate and cell yield

The batch culture has a continually changing environment o nutrients are depletedo products producedo cells change

Ultimately the culture quits growing due to nutrient limitation or product accumulation

e.g., test tube to flask to 100,000 L fermenter

ii. Fed BatchA nutrient stock (limiting nutrient) is added at intervals or continuously to a batch culture

iii. Continuous CultureSpent culture is replaced by fresh medium allowing continual growth of the culture.

Open system system can be manipulated to reach an equilibrium or steady state where the cell density and

nutrient status remain constant Can control culture growth rate as well as yield of cells by manipulating dilution rate and the

level of the limiting nutrient, respectively More sophisticated apparatus required Superior productivity possible because of reduced downtime.

e.g., Chemostat uses dilution rate and nutrient concentration to control growth and population density

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growth rate (adjust dilution rate) and yield (adjust limiting nutrient) can be controlled independently of each other

Compared to batch culture – the chemostat allows: experimenter to vary growth rate and population density independently of each other can maintain population in exponential phase at a known growth rate for long periods of time Can study microbial growth at very low nutrient concentrations – close to those present in

nature

3. Bacterial Growth Curve Growth of a batch culture population of cells can be monitored and plotted as a growth curve A typical batch culture growth curve can be divided into 4 phases (Fig 6.15)

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i) Lag PhaseInitial phase during which time cells are adjusting their metabolism to prepare for a new cycle of growth. There is no increase in cell number - increase in cell size The cells are transporting nutrients, synthesizing RNA and subsequently enzymes needed for

growth; replicating DNA The length of this phase depends on the history of the culture and growth conditions

Examples:

ii) Exponential Phase (Log phase) Cell are growing and dividing at the maximum growth rate possible given their genetic

potential, the nature of the medium and incubation conditions. One cell gives rise to two and so on: Cell number is increasing as an exponential function of

time Log transformation of data results in a linear curve During this phase the resulting cell population is most uniform with respect to chemical and

physiological properties; cells in this phase are most often used in biochemical and physiological studies

Exponential growth is said to be balanced growth because all cellular components are made at constant rates relative to each other. If the nutrient levels or some other environmental parameter changes then unbalance growth results: growth during which the rates of synthesis of the various cellular constituents vary relative to one another until a new balanced state is reached.

Shift-up (culture is transferred from a nutritionally poor medium to a richer medium) and shift-down (culture is moved to from a nutritionally rich medium to a poor medium) experiments produce unbalanced growth.

In the shift-up experiment there is a lag in while the cells first produce more ribosomes to enhance protein synthesis. There is then an increase in protein and DNA synthesis followed by the rise in productivity.

In the shift down experiment:

Determinants of growth rate Different nutrients and nutrient concentration allow for different growth rates. Growth rate

increases with increasing nutrient concentration. At some point nutrient transport systems are saturated and growth rate can increase no further

Temperature, pH, Oxygen and other physical parameters Genetic determinants Small cells generally grow faster than larger cells (surface area to volume ratio) Nutrient concentration affects maximum cell yield

iii) Stationary Phase Closed system - cells can’t grow indefinitely No further net increase in cell number Total number of viable cells remains unchanged because i) growth rate = death rate (i.e.,

some cells in the population grow while others die. This is known as cryptic growth) or ii) the population may not be dividing but remain metabolically active

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Stationary phase is entered because 1) nutrient limitation, 2) oxygen limitation, 3) build up of toxic wastes (e.g., organic acids), 4) a critical population level is reached, or 5) several of these factors acting together

Cellular composition and activity changes Prokaryotes have evolved a number of strategies to deal with starvation. A few genera will

produce endospores but most will reduce cell size, which is often accompanied by protoplast shrinkages and nucleoid condensation. Morphological changes can also occur

e.g., Arthrobacter - log cells - rods - stationary cells - coccoid

The most important changes are in gene expression and physiology. o Different genes are turned on (e.g., catalase, exonuclease and acid phosphatases; survival

genes (sur) have been identified for E. coli)o Most starving cells produced starvation proteins that make the cell more resistant to

environmental stresses (e.g., elevated temperature, osmotic pressure and toxic chemicals such as hydrogen peroxide and chlorine) and harder to kill. The cells increase peptidoglycan crosslinking and cell wall strength, produce proteins to protect their DNA (DNA binding protein from starved cells – Dps) and to prevent protein denaturation and renature damaged proteins (Chaperone proteins).

vi) Death Phase (Senescence phase) Exponential decline in viable cell numbers. Typically the rate of exponential decline is much

slower than that of exponential growth In many instances this phase can be reversed if modify the environmental parameters In many cases the decline is cell number is associated with a loss of intact cells. In other

cases this is not the case A decline in viable cell numbers may be explained by simple cell death associated with

starvation or build up of toxins. But two other hypotheses have been proposed

i) Not all cells are culturable = Viable but nonculturable (VBNC) cells.Cells are viable as demonstrated by the presence of metabolic activities but can't be cultivated in the lab - detected by discrepancies between indirect and direct counts. VBNC cells are genetically programmed to become dormant (genetic response triggered in starving stationary phase cells) and when appropriate conditions become available (e.g., change in temperature, passage through animals), the cells begin growing again.

ii) Programmed cell death. A fraction of the microbial population is genetically programmed to commit suicide – nonculturable cells are dead and the nutrients that they leak enable eventual growth of those cells in the population that did not commit suicide.

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4. Measurement of Growth Enumeration of microbial populations or measuring mass

i) Measurement of Cell Numbers

a) Direct Counting (counts all cells - viable and dead)

Direct microscopic counts with counting chambers (Fig 6.20) Use a chamber (e.g., Petroff-Hausser counting chamber) of defined volumes. Count cells the

aid of a microscope can also use samples dried onto slides

Advantages rapid counts all cells in a sample (can often count individual cells in clumps) can acquire cell morphology information with these methods

Disadvantages can't determine which cells are viable unless they are treated in a special manner

(e.g.,fluorescent live/dead cell stains). small cells are difficult to see affected by debris in samples not suitable for cell suspensions of low density (< 106/mL); precision difficult to achieve motile cells are difficult to count phase contrast microscopy required if sample not stained may require expensive pieces of equipment unable to perform further studies on the observed microbes without further cultivation

Filtration known volume of a suspension filtered onto a black polycarbonate filter membrane. cells are stained with fluorescent dyes and counted under the microscope

Coulter Counter automated method of counting cell. as cell pass through a aperture they disturb an electric field perturbations are transformed into number and size data. Most useful for larger cells

Fluorescence Activated Cell Sorter (FACS)

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b) Viable Counts (counts viable cells that can be cultured)

Viable Plate Count counts viable cultivable bacteria Viable count methods assume that each viable cell can grow and divide to yield one colony Serial dilutions of cultures are prepared and these suspensions of bacteria are plated onto agar

medium use spread plate or pour plate technique Following incubation - count number of colonies in order to determine the number of colony

forming units (CFUs) per unit volume. limit counting to plates with between 30 and 300 colonies plates containing less than 30 colonies are not acceptable for statistical reasons plates containing greater than 300 (TNTC) - plates are crowded and it becomes hard to

distinguish and count colonies. Problems with culturability of particular microbes on the medium - may be selective!!!!

Spread Plate (Fig 6.17) suspension of microbes is spread over the surface of agar medium. spreading separates cells that grow and give rise to isolated colonies assumes each colony arises from a single cell or clump of cells (CFU). suspension of cells must be dilute enough otherwise the plate will be overgrown - too

many cell get confluent growth or a lawn of cells with no discrete colonies. Usually spreading 0.1 mL of less on the plate

Pour Plate (Fig 6.17) suspensions of cells (0.1 to 1.0 mL) are added to molten agar (42 to 45C) Note - agar begins solidifies at approx. 42C. molten agar is poured into a petri dish, allowed to solidify and incubated; the hot agar

may kill or injure sensitive cells

Advantages of viable plate counts Counts only viable cells – widely used in food, dairy, medical industries and research Very sensitive – detect presence of very few cells Use of selective and/or differential media can restrict counts to a particular cell type the techniques require inexpensive materials once counts are completed you have viable cultures to use in subsequent experiments

Disadvantages of plate counts these methods are selective and count only viable cells or cells that can be grown with the

culture techniques used (i.e., they underestimate actual cell number) they do not distinguish between an individual cell and a cluster of cells and therefore

underestimate cell numbers takes time for data acquisition (i.e., Cells must grow for >12 h to be counted with the

viable count methods size of colonies vary and it is easy to miss small colonies subject to large errors if not done carefully – require adequate replication

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Most Probable Number (MPN) another technique for counting viable CFU dilute to extinction - such that not all aliquots transferred to tubes of growth medium will

contain a cell following incubation one checks for growth and compares results to a table of statistical

probability for obtaining the observed results.

Membrane filtration Aquatic samples are filtered through a membrane – trapping cells on the membrane The membrane is placed on an agar medium and incubated until each cell forms a colony Useful for analyzing water samples especially when the populations are low

c. Indirect estimation of Bacterial Numbers

Microbial Dry Weight Cells growing in liquid medium are collected by centrifugation or filtration, washed, dried in

a vacuum oven and weighed Time consuming, not very sensitive but good for filamentous fungi

Turbidity (Spectophotometry) rapid and sensitive method for obtaining estimate of culture density The more cells that are present the more light that is scattered by a suspension can measure transmittance of light and determine the optical density (OD) of a suspension

using a spectrophotometer growth results in increased turbidity and OD proportional to cell number for unicellular

organisms

Can generate a standard curve to relate OD to CFU's/unit volume or some other measure of growth (e.g., dry weight)

Metabolic Activity Measures a metabolic product and assumes there is a direct relationship between the amount

of the metabolic product and the cell number. Measurement of CO2 evolution

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Documents

Microbial Growth - U of L Personal Web Sitespeople.uleth.ca/~selibl/Biol3200/CourseNotes/GrowthCh6.docx · Web viewMicrobial Growth Why study growth? Important to understanding biology