MICROBIAL GROWTH

LECTURE

INTRODUCTION Growth = cell reproduction

Binary Fission = doubling viable cell number in popln

Growth is exponential - rate of increase dependant on doubling time

Growth curve (adaptation, reproduction, no net growth and decline)

Factors influencing bacterial growth rates - temp, salinity, pH, O2, other physical & chemical factors

Bacteria exhibit ranges of tolerance

GROWTH: steady increase in all chemical components of an organism, usually results in increase in size of cell and frequently results in cell division

Bacterial Cell Cycle: SIMPLE

Time of division of mother cell into 2 daughter cells then WHEN 1 daughter cell then divides into 2 more daughter cells

Characterized: continuous macromolecular synthesis

Cell elongation occurs along with genome replicated

Eukaryotic Cell Cycle: COMPLEX

Involves separate phases for cell enlargement, replication of the genome, separation of replicated genomes by mitosis and cell division (cytokenesis)

Cells grow by increasing cellular constituents and then dividing into 2 cells (ASEXUAL PROCESS)

Known as BINARY FISSION

Division is GEOMETRICAL (Population Doubles)

1 cell divides into 2 new cells

BACTERIAL GROWTH

BINARY FISSION

Involves 3 processes: Increase in cell size (cell elongation) DNA replication Cell division

NOTE: NOT ALL BACTERIA

Yeast-like budding

GROWTH RATE: time for cell to reproduce

KINETICS OF BACTERIAL REPRODUCTION

Binary fission results in doubling viable cell no’s

GENERATION TIME: Time required for a complete

fission cycle

i.e., time for 1 parent cell to form 2 new daughter cells

1st Generation = 2 cells

2nd = 4 cells

3rd = 8 cells

4th = 16 cells

5th = 32 cells

AND SO ON…….

GENERATION TIME

Formation of each new bacterial cell, its growth and eventual division into 2 cells

EXAMPLES: (Optimal conditions)

Bacillus stearothermophilus 11 mins

E.coli generation time 20 mins

Staphylococcus aureus 28 mins

Lactobacillus acidophilus 60-80 mins

Mycobacterium tuberculosis 360 mins

Treponema pallidum 1980 mins

QUANTITATIVE ASSEMENT

(1) Expressed as 21, 22, 23, 24 …..2n

The power value increases by 1 each generation

(the number of the generation)

Termed EXPONENTIAL

Log 10

Log10

No' of CellsCell No's

Time

Logrithmic graphs are preferred - more accurate cell numbers during early growth

(2) SO starting with 1 cell, the total popln N after n generations

N = 1 x 2n

(3) IF original popln was hundreds/thousands cells express as:

N = No x 2n

No represents original popln size at time zero

(4) Expression to determine no’ of generations (n)

(USE THE LOGARITHM)

HENCE Log N = Log No + nLog 2

(5) Rearrange to solve for (n)

n = Log N- Log No

Log 2

(6) Simplify by substituting Log of 2 = 0.301

n = Log N- Log No

0.301

(7) THUS, knowing No and N (initial popln and total popln respect) can calculate no’ of generations (n) occurred over elapsed time (t)

Generation time g = time elapsed

no’ of generations

g = t

n

(8) OR Using (6)

g = 0.301 x t

Log N - Log No

(9) Growth rate of culture (K) is no’ of generations per unit time i.e., reciprocal of g

K = Log N - Log No

0.301 x t

EGG SANDWICH EXAMPLE

Calculate the number of Staphylococcus aureus cells present your egg sandwich which you made at 8am and has been sitting on the back seat of you car on True Blue campus for the past 4 hrs

Length of Time (t) = 4 hours (240 minutes)

Inoculum no’ (No) = 10

Generation time (g) = 20 minutes

We know: N = No x 2n

No of generations (n) = t = 240 = 12

g 20

Therefore: N = No x 2n

= 10 x 212

= 10 x 4096

= 40960 cells present in sandwich after 4 hrs

PHYSIOLOGICAL EFFECT OF GROWTH

Various consequences Growth rate increases = increased cell mass

i.e., at faster growth rates they become larger

Contain increased cell components: DNA, RNA & protein (Increases at exponential rate)

RNA/PROTEIN: Ribosome No’s (Biosynthesis proteins, polymerize Aa’s)

DNA: fast growing cells initiate DNA replication prior to cell division

NORMAL GROWTH

IN REALITY

Popln does not maintain its potential growth rate,

i.e., does not double endlessly

• GROWTH CURVEIn a closed system: nutrients and space finite

no removal of waste products

(A) LAG PHASE

1. Newly inoculated cells, adjust to new environment

2. Cells not multiplying at maximum rate

3. Popln is sparse or dilute

(B) EXPONENTIAL (LOG) PHASE

Growth occurs at an exponential rate

Cells reach maximum rate of cell division

(Continues as long as nutrients and environment is favorable)

FACTORS: TEMPERATURE

E. coli @ 30oC gtime = 1hr, @ 37oC gtime = 30mins

(C) STATIONARY PHASE

Popln reaches maximum numbers

Rate of cell inhibition (death) = Rate of multiplication

FACTORS: pH changes, accumulation of waste, reduced O2

(D) DEATH PHASE

Decline in growth rate

Caused by depletion of nutrients , O2

excretion of toxic waste products

increased density of cells (limited space)

FACTORS: Same as STATIONARY PHASE

ENUMERATION OF BACTERIA

Assess rate of microbial reproduction Determine no’s of microbes present

Various Methods

Live bacteria (reproducing on media)

Dead & Live bacteria

SERIAL DILUTIONS

VIABLE COUNT PROCEDURES Viable Plate Count: living bacteria

STEP 1: Serial dilutions of suspension of bacteria

(addition of aliquot of specimen to sterile water)

If 1ml of sample added to 99ml of SW

Dilution = 1:100 (10-2)

(Same 0.1ml sample to 9.9ml SW)

Greater dilutions: 1ml first dilution (10-2) to 9ml SW = 10-3

1ml 10-3 dilution to 9ml SW = 10-4

AND SO ON

STEP 2: Plate onto growth media (Spread or Pour)

SPREAD: drop of suspension (known volume) placed onto center agar plate & spread over surface (sterile glass rod)

POUR: suspension (known volume) added to tubes molten agar (42o - 45oC) & poured into petri dish

Alternatively: known volume added to center of plate and molten agar poured into plate

Colonies form throughout agar

Reproduction on medium visible colonies (16-24hr)

(Assumption 1 colony arises from1bacterial cell)

STEP 3: Count colonies (CFU’s - Colony Forming Units)

Concentration of bacteria in original sample determined

(number bacteria/mL) (MUST account for dilution factors)

VIABLE COUNT BY SAMPLE DILUTION

POUR PLATE METHOD

10-1

10-2

10-3

10-410-5

10-6

10-7

10-8

SURFACE DROP (MILES + MISRA METHOD)

Record Results:

10-1, 10-2, 10-3, 10-4, 10-5 All show confluent growth TNTC (Too Numerous To Count)

10-6, 10-7 Look for colonies 30- 300 colonies

10-8 Has only 10 colonies TFTC (Too Few To Count)

10-7 Has countable no’ of colonies used in calculation

CALCULATION

IF No’ of colonies = 40

Dilution counted = 10-3

Volume of drop = 1

THEREFORE No’ of bacteria present in original sample

= 40 x 1 x 103 = 4 x 104/cm-3

ASSUMING EACH COLONY ARISES FROM

1 VIABLE CELL

Sample Time(mins)

DilutionCounted

Mean Count X 108 Log 10

0 -6 21.7 2.17 x 10-7 7.34

30 -6 75 7.5 x 10-7 7.88

60 -6 182 1.82 x 10-8 8.26

90 -6 229 2.29 x 10-8 9.36

120 -7 155 1.55 x 10-9 9.19

150 -6 112.6 1.12 x 10-8 9.04

180 -7 254 2.54 x 10-9 9.40

210 -8 51 5.1 x 19-9 9.70

240 -8 56.7 5.67 x 10-9 9.75

270 -8 207 2.07 x 10-9 9.32

9.32

CELL COUNT: Microscopic

NOT Distinguish DEAD & LIVE CELLS

Calibrated slide Petroff-Hauser Counter

(Hemocytometer (RBC’s count)

Aliquot of culture under cover slip

Depth known

Caculate: No’ orgs/unit volume

CELL DENSITY: Spectrophotometer

Record optical density (OD) or Absorbance (A) units

As popln increases, turbidity (density) increases

QUESTIONA research student obtained a set of data at set time intervals (every 30 minutes) from a culture growing in a fermenter. The number of viable cells in each sample was then counted using the pour plate method

TIME VIABLE COUNT RESULTS0 0.446 x 108

30 0.512 x 108

60 1.202 x 108

90 3.36 x 108

120 1.99 x 109

180 2.29 x 109

210 2.51 x 109

240 2.51 x 109

270 2.29 x 109

300 1.99 x 109

1. Plot data in most suitable form

(THINK FIRST) 2. Label each phase of growth on the graph Calculate the mean generation time

Take 2 popln values from each end of the LOG growth phase. Use these to calculate the MGT, using the following equation:

t x Log 2

Log b - Log a

Where:

t = time interval (mins)

Log b = Log cell numbers at end of Log Growth Phase

Log a = Log cell numbers at beginning of Log Growth Phase

PHYSICAL & CHEMICAL FACTORS AFFECTING GROWTH

ENVIRONMENTAL FACTORS

Physical factors affecting MICROBIAL GROWTH

TEMPERATURE

3 Cardinal Temperatures MINIMUM

Lowest temp, permits microbial growth + metabolism MAXIMUM

Highest temp permits growth + metabolism OPTIMUM

Small range of temp’s, (promotes fastest growth & metabolism rates)

Extremes of MIN & MAX beyond which growth is inhibited

TEMPERATURE RANGES (OPTIMA GROWTH) OF SOME BACTERIA

Bacterium Growth Temperature oC

Min Max Optimum

1. Pseudomonas fluorescens 2-4 36-38 25-30

2. Pseu. aeruginosa 10-15 41-44 c. 37

3. Escherichia coli 15-20 45 37

4. Bacillus polymyxa 5-10 35-45 30-32

5. B. stearothermophilus 30-45 65-75 c. 55

6. Thermus sp. 40 79 70-72

TEMPERATURE ADAPTATIONS

PSYCHROPHILE: Psychrophilic

Opt temp below 15oC

Capable of growth at 0oC

Cannot grow above 20oC

Found: SNOW FIELDS, POLAR ICE, DEEP OCEAN

EXAMPLES: Pseudomonas, Flavobacterium, Alcaligenes & Achromobacter sp.

FACULTATIVE PSYCHROPHILE:

Grow slowly in cold conditions

BUT have opt temp above 20oC

EXAMPLES: Staphylococcus aureus, L. monocytogenes

CONCERN: Contaminants of food/dairy products

MESOPHILE:

Opt temp 20-40oC

Capable of growth 10-50oC

Group containing HUMAN PATHOGENS (30-37oC)

EXAMPLE: E. coli

THERMOPHILE:

Opt temp >45oC

Capable of growth 45-85oC

Incapable of growth at usual body temp

(NOT INVOLVED in HUMAN INFECTIONS)

Found: VOLCANO, DIRECT EXPOSURE TO SUN

EXAMPLE: Bacillus stearothermophilus

(EXTREME THERMOPHILES: opt temp >80oC)

TEMPERATURE RANGES FOR MICROBIAL GROWTH

GAS REQUIREMENTS

Oxygen plays important role in MICROBIAL GROWTH

- terminal electron acceptor in respiration

Oxygen - limited solubility in water

Therefore can be limiting factor

Enzymes are required

Reduce Oxygen to water and toxic products

(hydrogen peroxide + superoxide)

Microbes convert toxic products to molec Oxygen by:

1. CATALASE

H2O2 H2O + O2

2. PEROXIDASE

H2O2 + NADH + H+ 2H2O + NAD+

3. SUPEROXIDE DISMUTASE

2O2- + 2 H+ H2O2 + O2

Peroxide is metabolized by Catalase (as above)

BASED ON OXYGEN REQUIREMENTS

microbes divided into 4 groups

OBLIGATE AEROBES

Totally dependant on O2 for growth

Requirement of 1 atmosphere (20%)

Produce H2O2 and O2- but possess catalase and

superoxide dismutase - can tolerate high [O2]

MICROAEROPHILES

Grow in presence of O2 BUT tolerate only 4%

Possess enzymes BUT if toxic products , enzyme systems overload INHIBITING GROWTH

FACULTATIVE ANAEROBES

Grow in presence or absence of O2

Presence use Aerobic respiration

Absence fermentation for energy prodn

Grow best under AEROBIC CONDITIONS

e.g. Enterobacteriacea

ANAEROBES

Grow ONLY in ABSENCE of O2

Effect of presence variable - LETHAL or TOLERANCE

LETHAL - org’s lack enzymes to remove toxic products

TOLERANT - org’s lack enzymes to reduce O2 to water or toxic products

e.g. LOWER GI ORG’S Clostridium sp, Bacteroides sp

OBLIGATE ANAEROBES:

Fermentative metabolism

EXAMPLE: Desulfovibrio, Archaebacteria & Protozoa

STRICT ANAEROBES:

Sensitive to O2

Brief exposure will KILL

Bacterial Enzymes that Protect the Cell Against Toxic Forms of Oxygen

Microorganism Catalase Superoxidedismutase

Aerobe + +

Facultative anaerobe + +

Microaerophile - +

Obligate anaerobe - -

OXYGEN & BACTERIAL GROWTH

HIGH

LOW

O2

Potential

1 2 3 4 5

1: Obligate Aerobe 2: Facultative Anaerobe3: Aero-tolerant Anaerobe 4: Strict Anaerobe

5: Microaerophilic

EFFECTS OF pH

pH - degree of acidity or alkalinity of a soln related to the [H+]

pH = -log H+ (1/log H+)

Neutral Solutions (pH 7)

Alkaline (Basic) Soln (pH >7)

Acidic Soln (pH <7)

GROWTH RATES INFLUENCED BY pH VALUES

(NATURE OF PROTEIN)

INFLUENCE ON GROWTH & SURVIVAL

Most live between pH 6-8 (Neutrophiles 6.5-7.5)

Fungi between pH 5-6 (acidic)generally wider range (5-9)

pH EXTREMES:

few microbes in stomach pH2

(Lactobacillus acidophilus, Helicobacter pylori)

ACIDOPHILE: growth at low pH Thiobacillus sp (pH2)

OBLIGATE ACIDOPHILE - Euglena mutabilis

(acid pools 0-1)

ALKALINOPHILES - high levels of minerals (salt) pH 9-11

WATER ACTIVITY (Aw)

ALL BACTERIA require water (growth & reproduction)

Essential solvent, biochemical reactions

Water activity = index amount of water free to react

Equivalent to atmospheric measure (Relative Humidity)

Absorption & Solution factors reduce availability ( Aw)

Pure distilled water (Aw =1)

E.g., Saturated soln NaCl (Aw = 0.8)

Seawater [NaCl] 3% (Aw = 0.98)

RH = 100 Aw Therefore, 90% RH = 0.90 Aw

Most bacteria Aw >0.9 (active metabolism)

Most microbes - grow opt Aw = 1.0

Aw = slow growth rate

Below Aw 0.9 Bacteria unable to grow

EXCEPTIONS:

XEROTOLERANT: lower Aw

Fungi able to grow Aw 0.60

Yeasts (conc sugar soln’s Aw = 0.60)

Salt-tolerant Bacteria - Halophiles (High [Solute], low Aw)

Effect of Aw on growth of Staphylococcus aureus in medium containing hydrolysate

The Interrelationships of Aw of various foods & susceptibility to microbial spoilage

OSMOTIC PRESSURE

Results from: water diffusing across cell membrane in response to [solute]

Association with [salt] = SALINITY

OSMOTOLERANT: withstand high osmotic pressure

high [solute]

OSMOPHILES: require high [solute] for growth

E.g., Xeromyces (opt Aw =0.9)

SALINITY:

Most microbes - HYPOTONIC or ISOTONIC conditions

HALOPHILES (exceptions) - Require High [NaCl]

Moderate Halophiles: Marine bacteria 3% [salt]

Require 1.5% NaCl (maintain membrane integrity)

Extreme Halophiles: saturated brine soln’s

OBLIGATE HALOPHILES Halobacterium, Halococcus sp

Both 25% NaCl

Drying, Salting, Jamming (achieve low Aw)

EFFECTIVE methods of preservation

HYDROSTATIC PRESSURE

Normal pressure: 1 atmosphere (atm)

Ocean Depth (1000m +) HP = 600-1100 atm (2-3oC)

Bacteria survive & adapt BAROPHILES

BAROTOLERANT: pressure not adversely affected

NUTRIENT CONCENTRATION

Must utilize various nutrients Required for: energy production & macromolecular biosynthesis

Hence:

growth limited by concentration of required nutrient

Final net growth (cell yield) increases with initial amount of limiting nutrient

a) Effect of changes in limiting concentration on total microbial yield

b) Effect on growth rate

Total growth(cells or mg/ml)

Nutrient concentration

Nutrient concentration

Growthrate(hr-1)

Hyperbolic Curve: rate of nutrient uptake by microbial transport systems

High Nutrient Level: transport system saturated NO FURTHER GROWTH (with increase in nutrients)

GROWTH YIELD: microbial mass produced from a nutrient

Y = mass of microorganisms formed

mass of substrate consumed

Expressed: grams of cells formed/ gram substrate used

(MOLAR GROWTH: grams cells/mole nutrient consumed)

MICROBIAL ASSOCIATIONS

Influence on growth

SYMBIOTIC: org’s iving in close nutritional relationship

3 Types: MUTUALISM

COMMENSALISM

PARASITISM

MUTUALISM: orgs live in an obligatory BUT mutually beneficial relationship

EXAMPLE:

Lactobacillus arabinosus requires phenylalanine

Streptococcus faecalis requires folic acid

EXAMPLE: MUTALISM (MICROBES & ANIMALS)

Mammals unable digest cellulose

Microbes & Ruminants: ability to digest cellulose

Cellulose degradation in RUMEN

1010 - 1011 bacteria, 105 - 106 protozoa/gram contents

EXAMPLE: MUTALISM (MICROBES & PLANTS)

Rhizobium sp. & Legumes (clover, soybeans, alfalfa, peas)

Bacterium: Nitrogen fixation

Plant provides environment required

COMMENSALISM: commensal benefits, other org’s neither harmed nor benefit

Unidirectional relationship between populations

1. 1 microbe may destroy/neutralize an antimicrobial factor (enables 2nd org to grow)

2. One popln makes metabolic by-product serves as source (carbon, energy or growth factor) for another microbe

EXAMPLE

GI TRACT millions of bacteria live on the waste products within Large Intestine Bacteroides, Fusobacterium etc.

ORAL CAVITY anaerobes survive due to removal of O2 by facultative anaerobes present

SKIN Staph. epidermidis reside on outer dead layer of skin in conjunction with Matassezia furfur

PARASITISM: host org provides parasitic microbe with nutrients and habitat. Multiplication of parasite HARMS HOST

EXAMPLE

Rickettsiae - obligate parasitic bacteria of humans.

Rocky Mountain Spotted Fever - children, young

adults. Org carried in tick, lice etc DOG TICK

NON SYMBIOTIC: org’s are free-living, relationship not required for survival

2 Types: SYNERGISM (PROTO-COOPERATION)

ANTAGONISM

SYNERGISM: relationship where org’s cooperate and share nutrients that are beneficial BUT not necessary for survival

SYNTROPHISM: form of synergism

Result of cross-feeding

2 popln’s supply each others nutritional needs

(Completion of metabolic pathway)

Compound A B C D

species 1 2 3

ANTAGONISM: 1 popln has harmful effect on growth of another popln

Competition between free-living species where some members are inhibited or destroyed by others

EXAMPLE

Lactobacillus sp in vagina produces an acidic environment which prevents against infection by other microbes

QUESTIONA research student obtained a set of data at set time intervals (every 30 minutes) from a culture growing in a fermenter. The number of viable cells in each sample was then counted using the pour plate method

TIME VIABLE COUNT RESULTS0 0.446 x 108

30 0.512 x 108

60 1.202 x 108

90 3.36 x 108

120 1.99 x 109

180 2.29 x 109

210 2.51 x 109

240 2.51 x 109

270 2.29 x 109

300 1.99 x 109

Experimental Growth Curve

7

7.5

8

8.5

9

9.5

10

0 30 60 90 120 180 210 240 270 300

Time (mins)

Log

10

Cel

l Num

bers

t x Log 2

Log b - Log a

t = 120 - 60 = 60 mins

Log b = 9.30

Log a = 8.08

Therefore 60 x 0.301

9.30 - 8.08

= 18.06

1.22

MGT = 14.80 minutes