Copyright © 2013 Pearson Education, Inc.Lectures prepared by Christine L. Case

Chapter 6

Microbial Growth

© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

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Microbial Growth

Increase in number of cells, not cell size

Populations

Colonies

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The Requirements for Growth

Physical requirements

Temperature

pH

Osmotic pressure

Chemical requirements

Carbon

Nitrogen, sulfur, and phosphorous

Trace elements

Oxygen

Organic growth factor

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Physical Requirements

Temperature

Minimum growth temperature

Optimum growth temperature

Maximum growth temperature

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Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature.

PsychrophilesPsychrotrophs

Mesophiles

Thermophiles

Hyperthermophiles

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Affect enzyme reaction

Denature enzyme, carrier & protein

Disrupt membrane

1. psychrophile: 0-20°C, opt. 15°C

2. psychrotroph: 0-35°C, opt. 20-30°C

3. mesophile: (15-20°C)-45°C, opt. 20-45°C

most MO in this group

4. thermophile: bacteria,min 45°C, opt. 55-65°C

5.hyperthermophile: archaea, opt.80°C or greater

have much more heat-stable en. & protein

membrane lipid more saturated

Temperature vs. Microorganisms

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Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent.

Water is being emitted through the ocean floor at temperatures above 100°C.

Chemoautotrophic bacteria in

deep-sea water use chemical

energy from H2S as a source of

energy to fix CO2.

Hydrothermal Vent-

•Next frontier for new drugs

•Produce alternative fuels, H2

and butanol

•Provide heat-resistant

enzymes, eg. DNA polymerase

used for PCR

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Figure 6.2 Food preservation temperatures.

Temperatures in this range destroy most microbes,

although lower temperatures take more time.

Very slow bacterial growth.

Rapid growth of bacteria; some may produce toxins.

Many bacteria survive; some may grow.

Refrigerator temperatures; may allow slow growth

of spoilage bacteria, very few pathogens.

No significant growth below freezing.

Danger zone

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Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.

Refrigerator air

5 cm (2′′) deep

15 cm (6′′) deep

Approximate temperature

range at which Bacillus cereus

multiplies in rice

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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

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Osmotic Pressure

Hypertonic environments, or an increase in salt or

sugar, cause plasmolysis

Extreme or obligate halophiles require high

osmotic pressure

Facultative halophiles tolerate high osmotic

pressure

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pH

Most bacteria grow well in neutral pH (pH 6.5 - 7.5)

(neutrophile). Few are acidophiles, eg. One in the

drainage water from coal mines oxidezes sulfur to form

sulfuric acid and can survive at pH 1.0.

Molds and yeasts: pH 1-11, opt. pH 5- 6

Acidophiles: pH 1-5.5

Alkalophile: pH 8.5-11.5

The Requirements for Growth: Physical Requirements

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Figure 6.4 Plasmolysis.

Plasma

membraneCell wall

Cytoplasm

H2O

NaCl 10%

Cytoplasm

Plasma

membrane

Cell in isotonic solution. Plasmolyzed cell in hypertonic

solution.

NaCl 0.85%

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Chemical Requirements

Carbon

Structural organic molecules, energy source

Chemoheterotrophs use organic carbon sources

Autotrophs use CO2

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Chemical Requirements

Nitrogen

In amino acids and proteins

Most bacteria decompose proteins

Some bacteria use NH4+ or NO3

–

A few bacteria use N2 in nitrogen fixation

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Chemical Requirements

Sulfur

In amino acids, thiamine, and biotin

Most bacteria decompose proteins

Some bacteria use SO42– or H2S

Phosphorus

In DNA, RNA, ATP, and membranes

PO43– is a source of phosphorus

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Chemical Requirements

Trace elements

Inorganic elements required in small amounts

Usually as enzyme cofactors

Na, K, Mg, Ca, S, P, in mg/ml scale

Fe, Cu, Mo, Zn in ng/ml scale

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

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Singlet oxygen: 1O2− boosted to a higher-energy

state

Superoxide free radicals: O2

Peroxide anion: O22–

Hydroxyl radical (OH•)

Toxic Oxygen

Superoxide dismutaseO2 + O2 + 2 H+ H2O2 + O2

Catalase2 H2O2 2 H2O + O2

PeroxidaseH2O2 + 2 H+ 2 H2O

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Both obligate aerobe and facultative anaerobe： have SOD

and catalase

Obligate anaerobe: without SOD & catalase, O2 sensitive

Aerotolerant anaerobe: not use O2 for growth, tolerate O2,

have SOD or equivalent enzyme to neutralize O2 toxicity,

lactobacilli

Microaerophile: require less O2, sensitive to superoxide anion

and peroxide produced under O2-rich condition

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Organic Growth Factors

Organic compounds obtained from the environment

Vitamins, amino acids, purines, and pyrimidines

© 2013 Pearson Education, Inc. Figure 6.5

Biofilms

Microbial communities

Form slime or

hydrogels

Bacteria attracted by

chemicals via quorum

sensing

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Biofilms

Share nutrients

Sheltered from harmful factors

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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.

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Biofilms

Patients with indwelling catheters (留置導尿管)

received contaminated heparin

Bacterial numbers in contaminated heparin were too

low to cause infection

84–421 days after exposure, patients developed

infections

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Culture Media

Culture medium: nutrients prepared for microbial

growth

Sterile: no living microbes

Inoculum: introduction of microbes into medium

Culture: microbes growing in/on culture medium

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Agar

Complex polysaccharide

Used as solidifying agent for culture media in Petri

plates, slants, and deeps

Generally not metabolized by microbes

Liquefies at 100°C

Solidifies at ~40°C

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Culture Media

Chemically defined media: exact chemical

composition is known

Complex media: extracts and digests of yeasts,

meat, or plants

Nutrient broth

Nutrient agar

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

coli

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Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

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

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Anaerobic Culture Methods

Reducing media

Contain chemicals (thioglycollate or oxyrase) that combine

O2, oxyrase is a respiratory enzyme that can reduce O2 to

water

OxyPlate: plate with oxyrase, used in clinical lab.

Heated to drive off O2

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Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.

Lid with

O-ring gasket

Envelope containing

sodium bicarbonate

and sodium

borohydride

Anaerobic indicator

(methylene blue)

Petri plates

Clamp with

clamp screw

Palladium

catalyst pellets

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Figure 6.7 An anaerobic chamber.

Arm

ports

Air

lock

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Capnophiles

Microbes that require high CO2 conditions

CO2 packet

Candle jar

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Candle jar

Capnophiles require high CO2

Figure 6.7

•CO2-packet

Low O2, high CO25% O2, 10% CO2

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BSL-1: no special precautions

BSL-2: lab coat, gloves, eye protection

BSL-3: biosafety cabinets to prevent airborne

transmission

BSL-4: sealed, negative pressure Exhaust air is filtered twice

Biosafety Levels

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

© 2013 Pearson Education, Inc. Figure 6.10

Selective Media

Suppress unwanted

microbes and encourage

desired microbes

Eg. Bismuth sulfite agar for

Salmonella typhi, Bismuth

sulfite: inhibit G(+) and

most G(-) intestinal bact.

Brilliant green agar for G(-)

Salmonella

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Eosine methylene

blue (EMB) agar:

E. coli: black-center

colony, surrounded

by metallic green

sheen

Enterbacter aerogenes:

Dark-center colony

Differential media : Make it easy to distinguish

colonies of different microbes.

Figure 6.9b, c

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Figure 6.9 Blood agar, a differential medium containing red blood cells.

Bacterial

colonies

Hemolysis

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Enrichment Culture

Encourages growth of desired microbe

Assume a soil sample contains a few

phenol-degrading bacteria and thousands of

other bacteria

Inoculate phenol-containing culture medium with the soil,

and incubate

Transfer 1 ml to another flask of the phenol medium, and

incubate

Transfer 1 ml to another flask of the phenol medium, and

incubate

Only phenol-metabolizing bacteria will be growing

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Obtaining Pure Cultures

A pure culture contains only one species or strain

A colony is a population of cells arising from a

single cell or spore or from a group of attached cells

A colony is often called a colony-forming unit (CFU)

The streak plate method is used to isolate pure

cultures

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Figure 6.11 The streak plate method for isolating pure bacterial cultures.

Colonies

1

2

3

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Preserving Bacterial Cultures

Deep-freezing: –50° to –95°C

Lyophilization (freeze-drying): frozen

(–54° to –72°C) and dehydrated in a vacuum

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Binary fission

Budding

Conidiospores (actinomycetes)

Fragmentation of filaments

ANIMATION Bacterial Growth: Overview

Reproduction in Prokaryotes

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Figure 6.12a Binary fission in bacteria.

Cell elongates and

DNA is replicated.

Cell wall and plasma

membrane begin to constrict.

Cross-wall forms,

completely separating

the two DNA copies.

Cells separate.

Cell wall

Plasma

membrane

DNA

(nucleoid)

(a) A diagram of the sequence of cell division

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Figure 6.12b Binary fission in bacteria.

(b) A thin section of a cell of Bacillus licheniformis

starting to divide

Cell wallDNA (nucleoid)

Partially formed cross-wall

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Figure 6.13a Cell division.

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Figure 6.13b Cell division.

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If 100 cells growing for 5 hours produced

1,720,320 cells:

ANIMATION Binary Fission

Generation Time

Number of generations =Log number of cells (end) − Log number of cells (beginning)

0.301

Generation time =60 min × hours

Number of generations= 21 minutes/generation

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Nt = No x 2n

Log Nt = Log No +n Log 2

n Log 2 = Log Nt – Log No

n =(Log Nt-Log No)/Log 2

n= number of generation =incubation time(t) /generation time (tg)

No : initial number

Nt : final number after t time incubation

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The Growth Curve

observed when microorganisms are

cultivated in batch culture

culture incubated in a closed vessel with

a single batch of medium

usually plotted as logarithm of cell

number versus time

usually has four distinct phases

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Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line)

and arithmetically (solid line).

Log10 = 1.51

Log10 = 3.01

Log10 = 4.52

Log10 = 6.02

(1,048,576)

Generations

Lo

g1

0 o

f n

um

be

r o

f cell

s

Nu

mb

er

of

cell

s

(32) (1024)

(32,768)

(65,536)

(131,072)

(262,144)

(524,288)

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Lag PhaseIntense activity

preparing for

population growth,

but no increase in

population.

Log PhaseLogarithmic, or

exponential,

increase in

population.

Stationary PhasePeriod of equilibrium;

microbial deaths

balance production of

new cells.

Death PhasePopulation Is

decreasing at a

logarithmic rate.

The logarithmic growth

in the log phase is due to

reproduction by binary

fission (bacteria) or

mitosis (yeast).

Figure 6.15 Understanding the Bacterial Growth Curve.

Staphylococcus spp.

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Bacterial growth phase

Lag phase: little or on cell division

prepare to synthesize enzymes and molecules for adaptation to a new environment

Log phase: cell number increases logarithmically

cells are most active metabolically, very sensitive to adverse conditions

Stationary phase: cell growth and cell death balance the total number

exhaustion of nutrients, accumulation of waste, harmful pH

Death phase: numbers of death exceeds the number of new formed

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Lag Phase

cell synthesizing new components

varies in length

in some cases can be very short or even

absent

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Exponential Phase

also called log phase

rate of growth is constant

population is most uniform in terms of chemical and

physical properties during this phase

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Effect of nutrient concentration on growth

Figure 6.7

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Stationary Phase

total number of viable cells remains constant

may occur because metabolically active cells

stop reproducing

may occur because reproductive rate is

balanced by death rate

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Possible reasons for entry into stationary phase

nutrient limitation

limited oxygen availability

toxic waste accumulation

critical population density reached

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Death Phase

two alternative hypotheses

Cells are Viable But Not Culturable (VBNC)

Cells alive, but dormant

programmed cell death

Fraction of the population genetically programmed to die

(commit suicide)

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Measurement of Cell Numbers

direct cell counts

counting chambers

electronic counters

on membrane filters

viable cell counts

plating methods

membrane filtration methods

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Plating methods…

simple and sensitive

widely used for viable counts of microorganisms in food, water, and soil

inaccurate results obtained if cells clump together

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Figure 6.16 Serial dilutions and plate counts.

Original

inoculum

1 ml 1 ml 1 ml 1 ml 1 ml

9 m broth

in each tube

Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000

1 ml 1 ml 1 ml 1 ml 1 ml

1:10 1:100 1:1000 1:10,000 1:100,000(10-1) (10-2) (10-3) (10-4) (10-5)

Plating

Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml

(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)

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After incubation, count colonies on plates that have

25–250 colonies (CFUs)

Plate Counts

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Figure 6.17 Methods of preparing plates for plate counts.

The pour plate method The spread plate method

Inoculate

empty plate.

Add melted

nutrient

agar.

Swirl to

mix.

Colonies

grow on

and in

solidified

medium.

1.0 or 0.1 ml 0.1 ml

Bacterial

dilution

Inoculate plate

containing

solid medium.

Spread inoculum

over surface

evenly.

Colonies grow

only on surface

of medium.

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Another Viable Count Method - Membrane filtration

especially useful for analyzing aquatic samples

Figure 6.13

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Figure 6.18 Counting bacteria by filtration.

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Figure 6.14

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Multiple tube MPN test

Count positive tubes

Most Probable Number

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Figure 6.19a The most probable number (MPN) method.

Volume of

Inoculum for

Each Set of

Five Tubes

(a) Most probable number (MPN) dilution series.

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Compare with a statistical table

Most Probable Number

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Figure 6.19b The most probable number (MPN) method.

(b) MPN table.

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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 here and fills the

shallow volume over the squares by capillary

action.

Bacterial

suspension

Cover glass

Slide

Cross section of a cell counter.

The depth under the cover glass and the area

of the squares are known, so the volume of the

bacterial suspension over the squares can be

calculated (depth × area).

Microscopic count: All cells in

several large squares are

counted, and the numbers are

averaged. The large square shown

here has 14 bacterial cells.

The volume of fluid over the

large square is 1/1,250,000

of a milliliter. If it contains 14 cells,

as shown here, then

there are 14 × 1,250,000 =

17,500,000 cells in a milliliter.

Location of squares

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Direct Microscopic Count

Number of bacteria/ml =Number of cells counted

Volume of area counted

14

8 × 10−7= 17,500,000

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Figure 6.21 Turbidity estimation of bacterial numbers.

Light source

Light

Blank

Spectrophotometer

Light-sensitive

detectorScattered light

that does not

reach detector

Bacterial suspension

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Measuring Microbial Growth

Direct Methods

Plate counts

Filtration

MPN

Direct microscopic count

Indirect Methods

Turbidity

Metabolic activity

Dry weight