Chapter 6:Microbial Growth

1. Requirements for Growth

2. Culturing Microorganisms

3. Patterns of Microbial Growth

4. Measuring Microbial Growth

1. Requirements for GrowthChapter Reading – pp. 166-174

Factors that affect Microbial GrowthMicrobial growth depends on physical factors…

• pH• osmotic pressure

…and chemical factors

• availability of a useable carbon source

• temperature

• useable sources of nitrogen, sulfur & phosphorus

• availability of trace elemental nutrients (Fe, Mg…)

• presence (or absence) of oxygen gas (O2)

Psychrophiles

Mesophiles

Gro

wth

rate

Thermophiles

Hyperthermophiles

120Temperature (°C)

1101009070 806050403020-10 0 10

Temperature & Microbial Growth

Microorganisms can be grouped based on the temperature range in which they can grow…• each has an optimal temp. & minimum, maximum growth temps.

Osmotic PressurepH• most microorganismsgrow best at pH levelsnear neutral (6.5-7.5)

• few microorganismsgrow at the moreextreme pH levels(below 4.0, above 10.0)

• microbial growth tendsto acidify the growthmedium, inhibitingfurther growth

Hypertonic solutions can draw water out of cells via osmosis:• causes membrane to detachfrom cell wall (plasmolysis)• caused by high salt, sugar…• inhibits bacterial growth

Oxygen (O2)As we’ve learned, oxygen can promote growth(via respiration in aerobes) or inhibit growth(of obligate anaerobes).Why is oxygen so toxic to some organisms?

superoxide radical (most toxic!): O2-

• aerobic organisms, unlike obligate anaerobes, have enzymes to eliminate these dangerous radicals

e.g. superoxide dismutase (SOD), catalase, peroxidase

peroxide anion: O22-

hydroxyl radical: OH

singlet oxygen: O2 (“energized” O2)

• O2 can undergo changes resulting in the formation of very toxic reactive oxygen species (ROS):

High

Oxygenconcentration Loose-

fitting cap

Obligateaerobes

LowObligate

anaerobesFacultativeanaerobes

Aerotolerantanaerobes

Oxygen & Microbial Growth

• thioglycollate medium produces an O2 gradient• a given bacterial species will grow only in the

regions it can tolerate (e.g., anaerobes at bottom)

Chemical Factors for GrowthSource of Carbon• autotrophs simply need access to CO2 to grow• heterotrophs require an organic carbon source

• proteins, carbohydrates, lipids

**The carbon source a given organism can use dependsdepends on its metabolic abilities (i.e., its enzymes!)**

Trace Elemental Nutrients• all organisms need trace (small) amounts of

many so-called “mineral” elements:iron (Fe), zinc (Zn), magnesium (Mg), calcium (Ca)…

• most are essential cofactors for various enzymes

Nitrogen, Sulfur & Phosphorus• all organisms need access to nitrogen, sulfur &

phosphorus to make proteins, nucleic acids, vitamins

• some organisms require organic sources of theseelements, others are more flexible:

• e.g., nitrogen fixers are unique in being able toobtain nitrogen from the atmosphere (N2), mostother organisms need Nitrogen in other forms

*One can effectively promote or inhibit the growthof a microorganism of interest (or concern) bycontrolling its physical & chemical environment!*

BiofilmsIt is estimated that the majority of bacteria in nature live in biofilms, and that most bacterial diseasesare due to bacteria in a biofilm.

• a gelatinous extracellular matrix (ECM) consistingprimarily of polysaccharides in the glycocalyces of the bacteria in the biofilm

SO WHAT’S A BIOFILM?

• forms on hard surfaces (rocks, teeth, prosthetics…)

• multiple bacterial species live synergistically in a biofilm

• when sufficient bacterial numbers are present, a signaling process called quorum sensing induces biofilm

**bacteria in biofilm are MUCH harder to get rid of than isolated bacteria**

Free-swimming microbes are vulnerable to environmentalstresses.

Bacteria

Some microbes land on a surface, such as a tooth, and attach.

The cells begin producing an extracellular matrix and secrete quorum-sensing molecules.

Quorum sensing triggers cells to change their biochemistry and shape.

New cells arrive, possibly including new species, and water channels form in the biofilm.

Some microbes escape from the biofilm to resume a free-living existence and, perhaps, to form a new biofilm on another surface.

Chemical structure of one type of quorum-sensing molecule

Matrix

Water flow

Water channelEscapingmicrobes

1

2 3 4 5 6

Biofilm Formation

2. Culturing MicroorganismsChapter Reading – pp. 174-182

Culture MediumThe culturing of microorganisms requires anappropriate growth medium:

• material containing all nutrients required forthe desired organism to grow

• can be liquid or solid (i.e., solid agar)

• media can be sterilized by heat or by filtration

• must initially be sterile (i.e., no live organisms)

• growth should only occur following inoculationof the medium with the desired organism

Defined vs Complex Medium

Defined medium hasa precisely known chemical composition• used for assessing metabolic characteristics

Complex medium is rich innutrients though chemical composition is not known• used to sustain rapid growth

Selective & Differential MediaSelective media promote the growth of desiredorganism(s), suppress growth of others:

• include something in the growth medium thatdesired organism can tolerate, most otherorganisms cannot (e.g., antibiotic, low pH, high salt)

• use defined media that sustain growth of desiredorganism, not others (e.g., lactose as carbon source)

On differential media, microorganisms can be distinguished based on appearance

• e.g., contain substances that change color due topH change, production of particular by-product

Selective mediumA B

C D

• compare A(non-selective) with B (selective)

Differential medium

• C illustrates differential growth

• D is differential& selective

Culturing Obligate Anaerobes

• special chambers are used to remove and exclude any oxygen (O2) that would otherwise kill such organisms

How to Obtain a Pure Culture

• inoculate an isolated colony (derived from a single original cell) into liquid medium to obtain a pure culture

“quadrant streak” or “streak plate” will yield isolated colonies if done correctly

Plating Bacteria 2 basic methods:

2) spread small volumeof culture (0.1 ml) on solid agar surface • best method!

1) mix 1 ml of culturewith molten agar (not too hot, ~45-50o C.)& pour in plate

• colonies grow INas well as ON agar • some cells may be

harmed by higher temp.

1 2

**Each colony starts with 1 CFU!**

3. Patterns of Microbial GrowthChapter Reading – pp. 182-185

Bacterial Growth • most bacteria divide by binary fission ( a few by budding)

• increase in cell numbers isexponential

1 bacterium can become 1 billion in just 30 generations!!!

1

70

Num

ber o

f cel

ls

Species A

Time (hours)

60

50

40

30

20

10

20 1

70

Num

ber o

f cel

ls

Species B

Time (hours)

60

50

40

30

20

10

20

Arithmetic vs Exponential Growtharithmetic exponential

• real livingorganismsreproduceexponentially

Rates of Microbial GrowthThe rate of microbial growth depends on thegeneration time:• the time for a microbial cell to divide• depends on the type of microorganism• also depends on the growth medium

• a practical measure is the the time it takes a microbial population to double in size (doubling time)

• i.e., when every cell divides once!

***can be as short as 20 minutes (E. coli) or >24 hr***

Microbial Growth Patterns Microorganisms cannot undergo unlimited growth, eventually the chemical and physicalenvironment in which they’re growing will nolonger be able to sustain such numbers:

• sources of carbon, nitrogen, etc, get used up

• waste products accumulate, pH may change

Therefore, microbial growth tends to follow a characteristic pattern:

Lag phase > Log phase > Stationary phase > Death phase

Phases of Microbial Growth

Lag phase: cells adjust to medium before dividingLog phase: exponential growth (unrestricted)Stationary phase: growth = death (wastes, lack of nutrients)Death phase: poor environment results in death > growth

log phase growth is “linear”(straight line) on a logarithmic plot

4. Measuring Microbial GrowthChapter Reading – pp. 185-190

How to Measure Microbial Growth?There are a number of methods used tocount microorganisms and thus determinethe growth rate.

The method used depends on several things:

• the organism being analyzed

• how quickly one needs the result

• the degree of accuracy needed

• the nature of the sample being tested

1 ml originalculture

Too numerous to count(TNTC)

9 ml broth +1 ml original

culture

1.0 ml

1:10dilution

1.0 ml

1:1000dilution

1.0 ml

1:10,000dilution

1.0 ml

1:100,000dilution

1:100dilution

0.1 ml of eachtransferred to

a plate

0.1 ml

0 colonies

0.1 ml 0.1 ml0.1 ml

Incubationperiod

6 colonies65 coloniesTNTC

Counting by Serial Dilution

resulttakes ~24 hr

***ea colony starts w/1 CFU**

Counting by Filtration

Direct Microscopic Counts

• place sample of culture “counting chamber” slide

• count cells within grid and calculate the cell density

• the volume covering each grid or square is known so the number of cells per unitvolume is easily determined

• gives immediate and relatively accurate results!

SpectrophotometryOne of the quickest, most convenient methods to determine cell density iswith a spectrophotometer.

• measures how much light is transmitted through a liquid culture sample

• more light blocked = greater cell density (i.e., turbidity)

• % transmittance can be usedto calculate cell density

**Less precise, but gives immediate results!**

Key Terms for Chapter 6

• defined, complex, selective & differential media

• superoxide dismutase, catalase, peroxidase

• binary fission, exponential growth, generation time

• psychrophile, mesophile, thermophile, hyperthermophile

• Lag, Log, Stationary and Death phases

Relevant Chapter Questions MC: 1-5, 7, 9-12, 15 FB: 1-7, 9-11

• serial dilution, spectrophotometry

• biofilm, extracellular matrix, quorum sensing