Chapter 6: Microbial Growth - Los Angeles Mission College Chapter 6.pdf · Chapter 6: Microbial...

Chapter 6: Microbial Growth

1. Requirements for Growth

2. Culturing Microorganisms

3. Patterns of Microbial Growth

1. Requirements for Growth

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)

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.

Temperature is the easiest wayto control Microbial Growth

If you want microbial growth (e.g., in the lab), then you incubate them at the optimal temperature.

If you don’t (e.g., food),you can incubate attemperatures abovethe range of tolerance(cook!), and/or store at temperatures belowthe range of tolerance(freeze) or close to it(refrigerate) .

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?• O2 is a very reactive molecule which can result in

the formation of very destructive free radicals(molecules or atoms with an unpaired electron)

e.g. superoxide (O2-), peroxide anion (O2

2-)

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

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

Oxygen & Microbial Growth

• in thioglycollate medium (binds O2), the top is O2-rich, the middle O2-poor, & the bottom lacks O2

• 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!*

2. Culturing Microorganisms

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

Complex

• used for assessing metabolic characteristics

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

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 beharmed by higher temp.

1 2

**Each colony starts with 1 cell!**

How to Obtain a Pure Culture

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

• streak as shown to obtain isolated colonies

3. Patterns of Microbial Growth

Bacterial Cell Division

Most bacteria divide by binary fission ( a few by budding)

Microbial Growth is Exponential Increase in numbers by the same factor (i.e., x 2)each generation

• also referred to as logarithmic growth

• differs from arithmetic growth, increase by the sameamount each generation (e.g., + 2)

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

How Fast do Microorganisms Grow?The 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

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

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

Counting by Serial Dilution

Dilute culture sample in series & plate each dilution:ea colony starts w/1 cell

• (# of colonies x dilution factor) / sample vol. = cell density

*

• count a plate with a “practical” # of colonies (~30-300*)

resulttakes ~24 hr

Counting by Filtration Specific volume of a test solution (e.g., a water sample)is filtered to trap microbes

• e.g., 200 colonies from100 ml of filtered test sample= 200 cells/100 ml or 2/ml

Grow filter on agar plate,# of colonies = # of microbes in test sample

**Not all cells can grow on culture medium!

Direct Microscopic Counts

• gives immediate results!

• known volume covering each grid or square is used to calculate cell density

• place sample on “counting chamber” slide

Cell Density via SpectrophotometryOne of the quickest, most convenient methods isto measure the optical density (OD) of a culture.

• a spectrophotometer is used to measure how much lightis “absorbed” by aliquid culture sample

• more light blockedor “absorbed” =greater cell density(i.e., turbidity)

• the absorbance orOD value is used to calculate cell density (e.g., 1 ODunit = 108 cells/ml)Less precise, but gives immediate results!

Key Terms for Chapter 6

• superoxide & peroxide ions

• defined, complex, selective & differential media

• superoxide dismutase, catalase, peroxidase

• logarithmic (exponential) growth, generation time

• psychrophile, psychrotroph, mesophile, thermophile & hyperthermophile

• Lag, Log, Stationary and Death phases

Relevant Chapter Questions rvw: 1-6, 9-13 MC: 1-8

• serial dilution, spectrophotometry