MCB100

Introductory Microbiology

2010

Microbial Nutrition

(Chapter 6 of Microbiology by Bauman)

Microbial Growth Requirements
7 Basic Nutrients
that all living organisms need
(besides water)
1. an energy source
2. carbon source
3. nitrogen
4. sulfur
5. phosphate
6. trace elements
7. essential
growth factors
(vitamins)
Oxygen
- All organisms require oxygen.
- But not every organism requires elemental oxygen.
(Which is also known as non-combined oxygen or O2.)
- Combined oxygen is oxygen that is chemically joined
to another element.
Examples of combined oxygen sources that
microorganisms can use include:
water H2O
carbon dioxide CO2 bicarbonate HCO3-
nitrate NO3- nitrite NO2-
sulfate SO42- sulfite SO32-
Anaerobic: means “without air”, the term refers
to living without elemental oxygen.
Why do living organisms need nitrogen?
Nitrogen is found in
amino acids, so proteins
cannot be made
without nitrogen.

Nitrogen is also an important

component of nucleic acids,
DNA and RNA cannot be
made without nitrogen.

Some modified sugars, such as those in the bacterial cell

wall material peptidoglycan, also contain nitrogen atoms.
Common Nitrogen Sources:
Organic Nitrogen from amino acids, nucleotides etc.
Inorganic Nitrogen: ammonia NH3, nitrate, nitrogen N2
Phosphorous
and Sulfur
Phosphorous is needed in the oxidized
state for nucleotides and phospholipids.

Some protein molecules have a phosphate group or two that are attached
after translation. The addition or removal of phosphate groups is a
common way of controlling the activity of enzymes.
-----------
Sulfur is found in a reduced state (-SH) in the amino acid cysteine and in
important vitamins that act as enzyme cofactors such as biotin, thiamine
and coenzyme-A.

Hydrogen sulfide is a gas. Most of the sulfur that is found dissolved in

water is in an oxidized state as either sulfate or sulfite anions.
Most organisms can reduce sulfate and sulfite to a form that can be
assimilated into organic molecules.
Trace Elements and Growth Factors
Trace Elements are inorganic elements needed in small amounts.
Important metals include: iron, copper, magnesium and zinc.
Ions: calcium, potassium, sodium and chloride are needed, but rarely limiting.

Growth factors are organic compounds including:

vitamins, amino acids, fatty acids etc.
Trace elements and growth factors are needed as components of
biological macromolecules or enzyme cofactors.
Some microorganisms require few or no exogenous growth factor.
Fastidious species require many specific growth factors in their diet.
Energy and Carbon
Autotrophic Organisms vs. Heterotrophic Organisms
An autotrophic organism is able to obtain the carbon needed for
growth from an inorganic source, usually carbon dioxide.
(auto-: self, -troph: feed or nourish)
A heterotrophic organism needs organic carbon to grow.
Organic compounds come from other living organisms.
(hetero-: other)

Plants are autotrophic, animals are heterotrophic

Phototrophic Organisms vs. Chemotrophic Organisms
A phototrophic organism can derive the energy needed to sustain
it's metabolism from light.
A chemotrophic organism requires a certain mix of chemicals
that allows it to conduct redox reactions that yield energy.
Phototrophic organisms cannot grow in the dark, while
chemotrophic organisms cannot grow unless they have
the proper mix of chemical compounds available.
The Two Types of Chemotrophic Organisms
Organotrophs vs. Lithotrophs
- Organotrophs derive energy from chemical reactions that
use organic compounds.
(Organotroph is a synonym for chemoheterotroph.)
- Lithotrophs derive energy from chemical reactions that do not
require carbon based compounds. (litho-: (G) stone or mineral)
Organotrophs acquire organic compounds that are required for
energy producing metabolism from the environment.
Most organic compounds found on Earth today were produced by
living organisms, but some organic compounds can be produced
by volcanoes and lightening in the right type of atmosphere.
The oxidation of sugar to form carbon dioxide and water is a form of
chemotrophic energy production that occurs in animals and many
bacteria.
Fungi and protozoa are organotrophs.
Many Bacteria, including those that are animal or plant pathogens
are organotrophs.
Lithotrophs derive energy from chemical reactions that do not
require carbon based compounds. (litho-: (G) stone or mineral)
Examples of Lithotrophic forms of metabolism include:
- the oxidation of ammonia to nitrate,
- the oxidation of hydrogen sulfide to sulfate and
- the oxidation of iron to iron oxide.
Lithotrophic organisms require some substance that is
relatively reduced and some oxidizing agent.

Photograph of cave passage showing

microbial mats growing in sulfidic stream
channel formed downstream of the Upper
Spring orifice in Lower Kane Cave, Wyoming.
Sulfide oxidizing bacteria produce acid that
contributes to cave formation.
source:
Annette Summers Engel, Natuschka Lee, Megan L . Porter,
Libby A . Stern, Philip C . Bennett, and Michael Wagner
Research Group for Microbial Geochemistry, Department of
Geological Sciences, University of Texas at Austin
 Pure Culture Techniques
A "culture" is a population of microbial cells in some environment. A
"pure culture" is a highly artificial (made by humans) condition where all
of the microorganisms in the culture are members of a single species or a
strain. This is in contrast to a "mixed culture" which contains two or
more species of microorganisms. A "contaminated culture" is a mixed
culture that contains one or more microorganisms that are undesirable
from a human perspective.

Who gets credit for developing many of the techniques that are used to
grow pure cultures of microorganisms in the laboratory?

A. Antony Van Leewoenhoek

B. Ignaz Semmelweis
C. Louis Pasteur
D. Robert Koch
E. Carl Woese
SOLID MEDIA
Why use solid media?
When bacteria grow in liquid culture they are free to move around and mix
with each other. On solid media the bacteria are unable to move around so
freely and thus an isolated cell will grow and reproduce for many
generations in one place.
This can produce a pile of bacteria that all descended from a single cell and
are thus all genetically identical.
A colony is a visible pile of bacteria that all descended from a single cell.
A well isolated colony of a microorganism is a small pure culture.

Agar
Agar is a complex carbohydrate that is
derived from the cell walls of certain
types of sea weed. It is indigestible
by animals and most microorganisms
and so by itself provides no nutritional
value to the medium.
 OXYGEN REQUIREMENTS

Tubes of a rich soft agar medium were inoculated with bacteria by stabbing
to the bottom of the tube with an inoculating needle.
Which one of the tubes would indicate that the bacterium is a microaerophile?
Which one is a facultative anaerobe?
Which one is an obligate aerobe?

A. B. C. D.

What is the difference between an aerotolerant organism and an aerobic organism?

 TEMPERATURE REQUIREMENTS
All bacteria have:
an optimum growth temperature, (fastest rate of growth)
a minimum growth temperature and (cold. = slow growth)
a maximum growth temperature. (too warm = slow growth)
Above their maximum growth temperature the bacteria may die.

Generation time: the time it takes for a bacterial population to double

Generation time = 1 / (growth rate)
TEMPERATURE REQUIREMENTS
Psychrophile: cold-loving, a bacterium that grows best
at less than 20o C.
Mesophile: loves the middle, a bacterium that grows best
at temperatures that humans consider comfortable,
generally about 25 – 45oC.
Thermophile: heat-loving, a bacterium that grows best
at temperatures above 60oC.
Snow algae The mammalian gut is a home Mammoth Hot Springs in
in a glacier for mesophilic bacteria. Yellowstone National Park
Thermoduric vs. Thermophilic Bacteria
A thermophilic bacterium grows at a high temperature.
A thermoduric bacterium grows at a moderate temperature,
but is not killed rapidly by high temperatures.

Example:
The endospores can survive exposure to high temperatures.
Boiling for 20 minutes will not
kill all of the anthrax spores.

However anthrax does not grow

at high temperatures.

It grows best at the temperature

of a mammalian body, about 40oC.
The Growth of Microbes is affected by pH
Most microorganisms grow best when the pH is near neutrality or
slightly acidic.
Acidophiles: Grow best at a low pH
Alkalinophiles: Grow at a higher pH
Other Factors That Affect the Growth of Some Microbes
Capnophiles: Grow best when the concentration of carbon dioxide is
elevated
Halophiles: Grow best at high concentrations of salt

The pink color of Owens Lake, California is

caused by a bloom of halophilic, photosynthetic
bacteria. The water is saturated with salt at a
temperature of about 60oC, and yet the lake is
teeming with life.
The white material around the lake is sodium
carbonate. (photo from NASA)
 WATER ACTIVITY OR OSMOLARITY
AND BACTERIAL GROWTH
One of the most important substances in cytoplasm is water.
If the concentration of dissolved salts, proteins etc. in a solution is
high, then the concentration of water molecules is lower.

For example, in distilled water there are 55.5 moles of water molecules
per liter but in a salty solution some of the space is occupied by salt
ions, so there is less water per liter. The concentration of water in a
solution is called the water activity.

Distilled water has a high water activity while salt water or cytoplasm
has a lower water activity.

There is a limit to how much stuff can be dissolved in a given volume of

water. Distilled water is a good solvent. A solution with a low water
activity is a poor solvent for additional solutes. If the water activity of
cytoplasm becomes too low, enzymes may become insoluble and form
precipitates.
Why is it possible to store
maple syrup at room
temperature without spoilage?
(Why is this an issue?)
Many bacteria can use sugar
as a food source.
Maple syrup is mostly water and sugar.

Maple syrup has a high concentration of sugar and

thus a low water activity.
Maple syrup is a hypertonic solution.
If a bacterial cell should accidentally find itself in a bottle of
maple syrup, water would diffuse out of the cell and into the
surrounding solution. This would cause the cytoplasm of the
cell to shrivel up.
If you dilute maple syrup with water and leave it at room temperature,
microorganisms will grow in it and cause it to spoil.
Culturing Microorganisms - Important Terms and Concepts

Culture - population of microorganisms growing in an environment

Medium - the food that microorganisms are grown on
Broth - liquid medium, there are many different recipes
Agar - an algal extract used to solidify media
Colony - a visible pile of microorganisms on a solid medium
Inoculate – deliberately introduce microorganisms into an
environment
Media for Culturing Microorganisms
Defined Media vs. Complex Media
- Defined Medium: The microbiologist knows the complete
chemical composition of the medium
ingredient examples:
glucose, ammonium nitrate, ammonium phosphate
sodium chloride, magnesium sulfate, trace elements
specific vitamins, amino acids, fatty acids, sugars, etc.

- Complex Medium: contains substances that have not been

completely chemically analyzed,
such as:
milk, blood, yeast extract, meat, eggs, plant extracts

Broth vs. Solid Media

- Broth = liquid media
- Solidifying agents: gelatin (protein that can be degraded by many bacteria)
starch or cellulose (potato slice or paper)
agar (a complex polysaccharide from seaweed)
 Media for Culturing Microorganisms

General Purpose Media - Used to grow a wide variety of microorganisms, rich

Selective Media - Some species of microorganisms grow, but others don’t
Differential Media - Used to reveal a metabolic difference between species
Enrichment Media - Used to enhance the growth of some species over others
MATHEMATICAL CONSIDERATIONS OF
BACTERIAL GROWTH
Methods of Enumerating Microorganisms (or Counting the Bacteria in a Sample)
Total Bacterial Cell Count VS. Viable Cell Count
The total count includes dead bacteria,
the viable count enumerates only those that can grow and reproduce.
Methods of Enumerating Microorganisms
- Direct microscopic count
A direct microscopic count is relatively fast but labor intensive.
Microscopy works best for things that are larger than bacteria, like blood cells.
Works best for samples with a concentration of between 10 -200 million cells per ml.

A dead cell may look the same as a living cell, especially for bacteria.
EXAMPLE: The Breed smear is a direct count technique that is used in the dairy
industry. A sample of a given size is smeared on a slide and examined. A count is
made of the concentration of bacteria and particulate matter such as dirt and straw.
This is used to quickly determine if a sample of milk from a farm is clean enough to
accept at the dairy.
See figure 6.23 on page 192 of your textbook.
Turbidity
Turbid means "cloudy".
Liquid broth culture that contains a lot of bacteria looks cloudy
whereas a sample of sterile broth medium looks clear.
There is a direct relationship between the concentration of bacteria in a
liquid culture and the absorbance or scattering of light as it passes
through the sample, one can use a spectrophotometer to measure
the number of bacteria in a liquid sample.

A tube of sterile The more bacteria there are in the culture

broth looks clear. the more cloudy the liquid appears.
See figure 6.25 on page 194 of your textbook.
- Viable Plate Count
A known volume of a liquid sample, usually 0.10 ml, is spread on the
surface of an agar plate that will allow the bacteria to grow.
After an appropriate incubation period, visible colonies of bacteria form.
One can estimate the number of bacteria that were in the sample
by counting the number of colonies that appear on the plate.
Concentration of Bacteria = _____(number of colonies)______
(amount plated) (dilution)

See figure 6.21 on page 188 of your textbook. (pg 190 in 1st ed.)
Counting Bacteria in a Sample

Membrane Filter method

Similar to the viable plate count except it is used for samples with a
low concentration of bacteria. A known volume of water is run
through a sterile filter. Microbes are trapped on the filter. The filter is
placed on the medium and allowed to incubate.

Concentration of Bacteria = (number of colonies)

(amount filtered)
Counting Bacteria in a Sample
Most Probable Number
MPN is a statistical way of estimating the viable number of
bacteria in a liquid sample by inoculating tubes of broth and
counting the number of tubes that show growth.
See figure 6.24 and Table 6.6 of the text book.

Fluorocult® Lauryl Sulfate Broth (EMD Chemicals)

Mode of Action
The lauryl sulfate largely inhibits the growth of undesirable
microbial flora. The presence of E. coli is indicated by
fluorescence under a long wavelength UV lamp. A positive
indole reaction and gas formation due to fermentation of
lactose confirm the results.
SCHINDLER (1991) recommended the use of this medium
in the quality control of bathing water.

Typical Composition (g/liter)

Tryptose 20.0; lactose 5.0; sodium chloride 5.0; sodium
lauryl sulfate 0.1; di-potassium hydrogen phosphate 2.75;
potassuim dihydrogen phosphate 2.75; L-tryptophan 1.0; 4-
methylumbelli- feryl-β-D-glucuronide 0.1.
MPN Test Results
- One coliform is enough to produce a positive result after incubation.
- A bubble in the Durham tube indicates the presence of coliform bacteria.
- The bubble is carbon dioxide produced by the fermentation of lactose.
- A higher concentration of coliforms in the sample gives more positive tubes.
 5 – Tube MPN Table
10 ml samples 1 ml samples 0.1 ml samples MPN (per 100 ml)
0 0 0 <1
1 0 0 2
1 1 0 4
2 0 0 5
2 1 0 7
3 0 0 8
3 1 0 11
3 2 0 14
3 2 1 17
4 0 0 13
4 1 0 17
4 2 0 22
5 0 0 23
5 1 0 33
5 1 1 49
5 2 0 46
5 2 1 70
5 3 0 79
5 3 1 110
5 4 0 130
5 4 1 170
5 4 2 220
5 5 0 240
5 5 1 350
5 5 2 540
5 5 3 920
5 5 4 1600
5 5 5 > 2400
Counting Bacteria in a Sample
Dry-weight method
- A liquid sample is run through a filter that has been previously
weighed.
- Microorganisms are trapped on the filter.
- The filter is baked dry to remove the water and reweighed.
- The increase in weight is proportional to the number of
microorganisms that are trapped in the filter.
- Advantage of the dry weight method is that some microorganisms
are difficult to count by other methods because they form clumps
of cells that are hard to separate.
- Disadvantage is that dirt and other particulate contaminants will
also be trapped by the filter.
MATHEMATICAL CONSIDERATIONS OF BACTERIAL
GROWTH

Bacterial growth is not so much about the growth of an individual,

but the growth of the bacterial population.
Bacteria reproduce by binary fusion.
When the conditions are good for growth the bacterial population
increases at an exponential rate.

Generation time = Time it takes for a microbial population to

double
Example: 30 minutes per generation

Growth rate = Generations per unit of time [1/(generation time)]

Example: 2 generations per hour
Bacterial Growth Over Ten Generations

generations (x): 0 1 2 3 4 5 6 7 8 9 10

number of bacteria (y): 1 2 4 8 16 32 64 128 256 512 1024

MATHEMATICAL CONSIDERATIONS OF BACTERIAL GROWTH
Plotting on semi-logarithmic graph paper allows you to draw a straight
line for an exponential function.
If you inoculate a tube of broth with 350,000 bacterial cells and
you incubate it until 8 generations have passed, how many cells
will be in the culture?

The formula for determining the number of bacteria in a growing

population is:

Nf = Ni (2n)

where:
Nf = the total number of bacteria,
Ni = the initial number of bacteria
n = the number of generations

For this problem: Nf = (3.5 x 105 ) x (28 )

After 8 generations (doublings), there will be about

8.96 x 107 cells in this culture.
 The 4 phases of a bacterial growth curve

Phase 1: Lag phase, No detectable growth of the population of bacteria

Phase 2: Log phase or Exponential phase, The population of bacteria is growing at a steady, fast, exponential rate.

Phase 3: Stationary phase, The population stops growing because the medium has run out of some essential nutrient
or is just too crowded. New cells are still forming, but the number of cells that form is equaled by the number of cells
that die.

Phase 4: Decline or Death phase. The population of cells declines because toxic waste products have accumulated or
essential nutrients are used up. Phase 4 may be difficult to detect by turbidity readings.