MCB 100 chap 6

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 SO3

2-

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 SulfurPhosphorous 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 FactorsTrace 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

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 chemicalsthat 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.

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

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.

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

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:

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 SemmelweisC. 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.

AgarAgar 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 pHAlkalinophiles: Grow at a higher pH

Other Factors That Affect the Growth of Some Microbes

Capnophiles: Grow best when the concentration of carbon dioxide is elevatedHalophiles: 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 environmentMedium - the food that microorganisms are grown onBroth - liquid medium, there are many different recipesAgar - an algal extract used to solidify mediaColony - a visible pile of microorganisms on a solid mediumInoculate – 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, richSelective Media - Some species of microorganisms grow, but others don’tDifferential Media - Used to reveal a metabolic difference between speciesEnrichment Media - Used to enhance the growth of some species over others

MATHEMATICAL CONSIDERATIONS OF BACTERIAL GROWTHMethods 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 countA 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.

TurbidityTurbid 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 culturebroth 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 ActionThe 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 Table10 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 doubleExample: 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 GROWTHPlotting 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.