Micro.: Ch. 4-Microbial Growth and its control

Nutrients

Supply of monomers (precursor molecules) required by cells for growth.

What are two types of nutrients for microbes?

Macronutrients and Micronutrients

Macronutrients

Nutrients required in large amounts.

Micronutrients

Nutrients required in minute amounts.

What are types of micronutrients?

Trace metals and growth factors

Dry weight

-Weight of elemental compounds with the microbe.

What cells need carbon for nutrients?

-ALL cells

How much of the elemental composition of a typical bacterial cell is carbon?

about 50% (dry weight)

What is carbon used for?

-Major element in ALL classes of macromolecules.

Most microorganisms are _______.

Heterotrophs

What are heterotrophs?

Use organic carbon

What are autotrophs?

-Self feeders that use inorganic carbon.

-Mainly CO2

How much of the elemental composition of a typical bacterial cell is nitrogen?

Typical bacterial cell is 13% (dry weight)

What is nitrogen used for?

Key element in proteins, nucleic acids, any many more cell constituents.

What microbes use nitrogen?

Nearly all microorganisms can use NH3 as a nitrogen source.

How is the majority of N2 broken down?

Nitrogen fixing bacteria and archaea.

What other nitrogen source do some microbes use other than N2?

-Some microbes use NO3-

-Only certain Bacteria and Archaea can use N2

What are other macronutrients?

-Phosphorous

-Sulfur

-Potassium

-Magnesium

-Calcium

-Sodium

What is phosphorous needed for?

Required for nucleotides (including ATP) and phospholipids.

What is sulfur required for in cells?

Required for sulfur-containing amino acid and iron-sulfur proteins.

What are some sulfur-containing amino acids?

Cysteine and methionine

What is sulfur present in?

Present in some vitamins (ex: thiamine, biotin, lipoic acid) and coenzyme A.

What is potassium required for in the cell?

Required by some enzyme for activity.

How does magnesium get used in the cell?

Stabilizes ribosomes, membranes, and nucleic acids.

Why is magnesium required for the cell?

Required for many enzymes and pigments.

How does calcium get used in the cell?

-Helps stabilize cell walls in microorganisms.

-Plays key role in heat stability of endospores.

Where are microorganism located that use sodium?

Marine or other saline environments.

What microorganisms need sodium?

Required by some microorganisms.

What are micronutrients?

Iron

Why is iron needed in the cell?

Key component of cytochromes and FeS proteins that facilitate electron transport.

What form is iron under anxoic conditions?

Iron is generally in ferrous form (Fe²⁺) and soluble.

What form is iron in oxic conditions?

Iron is usually in ferric (Fe³⁺) and exist in insoluble mineral.

Siderophores

Iron binding agents that are used to obtain iron from insoluble minerals.

When are siderophores used?

Oxic conditions

Growth factors

Organic compounds required in small amounts by certain organisms.

What are examples of growth factors?

Vitamins, amino acids, purines, and pyrimidines.

What is the most required growth factors?

Vitamins

Vitamins

Most function as coenzymes

Culture media

Nutrient solutions used to grow microbes in the laboratory.

How are culture media sterilized?

Typically sterilized in an autoclave.

What are two classes of culture media?

Defined media and complex media

Defined media

Exact chemical composition known.

Complex media

Composed of digests of microbial, animal, or plant products.

What are examples of plant products used in a complex media?

Yeast and meat extracts

How can you count microbial cells?

Enumerated by direct microscopic observations using a Petroff-Hausser cell counter.

How does the Petroff-Hausser cell counter work?

1. Sample is added to a platform with ridges. About 0.02 mm space between coverslip and slide.

2. All cells are counted in large squares (16 small squares)

How do you calculate the number of cells?

#of cells x 25 large squares x 50 × 10³

What are the limitation of a microscopic cell counts?

-Can’t distinguish between live and dead cells without special stains.

-Small cells can be overlooked.

-Phase-contrast microscope required if a stain is not used

-Cell suspensions of low density (<10^6 cells/mL) are statistically unreliable.

-Motile cells need to be immobilized

-Debris in sample can be mistaken for cells

Viable cell counts (plate counts)

Measure living, reproducing cells in a population; two main methods.

What are two methods for viable cell counts?

-Spread-plate method

-Pour-plate method

Spread plate method

1. Sample is pipetted onto surface of agar plate (0.1 mL or less)

2. Sample is spread evenly over surface of agar using sterile glass spreader.

3. After incubation: typical spread-plate results are surface. colonies.

What is the result of spread-plate method?

Surface colonies

Pour-Plate method

1. Sample is pipetted into sterile plate.

2. Sterile medium is added and mixed well with inoculum.

3. After solidification and incubation

What are the typical pour-plate results?

Surface and subsurface colonies

In order to obtain a countable colony, what should you do?

The sample should always be diluted.

What is a countable number of colonies?

30-300 colonies per plate

Turbid means _____.

Cell suspension is this because the cells scatter light.

_______ cells=_______ turbidity=_______scattered light

More, more, more

Turbidity Measurements

An indirect and rapid method of measuring microbial growth.

How is turbidity measured?

Spectrophotometer

What is the measurement for a spectrophotometer?

Optical density (OD) at a specified wavelength

How to extrapolate a cell count from a turbidity value?

A standard curve must be established.

When using a spectrophotometer, what wavelength should we not use?

Don’t use a color wavelength that is absorbed.

When there are more cells in a solution, what is the result with the spectrophotometer?

-Less light hits the detector

-If the light hits a cell, doesn’t hit the photocell.

When should you do distill count and spectrophotometer?

-Count the cells with distilled first and then get optimal density with the spectrophotometer.

How many times do you have to do the distilled counting?

once

What are the advantages of turbidity measurements?

-Quick and easy to perform

-Typically, do not require destruction or significant disturbance of sample.

-The same sample can be checked repeatedly.

What are the disadvantages to turbidity measurements?

-Microorganisms that form clumps or biofilms in liquid medium

-Microorganisms that produce pigments.

Growth

Increase in the number of cells, not size of cell.

Binary Fission

Cell division following enlargement of a cell to twice its minimum size.

What does binary fission coincide with?

DNA replication

Generation time

Time required for microbial cells to double in number.

What does generation time depend on?

-Nutritional, physical (temperature, pH, osmolarity), and genetic factors.

Does generation time vary among species of different and individual species?

Yes

What are the phases for a typical growth curve for a population?

1. Lag phase

2. Exponential phase

3. Stationary phase

4. Death phase

Lag Phase

Interval between when a culture is inoculated and when growth begins.

Exponential phase

Cells in this phase are typically in the healthiest state and are growing most rapidly.

Stationary phase

-Growth rate of a population is zero

Death Phase

If incubation continues after cells reach stationary phase, the cells will eventually lose viability.

Why would a stationary phase happen?

-Decrease in space and nutrients

-Increase in waste

Exponential growth

Growth of microbial population in which cell numbers double within a specific time interval.

What is an example of exponential growth?

Generation time

How does an exponential graph look like?

Initially slow, but population soon increases dramatically.

What is a major environmental factor when controlling microbial growth?

Temperature

Cardinal Temperature

The minimum, optimum, and maximum temperatures at which an organism grows.

What happens at a bacteria’s minimum temperature?

Membrane gelling: transport processes so slow that growth cannot occur.

What happens at a bacteria’s maximum temperature?

Protein denaturation; collapse of the cytoplasmic membrane; thermal lysis.

What happens at a bacteria’s optimum temperature?

Enzymatic reactions occurring at maximal possible rate.

What happens to a bacterium between minimum and optimum temperature?

Enzymatic reactions occurring at increasingly rapid rates.

What organisms have cardinal temperatures?

ALL organisms.

What is the optimal temperature for pathogens?

37 Celsius: human temperature

What are some classifications for microorganisms based on their growth temperature?

-Psychrophile, mesophile, thermophile, hyperthermophile, psychrophile,

Psychrophile

Low temperature; found in permanently cold environments.

Mesophile

Midranges temperature; found in temperate environments and in warm-blooded animals.

What temperature class are human pathogens?

Mesophiles

Thermophile

High temperature; found in permanently hot environments.

Hyperthermophile

Very high temperature; found in extremely hot habitats, such as boiling hot springs and deep-sea hydrothermal vents.

What does the suffix “phile” mean?

love something

Are mesophiles also extremophiles?

No, since they are around normal temp.

What is the optimal temperature for psychrophiles?

4 celcius