ch 4-6 microbio

microbial growth

an increase in the number of cells in a population

binary fission

one cell divides into two identical cells. This is an exponential process

1    - 2    -   4   -     8   -     16

streak plate

• —A sterilized inoculating loop is dipped in solution containing organisms.

• —The loop is passed across 1/3 of the plate in parallel streaks.

• —The loop is then sterilized (with fire).

• —The loop is passed across the next 1/3 of the plate, touching the first 1/3 of the plate only at the start.

• —The loop is sterilized again.

• —The loop is passed across the last 1/3 of the plate, again only touching the 2^nd 1/3 of the plate at the start.

advantages of agar

• —Not degraded by most bacteria

• —Not destroyed at high temperatures (can be sterilized)

• —Solid up to 95°C

Lag Phase

when cells are added to a media they must begin synthesizing macromolecules and obtaining nutrients to divide. No growth in this phase

Log Phase or exponential phase

—cells divide at a constant rate, which is exponential. This is when the generation time is calculated.

Stationary Phase

—as nutrients diminish, the rate of growth decreases to the point that it equals the rate of death.

birth=deathrate

Death Phase

—nutrients are depleted to a point that the constant rate of cell death is greater than that of cell growth.

cell death > growth

Prolonged Decline

the final 1% of cells will continue to decline in number at a slower rate, lasting days to years. The “fittest” cells survive the longest.

closed system

nutrients are not renewed and waste is not removed the organisms display a predictable growth pattern

agar is an example of this

open system or continuous culture.

Cells kept in a state of continuous growth if nutrients are continuously added and waste products are removed.

Growth on agar plate

Cells at the outer most part of the colony are most likely to be found in the log phase

Those in the very center are most likely in the death phase.

In between are cells in the stationary phase.

Psychrophile

—optimum temperature between -5 – 15°C

Mesophile

—optimum temperature between 25 – 45°C

infect human and cause disease

Thermophile

—optimum temperature between 45 – 70 °C

Hyperthermophile

optimum temperature 70°C or greater

mostly archaea

proteins resistant to denaturing from heat

Temperature

Mycobacterium leprae and Treponema pallidum(syphilis) prefer cooler regions of body ex feet, hands, fingers

Immune system triggers fever to kill or reduce bacterial growth

—Obligate anaerobes

—Cannot multiply in the presence of oxygen, or are killed by O₂ toxic derivatives (superoxide and hydrogen peroxide).

lack the enzymes superoxide dismutase (needed to convert superoxide to hydrogen peroxide) and catalase (needed to convert hydrogen peroxide to water and O_2).

_{C botulinum and C tetani}

—Aerotolerant Anaerobes

Able to grow in the presence of oxygen, but do not use it to produce energy

ex —Streptococcus pyogenes

—Facultative Anaerobes

—Do not require O₂ to grow

However, if oxygen is present these organisms switch to aerobic respiration and grow FASTER

ex e coli

—Microaerophiles

—Require small amounts of O₂ in order to grow

—Growth is inhibited by higher concentrations of oxygen

—Example: Helicobacter pylori

—Obligate Aerobes

—Have an absolute requirement for O₂ in order to grow

pH

Most bacteria live and grow in a pH range of 5-8, and grow best at a pH at 7 (neutrophiles)

Acidophiles

prefer pH below 5.5

Alkalophiles

—prefer pH above 8.5

neutrophiles

prefer pH of 7

Helicobacter pylori is an ex

neutralizes stomach acid in its vicinity by releasing the enzyme urease

halotolerant

bacteria able to resist loss of water and grow in high salt environments

ex Staphylococcus aureus

Autotrophs

use inorganic carbon (CO₂) to produce organic compounds through carbon fixation

Heterotrophs

use organic molecules as their carbon source

phototroph

Organisms that harvest energy from sunlight are

chemotrophs

organisms that obtain energy by breakdown(oxidizing) chemical compounds are

• can be organic(organo)

• inorganic(litho)

—chemo organotrophs

breakdown (oxidize) organic chemical compounds

chemolithotrophs

breakdown(oxidizing) of chemical compounds that are inorganic

Complex media

• —nutrient-rich growth mediums with an unknown, variable chemical composition

• contains a variety of ingredients

• These generally contain meat extract or other cells (example: Blood agar)

Chemically defined media

a growth broth/agar with a precisely known composition, using pure, identified chemicals (salts, sugars, amino acids, vitamins) for exact nutrient control

• These are used to study the nutritional requirements of bacteria.

• exact composition

Selective Media

inhibit the growth of organisms other than the one being sought

Differential Media

contain a substance that certain bacteria change in a recognizable way, changes color

Example: Blood agar contains RBCs that are lysed by Streptococcus pyogenes to produce clear zones on the red plate.

Direct Cell Counts

Total number of cells, living AND dead

Direct Microscopic Count

using a microscope to count cells

Coulter Counter

using electrical current to count cells

Flow Cytometer

using light to count cells

Viable Cell Counts

Total number of cells that are able to grow (living cells)

Plate Count

Small number of cells are plated on agar, and the number of colonies is counted (each colony represents one cell from original sample)

Membrane Filtration

Cells are collected with a membrane filter from dilute water. The cells are then plated and colonies are counted (similar to plate count).

Most Probable Number

Statistical estimate of cell number. Not precise.

Turbidity (cloudiness)

The amount of scattered light in a broth is measured using a spectrophotometer.

As the number of cells increases, the amount of light that passes through the broth decreases.

This requires a very large number of cells to make a broth turbid.

total weight

This is tedious and time consuming, but is most effective for measuring growth in organisms that do not readily separate into individual cells (filamentous organisms)

Measuring Biomass

counting the mass or size of the population in general

Keep in mind that if the mass of a sample doubles, then the number of the cells doubled

Sterile

completely free of all viable microbes (bacteria, virus, etc.)

Disinfect

eliminate most or all pathogens on a material

Bacteriostatic

—prevents bacterial growth, does not kill bacteria

Antiseptic

a disinfectant that is non-toxic enough to be used on skin

Moist heat

Denatures proteins

very effective at killing bacteria

Boiling

kills most microorganisms in 5 minutes. Notable exception: endospores

MOIST HEAT

Pasteurization

brief heating used to reduce the number of spoilage organisms and to kill disease-causing organisms.

Used for milk, juice, wine, and other products that are damaged by boiling. Example: milk is heated to 72°C (160°F) for 15 seconds.

MOIST HEAT

Autoclaving

—heat water in an enclosed vessel.

As steam is produced, the pressure in the vessel increases beyond atmospheric pressure and allows for the temperature to reach >100°C

MOIST HEAT

Dry Heat

not as effective as moist heat. Requires longer times and higher temperatures

Incineration

fire oxidizes cell components to ashes

dry heat

Dry heat ovens

oxidizes cell components and denatures proteins.

• Often used for laboratory glassware

• Dry heat

filtration

—removes microbes from fluid or air

Radiation

damage to cell dependent upon wavelength

—Ionizing radiation- destroys DNA and cytoplasmic membrane. Used to sterilize heat-sensitive materials and items that have been packaged.

Ultraviolet light- damages DNA, but penetrates poorly. Used to destroy microbes in the air, drinking water, and on surfaces

Pressure

extremely high pressure may denature proteins and alter permeability of cell membrane.

Used to extend the shelf life of certain commercial foods

Alcohols

—coagulates enzymes and proteins, and damages lipid membranes

—Kill vegetative bacteria and fungi. Not effective against endospores and some viruses.

—Relatively non-toxic and inexpensive

disinfectant

—Aldehydes

—Inactivate proteins and nucleic acids

• —Can kill all forms of microbial life, including endospores and viruses

• —Irritating to the respiratory tract, skin and eyes

—Ethylene Oxide Gas

—Commonly used to sterilize medical devices

—Destroys all microbes by reacting with proteins

—Carcinogenic

—Halogens

—Chlorine and Iodine are common disinfectants that act by oxidizing proteins and other cell components

—Chlorine is used to disinfect inanimate objects, surfaces, and drinking water. Too irritating to use on skin

—Iodine can be used as an antiseptic on the skin, but is not as effective against endospores

—Metals

—Metal containing compounds, such as silver nitrate, bind to proteins and interfere with their function

• —Can not be used medically because of toxicity to humans

• —Act as preservatives in industry

—Peroxygens

—Powerful oxidizing agents that can be used as sterilants

—Readily biodegradable and less toxic to humans than ethylene oxide and aldehydes

—Lower concentrations of hydrogen peroxide can be used as antiseptic on skin

preservation of perishable products

• —Weak organic acids may be added to food (bread, cheese, juice) to prevent microbial growth

• —Nitrate is added to cured meats (cold cuts, bacon, smoked fish) in order to prevent the growth of Clostridium botulinum

• —Refrigeration slows enzyme activity and bacterial growth, with the exception of Psychrophilic organisms

• —Freezing preserves food by stopping microbial growth

dry preservation

• —Adding sugar and salt draws water out of cells, dehydrating them. Used in jams/jellies and cured meats.

• —Desiccation, or the removal of water, is used to produce jerkies.

• —Lyophilization (freeze-drying) is used to preserve coffee, milk, meats, and vegetables. This stops microbial growth but does not kill bacteria or fungi.

Oxidation

a process in which an electron is removed from a molecule. This often results in breaking a chemical bond

Reduction

a process in which an electron is gained by a molecule.

This often results in creating a chemical bond

Metabolism

— the sum total of chemical reactions used for biosynthetic and energy-harvesting processes

• —Anabolism- processes that uses energy (ATP) to synthesize and assemble subunits of macromolecules; biosynthesis

• —Catabolism- processes that harvest energy released during the breakdown of compounds to synthesize ATP

Enzymes

biological catalysts, accelerating the conversion of one substance, the substrate, into another, the product. reaction happen faster

• —Reaction rates are increased because enzymes lower the activation energy of the reaction. This is the energy needed to initiate a chemical reaction.

active site

a specific groove or pocket where a substrate binds and a chemical reaction is catalyzed

Organic cofactor (coenzyme)

derived from vitamins

help enzymes catalyze biochemical reactions, acting as temporary carriers for atoms or electrons

inorganic cofactor

metals like magnesium, zinc, copper, and other trace elements

allosteric regulation

process in which a molecule binds to the back of enzyme and changes activity by decreasing or increasing activity

a molecule binds away from the active site, which can include both inhibition (like noncompetitive) and activation, affecting enzyme activity.

• not a substrate

• makes product when we need it, stops when we have it(feedback mech)

Competitive Inhibition

—A molecule that is similar in structure binds at the active site of the enzyme and blocks the substrate from entering.

• binds to active site

• looks similar to substrate

• blocks substrate from binding

• competes with substrate

Non-Competitive Inhibition

A molecule binds elsewhere on the enzyme( the back), changing its shape and preventing the substrate from entering the active site.

form of allosteric inhibition where the inhibitor binds to an allosteric site (the back of enzyme)

• Always reduces enzyme activity (inhibits). 

• not competing with substrate

Ionizing radiation

destroys DNA and cytoplasmic membrane.

Used to sterilize heat-sensitive materials and items that have been packaged.

Ultraviolet light

damages DNA, but penetrates poorly.

Used to destroy microbes in the air, drinking water, and on surfaces