Prevention And Control Of Microbial Growth

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Psychrophiles

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Optimum < 15
*Snow fields, polar ice pockets

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Psychrotrophs

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Optimum 20-30
*Food Spoilage

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Mesophiles

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Optimum 25-40
Range 10-50
*Most pathogens (E. coli)

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Extreme (hyper) thermophiles

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Optimum<80
Archea
Deep sea vent, volcanoes

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pH Requirements

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Most thrive around neutrality.
*Acidophiles have optimal pH as low as 1.

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Osmotic Pressure Requirements

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Bacteria are protected from lysis by their cell walls if they are in a hypotonic environment. Hypertonic environments often cause plasmolysis.
*Halophiles can exist in extremely hypertonic environments; Dead sea bacteria actually require 30% salt!!!

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Carbon Requirements (SPONCH)

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-Carbon is needed for energy & molecule building.
*Heterotrophs - Carbohydrates, proteins, lipids.
*Autotrophs- CO2 (inorganic)

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Nitrogen Requirements
(SPONCH)

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-Nitrogen is needed for proteins, DNA, RNA
*Heterotrophs - proteins, DNA, RNA.
*Autotrophs- Ammonia, Nitrite/Nitrate.
*Nitrogen Fixers-atmospheric nitrogen (N2)

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Phosphorus Requirements
(SPONCH)

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-Phosphorus is needed for DNA, RNA, ATP, and phospholipids.
*Heterotrophs- DNA, RNA, ATP, phospholipids
*Autotrophs- inorganic phosphate (PO4^-3)

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Sulfur Requirements

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-Sulfur is needed for certain amino acids (cysteine, methionine)
*Heterotrophs- proteins
*Autotrophs- hydrogen sulfide (H2S), sulfate (SO4^-2)

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Trace Chemicals

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-Many minerals (inorganic, inc. K, Na, Ca, Mg, Fe)
-Various vitamins (organic). Some microbes can synthesize them, some must ingest them, in which case they are termed "essential."

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Obligate Aerobes

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Need oxygen to live.

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Facultative Anaerobes

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Will use oxygen if present, but can exist (usually by fermentation) without it.

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Obligate Anaerobes

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Cannot tolerate oxygen.

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Aerotolerant Anaerobes

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Do not use oxygen, but it won't kill them.

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Microaerophiles

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Need oxygen, but can only grow under low oxygen concentrations (soil, water)

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Byproducts of Cellular Respiration?
*Why oxygen can be "dangerous" to life?

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-H2O2 (Hydrogen Peroxide)
-O2- (Superoxide)

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Superoxide Dismutase

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Converts O2- to H2O2

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Catalase

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Converts H2O2 to H2O and O2

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Peroxidase

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Converts H2O2 to H2O

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Why are these enzymes (superoxide dismutase, catalase, peroxidase) functional to organisms that can tolerate oxygen?

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They help detoxify dangerous byproducts of oxygen metabolism.
*Obligate anaerobes lack all these enzymes!

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2 Types of Culture Media

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*Solid = Agar is poured into plates & solidifies; bacteria grow in colonies on the surface.
*Liquid = Culture is suspended in the liquid.

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Streak Plate Method

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-Grow desired bacteria in liquid medium
-Plate onto agar plate, using serial dilutions
-Pick colony & put back in liquid medium
-Keep plate in regrigerator

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Binary Fission

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1) Cell elongates and DNA is replicated

2) Cell wall and plasma membrane begin to divide

3) Cross-wall forms completely around divided DNA

4)Daughter cells separate

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Generation Time

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The time it takes for one cell to become two; in E.coli under optimum conditions - it is about 20 minutes.
*Bacteria grow exponentially!
*Bacteria plotted in logarithm so it looks like a straight line.

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What are the (Binary Fission) Phases of Growth?

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1) The Lag Phase - Little growth, high metabolic activity.

2) The Log Phase - Exponential growth is occurring

3) The Stationary Phase - the # of cells remains the same, but the rate of death = the rate of growth (depletion of nutrients, buildup of wastes)

4) The Death Phase - Exponential Decline

*Know the graph pg.92

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The plate count method for determining the number of bacterial cells present?

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-Serially dilute your sample, and plate out the dilution until you get a countable plate.
-Count the colonies (which came from one cell) and multiply by the dilution factor.

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Why do we Control Microbial Growth?

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1)Prevention/treatment of infection (general)
2) Prevention of infection (aseptic surgery)
3) Prevention of food spoilage

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Sterilization

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The complete destruction of all microbes on an object

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Disinfection

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The reduction or inhibition of microbial growth on a non-living surface (i.e., disinfectants)

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Antisepsis

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The reduction or inhibition of microbial growth on living tissue (i.e., antiseptics)

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Bacterial death.

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Die at a fixed exponential rate!

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How do various agents harm bacteria?

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-Damage to the cell wall
-Alteration of membrane permeability (or membrane destruction)
-Damage to Proteins and/or Nucleic acids: this can cause enzymes to malfunction or inhibit the processes of DNA replication and/or protein synthesis.
**Since bacteria have both physical and chemical needs, they can be controlled by either physical or chemical agents!

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HEAT: How does it generally harm bacteria?
(Physical Agents)

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Quickly denature proteins; also may damage the membrane (make it more fluid) and denature DNA and RNA.

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MOIST HEAT: What are the 2 kinds of moist heat?
(Physical Agents)

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Boiling & Autoclaving

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BOILING
(Moist Heat)

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-Kills most bacteria in 10 minutes, but endospores and some viruses are resistant (30 minutes recommended for drinking water)

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AUTOCLAVING
(Moist Heat)

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-Moist heat under high pressure allows the temperature to increase to 121 degrees C in most autoclaves (under 2 atm). Very popular in sterilizing culture media and lab instruments. In essence, a "pressure cooker."

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PASTEURIZATION
(Moist Heat)

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-Short duration/high temperature treatment of food (72 degrees C for 15 seconds.) Does not sterilize, but kills many undesirable bacteria and does not alter the flavor or texture of the food.

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How does DRY HEAT harm bacteria? 2 methods?
(Physical Agent)

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-Dry Heat kills by oxidizing effects, can denature proteins and DNA.
-1) Incineration (combustion)
-2) Dry ovens

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How does COLD effect bacteria? 2 methods?
(Physical Agent)

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-COLD inhibits bacterial growth, but usually does not kill the microorganism!!!
-1) Refrigeration
-2) Freezing

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How does DESICCATION effect bacteria? 2
(Physical Method)

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-Removal of water; often stops growth, but then again doesn't necessarily kill.
-Many endospores are resistant to desiccation.
-1) Freeze-drying, AKA Lyophilization

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FREEZE-DRYING (AKA: Lyophilization)
(Desiccation)

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-The simultaneous combination of freezing and desiccation. A great way of preserving samples of microbes or even food!

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FILTRATION
(Physical Method)

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-The passage of a liquid or gas through a filter with pores small enough to retain microorganisms
-Membrane filters are available that can eliminate most bacteria; air filters can do the same.
-Cannot remove toxins that might be present.

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Osmotic Pressure Treatment
(Physical Method)

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*Use of salts*
-Causes plasmolysis, and is often used in preservation of food. Salts and sugar solutions are used to "cure" meats and preserve fruits, respectively.

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Radiation & 2 Types?
(Physical Method)

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-Radiation: kills bacteria in many ways depending on type and dosage.

-1) Ionizing Radiation (gamma rays, X-rays): can cause large scale mutations in DNA. Often used in disinfecting food (somewhat controversial).

-2) Non-ionizing Radiation (UV, microwaves): can cause abnormal base linkages in DNA (e.g., thymine dimers), and heat (microwaves).

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Disinfectants

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-antimicrobial agents that are applied to non-living objects to destroy microorganisms

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Chlorine. 3 Kinds? Pros & Cons?
(HALOGENS; Chemical Disinfectant)

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-When chlorine in various forms is added to water, hypochlorous acid forms, which is a strong oxidizing agent and disrupts enzymes.
1)-Compressed chlorine gas is used in disinfection of public drinking water and pools
2)-Hypochlorite is the chemical of choice in treatment of swimming pools.
3)-Bleach (sodium hypochlorite) (Clorox) is a common household disinfectant.
-Pros: Kills not only bacterial cells but most endospores, fungi, viruses, and algae.
-Cons: Not effective at basic pHs; relatively unstable, especially if exposed to light, including sunlight.

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Iodine. 2 Kinds?
(HALOGENS; Chemical Disinfectant)

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-Acts similarly to chlorine, by denaturing enzymes.
1)-In tincture form, which is a solution in alcohol, it is often used as an antiseptic.
2)-Iodine tablets can be used to disinfect drinking water (while camping); the water doesn't taste so great thereafter!

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Why aren't Fluorine and Bromine used as chemical (halogen) disinfectants?

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-Fluorine and Bromine are potentially dangerous to handle, and not generally used for these purposes, although some Bromine-based pool cleaning systems exist.

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Alcohols
(Chemical Disinfectants)

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-Work by dissolving membrane lipids and denaturing proteins
-Ethanol and Isopropyl alcohol (rubbing alcohol) are most popular.
-They can be used as antiseptics, or for wiping down surfaces: they then quickly evaporate.

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Surface acting agents AKA Surfactants (Chemical Disinfectants)

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-Detergents and soaps
-Chemical structure: long chain hydrocarbon with a charged "head"; they are amphipathic, like phospholipids.
-Detergents act by disrupting cell membranes, and are also good cleansing agents and emulsifiers.
-Soap (an anionic detergent) isn't much of a disinfectant, but through emulsification of oils and scrubbing action, its use removes many germs from skin surfaces.

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Quaternary Ammonium Salts; aka QUATS
(SURFACTANTS->Chemical Disinfectants)

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-They are cationic detergents (ammonium ion bound to four other groups) and work by disrupting plasma membranes.
-Very popular in household disinfectants, like Lysol, bathroom cleaners, even mouthwashes, in which case we must call them antiseptics.
-Popular example: n-Alkyl dimethyl benzyl ammonium chloride (where the alkyl groups are a mixture of long chain hydrocarbons [12-18Cs]). Common household cleaners may use QUATS with alcohol.

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Phenol and Phenolics (Chemical Disinfectants)

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-Disrupt plasma membranes and denature proteins
-First chemical used by Lister to disinfect wounds
-Toxic, so not used often as an antiseptic, except in throat lozenges and sprays (at low concentration!)
-Phenolics are related compounds (usually an aromatic ring with a different functional group or added groups) that are less toxic and or smelly - some of these are used in some formulations of Lysol and other household disinfectants.

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Aldehydes. 3 types?
(Chemical Disinfectants)

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-Organic compounds with a terminal CHO group
1)-Formaldehyde is the most famous and very effective. They work by inactivating proteins and nucleic acids. Formaldehyde is very toxic, which limits its general usefulness.
2)-Formalin is the commonly used liquid preservative made by dissolving formaldehyde gas in water.
3)-Gluteraldehyde (with two terminal CHO groups) is less toxic and very effective and commonly used (e.g. for sterilization of medical instruments).

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Heavy Metals
(Chemical Disinfectants)

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-They work by inactivating proteins by interacting with functional groups and include mercury mercury, silver, copper, and zinc.
-An antiseptic example would be "mercurochrome", a solution of mercury often used similarly to tincture of iodine, which is no longer considered a good antiseptic due to the toxicity of mercury.

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Chemical Food Preservatives.

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-Many are weak organic acids. Most popular are benzoic acid and sorbic acid, usually encountered as sodium salts, (e.g. sodium benzoate.)
-Nitrite salts prevent germination of C. botulinum endospores in meat and so often are used as preservatives in bacon, etc.

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Who's more susceptible to PHYSICAL/CHEMICAL AGENTS?
(Gram + or Gram - ?)

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-Gram-positive bacteria are generally more susceptible than gram-negative bacteria.
-Endospores are very resistant.
-Enveloped viruses are often MORE SUSCEPTIBLE to chemicals, than non-enveloped ones.