Microbiology Exam 2

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Microbes are part of the natural environment. Most are ________ or ________.

benign or beneficial

Examples of benign and beneficial microbes

-gut microbes assist with digestion of carbohydrates and proteins
-gut microbes assist with synthesis of folic acid, vitamin k
-microbes in sanitation systems decompose human wastes
-microbes produce useful fermentation products (bread, beer, etc.)

Why is it important to understand mechanisms of growth control?

-some microbes are pathogens
-important to understand how to effectively disinfect surfaces or equipment in a hospital setting
-important for control of foodborne illnesses
-important for safe home canning
-important for understanding food, drug, home product labels

Sterilization

destruction of all microbes, including endospores

Commercial Sterilization

destruction of most but not all microbes including endospores of Clostridium botulinum

Disinfection

destruction of vegetative (growing) pathogens, doesn't kill endospores

Antisepsis

destruction of vegetative pathogens on living tissue

Degerming

Removal of microbes from a limited area

Sanitization

treatment to lower microbial counts on eating and drinking utensils

Nosocomial infections

hospital-acquired infections

Biocide or germicide

treatment or agent that kills microorganisms

Fungicide

treatment or agent that kills fungi

Virucide

a treatment or agent that kills viruses

Tuberculocide

a treatment or agent that kills mycobacterium tuberculosis (TB)

Bacteriostasis

a treatment designed to stop growth (but doesn't sterilize). if you remove treatment, growth may resume

Sepsis

indicates bacterial contamination, also used to describe disseminated bacterial disease

Microbial Death Rate

number of dying cells per unit of time

Factors that affect microbial death rate:

-number of microbes
-presence of organic matter in human wastes
-temperature
-fats and proteins
-pH
-time of exposure to agent
-specific characteristics of the microorganism

Factors that affect microbial death rate: number of microbes

high population load, longer period of time required to kill all the organisms

Factors that affect microbial death rate: presence of organic matter in human wastes, etc.

presence of organic matter in human wastes and bodily fluids inhibits death induced by antimicrobial growth

Factors that affect microbial death rate: temperature

some disinfectants work better at warm temperatures

Factors that affect microbial death rate: fats and proteins

fats and proteins can protect the microorganism from exposure to disinfectants

Factors that affect microbial death rate: pH

some antimicrobials work better at acidic pHs

Factors that affect microbial death rate: time of exposure to agent

ex: pasteurization uses lower heat for a longer time to achieve the same effect that high heat for a shorter time can achieve in the destruction of microorganisms in food products

Factors that affect microbial death rate: specific characteristics of the microorganisms

they affect death rate. gram positive organisms are more susceptible to many agents than gram negative organisms

Why are gram positive bacteria more susceptible to biocides than gram negative bacteria?

they have the extra outer membrane--porin proteins inhibit movement of mircrobicides across the membrane

Mechanisms of action of antimicrobial control agents

-damage to plasma membrane
-damage to enzymes and other structural proteins
-damage to nucleic acids

Thermal death point (TDP)

the lowest temp at which all microorganisms are killed in 10 minutes time

Thermal death time (TDT)

the minimal amount of time necessary to kill all organisms at a given temp

Decimal reduction time (DRT or "D" value)

the time in minutes necessary to kill 90% of the bacteria at a given temp

Moist Heat (boiling, autoclaving) kills how?

by coagulating proteins (similar to a fried egg)
-most organisms (including viruses and fungi) are killed by 10 minutes of boiling
-more heat resistant organisms and bacterial endospores are killed by moist heat under pressure (pressure cooker/autoclave)

Autoclaving--high pressure and heat

-the higher the pressure, the greater the temp
-sterilization occurs by contact of the organisms to steam or in liquids
-15 lbs/sq.in (psi) = 121*C kills all organisms and endospores in 15 minutes
-this works well for small volumes. larger volumes require more time
-to sterilize surfaces, the steam in an autoclave must contact ALL surfaces
-instruments are wrapped in paper that can be wtted and allow the steam to contact them

Home canning

-heat under pressure can effectively preserve food
-follow directions carefully. a good guide is Ball Canning Guide (?)
-need to drive off cold air before cooker is sealed
-quarts require more time than pints

Pasteurization

-another example of antimicrobial treatment using moist heat
-process developed by Louis Pasteur to preserve wine
-kills pathogenic organisms
-classical treatment for milk--63*C for 30 minutes

Modern method of Pasteurization

High temperature short time (HTST), pasteurization @ 72*C for 15 sec

Ultra high temperature treatment (UHT)

kills everything so it can be stored at room temp. ex: coffee creamers treatment this way. milk heated to 74C, then passed in a film over steam, raised temp to 140C for 3 sec, cooled rapidly to 74*C

Dry heat sterilization

-kills by oxidation
-example flaming
-example oven heat 170*C for 2 hrs

Other physical methods of microbial control:

-filtration
-low temps
-desiccation
-osmotic pressure differences
-radiation

Filtration

-removes bacteria or other large cells
-this method is used to sterilize heat-sensitive media, protein solutions, etc.
-filters are 0.22 micrometers and 0.45 micrometers in size
-HEPA (high efficiency particulate air filters) in biosafety hoods remove bacteria from the air

Low Temperatures

-ordinary refrigeration temperature (-7*C) is bacteriostatic and not bacteriocidal
-inhibits growth of most pathogens, but not other psychrotropic organisms
-freezer temps are bacteriostatic, not bactericidal, but some organisms such as parasitic worms are killed by freezing

*What is the name of one important human pathogen that can grow in refrigerators?

Listeriosis (Listeria monocytogenes)

Desiccation

(drying out), bacteriostatic

Osomotic pressure differences

-high salt to preserve meat
-high sugar to preserve fruits
-like desiccation, this is only bacteriostatic

Radiation: ionozing raditation (gamma rays, electron beams, X-rays)

-short high energy waves (< 1nm)
-radiation effects penetrate deeply
-water molecules in the microorganisms are ionized and form reactive hydroxyl radicals (OH*) that are toxic to cells
-used to sterilize pharmaceuticals, laboratory plastic ware, disposable medical supplies
-this method may become an important method for sterilization of food (meats and vegetables)

Radiation: nonionizing radiation (uv light)

-best wavelength for germicidal effects is 260 nm
-UV light damages DNA
-limitations:
1. doesn't penetrate, kills only on surface
2. harmful to eyes, skin
3. sunlight has some uv, but most of the germicidal wavelengths are filtered out by ozone in the atmosphere

Radiation: microwaves

-not effective for germicidal treatment
-food is cooked by the heated moisture in food, but surfaces are not sterile inside a microwave oven

Chemical methods of microbial control

-very few chemical methods are capable or sterilization, but most reduce the number of microbes
-each agent is useful in a specific setting. it is important to understand which one works best in each situation
-concentration is CRITICAL. (use as directed!!)
-organics and pH can interfere

Tests to determine effectiveness of chemical control methods: Use-dilution test

-tests effectiveness of each agent on 3 species of pathogenic bacteria
-metal rings dipper in bacteria
-dried at 37*C
-exposed to disinfectant 10 minutes 20*C, wash
-transfer rings to culture medium
-degree of growth gives you a measure of the effectiveness of the disinfectant

Use-dilution test: Salmonella cholerausuis

a serovar of Salmonella typhimurium that binds to blood vessels

Use-dilution test: Staphylococcus aureus

a skin pathogen that often infects surgical wounds

Use-dilution test: Pseudomonas aeruginosa

a skin pathogen that is an opportunist, problem for burn patients

Tests to determine effectiveness of chemical control methods: Disk-diffusion test

-disc of filter paper is soaked in the disinfectant
-placed on a "lawn" of bacteria on an agar plate
-effectiveness is judged by zone of inhibition

Chemical Disinfectants: Phenol and phenolics

-first used by Joseph Lister (phenol is carbolic acid)
-rarely used now
-at concentrations above 1%, they have a significant antibacterial effect
-stable, long-lasting, active in presence of organic compounds
can be used to disinfect pus, saliva, feces

What is the main ingredient in Lysol?

a derivative of phenol called O-phenylphenol

What is the machanism of action for phenol and phenolics?

they act by disturbing the plasma membrane

Chemical Disinfectants: bisphenols

-related to phenol, but molecular sturcture has 2 phenolic groups
-good agent to kill gram positive staphylococci and streptococci
-broad spectrum activity

2 examples of bisphenols

hexachlorophene (pHisohex) - used for control of microbes in surgical wards
triclosan - agent in antibacterial soaps. this agent has widespread use and triclosan resistant bacteria are now appearing

Chemical disinfectants: biguanides

-broad spectrum (bacteria, fungi, some enveloped viruses, but not myobacteria, endospores, or protozoa)
-used as antimicrobial for skin (surgical hand scrub)

Example of biguanide

chlorohexidine

Mechanism of action for biguanides

acts by disturbing plasma membrane of bacteria

Chemical disinfectants: halogens (iodine)

-broad spectrum (most bacteria, fungi, some viruses, many endospores are killed)
-iodine used as alcohol solution (tincture of iodine)
-iodine also combined with organic molecules as iodophor
-used for disinfection of skin

Iodophores: what do they do, and what are some examples?

they release more iodine more slowly; Betadine and isodine are examples

Mechanism of action for iodine

inhibits protein function, strong oxidizing agent

Chemical disinfectants: halogens (chlorine)

-hypochlorous acid (HCl) is formed when chlorine gas is combined with water
-disinfection of drinking water, swimming pools, sewege

Calcium hypocloride

caled chloride of lime, used by Semmelweis in 1840s to control childbirth fever

Sodium Hypochlorite

Chlorox bleach

How can Chlorox be used as an emergency disinfectant of drinking water?

2 drops bleach per liter of water for 30 min--water is safe for drinking

Mechanism of action for Chlorine

strong oxidizing agent, alters cells

Chemical disinfectants: alcohols

-kill bacteria and fungi, enveloped viruses, but not endospores
-ethanol and isopropyl alcohol more often used
-enhances activity of other agents (ex. benzalkonium chloride)

Why is 70% ethanol more effective than 100%?

allows all plasma membrane lipids to be destroyed, water breaks cells

Mechanism of action for alcohols

protein denaturation and disruption of plasma membranes

Chemical disinfectants: heavy metals

examples: silver, mercury, copper

What was 1% silver nitrate once used for?

to disinfect newborn babies' eyes to protect against acquired Neisseria gonorrhoeae

What is silver-sulfadiazine now used for?

to treat antibiotica resistant material

What is cpper sulfate used as?

an algaecide

What is zinc chloride used in?

mouth washes

Mechanism of actin for heavy metals

combine with sulfhydryl groups of proteins to denature proteins

Chemical disinfectants: surface active agents

-ex: soap, detergents
-not useful as antiseptic, but remove microbes by scrubbing
-good demerging agents
-many soaps contain triclosan--active against gram positive bacteria

Chemical disinfectants: quaternary ammonium compounds (quats)

-surface active
-cationic (positively charged)
-active against gram positive and some gram negative bacteria, fungi, protozoa, and enveloped viruses
-not active against endospores or microbacteria
-not active against Pseudomonas

2 examples of quats

Zephiran (benzalkonium chloride)
Cepacol (brand name for cetylpridinium chloride)

Mechanism of action for quats

disruption of plasma membranes, protein denaturation

Food preservatives: Inhibit molds

-sorbic acids
-potassium sorbate
-sodium benzoate
-calcium propionate

Preservatives of meats such as ham, bacon, hot dogs

-sodium nitrate/sodium nitrite
-added to prevent growth of agents of botulism (Clostridium botulinum)

Preservatives in cheese and other food products

-nisin (natural antimicrobial peptide made by Streptococcus lactis)
-natamycin
-antifungals and antibacterial agents

Aldehydes: formaldehyde and glutaraldehyde

used for disinfections of hospital instruments
-sterilize as 2% solution
-they are bactericidal, tuberculocidal, virucidal, sporicidal in 3-10 hours
-morticians use for embalming

Mechanism of action for aldehydes

protein denaturation

Ethylene Oxide and propylene Oxide

-chemical gases used for sterilizing
-act to denature proteins
-kill all bacteria and endospores, but takes 4-18 hrs
-penetrates well
-STERILIZES WITHOUT HEAT

Peroxygens (oxidizing agents): Ozone

-O3
-added to chlorine in water

Peroxygens (oxidizing agents): hydrogen peroxide

-H2O2
-household use to disinfect puncture wounds and interfere with growth of anaerobic bacteria

Benzoyl peroxide

acne treatment

Peracetic acid

-very effective to sterilize
-kills bacteria and fungi in 5 min
-kills endospores and viruses in 30 minutes

Decreasing Order of Resistance of microorganisms to Chemical Agents

-prions most difficult to kill
-endospores of gram positive bacteria next most difficult to kill
-gram positive bacteria killed more easily than g-negative
-LPS in outer membrane of gram negatives is protective
-mycobacteria also resistant because of their waxy coat that prevents penetration of many agents

Ability to kill mycobacteria

tuberculocidal

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