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181 Cards in this Set
- Front
- Back
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= number of cells, not the size of the cells.
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Microbial growth
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= increasing in number, accumulating into colonies (groups of cells large enough to be seen without microscope) of hundreds if thousands of cells, or populations of billions of cells.
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Microbial growth
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By understanding the conditions necessary for microbial growth =
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we can det. How to control the growth of microbes that cause diseases and food spoilage, encourage growth of helpful microbes.
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2 main categories of the requirement of microbial growth
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1. physical
2. chemical |
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Physical
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1.temperature
2.ph 3.osmotic pressure |
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chemical
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1.carbon
2.nitrogen 3.sulfur 4.phosphorous 5.trace elements 6.oxygen 7.organic growth factors |
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1. temperature
– cold loving – found in ocean’s depth or in certain polar regions such organism seldom cause problems in food preservation. |
psychrophiles (0 to 15)
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temperature
– common in low temp food spoilage bec they grow fairly well at refrigerator temp., sometimes called moderate psychrophiles or facultative psychrophiles. (degrade food – mold mycelium, slime on food surfaces, off-tastes, off-colors) |
Psychotrophs (20 to 30)
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temperature
– moderate temp.loving microbes, include most of the common spoilage and disease organisms |
Mesophiles (25 to 40)
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temperature
– heat-loving microbes, temp of water from a hot water tap, sunlit soil and in thermal waters such as hot springs, but cannot grow at temp. below 45C. Endospores formed by thermophilic bacteria are unusually heat resistant and may survive the usual heat treatment given canned goods. Although elevated storage temp. may cause surviving endospores to germinate and grow, thereby spoiling the food, these thermophilic bacteria is not considered a public health problems. Important in organic compost piles. |
thermophiles (50 to 60)
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temperature
– live in hot springs associated with volcanic activity, sulfur is usually important in their metabolic activity |
Hyperthermophiles (extreme thermophiles) – (80C)
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– known high temp near deep sea hydrothermal vent. The immense pressure in the ocean depths prevents water from boiling even at temp. well above 100C
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121C
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– optimum temp for many pathogenic bacteria & incubators for clinical cultures.
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37C
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(human temp.) – largest growing bacteria affecting humans
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mesophilic bacteria
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– favors cold environment, cheese/meats (esp. soft cheese), causes SS in infant/mother, septic abortion, tolerant psychotroph – tolerates low temps.
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listeria menocytogenes
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Appropriate food storage conditions –
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“keep hot, hot; cold foods, cold”
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– common in rice/cereal
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Bacillus becerus
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2. Ph
- Most organisms grow at neutral ph = 6.5 and 7.5 - Yeasts are acidophilic, bacteria not grow in acidic (fungus do) - Very few bacteria grow at an acidic ph < 4, this is why a number of foods such as sauerkraut, pickles and many cheeses, are preserved from spoilage by acids produced by bacterial fermentation |
true
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ph
- Acidophiles – bacteria that are remarkably tolerant of acidity. - Chemoautotrophic bacteria – found in drainage water from coal mines and oxidizes sulfur to form sulfuric acid, can survive at a ph = 1 |
true
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ph
- Molds and years will grow over a greater ph range than bacteria will, but the optimum ph of molds and yeasts is generally below that of bacteria, ph = 5 to 6 - alkalinity also inhibits microbial growth but is rarely used to preserve foods. |
true
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ph
- Peptones & amino acids – act as buffers, and many media also contain phosphate salts; used to neutralize the acids and maintain proper ph - phosphate salts - have advantage of exhibiting their buffering effect in the ph growth range of most bacteria. They are also nontoxic; in fact they provide phosphorous, an essential nurtrient. |
true
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4. osmotic pressure
Microorganisms obtain almost all their nutrients in solution from the surrounding water. High osmotic pressure have the effect of removing necessary water from a cell. |
true
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ph
– when a microbial cell is in a solution that has a higher concentration of solutes than in the cell. -this osmotic loss of water causes plasmolysis or shrinkage of the cell’s cytoplasm -example… salted fish, honey and sweetened condensed milk: this mechanisms – the high salt or sugar concentrations draw water out of any microbial cells that are present and thus prevent their growth. These effects of osmotic pressure are roughly related to the number of dissolved molecules and ions in a volume of solution |
Hypertonic
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ph
– have adapted so well to high salt concentration that they actually require them for growth. Termed obligate halophiles – Organisms from such saline waters as the Dead Sea often require nearly 30% salt and the inoculating loop used to transfer them must first be dipped into saturated salt solution. |
extreme halophiles
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ph
– do not require high salt concentration but are able to grow at salt concentration up to 2%, a concentration that inhibits the growth of many others organisms. |
Facultative halophiles
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ph
– osmotic pressure is unusually low, like distilled water – water tends to enter the cell rather than leave it. Some microbes that have a relatively weak cell wall may be lysed by such treatment |
Hypotonic
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ph
– increase salt or sugar – cause plasmolysis |
Hypertonic environments
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ph
– increase salt or sugar – cause plasmolysis |
Hypertonic environments
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ph
– require high osmotic pressure, not cause infections in humans because in extreme salt content/conditions, wont grow without those conditions |
Extreme or obligate halophiles
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ph
– e.g. vibrio cholera, tolerate high osmotic pressure |
Facultative halophiles
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creates hyperosmotic milieu, causes plasmolysis
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Salt
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Microaerophiles grow best in reduced oxygen, -- carbon dioxide
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true
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O2 toxic, hyperactive free radical – breaks up membranes. Explains aging, cell death (apoptosis)
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true
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Chemical
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1.carbon
2.nitrogen, sulfur and phosphorous 3.Trace elements – inorganic elements required in small amounts; usually as enzyme cofactor 4.oxygen |
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Chemical
1.– is the structural backbone of living matter, it is needed for all the organic compounds that make up living cell -chemoheterotrophs – use organic carbon source -autotrophs – use carbon dioxide |
carbon
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carbon
-protein synthesis requires considerable amt of nitrogen and sulfur. The synthesis of DNA and RNA also require nitrogen and some phosphorous, as does the synthesis of ATP. |
nitrogen, sulfur and phosphorous
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: in amino acids, proteins
-most bacteria decompose proteins -some bacteria use NH4 or NO3 -a few bacteria use N2 in nitrogen fixation |
Nitrogen
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chemical
-in amino acids, thiamin, biotin -most bacteria decompose proteins -some bacteria use So4, H2S |
Sulfur
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-in DNA, RNA, ATP and membranes
-PO4 is a source of phosphorous |
phosphorous
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– cyanobacteria use gaseous nitrogen directly from atmosphere
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Nitrogen fixation
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– live cooperatively with the roots of legumes, clover, soybeans, alfalfa, beans and peas
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Symbiosis
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3. – inorganic elements required in small amounts; usually as enzyme cofactor
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Trace elements
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4.oxygen -
– organisms that require oxygen to live; are at disadvantage because oxygen is poorly soluble in the water of their environment |
obligate aerobes
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oxygen
– (e.g. E. coli in human intestines and yeasts) - organisms that have capability to continue growing in the absence of oxygen, but the efficiency to produce energy decreases in the absence of oxygen |
facultative anaerobes
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Oxygen
–(e.g. Genus Clostridium – cause tetanus & botulism) - bacteria that are unable to use molecular oxygen for energy-yielding reactions, most are harmed by it. Extremely sensitive to oxygen |
obligate anaerobes
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– essential components of one of the body’s most important defenses against pathogens, phagocytosis. Ingested pathogens are killed by exposure to singlet oxygen, superoxide free radicals, peroxide anions of hydrogen peroxide and hydroxyl radicals.
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Toxic forms of oxygen
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1. – normal molecular oxygen that has been bosted into a higher-energy state and is extremely reactive.
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singlet oxygen
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2. – formed in small amts. During the normal respiration of organisms that use oxygen as the final electron acceptor forming water. In the presence of oxygen, obligate anaerobes also appear to form some superoxide free radicals, which are so toxic to cellular components that all organisms attempting to grow in atmospheric oxygen must produce an enzyme, superoxide dismutase (SOD), to neutralize them.
Aerobic bacteria, facultative anaerobes and aerotolerant anaerobes – produce SOD with which they convert the superoxide free radical into molecular oxygen |
superoxide free radicals
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3Hydrogen peroxide produced in this reaction contains peroxide anion which is also toxic. Catalase (human has it) – enzyme to neutralize the peroxide anion.
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true
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– another enzyme that breaks down hydrogen peroxide
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Peroxidase
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4. – another intermediate form of oxygen and probably the most reactive. It is formed in the cellular cytoplasm by ionizing radiaton.
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hydroxyl radical
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(e.g. lactobacilli – used in the production acidic fermented foods: pickles, cheese) – cannot use oxygen for growth, but tolerate it fairly. Ferment carbohydrates to lactic acid.
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Aerotolerant anaerobes
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– aerobic, require oxygen, but reduced oxygen.
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Microaerophiles
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– only aerobic growth, oxygen required, growth occurs only where high concentration of oxygen have diffused into the medium, presence of enzyme catalase and superoxide dismutase (SOD) allows toxic forms of oxygen to be neutralized, can use oxygen
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Obligate aerobes
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– both aerobic and anaerobic growth, greater growth in presence of oxygen, growth is best where most oxygen is present, but occurs throughout the tube, presence of enzymes catalase and SOD allows toxic forms of oxygen to be neutralized, can use oxygen
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Facultative anaerobes
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– only anaerobic growth, ceases in presence of oxygen, growth occurs only where there is no oxygen, lacks enzyme to neutralize harmful forms of oxygen, cannot tolerate oxygen
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Obligate anaerobes
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– only anaerobic growth, but continues in presence of oxygen, growth occurs evenly, oxygen has no effect, presence of one enzymes (SOD), allows harmful forms of oxygen to be partially neutralized, tolerates oxygen
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Aerotolerant anaerobes
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– only aerobic growth, oxygen required in low concentration, growth occurs where a low concentration of oxygen has diffused into medium, produced lethal amounts of toxic forms of oxygen if exposed to normal atmospheric oxygen
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Microaerophiles
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5. – organic compounds unable to synthesize . organic compounds obtained from the environment, vitamins, amino acids, purines, pyrimidines
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organic growth factors
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– nutrient material prepared for the growth of microorganism in the lab.
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Culture media
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– term when microbes are introduced into a culture medium to initiate growth
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Inoculum
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– microbes that grow and multiply in or on a culture medium
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Culture
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Criteria to be met by culture medium to grow a culture
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1.right nutrients for specific organism
2.have sufficient moisture 3.properly adjusted ph 4.suitable level of oxygen or perhaps none at all 5.medium must initially sterile 6.incubated at the proper temperature |
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– used as thickener in foods such as jellies and ice cream, complex polysaccharide from a marine alga
- solidifying agent is added to medium when a bacterium is wished to grow on a solid medium - liquefies at about 100C and at sea level remains liquid until the temp. drops to about 40C; generally not metabolized by microbes |
agar
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- for laboratory, agar is held in water baths at about 50C, at this temp, it does not injure most bacteria when it is poured over them, once the agar has solidified, it can be incubated at temp. approaching 100C before it again liquefies, this is important when bacteria are being grown
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true
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– agar in test tubes, when they are allowed to solidify with the tube held at an angle so that a large surface are for growth
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Slants
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– agar in vertical tube
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Deep
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– named for their inventor, shallow dishes with a lid that nests over the bottom to prevent contamination, when filled they are called Petri (culture) plates
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Petri dishes
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= growth environment which bacteria live
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Medium
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= solid, slants, broths, plates, agar
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Media
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= gelatin
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Agar
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– useful in growing canophiles
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Candle jars
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– contains only 1 organism
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Pure culture
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– contain more than 1 organism
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Quadrant streak media
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To Support microbial growth a medium :
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1.must provide an energy source
2.sources of carbon, nitrogen, sulfur, phosphorous 3.organic growth factor |
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– one whose exact chemical composition is known.
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chemically defined medium
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– growth factors include glucose
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E.Coli
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– many organic growth factors must be provided in the chemically defined medium
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Neisseria
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– organisms that require many growth factors e.g. lactobacillus – used in tests that determine the concentration of a particular vitamin in a substance.
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Fastidious
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– to determine vitamin in a substance, a growth medium is prepared contains all the growth requirements of the bacteria except the vitamin being assayed.
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Microbial assay
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– heterotrophic bacteria and fungi, usually use in the prep laboratory. Made up of nutrients, including extracts from yeasts, meat, or plants, or digests of proteins. Peptone (proteins-energy, carbon, nitrogen and phosphorous), beef extract – vitamins and other organic growth factors, sodium chloride, agar, water
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Complex media
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– if complex medium is liquid
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Nutrient broth
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– when agar is added to nutrient broth
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Nutrient agar
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Anaerobic Growth Media and Methods
– used in cultivation of anaerobic bacteria. Medium contains ingredients such as sodium thioglycolate, chemically combine with dissolved oxygen and deplete the oxygen in the culture medium. |
Reducing media
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Anaerobic Growth Media and Methods
– used when the culture must be grown in Petri plates to observe individual colonies |
Special jars
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– enzyme used to provide an anaerobic environment, respiratory enzyme derived from the plasma membranes of certain bacteria.
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Oxyrase
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Special culture technique
Examples. |
1. Mycobacterium leprae – leprosy bacillus, now usually grown in armadillos, which has relatively low body temp. that matches the requirements of the microbes.
2. syphilis spirochete 3. rickettsias and the chlamydias |
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– microbes that grow better at high CO2, can be obtained with candle inside the jar. Low oxygen, high CO2 : intestinal tract, respiratory tract, and other body tissues where pathogenic bacteria grow
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Capnophiles e.g. microaerophilic Campylobacter – precise oxygen and carbon dioxide
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Selective and Differential Media
– designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes. |
Selective media E.g. bismuth sulfite agar – medium used to isolate the typhoid bacterium, the gram(-) Salmonella typhi from feces
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– has ph of 5.6, used to isolate fungi that outgrow most bacteria at this ph
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Sabouraud’s dextrose agar
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– make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. Pure cultures of microorganisms have identifiable reactions with differential media in tubes or plates.
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Differential media
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– (contains red blood cells) – a medium that microbiologists often use to identify bacterial species that destroy red blood cells. E.g. streptococcus pyogenes – bacterium that causes strep throat, show clear rind around their colonies (beta-hemolysis) where they have lysed the surrounding blood cells.
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Blood agar
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– used selective and differential media to culture
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Staphylococcus aureus
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-used to culture small numbers of bacteria, often case for soil and fecal samples.
-Usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others. -Also a selective medium but designed to increase very small numbers of the desired type of organisms to detectable levels |
Enrichment Culture
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-Encouraged growth of desired microbe
-Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria (inoculate phenol-containing culture medium with the soil and incubate, transfer 1 ml to another flask of the phenol medium and incubate, transfer 1 ml to another flask of the phenol medium and incubate, only phenol-metabolizing bacteria will be growing) |
Enrichment Culture
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– Types of Culture Media with growth of chemoautotrophs and photoautotrophs; microbiological assay
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Chemically defined
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– Types of Culture Media with growth of most chemoheterotrophic organisms
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Complex
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– Types of Culture Media with growth of obligate anaerobes
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Reducing
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– Types of Culture Media with suppressing if unwanted microbes, encouraging desired microbes
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Selective
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– Types of Culture Media with differentiation of colonies of desired microbes from others
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Differential
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– Types of Culture Media with similar to selective media but designed to increase numbers of desired microbes to detectable levels
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Enrichment
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– theoretically arise from a single spore or vegetative cell or from a group of the same microorganisms attached to one another in clumps or chains.
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Visible colony
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– isolation method commonly used to get pure culture
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Streak plate method
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– contains only one species or strain
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Pure culture
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– often called a colony-forming unit (CFU)
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colony
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-refrigeration can be used for the short term storage of bacterial cultures.
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Preserving Bacterial Cultures
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1. – process in which a pure culture of microbes is placed in a suspending liquid and quick-frozen at temp. ranging from -50C to -95C. The culture can usually be thawed and cultured even several years later
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deep-freezing
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2.– (freeze drying) – a suspension of microbes is quickly frozen at temp. ranging from -54C to -72C and the water is removed by a high vacuum (sublimation). While under vacuum, the container is sealed by melting the glass with a high temp. torch. The remaining powderlike residue that contains the surviving microbes can be stored for years. The organisms can be revived at any time by hydration with a suitable liquid nutrient medium.
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lyophilization
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– bacteria normally reproduce
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Binary fission
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– methods where few bacteria reproduce; they form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates. Some filamentous bacteria (certain actinomycetes) reproduce by producing chains of conidiospores carried externally at the tips of filaments.
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Budding
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– time required for a cell to divide, varies considerably among organisms and with environmental conditions such as temp.
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Generation time
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– shows the growth of cells over time
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Bacterial growth curve
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4 basic phases of growth
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1. lag
2. log 3. stationary 4. death |
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– period of little or no cell division and can last for 1 hr or several days. Cells are not dormant, microbial population is undergoing a period of intense metabolic activity – synthesis of enzymes and various molecules.
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lag
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– (exponential growth phase) – phase where cells begin to divide and enter a period of growth or logarithmic increase, cellular reproduction is most active during this period and generation time reaches a constant min. Because the generation time is constant, a logarithmic plot of growth during the log phase is straight line.
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log
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- it is time when cells are most active metabolically and is preferred for industrial purposes where for ex. A product needs to be produced efficiently, organisms are sensitive to adverse conditions such as radiation and many antimicrobial drugs
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log
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( – exert their effect by interfering with some important step in the growth process and are therefore most harmful to cells during this phase)
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antibiotic penicillin
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. – period of equilibrium. The number of microbial deaths balances the number of new cells
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stationary
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. – (logarithmic decline phase) – number of deaths exceeds the number of new cells. This phase continues until the population is diminished to a tiny fraction of the number of cells.
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death
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Measure of microbial growth
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1.plate count
2.serial dilution 3.pour plates and spread plates – 4.Filtration 5.the most probable number (MPN) method 6.Direct Microscopic Count |
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1.– frequently used method of measuring bacterial population, adv measures the number of viable cells. Disadv. Takes some time.; usually 24 hours or more for visible colony to form
-often reported as colony forming unit (CFU) |
plate count
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3.– methods to do plate count
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pour plates and spread plates
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– problem can damage some relatively heat-sensitive microorganisms by the melted agar and will therefore be unable to form colonies. To avoid these problems – spread plates are used and inoculum is added to the surface of a prepoured, solidified agar medium.
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pour plate method
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– inoculate empty plate, add melted nutrient agar, swirl to mix, colonies grow in and on solidified medium
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Pour plate method
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– inoculate plate containing solid medium, spread inoculum over surface evenly, colonies grow only on surface of medium
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Spread plates
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4.– method used when the quantity of bacteria is very small. 100ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass; - used to detection and enumeration of coliform bacteria, which are indicators of fecal contamination of food or water.
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Filtration
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5. – useful when the microbes being counted will not grow on solid media (chemoautotrophic nitrifying bacteria). It is also useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (coliform bacteria). A statement that there is a 95% chance that the bacterial population falls within a certain range and that the MPN is statistically the more probable number
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the most probable number (MPN) method
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6. – measured volume of a bacterial suspension is placed within a defined area on a microscope slide. Breed count method – used to count bacteria in milk.
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Direct Microscopic Count
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– used in direct microscopic counts
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Petroff-hausser cell counter
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– automatically count the number of cells in a measure liquid volume.
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Electronic cell counters (coulter counters)
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– no incubation time is required, reserved for application in which time is the primary consideration.
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Advantage of microscopic count
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Indirect Methods (Estimating Bacterial Numbers)
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1.Turbidity
2.metabolic activity 3.dry weight |
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Question: At what phase of bacterial growth do you think antibiotics are usually the most effective?
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Answer: The best time to treat bacterial infection is during log (logarithmic exponential growth phase): spore is declining, metabolically active, metabolic waste is accumulating, lack of nutrients, restriction based on media – spores on this phase. Patient will present with symptoms when there’s onset of presence of bacteria
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Spore stage not normal phase of growth: spores formed when bacteria deprived of nutrients (usually in declining phase) Spores are metabolically active, therefore resistant to Abs.
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true
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Place spore in growth media, will produce 2 rod, which will divide and start log phase
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true
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Decline phase due to running out of nutrients, metabolic wastes accumulating, nutrition based on volume
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true
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- (synchronous cultures) all of organisms are in same phase of growth. But do not use synchronous cultures in microbial biology. Only use in experimental/research conditions to examine. Start off with a pure culture and monitor conditions to see adaptive response of organisms.
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Continuous culture
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method to isolate pure culture
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Quadrant streak
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Essential central concept of control: to limit/prevent/restrict replication/growth so there is no increase in number.
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true
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*Necessary requirements to achieve growth
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-Complete elimination of microorganisms from particular site not essential to achieve control. Abs eliminate the #s to point where host defenses eliminate the pathogen. Complete elimination of normal flora predisposes to secondary overgrowth, decreases colonization resistance, makes infection more likely.
- Ex: problem with overuse of hand sanitizers. Normal flora on skin provides natural barrier to pathogen. |
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- Antibiotic-induced changes in normal flora cause 2 prominent 2ndary infections:
1) fungal vaginitis following Ab-tx. 2) C. difficile (Ab-associated colitis) - But, in immunocompromised hosts, infectious organism must be eliminated b/c host defenses are deficient. |
true
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– has triclosyn; in gram-neg same xport porin used to xport Abs as triclosyn. => ultimately lead to Ab-resistance by bac.
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Antibacterial soaps
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Question: At what phase of bacterial growth do you think antibiotics are usually the most effective?
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Answer: The best time to treat bacterial infection is during log (logarithmic exponential growth phase): spore is declining, metabolically active, metabolic waste is accumulating, lack of nutrients, restriction based on media – spores on this phase. Patient will present with symptoms when there’s onset of presence of bacteria
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Spore stage not normal phase of growth: spores formed when bacteria deprived of nutrients (usually in declining phase) Spores are metabolically active, therefore resistant to Abs.
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true
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Place spore in growth media, will produce 2 rod, which will divide and start log phase
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true
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Decline phase due to running out of nutrients, metabolic wastes accumulating, nutrition based on volume
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true
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- (synchronous cultures) all of organisms are in same phase of growth. But do not use synchronous cultures in microbial biology. Only use in experimental/research conditions to examine. Start off with a pure culture and monitor conditions to see adaptive response of organisms.
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Continuous culture
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to isolate pure culture
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Quadrant streak
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Essential central concept of control: to limit/prevent/restrict replication/growth so there is no increase in number.
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true
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*Necessary requirements to achieve growth
-Complete elimination of microorganisms from particular site not essential to achieve control. Abs eliminate the #s to point where host defenses eliminate the pathogen. Complete elimination of normal flora predisposes to secondary overgrowth, decreases colonization resistance, makes infection more likely. - Ex: problem with overuse of hand sanitizers. Normal flora on skin provides natural barrier to pathogen. |
true
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- Antibiotic-induced changes in normal flora cause 2 prominent 2ndary infections:
1) fungal vaginitis following Ab-tx. 2) C. difficile (Ab-associated colitis) - But, in immunocompromised hosts, infectious organism must be eliminated b/c host defenses are deficient. |
true
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– has triclosyn; in gram-neg same xport porin used to xport Abs as triclosyn. => ultimately lead to Ab-resistance by bac.
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Antibacterial soaps
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Ab soaps response to most HCPs not routinely washing hands effectively leading to nosocomial infections.
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true
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Effectiveness of Chemical Antimicrobial Against Endospores and Mycobacteria
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-Mercury has no activity
-Phenolics -Bisphenols no activity -Chlorhexidine – used to clean babies when born but stopped -Mycobacteria and endospores are being used for chemical agent -Endospores can survive 100% alcohol for several months |
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Chemical Agents used to Control Microbial Growth
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1.Phenol, Phenolics and bisphenols – disruption of plasma membrane, denaturation of enzymes (except for bisphenols)
a.phenol – rarely used due to irritating qualities and disagreeable odor b.phenolics – surfaces, instruments, skin, mucous membranes. e.g. o-phenylphenol c.bisphenols – hand soaps and skin lotions. e.g. triclosan (effective against gram(+) 2.biguanides (chlorhexidine) – disruption of plasma membrane . e.g. skin disinfection, surgical scrubs 3.halogens – iodine exhibits protein function and strong oxidizing agent, chlorine forms strong oxidizing agent hypochlorous acids – alters cellular components. Iodine – effective antiseptic as a tincture and iodophor; chlorine used to disinfect dairy equipment 4.alcohols – protein denaturation and lipid dissolution, thermometers and other instruments; bactericidal and fungicidal but not effective on endospores or nondeveloped viruses (ethanol & isopropanol) 5.heavy metal and their compounds – denaturation of enzymes and other essential proteins. 6.surface-active agents a.soaps and detergents – mechanical removal of microbes through scrubbing b.acid-anionic sanitizers – not certain, may involve enzyme inactivation or disruption c.quarternary ammonium compounds – enzyme inhibition, protein denaturation, and disruption of plasma membrane 7.chemical food preservatives a.organic acids – metabolic inhibition, mostly affecting molds, action not related to their acidity b.nitrates/nitrites – active ingredient is nitrite (produced by bacterial action on nitrate) 8.aldehydes – protein denaturation 9.gaseous chemosterilizers – protein denaturation 10.peroxgens (oxidizing agents) – oxidation |
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Antibacterial Drugs ( Inhibitors of Cell Wall Synthesis)
1.Antimycobacterial antibiotics – inhibits synthesis of mycolic acid component of cell wall of Mycobacterium spp |
isoniazid
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Antibacterial Drugs ( Inhibitors of Cell Wall Synthesis)
1.Antimycobacterial antibiotics – inhibits incorporation of mycolic acid into cell wall of Mycobacterium spp |
ethambutol
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streptogramins
– alternative for treating ancomycin-resistant gram (+) bacteria |
quinupristin and dalfopristin (synercid)
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– useful primarily against penicillin-resistant gram (+) bacteria
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oxazolidinones (Linezolid, Zyvox)
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agents affecting fungal sterols (plasma membrane)
– injury to plasma membrane (systemic fungal infection: fungicidal) |
polyenes (amphotericin B)
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agents affecting fungal sterols (plasma membrane)
– inhibit synthesis of plasma membrane(topical use) |
azoles (clotrimazole, miconazole)
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agents affecting fungal sterols (plasma membrane)
– inhibits synthesis of plasma membrane (orally for systemic fungal infections) |
ketoconazole
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Antiviral Drugs
– inhibit DNA or RNA synthesis(herpes) |
acyclovir, ganciclovir, ribavirin, lamivudine
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Antiviral Drugs
– inhibits DNA or RNA synthesis(smallpox) |
Cidofovir
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Antiviral Drugs
(hepsera) |
Adefovir dipivoxil
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Attachment & Uncoating
– inhibit neuraminidase on influenza virus |
xanamivir, oseltamivir
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Attachment & Uncoating
– inhibit uncoating |
amantadine, simantadine
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Interferons
– inhibits spread of virus to new cells |
alpha-interferon
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Antiprotozoan drugs
– interferes with anaerobic metabolism |
Metronidazole, tinidazole (anaerobic bacteria) protozoa
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Antihelminthic drugs
– inhibits absorption of nutrients |
mebendazole, albendazole
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– inhibit extended spectrum
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beta-lactamase
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Evaluation of Antibiotic Efficacy
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1)Kirby-Bauer disc-diffusion susceptibility (qualitative concentration)
2)“E” Test 3)Agar dilution 4)Broth Dilution |
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1)Kirby-Bauer disc-diffusion susceptibility (qualitative concentration)
Make a lawn of bacteria, place Ab-disc, incubate, measure zone of inhibition, including the disc (mm of diameter). By itself, the zone size does not mean anything. Zone sizes interpreted and xlated into resistance by reference to the NCCLS (interpretive) chart. Where “R”(resistant), “I”(intermediate), and “S”(susceptibility) on C&S report comes from. Sensitivity zone size helps determine R, I, or S. Control Zone Diameter Data/Limits = must be within the range indicated for the test to be deemed valid and evaluated. Must set up many controls to make sure test is valid. |
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Ex: Kirby-Bauer disc-diffusion susceptibility - Ab X with S size >12. Control zone measured 100mm - inaccurate/not acceptable. Test not valid.
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Step for Kirby-Bauer disc-diffusion
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a. K-B disc-diff test is rigidly controlled and set up based on standards recommended.
b. The agar plate must be 4mm thick. c. The inoculum (bacteria) must conform to a known standard. d. The plates must be free of visible moisture and must be used within a specific time to prevent drying out which influences zone sizes (dryness and moisture 2 factors that will influence zone sizes). The medium used is Mueller-Hinton. |
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why control is necessary in an experiment?
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Controls to ensure testing standards are done properly to ensure valid results.
R = bacteria not inhibited; I = indicative of efficacy when there’s evidence of Ab accumulation in body fluids (blood, CSF, urine); S = susceptible, Ab effective in inhibiting bac/organism |
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2) “E” Test (pg 602)
Uses Ab strip containing graded/different amts of antibiotics. Requires a greater Bacteria. The zone inhibition 0.064; .25 (MIC); establish from E test.series of antibiotics put on a lawn of bacteria, plated on Mueller-Hinton plate. Zone of inhibition where it intersects with the Abs is the MIC (minimum inhibitory concn). Gives actual/quantitative concentration (vs K-B which gives qualitative I.R.S). “E” = epsilon. Procedure allows the MIC to be established to several Abs simultaneously and valuable guide to the concentrations to be achieved to inhibit organism. |
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3)Agar dilution
-Used to evaluate efficacy in treating fungi and anaerobic bacteria. These are not typically tested on a daily basis b/c the susceptibilities are predictable (resistance is not commonly seen). Test procedure consist of a series of agar plates w/ varying dilutions of antifungals/antibiotics. Several different organisms (fungi/bac) are seeded to each plate. Growth is then observed on the plate after incubation. This detects the concentration required to inhibit the different organisms and establishes whether resistance is occurring or changing. |
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4) Broth Dilution – MIC, MBC (pg 603 Fig 20.19)
Consist of setting up series of tubes w/ different concns of Abs. In microtiter plates, control has no Abs. Diff concns of Abs in tubes (mcg/mL). Inoculate all w/ same bacterium. Incubate then look for growth. Obviously, lower concns will not inhibit organism, (+ equals growth; - equals no growth), higher concns will inhibit organism. MIC is minimum concn to inhibit growth of organism. If growth in all, organism is resistant. MBC = taking negative cultures from tubes/microtiters (w/ no growth) then subculturing to Ab- Free media. Look for growth/no growth. If growth (not MBC), if growth (MBC). Want to determine if organisms are killed or inhibited. Put in media w/ no Abs to determine if killed or not. Could be that organism was inhibited. |
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Uses and Applications of MIC and MBC Data
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1)An MBC that is 64x or greater than MIC indicates tolerance. Typically means that the Ab is not useful therapeutically b/c have to use such a high concentration of Ab to kill organism, such that it will be toxic to the pt. The bacteria is tolerant/resistant to the Ab. If MIC and MBC are close and/or equal in value, Ab is a very effective therapy.
2) MIC and MBC data are essential in providing the basis for efficacy to warrant approval by FDA. FDA evaluates efficacy based on MIC and MBC data. MIC 50 and MBC 90 means show inhibit 50%; kills 90% MIC and MBC data used to show relative efficacy. MBC data is used relative efficacy that is efficacy of isolated test 3) MIC and MBC data detects changes in susceptibility serving as guide to use of antibiotic. Ex: Increases in MIC50 and MIC90 typically indicates changing patterns of susceptibility suggesting emergence of resistance. 4) MIC and MBC data are used to establish the concentrations required at a particular site to inhibit the bacteria tested. Ex: if the MIC of an organism is 5mcg/mL, and the Ab achieves a concn <5mcg/mL in blood it is not likely to be useful in tx’ing sepsis/septic shock. Ab must achieve concns above the MIC to be effective, otherwise, will be subtherapeutic. 5) Can also be used regionally/nationally to detect emerging patterns of resistance. 6) An essential component of FDA approval and evaluation by pharma comps. |
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Cmment on mechanisms of Ab resistance. Give/List factors contributing to emerging resistance.
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a.mutation in target organism
b.poor patients compliance (failure to take, or taking someone Abs) c.widespread access on the internet d.availability in foreign country e.medical practice (treating viral respiratory tract) |
