Lecture 18, 10/24 (Maybruck 3); Control of Microbial Growth

Audio for today's lecture is available here.

• • Slide 1 (handout from 10/22)
• Alcohols characterized as a hydrocarbon group linked to one or more hydroxel groups
• Alcohol has hydrophobic an hydrophilic end. The hydrophilic end is the reactive end - it actively bonds to other molecules.
• Surfactants enter into phospholipids bylayer of a cell membrane. This screws up selectively permeable layer of the cell membrane preventing the cell from functioning properly.
• Alcohol has the surfactant ability at 50% or greater concentration of alcohol.
• Also involved with the denaturing of proteins. If there is low water present then the alcohol will be less effective.
• Viruses with cell envelope are susceptible to alcohol
• • Slide 2
• Oxygen radicals – their metabolism requires oxygen to form energy. The oxygen used sometimes transforms into these oxygen radicals. Highly reactive compounds – create chemical bonds with whatever they can . . . RNA, protein, DNA
• Hydrogen peroxide is an example of oxygen radical
• At 3% can be an effective antiseptic
• At 35% - sterilant
• Oxygen radicals in general are good as a chemical decontaminant for anaerobic organisms.
• • Slide 3
• Surfactants (similar to detergents) – hydrophilic end has an overall positive charge. This is effective as most of our cells have an overall negative charge. This allows the bonding of the surfactant to the cell membrane.
• Soaps - mechanical removal of debris
• • Slide 4
• Main effect of heavy metals is the denaturing of proteins (causes them to unfold) if that metal is not typically associated with that protein.
• Heavy metal compounds
• Mercury, silver, gold, copper, arsenic, zinc
• Cofactor – metal ion that helps enzyme function
• Intermediate activity level
• Anaerobic organisms are susceptible to hydrogen peroxide
• • Slide 5 (he skipped a few)
• Aldehydes –
• Molecules that contain CHO group
• Two types: glutaraldehyde and formaldehyde
• Sugars are characteristic of an aldehyde
• Glutaraldehyde
• Cross link proteins – makes it more toxic. Binds quickly to surface proteins and such. It will also make its way into the cell itself. This messes up function of protein.
• Effective way to kill EVERYTHING. Kills vegetative cells in a few minutes and formaldehyde in a few hours.
• Effective preservitive because it does not lyce the cells. This allows the structure of the organism to be preserved.
• Formaldehyde
• Binds to proteins and nucleic acids
• Effective preservative because it does not lyce the cells. This allows the structure of the organism to be preserved.
• • Slide 1 (handout 10/24/07) Antimicrobial Chemotherapy – using drugs to destroy or remove microorganisms
• What is the perfect drug? (FYI: none exist, so we comprimise)
• Easily administered
• Upon administration it goes straight to the problem spot
• Easily excreted from the body
• Selectively toxic – goes after the bad stuff and not the good stuff
• • Slide 2
• Chemotherapy
• Prophylaxis – drug used to prevent the infection from occurring. (administered to office workers during anthrax scares)
• Antibiotics – chemicals released from microorganisms and fungi. They destroy and kill other microorganisms and fungi.
• Narrow spectrum antibiotic – specific in its target. Narrow range of microorganisms that it can eliminate.
• Broad spectrum antimicrobial – destroys wider range of cells
• • Slide 3
• Primary goal of antimicrobial chemotherapy – creating a drug effective in its removal of microorganisms.
• Microbocidal effect – kill unwanted microorganism
• Selectively toxic
• How is this goal achieved
• Make list of characteristics that allows it (pathogen) to survive
• make list of characteristics that allows our cells to survive
• compare theses two lists and determine the difference.
• Various things that you can target
• inhibit cell wall synthesis
• inhibit nucleic acid synthesis and structure
• inhibit protein syntheses
• alter cell membrane structure
• inhibit folic acid synthesis
• • slide 4: antimicrobial target: cell wall
• cell walls (peptidoglycan) protect cell from osmotic shock. The cell would lyce if it were not protected.
• subslide (drugs that target cell wall synthesis)
• cycloserine – inhibits formation of basic subunits of peptidoglycan.
• vancomycin – prevents elongation of peptidoglycan
• penicillin and cephalosporins – target the peptide bond between glycan sugars. Prevent cross linking of glycan molecules
• those drugs that target the cell wall are narrow in spectrum. This is because the cell wall they are attacking is found in gram (+) bacteria.
• In order for these things to work the organism has to be vegetative. A vegetative organism is always adding to the peptidoglycan layer, if it is not producing peptidoglycan then the drugs are useless (they also have no effect on endospores)
• • slide 5 targeting nucleic acids
• In the context of bacterial microorganisms this is a broad spectrum approach.
• Replication – DNA synthesis
• transcription – formation of RNA
• antibiotic Rifampin – blocks transcription. Binds to RNA polymerase and prevents the RNA polymerase from transcribing.
• Hydroxyurea – prevents formation of nucleotides (found in DNA and RNA: guanine, cytosine, thymine, adenine). It does this by binding to Ribonucleotide reductase.
• • Slide 6
• Mitomycin blocks DNA synthesis → cross linking guanines preventing DNA
• ATGGTCAG
• TACCAGTC

Lecture 17, (Maybruck 2) 10/22; Control of Microbial Growth (cont.)

Audio for lecture.

• • Slide 1 (slide labeled number 12 on the handout from 10/17)
• Sterilization with steam under pressure, this is the only way it can get up to 121ºC
• • Slide 2
• pasteurization does not remove endospore forming bacteria
• removes all vegetative forms of bacteria
• juice, beer, wine, milk are all pasteurized
• two methods used to pasteurize
• • flash method –
• • batch method – exposes microorganisms between 63-66º C for a longer exposure time of 30minutes
• two infections
• • salmonelosis – upon ingestion it will coat the lining of the large intestine (large intestine absorbs extra water) and prevents absorption of extra water.
• • brucellosis – mucus covers are GI tract which aids movement of food. It also traps bacteria and brings it to the stomach in order for it to be destroyed. Brucella escapes this mucis and penetrates to the blood vessels and has a grand ol’ time.
• • Slide 3
• Cold has primarily microbistatic effects
• Food inadequately cooked after being thawed may harbor pathogens
• • Slide 4
• Ionizing radiation (loss or gain of electrons)
• • As the wavelengths decrease the energy associated with that wave increases (enough to break chemical bonds)
• • Ionization causes atoms to lose their electrons and causes them to not be bound to one another anymore.
• • Ionizing radiation can be considered a Sterilant as a removal of endospores
• • Types of radiation that are effective as sterilants are: some UV, x-rays, and gamma rays. (they all attack DNA)
• Ionization radiation acts indirectly and forms oxygen radicals which are very reactive. They are always seeking to make chemical bonds.
• Radiation helps to remove contaminants and prolong shelf life.
• • Slide 5
• Sterilization through filtration (used only on liquid or air)
• Bacteria are .2um – 2um
• Protozoa and algae are 2um – 200um
• Create filter with a pore size less than .2um
• Filtration = sterile
• • Slide 1 (handout from 10/22/07)
• Desirable qualities of chemical antimicrobial agents for decontamination in health professions
• • Rapid action at low concentrations
• • Soluble in water or alcohol
• • Destruction of MOs without harm to animal tissue
• • Penetrates surfaces
• • Resistance to inactivation by organic matter
• • Noncorrosive or nonstaining properties
• • Affordable
• • Slide 2 (list found in book)
• High activity – acts as sterilant
• Intermediate activity – acts as disinfectant
• Low activity – acts as disinfectant to an antiseptic.
• • Slide 3
• How to choose an effective chemical antimicrobial agent
• • Look at characteristics – ex. Are they spore formers or not?
• • Characteristics of surface being treated – ex. Is there organic matter that could interfere. Is the surface porous or nonporous.
• • Initial contamination amount
• • Antimicrobial exposure time
• • Strength of antimicrobial chemical
• • Slide 4
• Halogens (group 17) – non-metals that are readily ionized. They love to gain electrons. When they gain that electron they become halides (the ionized state of a halogen). If iodine is ionized it becomes iodide. The non-ionized state is the more effective antimicrobial state.
• Chlorine is an effective antimicrobial agent.
• • When mixed with water it creates hypochlorous acid which reacts with cystine amino acids (breaks up disulfide bridge)
• • Cystine amino acids have a side chain group that contains sulfur. That sulfur will bind with other cystine amino acids. When they bind they form a covalently bonded disulfide bridge.
• Insulin helps cells take up glucose. Glucose is a carbohydrate we use for energy. Break the disulfide bridge, break up the cell.
• Iodine (another example of halogen) interferes in disulfide bridges and hydrogen bonds.
• Act at intermediate activity level
• • Slide 5
• Phenol – 6 carbon sugar - hydroxel group (OH group)
• Phenolic – any molecule that contains one or more phenol groups
• • Bind to proteins and interferes with their function. They make proteins hydrophobic. Hydrophobic – don’t like water.
• Chlorohexidine is rendered useless by toothpaste as it creates a surfactant
• You can adjust disinfectants by altering their concentrations to turn them into antiseptics.
• • Slide 6
• Alcohols – contain hydrocarbon group with one or more hydroxal groups
• Alcohols act as surfactant. Integrate themselves into cell membrane because they have similar characteristics of the phospholipids. Once there they destabilize membrane and allow things in and out of the cell that shouldn’t be ther.
• Alcohols also Denature proteins
• Decontamination characteristics of alcohol will be effective at >50% concentration.
• • Most effective at 70% alcohol and 30% water
• • It needs water to make it effective. If all the water is removed the proteins will remain stable.
• Alcohol is generally considered as a disinfectant. can be used as an antiseptic but can have adverse affects if it is absorbed through the skin.

Lecture 16, 10/17 (Maybruck 1); Control of Microbial Growth

Dr. Maybruck has slide handouts outlining the lecture.
The audio cut off with about 15-20 minutes left in the lecture. But the first part can be heard here.

• SLIDE 1
• Microbial regulation of organisms that are pathogenic
• Ancient civilizations filtered water and preserved the dead using salts and oils that microorganisms don’t like.
• Epidemic – wide spread disease in a community
• We are interested in regulating pathogens that can cause harm to human health.
• Romans figured out that burning dead bodies kept disease down. Also storage of water in copper and silver kept microbe population down.
• • Slide 2
• Decontamination methods – methods employed that will remove or destroy microorganisms on a surface (including water)
• Physical decontamination method: temp extremes and radiation. mechanical methods: filtration
• Results: sterilization or disinfection (removes all vegetative microorganism)
• Chemical methods
• liquids gases and solids
• results of method: sterilization (kills living and non-living (non-living → like endospores) organisms), disinfection (removes all vegetative microorganism) can only be applied to nonliving surfaces , antisepsis (this can be applied to our skin as it will not kill everything) is usually targeted toward specific microorganisms.
• aqueous chemical decontaminant. Mix of solid or gas with water. If you mix it with a alcohol it is called tincture.
• • Slide 3
• Bacterial endospores (very resistant)
• Two phase life cycle: vegetative (metabolically active and growing) and endospore (keeps them alive a long time and helps them survive in extreme conditions)
• Examples: Bacillus, clostridum, and thermoactinomyces
• How effective is this method of survival? → very effective, these organisms can be considered essentially immortal.
• Layers of sediment is a varve which is an annually deposited layer of sediment. Microorganims will live in these layers and then be able to come back to life once they get the appropriate nutrients.
• • Slide 4
• Antimicrobial agents (physical, mechanical, chemical) fall into two categories:
• Microbicidal agents [“cide”=to kill]
• Bacteriocide, fungacide, virucide and sporicide (could be considered a sterilant)
• Microbistatic agents [-static or –stasis=to prevent growth]
• Used on living tissue, gives our body enough time to get the immune system working
• Bacteriostatic, and fungastatic
• • Slide 5
• The mode of action of antimicrobial agents. More specific=less effective --- less specific=more effective
• Cell wall target – gram positive . . . specific.
• Cell membrane target – this is a less specific way of going about it = more effective.
• Detergents (cell membrane target) - called surfactant. A surfactant is a molecule with hydrophilic and hydrophobic ends. Phospholipids bylayer in cell membrane have a hydrophilic and hydrophobic end as well. The surfactants make their way into the phospholipids bylayer and destabilizes the cell.
• • Slide 6
• The mode of action of antimicrobial agents
• Nucleic acid and protein synthesis prevention
• UV radiation – targets pyrimidine nucleic acids (RNA, DNA). Has greatest effect on DNA. Pyrimidines includes cytosine and thymine. UV radiation “loves” these two nucleotides. For the radiation to occur the pyrimidine bases HAVE to be next to each other. Once hit with UV radiation the thymines will bond to each other – called thymine dimer. This prevents replication which brings about the death of the cell.
• Antibiotic binds to ribosome. Example: Chloramphenicol (an antibiotic) binds to ribosomes in such a way that protein synthesis is inhibited. TRNA cant add amino acids to growing protein strand. Doesn’t inhibit growth of protozoans and fungus.
• • Slide 7
• The mode of action of antimicrobial agents
• Hexokinase helps attach glucose and phosphate
• Alteration in protein conformation
• If pH is altered protein will unfold – called denaturing
• • Slide 8
• Temperature as controller
• Two physical states of heat used
• Moist heat – ex. heat created from boiling water/steam. Keeps some organisms from stabilizing, this is the best way to do mass sterilization.
• Dry heat – ex. Flames used to disinfect loops in lab.
• • Slide 9
• C. botulinum – interferes with nerve connection for muscle contraction
• Practical concerns: thermal death time (TDT)
• Food canning process
• Prevents microbial contamination including spore forming C. botulinum: botulism
• TDT for low-acid foods is 121C for 30 minutes
• • Slide 10
• Sterilization with seen under pressure
• At sea level pressure 15psi will boil water at 100C
• To kill all MOs, pressure at 30 psi and 121C (standard autoclave conditions)
• As pressure is increased temperature is increased