MMG 301 Dr. Frank DazzoControl of Microbial Growth: Chemical Antimicrobials3. Antibiotic disc agar diffusion method: The test organism is spread on the culture medium in an agar plate and then sterile antibiotic discs are applied. After incubation, the organism produces a confluent “lawn” of growth except in zones of inhibitionThe sulfa drug, sulfanilamide, is an analog of p-aminobenzoic acid, and inhibits the biosynthetic pathway that uses p-ABA as an intermediate in the synthesis of the vitamin, folic acid. Bacteria synthesize folic acid, humans don’t ( we must obtain it inMMG 301 Dr. Frank DazzoControl of Microbial Growth: Chemical Antimicrobials• Previously described physical factors (e.g., temperature) affecting microbialgrowth. Here focus on chemical antimicrobials affecting microbial growth.• Two main categories of antimicrobial agents:• non-chemotherapeutic agents: (e.g., antiseptics, disinfectants); used toinhibit or kill microbes on living sufaces (topically) or inanimate objects,but are too toxic for internal use inside the human body.• chemotherapeutic agents: (growth factor analogs, antibiotics) can beused internally to control microbes that cause infectious diseases.• Based on degree of selective toxicity to the human or domestic animal host.• Some Important (suffix) Terminology:• “-static”: antimicrobials that inhibit microbial growth without killing them,e.g., bacteriostatic, fungistatic.• “-cidal”: antimicrobials that kill microbes, e.g., bacteriocidal, fungicidal.• “-lytic”: antimicrobials that kill microbes by lysing them, e.g., bacteriolytic.Distinguish these 3 different categories by examining the effect of theantimicrobial agent on a growing bacterial culture:3 types of action of antimicrobialagents. At the time indicated by thearrow, a growth-inhibitoryconcentration of the agent wasadded to the exponentially growingculture. Note that the bacteriostaticagent inhibits growth without killingcells, bacteriocidal and bacteriolyticagents kill. Cell # decline when lysed.Many factors influence the effectiveness of an antimicrobial agent:• microbial population size• microbial population and community composition• microbial encasement within surface biofilms• concentration of the antimicrobial agent; may be “-static” at certainconcentrations and “-cidal” at higher concentrations• duration of exposure of the microorganisms to antimicrobial agent• temperature of treatment• presence of other organic materials• level of microbial resistance to the antimicrobial agent• other characteristics of the local environment.Various ways to measure antimicrobial activity:1. Tube dilution assay to detect the minimal inhibitory concentration (MIC).A series of increasing concentrations of the antimicrobial agent areprepared in the culture broth medium. Each tube is equally inoculated andincubated to allow microbial growth to proceed. Growth (turbidity) occursin those tubes containing the antimicrobial at concentrations below theMIC. Tube cultures are non-turbid (clear, no growth) at MIC and higherconcentrations of the antimicrobial agent.2. Phenol coefficient test: standardizes the potency of bacteriocidalagents in comparison with phenol using specific test microorganisms.1. A series of dilutions of phenol and the experimental antimicrobialagent are prepared in sterile tubes.2. Viable test bacteria (Staphylococcus aureus and Salmonella typhi)are inoculated into each tube and incubated at 37 °°°°C.3. After 10 minutes, samples of inoculated tubes are transferred intofresh culture medium without disinfectant and incubated for severalmore days.4. The broth culture tubes are evaluated for growth by turbidity.5. The highest dilutions of the test antimicrobial agent and of phenolthat effectively kill the test organisms within 10 minute exposure areused to compute the phenol coefficient as follows:1 / [Effective dilution of test antimicrobial agent]1/ [Effective dilution of phenol] Example: If the highest effective dilution of the test antimicrobial =1/500 and that of phenol = 1/100, the phenol coefficient of the testantimicrobial = 500/100 = 5.Agents with a phenol coefficient > 1 are more effective than phenol.The higher the phenol coefficient value, the more effective the testantimicrobial is in killing bacteria under the test conditions.Note that phenol coefficient tests are only valid for bacteriocidal (notbacteriostatic) antimicrobials, since cells inhibited (but not killed) bythe latter would resume growth when transferred to fresh medium.3. Antibiotic disc agar diffusion method: The test organism is spreadon the culture medium in an agar plate and then sterile antibiotic discsare applied. After incubation, the organism produces a confluent“lawn” of growth except in zones of inhibition around discscontaining antibiotics to which the organism is susceptible.Non-chemotherapeutic antimicrobials: antiseptics and disinfectants:(too toxic for internal use)• Antiseptics are antimicrobial agents that are sufficiently nontoxic tobe useful in decontaminating the surface of living tissues.• Disinfectants (also called “germicides”) are antimicrobial agentsthat are too toxic even for topical use on human tissue but can beused to decontaminate or sterilize nonliving materials.Agar diffusion of antibioticsproducing zones ofinhibition around discs onplate cultures containing aconfluent lawn of growth.• Major use of disinfectants in the water purification industry: usechlorine to kill human fecal pathogens in the potable water supply• Disinfectants are commonly used in many industrial applications,which can lead to toxic waste problems. Examples:1. Paper-pulp industry: organic mercurials & phenols are used toprevent microbial growth during manufacture2. Textile industry uses heavy metals & phenols to prevent microbialdeterioration of fabrics exposed to the outside environment3. Wood industry uses phenols to prevent deterioration of wood in theoutside environment4. Petroleum industry uses mercurials, phenols, & cationic detergentsto prevent bacterial growth during recovery and storage ofpetroleum and petroleum products5. Electrical power industry uses chlorine to prevent bacterial growthin condensers and cooling towers6. Nuclear power industry uses chlorine in nuclear reactors to preventgrowth of radiation-resistant bacteria (Deinococcus radiodurans).Chemotherapeutic Antimicrobials: compounds
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