General Microbiology Laboratory Microbial control agents

Terminology of Microbial Control  Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object.  Disinfection: destruction of vegetative pathogens on inert substances  Antisepsis: destruction of vegetative pathogens on living tissue

 Biocide or germicide: kills microorganisms.  Fungicide: kills fungi.  Bacteriostatic agent: stops growth of bacteria.  Aseptic technique minimizes contamination.  Degerming: mechanical removal of microbes from limited area.

Microbial Death  Microbes die at a constant rate  Factors affecting how long it takes to kill bacteria  number of microbes  environment (slowed by organic materials, biofilm).  time of exposure  characteristics of microbes: most resistant are spores thick lipid coats protozoan cysts

Actions of Microbial Control Agents  Cell wall.  Cell membrane.  Nucleic acid synthesis.  Protein synthesis.  Protein function.

Cell wall  Bacteria and fungi.  Block synthesis.  Degrade cellular components.  Destroy or reduce stability.  Agent: Penicillin, detergents, alcohols Cell membrane  All microbes and enveloped viruses.  Bind and penetrate lipids.  Lose selective permeability (leakage).  Agent: Polymyxin B

Nucleic acid synthesis  Irreversible bind to DNA.  Stop transcription and translation.  Mutations.  Agent:  Chemical agent – formaldehyde  Physical agent – radiation

Protein synthesis  Binds to ribosome's.  Stops translation.  Prevents peptide bonds.  Agent: chloramphenicol Protein function  Block protein active sites.  Prevent binding to substrate.  Denature protein.  Agent  Physical – Heat, pH change  Chemical – alcohols, acids, phenolics, metallic ions

Physical Methods of Microbial Control 1.Heat – moist and dry 2.Cold temperatures 3.Radiation 4.Filtration

Mode of Action and Relative Effectiveness of Heat  Moist heat: lower temperatures and shorter exposure time; coagulation and denaturation of proteins  Dry heat: moderate to high temperatures; dehydration, alters protein structure.

Pasteurization  Pasteurization:  Pasteurization: heat is applied to kill potential agents of infection and spoilage without destroying the food value.  Dry ovens – o C- coagulate proteins

Radiation Ionizing radiation:Ionizing radiation: deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA,  gamma rays, X-rays, cathode rays  used to sterilize medical supplies and food products

Radiation Nonionizing radiation:Nonionizing radiation: little penetrating power must be directly exposed UV light creates thymine dimers, which interfere with replication.

USING USING ANTIMICROBIAL CHEMOTHERAPY TO CONTROL MICROORGANISMS

Antimicrobial Drugs  Chemotherapy  Antibiotics  Antimicrobial chemotherapeutic chemicals  Selective toxicity The use of drugs to treat a disease produced by a microbe that inhibits another microbe kills harmful microbes without damaging the host chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

 In fact, only 3 major groups of microorganisms have yielded useful antibiotics: the actinomycetes (filamentous, branching soil bacteria such as Streptomyces), bacteria of the genus Bacillus, and the saprophytic molds Penicillium and Cephalosporium.  To produce antibiotics,  To produce antibiotics, manufacturers inoculate large quantities of medium with carefully selected strains of the appropriate species of antibiotic-producing microorganism. After incubation, the drug is extracted from the medium and purified. Its activity is standardized and it is put into a form suitable for administration.

 Some antimicrobial agents are:  cidal in action  cidal in action: they kill microorganisms (e.g., penicillins, cephalosporins, streptomycin, neomycin). static in action  Others are static in action: they inhibit microbial growth long enough for the body's own defenses to remove the organisms (e.g., tetracyclines, erythromycin, sulfonamides).

 Antimicrobial agents also vary in their spectrum. broad-spectrum  Drugs that are effective against a variety of both gram-positive and gram-negative bacteria are said to be broad-spectrum (e.g., tetracycline, streptomycin, cephalosporins, ampicillin, sulfonamides). narrow-spectrum  Those effective against just gram-positive bacteria, just gram negative bacteria, or only a few species are termed narrow-spectrum (e.g., penicillin G, erythromycin, clindamycin, gentamicin).

Antimicrobial Drugs  Antibiotic Resistance:  Antibiotic Resistance: bacteria gain ability to grow.  Antiretroviral:  Antiretroviral: act specifically against viruses  Combination of drugs: Synergism: action of two antibiotics greater Antagonism: action of drug is reduced; less effective

Five Modes of Antimicrobial Activity 1. Injury to Plasma Membrane polymixin B 2. Inhibition of Cell Wall Synthesis penicillins, bacitracin, vancomycin 3. Inhibition of Protein Synthesis (translation) 4. Inhibition of Nucleic Acid replication & transcription 5. Inhibition of essential metabolites

 Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen: 1.Tube dilution tests 2.The agar diffusion test (Bauer-Kirby test) Antibiotic Susceptibility Testing

Tube dilution tests minimum inhibitory concentration (MIC).  In this test, a series of culture tubes are prepared, each containing a liquid medium and a different concentration of a chemotherapeutic agent. The tubes are then inoculated with the test organism and incubated for hours at 35C. After incubation, the tubes are examined for turbidity (growth). The lowest concentration of chemotherapeutic agent capable of preventing growth of the test organism is the minimum inhibitory concentration (MIC). minimum bactericidal concentration (MBC).  Sub culturing of tubes showing no turbidity into tubes containing medium but no chemotherapeutic agent can determine the minimum bactericidal concentration (MBC). MBC is the lowest concentration of the chemotherapeutic agent that results in no growth (turbidity) of the subcultures. These tests, however, are rather time consuming and expensive to perform.

The agar diffusion test (Bauer-Kirby test)  A procedure commonly used in clinical labs to determine antimicrobial susceptibility is the Bauer-Kirby disc diffusion method. In this test, the in vitro response of bacteria to a standardized antibiotic-containing disc has been correlated with the clinical response of patients given that drug.  In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test).  The MIC for each agent was then compared to the usually- attained blood level in the patient with adequate dosage. Categories of "Resistant," "Intermediate," and "Susceptible" were then established.

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