General Microbiology Laboratory Microbial control agents Controlling Microorganisms

General Microbiology Laboratory Microbial control agents

Controlling Microorganisms v Many microorganisms are beneficial and necessary for human well-being. v However, microbial activities may have undesirable consequences, such as food spoilage and disease. v It is essential to be able to kill microorganisms or inhibit their growth to minimize their destructive effects. v Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area v Primary targets are microorganisms capable of causing infection or spoilage: § § § vegetative bacterial cells and endospores fungal hyphae and spores, yeast protozoan trophozoites and cysts worms viruses prions Mohammed laqqan

Terminology of Microbial Control v Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object. v Commercial sterilization: sufficient heat to kill Clostridium botulinum endospores (some nonpathogenic thermophilic bacteria may survive) v Disinfection: destruction of vegetative pathogens on inert substances v Antisepsis: destruction of vegetative pathogens on living tissue v Degerming: mechanical removal of microbes from limited area v Sanitization: lowering microbial counts on eating and drinking utensils to safe levels Mohammed laqqan

v. Biocide or germicide: kills microorganisms v. Fungicide: kills fungi v. Virocide: inactivates viruses § E. g. microbiocides for HIV used in spermacides v. Bacteriostatic agent: stops growth of bacteria v. Sepsis: bacterial contamination v. Asepsis: absence of significant contamination v. Aseptic technique minimizes contamination Mohammed laqqan

Microbial Death v Microbes die at a constant rate v Factors affecting how long it takes to kill bacteria § number of microbes § environment • slowed by organic materials, biofilms (e. g. , feces, sewage) • hastened by prior cleaning, heat v Factors affecting how long it takes to kill bacteria § time of exposure § characteristics of microbes: most resistant are • spores • thick lipid coats • protozoan cysts Mohammed laqqan

Actions of Microbial Control Agents v. Cell wall v. Cell membrane v. Nucleic acid synthesis v. Protein function Mohammed laqqan

Cell wall v Bacteria and fungi § Block synthesis § Degrade cellular components § Destroy or reduce stability v Agent § Penicillin, detergents, alcohols Cell membrane v All microbes and enveloped viruses § Bind and penetrate lipids § Lose selective permeability (leakage) v Agent § Surfactants Mohammed laqqan

Nucleic acid synthesis v. Irreversible bind to DNA § Stop transcription and translation § mutations v. Agent § Chemical agent – formaldehyde § Physical agent – radiation Mohammed laqqan

Protein synthesis v Binds to ribosomes § Stops translation § Prevents peptide bonds v Agent § chloramphenicol Protein function v Block protein active sites v Prevent binding to substrate v Denature protein v Agent § Physical – Heat, p. H change § Chemical – alcohols, acids, phenolics, metallic ions Mohammed laqqan

Physical Methods of Microbial Control 1. 2. 3. 4. 5. Heat – moist and dry Cold temperatures Desiccation Radiation Filtration Mohammed laqqan

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

Heat Resistance and Thermal Death v. Bacterial endospores most resistant – usually require temperatures above boiling Mohammed laqqan

Moist Heat Methods v. Steam under pressure – sterilization v. Autoclave 15 psi/121 o. C/10 -40 min v. Steam must reach surface of item being sterilized v. Item must not be heat or moisture sensitive v. Mode of action – denaturation of proteins, destruction of membranes and DNA Mohammed laqqan

Boiling Water v. Boiling at 100 o. C for 30 minutes to destroy non -spore-forming pathogens v. Disinfection Mohammed laqqan

Pasteurization v. Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value v 63°C - 66°C for 30 minutes (batch method) v 71. 6°C for 15 seconds (flash method) v. Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes Mohammed laqqan

Dry Heat Dry heat using higher temperatures than moist heat v. Incineration – flame or electric heating coil § ignites and reduces microbes and other substances v. Dry ovens – 150 -180 o. C- coagulate proteins Mohammed laqqan

Cold v. Microbiostatic – slows the growth of microbes v. Refrigeration 0 -15 o. C and freezing <0 o. C v. Used to preserve food, media and cultures Mohammed laqqan

Desiccation v. Gradual removal of water from cells, leads to metabolic inhibition v. Not effective microbial control – many cells retain ability to grow when water is reintroduced v. Lyophilization – freeze drying; preservation Mohammed laqqan

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 Mohammed laqqan

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

Filtration v. Physical removal of microbes by passing a gas or liquid through filter v. Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms Mohammed laqqan

USING ANTIMICROBIAL CHEMOTHERAPY TO CONTROL MICROORGANISMS Mohammed laqqan

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

v Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since then have come about by modifying the older forms. v 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. v 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. Mohammed laqqan

v. Some antimicrobial agents are: § cidal in action: they kill microorganisms (e. g. , penicillins, cephalosporins, streptomycin, neomycin). § 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). Mohammed laqqan

v Antimicrobial agents also vary in their 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). § 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). Mohammed laqqan

Antimicrobial Drugs v. Antibiotic Resistance - bacteria gain ability to grow § no longer sensitive to drug v Antiretroviral - act specifically against viruses v Combination of drugs: • Synergism -- action of two antibiotics greater • Antagonism -- action of drug is reduced; less effective Mohammed laqqan

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

Antibiotic Susceptibility Testing v For some microorganisms, susceptibility to chemotherapeutic agents is predictable. However, for many microorganisms (Pseudomonas, Staphylococcus aureus, and gram-negative enteric bacilli such as Escherichia coli, Serratia, Proteus, etc. ) there is no reliable way of predicting which antimicrobial agent will be effective in a given case. v This is especially true with the emergence of many antibiotic-resistant strains of bacteria. v Because of this, antibiotic susceptibility testing is often essential in order to determine which antimicrobial agent to use against a specific strain of bacterium. Mohammed laqqan

v 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) 3. Automated tests Mohammed laqqan

Tube dilution tests v 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 16 -20 hours at 35 C. 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). v Subculturing 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. Mohammed laqqan

The agar diffusion test (Bauer-Kirby test) v 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. v 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). v The MIC for each agent was then compared to the usuallyattained blood level in the patient with adequate dosage. Categories of "Resistant, " "Intermediate, " and "Susceptible" were then established. Mohammed laqqan

Procedure v a. Prepare a standard turbidity inoculum of the test bacterium so that a certain density of bacteria will be put on the plate. v Select 3 -5 isolated colonies of the bacterium that is being tested. v If the organism is a Staphylococcus or is fastidious and grows unpredictably in broth like the streptococci, suspend the colonies is saline, Mueller Hinton broth or trypticase soy broth. If the organism grows rapidly in broth, place the colonies in Mueller Hinton broth or trypticase soy broth and incubate 2 -8 hours. v Match the turbidity of the test suspension or culture with a 0. 5 Mc. Farland standard. (Mc. Farland standards are tubes containing either latex particles or barium sulfate and adjusted to a standard turbidity. ) § If the bacterial suspension is too turbid, add more saline or broth. § If the bacterial suspension is too light, pick off more colonies and suspend them in the broth. Mohammed or incubate longer. laqqan

v b. Inoculate a 150 mm Mueller-Hinton agar plate with the standardized inoculum so as to cover the entire agar surface with bacteria. v Dip a sterile swab into the previously standardized tube of the bacterium being tested. Squeeze the swab against the inner wall of the tube to remove excess liquid. v Swab the entire plate from top to bottom, edge-to-edge leaving no gaps. v Rotate the plate approximately 60 degrees and using the same swab, again swab the entire plate from top to bottom. v Rotate the plate approximately 60 degrees and using the same swab, and swab the entire plate from top to bottom a third time. Mohammed laqqan

vc. Place standardized antibiotic-containing discs on the plate. vd. Incubate the plate agar side upat 35°C for 16 -18 hours. ve. Measure the diameter of any resulting zones of inhibition in millimeters (mm) Mohammed laqqan

v f. Determine if the bacterium is susceptible, moderately susceptible, intermediate, or resistant to each antimicrobial agent using a standardized table § If there is a double zone of inhibition, measure the diameter of the innermost zone. § If there is a zone containing colonies, measure the diameter of the colony free zone. § If there is a feathered zone, measure the diameter of the point where there is an obvious demarcation between growth and no growth. § When testing swarming Proteus mirabilis, ignore the swaming. § When testing Staphylococcus aureus, the haze around an oxacillin should not be ignored. Measure the diameter of the zone free of growth or haze. Mohammed laqqan

v. The term intermediate generally means that the result is inconclusive for that drugorganism combination. v. The term moderately susceptible is usually applied to those situations where a drug may be used for infections in a particular body site, e. g. , cystitis because the drug becomes highly concentrated in the urine. Mohammed laqqan

Automated tests v. Computerized automated tests have been developed for antimicrobial susceptibility testing. These tests measure the inhibitory effect of the antimicrobial agents in a liquid medium by using light scattering to determine growth of the test organism. Results can be obtained within a few hours. v. Labs performing very large numbers of susceptibility tests frequently use the automated methods but the equipment is quite expensive. Mohammed laqqan

End of lecture Mohammed laqqan