Chapter 5 Microbial growth control Physical methods Chemical agents Food Preservation

Control of microorganisms by physical and chemical agents frequently used terms Sterilization: the process by which all living cells, viable spores, virus, and viroids are either destroyed or removed from an object or habitat. Disinfection: the killing, inhibition, or removal of microorganisms that may cause disease. Sanitization: the microbial population is reduced to levels that are considered safe by public health standards. Antisepsis: the prevention of infection or sepsis -cide: kill; -static: stop germicide: kill pathogens but not necessarily endospores. Bactericide; fungicide; algicide; viricide; bacteriostatic; fungistatic

Conditions influencing the effectiveness of antimicrobial agent activity 1. Population size. 2. Population composition. Spore, young/mature 3. Concentration or intensity of an antimicrobial agent. 4. Duration of exposure. 5. Temperature. higher 6. local environment. eg. 1. Heat kill more readily at an acid pH. 2. Organic matter protect microorganisms. 3. Biofilm protection.

Microbial Growth Control Physical ways Heat sterilization (including autoclave and pasteurization) Radiation (microwaves, UV, X-rays, g-rays and electrons) Filtration (depth filter, membrane filter and nucleation track (nucleopore) filter)

Heat(terms) TDP: the lowest temperature at which a microbial suspension is killed in 10 minutes. TDT: the shortest time needed to kill all organisms in a microbial suspension at a specific temperature and under defined conditions. Decimal reduction time (D) or D value: the time required to kill 90% of the microorganisms or spores in a sample at a specified temperature. D121

Moist heat sterilization Autoclave: a device somewhat like a fancy pressure cooker. The air initially present is forced out the chamber is filled with saturated stream 121oC or 15 pounds 15-30min 1. All air must be flushed out of the chamber, or it will not reach 121oC even though it may reach pressure of 15 pounds. 2. The chamber should not be packed too tightly.

Pasteurization Milk, beer, and many other beverages are treated with controlled heating at temperatures well below boiling. Not sterilize, just pasteurized. Past : 63oC, 30min HTST: 72oC, 15sec UHT: 140-150oC, 1-3sec

Dry heat sterilization 160-170oC 2-3h Suitable for glass petri dishes and pipettes Not suitable for heat-sensitive materials like many plastic and rubber items.

Filtration Heat-sensitive solution Depth filters: diatomaceous; unglazed porcelain. Membrane filters: membranes with pores about 0.2m in diameter are used to remove most vegetative cells, but not viruses. Air: 1. Surgical masks and cotton plugs on culture vessels. 2. Biological safety cabinets.

Radiation 1. Ultraviolet radiation: 260nm is quite lethal but does not penetrate glass, dirt films, water and other substances. uv lamps: the ceilings of rooms biological safety cabinets attention! uv lamp must be off 2 . Ionizing radiation: not always as effective against viruses. Co60: cold sterilization for antibiotics, hormones, sutures.

Microbial Growth Control Chemical ways (germicides) Agents that kill organisms are often called cidal agents (bactericidal, fungicidal and viricidal agents) Agents that do not kill but only inhibit growth are called static agents (bacteriostatic, fungistatic, and viristatic agents) Disinfectants are chemicals that kill microorganisms and are used on inanimate objects Antiseptics are chemical agents that kill or inhibit growth of microorganisms and that are sufficiently nontoxic to be applied to living tissues.

Ideal disinfectant 1. Must be effective against a wide variety of infectious agents at high dilutions and in the presence of organic matter. 2. Toxic for infectious agents no toxic to people or corrosive for common materials. 3. Odorless or with a pleasant odor, soluble in water and low surface tension. 4. Relatively inexpensive

Phenolics Denaturing proteins and disrupting cell membranes. Phenol, orthocresol(邻甲酚) Advantages: 1. Tuberculocidal 2. Effective in the presence of organic material. 3. Remain active on surfaces long after application. Disadvantages: 1. Disagreeable odor 2. Cause skin irritation

alcohols Bactericidal and fungicidal, not sporicidal 70-80% ethanol and isopropanol Denature proteins and dissolve membrane lipids. 10-15min soaking

Halogens Iodine and chlorine Iodine: oxidizing cell constituents iodinating cell proteins. 2% or more iodine in a water-ethanol solution of potassium iodide. Iodophor(碘载体): iodine is complexed with an organic carries to form. water soluble, nonstaining, release iodine slowly to minimize skin burns and irritation. Chlorine: municipal water supplies swimming pools. Not spores Cl2, HClO, Ca(OCl)2, oxidation

Heavy metals Mercury (Hg), silver (Ag), arsenic(As), zinc (Zn), copper (Cu) 1% AgNO3: the eyes of infants to prevent ophthalmic gonorrhea Silver sulfadizine is used on burns. CuSO4: algicide in lakes and swimming pools.

Aldehydes Combine with nucleic acids and proteins and inactivate them, probably by cross-linking and alkylating molecules. Sporicidal Formaldehyde; 2% glutaraldehyde(戊二醛)

Sterilizing gases Ethylene oxide gas 10-20% mixed with CO2 or dichlorodifluoromethane(二氯二荧光甲烷) Relative humidity: 40-50% 38oC 5-8h or 54oC 3-4h EtO con: 700mg/liter Toxic! Removed before use.

Evaluation of antimicrobial agent effectiveness The phenol coefficient test: The test bacteria: Salmonella typhi Staphylococcus aureus a series of dilutions of phenol and the experimental disinfectant. bacteria + agent; 5min, 10min, 15min; subculture to fresh medium, respectively; incubate 2-3 days. The highest dilutions that kill the bacteria after a 10mins exposure, but not after 5mins, are used to calculate the phenol coefficient. phenol coefficient =disinfect max dilution/phenol max dilution If >1: more effective than phenol

Microbial Growth Control: Chemotherapeutic agents Chemotherapeutic agents: to be used internally for control of infectious disease, they must have selective toxicity, these include: Growth factor analog: Sulfa drugs (磺胺药) Amino acid analogs Vitamin analogs DNA base, RNA base analogs

Chemotherapeutic agents: Antibiotics Antibiotics are chemical substances produced by certain microorganisms that inhibit and kill other microorganisms, they are natural products rather than synthetic chemicals Gram-positive bacteria are usually more sensitive to Gram-negative bacteria In bacteria, the important targets of antibiotic action are the cell wall, the cytoplasmic membrane and the biosynthetic processes of protein and nucleic acid synthesis b-lactam (内酰胺) group, which includes the penicillins and related compounds, has major clinical significance

Measuring antimicrobial activity Minimum inhibitory concentration (MIC) Agar diffusion method Tube dilution technique

Range of actions of antibiotics and other agents

Mode of action of major antibiotics

Food Preservation Sterilization Low temperature (best -20oC or 80oC) pH or acidity Low water availability Canning Chemical food preservation

Questions • several concepts Conditions influencing the effectiveness of antimicrobial agent activity? What are the physical- and chemical ways to halt microbial growth? • On what sites do antibiotics act on cells? • How to preserve food?