ANNOUNCEMENTS Seminar: Monday, Feb. 3rd at noon Be There February 5th – Five Year Celebration of the Legacy of Dr. Perkins. Dress to impress in Henderson Hall starting at 5pm February 7th – Founder’s Day Convocation 8am classes meet, 9:00am classes meet, 10am classes meet but let out early. 11am classes cancelled. But 1pm classes meet as scheduled along with others Class Website: http://vuumicrobiology.wordpress.com/

Chapter 05 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

A Glimpse of History British Medical Journal stated British physician Joseph Lister (1827–1912) “saved more lives by the introduction of his system than all the wars of the 19th century together had sacrificed.” Lister revolutionized surgery: introduced methods to prevent infection of wounds Impressed with Pasteur’s work, he wondered if ‘minute organisms’ might be responsible for infections Applied carbolic acid (phenol) directly onto damaged tissues, where it prevented infections Improved methods further by sterilizing instruments and maintaining clean operating environment

A Glimpse of History Until late 19th century, patients undergoing even minor surgeries were at great risk of developing fatal infections Physicians did not know their hands could pass diseases from one patient to the next Did not understand airborne microbes could infect open wounds Modern hospitals use strict procedures to avoid microbial contamination Most surgeries performed with relative safety

5.1. Approaches to Control Principles of Control Sterilization: removal of all microorganisms Sterile item is free of microbes including endospores and viruses (but does not consider prions) Disinfection: elimination of most or all pathogens Some viable microbes may remain Disinfectants used on inanimate objects May be called biocides, germicides, bactericides Antiseptics used on living tissues Pasteurization: brief heating to reduce number of spoilage organisms, destroy pathogens Foods, inanimate objects

5.1. Approaches to Control Principles of Control (continued…) Decontamination: reduce pathogens to levels considered safe to handle Sanitized: substantially reduced microbial population that meets accepted health standards Not a specific level of control Preservation: process of delaying spoilage of foods and other perishable products Adjust conditions Add bacteriostatic (growth-inhibiting) preservatives

5.1. Approaches to Control Situational considerations: Microbial control methods depend upon situation and level of control required

5.1. Approaches to Control Daily Life Washing and scrubbing with soaps and detergents achieves routing control Soap aids in mechanical removal of organisms Beneficial skin microbiota reside deeper on underlying layers of skin, hair follicles Not adversely affected by regular use Hand washing with soap and water most important step in stopping spread of many infectious diseases

5.1. Approaches to Control Hospitals Minimizing microbial population very important Danger of healthcare-associated infections Patients more susceptible to infection May undergo invasive procedures (e.g., surgery) Pathogens more likely found in hospital setting Feces, urine, respiratory droplets, bodily secretions Instruments must be sterilized to avoid introducing infection to deep tissues Prions relatively new concern; difficult to destroy

5.1. Approaches to Control Microbiology Laboratories Routinely work with microbial cultures Use rigorous methods of control Must eliminate microbial contamination to both experimental samples and environment Careful treatment both before (sterile media) and after (sterilize cultures, waste) Aseptic techniques used to prevent contamination of samples, self, laboratory CDC guidelines for labs working with microbes Biosafety levels range from BSL-1 (microbes not known to cause disease) to BSL-4 (lethal pathogens for which no vaccine or treatment exists)

5.1. Approaches to Control Food and Food Production Facilities Perishables retain quality longer when contaminating microbes destroyed, removed, inhibited Heat treatment most common and reliable mechanism Can alter flavor, appearance of products Irradiation approved to treat certain foods Chemical additives can prevent spoilage FDA regulates because of risk of toxicity Facilities must keep surfaces clean and relatively free of microbes

5.1. Approaches to Control Water Treatment Facilities Ensure drinking water free of pathogens Chlorine traditionally used to disinfect water Can react with naturally occurring chemicals Form disinfection by-products (DBPs) Some DBPs linked to long-term health risks Some organisms resistant to chemical disinfectants Cryptosporidium parvum (causes diarrhea) Regulations require facilities to minimize DBPs and C. parvum in treated water

5.2. Selection of an Antimicrobial Procedure Selection of effective procedure is complicated Ideal method does not exist Each has drawbacks and procedural parameters Choice depends on numerous factors Type and number of microbes Environmental conditions Risk of infection Composition of infected item

5.2. Selection of an Antimicrobial Procedure Type of Microorganism Multiple highly resistant microbes Bacterial endospores: only extreme heat or chemicals completely destroys Protozoan cysts and oocysts: resistant to disinfectants; excreted in feces; causes diarrheal disease if ingested Mycobacterium species: waxy cell walls makes resistant to many chemical treatments Pseudomonas species: resistant to and can actually grow in some disinfectants Naked viruses: lack lipid envelope; more resistant to disinfectants

5.2. Selection of an Antimicrobial Procedure Number of Microorganisms Time for heat, chemicals to kill affected by population size Fraction of population dies during given time interval Large population = more time Removing organisms by washing reduces time Decimal reduction time (D value) gauges commercial effectiveness Time required to kill 90% of population under specific conditions Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 108 Log decrease of 1 107 106 Logarithmic killing 105 Number of surviving cells (logarithmic scale) 104 103 Log decrease of 1 102 101 “D” “D” 1 30 60 90 120 150 Time (min)

5.2. Selection of an Antimicrobial Procedure Environmental Conditions Dirt, grease, body fluids can interfere with heat penetration, action of chemicals Important to thoroughly clean pH, temperature can influence effectiveness E.g., sodium hypochlorite (household bleach) solution can kill suspension of M. tuberculosis at 55°C in half the time as at 50°C Even more effective at low pH

5.2. Selection of an Antimicrobial Procedure Risk for Infection Medical instruments categorized according to risk for transmitting infectious agents Critical items come in contact with body tissues Must be sterile Include needles and scalpels Semicritical instruments contact mucous membranes but do not penetrate body tissues Must be free of viruses and vegetative bacteria Few endospores blocked by mucous membranes Includes endoscopes and endotracheal tubes Non-critical instruments contact unbroken skin only Low risk of transmission Countertops, stethoscopes, blood pressure cuffs

5.2. Selection of an Antimicrobial Procedure Composition of Item Some sterilization and disinfection methods inappropriate for certain items Heat inappropriate for plastics and other sensitive items Irradiation provides alternative, but damages some types of plastic Moist heat, liquid chemical disinfectants cannot be used to treat moisture-sensitive material

5.3. Using Heat to Destroy Microorganisms and Viruses Heat treatment useful for microbial control Reliable, safe, relatively fast, inexpensive, non-toxic Can be used to sterilize or disinfect Methods include moist heat, dry heat Moist heat: irreversibly denatures proteins Boiling destroys most microorganisms and viruses Does not sterilize: endospores can survive Pasteurization destroys pathogens, spoilage organisms High-temperature–short-time (HTST): most products Milk: 72°C for 15 s; ice cream: 82°C for 20 s Ultra-high-temperature (UHT): shelf-stable boxed juice and milk; known as “ultra-pasteurization” Milk: 140°C for a few seconds, then rapidly cooled

5.3. Using Heat to Destroy Microorganisms and Viruses Sterilization Using Pressurized Steam Autoclave used to sterilize using pressurized steam Increased pressure raises temperature; kills endospores Sterilization typically at 121°C and 15 psi in 15 minutes Longer for larger volumes Flash sterilization at higher temperature can be used Prions thought destroyed at 132°C for 1 hour Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Exhaust valve to remove steam after sterilization Valve to control steam to chamber Pressure gauge Safety valve Door Steam Air Jacket Thermometer Trap Pressure regulator Steam supply

5.3. Using Heat to Destroy Microorganisms and Viruses Commercial Canning Process Uses industrial-sized autoclave called retort Designed to destroy Clostridium botulinum endospores Reduce 1012 endospores to only 1 (a 12 D process) Virtually impossible to have so many endospores Critical because otherwise endospores can germinate in canned foods; cells grow in low-acid anaerobic conditions and produce botulinum toxin Canned food commercially sterile Endospores of some thermophiles may survive Usually not a concern; only grow at temperatures well above normal storage

5.3. Using Heat to Destroy Microorganisms and Viruses Dry heat Less effective than moist heat; longer times, higher temperatures necessary 200°C for 90 minutes vs. 121°C for 15 minutes Hot air ovens oxidize cell components, denature proteins Incineration a method of dry heat sterilization Oxidizes cell to ashes Used to destroy medical waste and animal carcasses Laboratory inoculation loop sterilized by flaming

5.4. Using Other Physical Methods to Remove or Destroy Microbes Some materials cannot withstand heat treatment Filtration retains bacteria Filtration of fluids used extensively Membrane filters Small pore size (0.2 µm) Thin Depth filters Thick porous filtration material (e.g., cellulose) Larger pores Electrical charges trap cells Filtration of air High-efficiency particulate air (HEPA) filters remove nearly all microbes from air Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Filter Flask Vacuum pump Sterilized fluid

5.4. Using Other Physical Methods to Remove or Destroy Microbes Radiation Electromagnetic radiation: radio waves, microwaves, visible and ultraviolet light, X rays, and gamma rays Energy travels in waves; no mass Wavelength inversely proportional to frequency High frequency has more energy than low frequency Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Wavelength (nm) 200 300 400 500 600 700 Ultraviolet (UV) light Visible light Ionizing radiation Gamma rays X rays UV Infrared Microwaves Radio waves 10–5 10–3 1 103 106 109 1012 Wavelength (nm) Increasing energy One wavelength Crest Trough Increasing wavelength

5.4. Using Other Physical Methods to Remove or Destroy Microbes Radiation (continued…) Ionizing radiation can remove electrons from atoms Destroys DNA Damages cytoplasmic membranes Reacts with O2 to produce reactive oxygen species Gamma rays and X rays important forms Used to sterilize heat-sensitive materials Generally used after packing Approved for use on foods, although consumer resistance has limited use FDA has approved for fruits, vegetables, and grains (for insect control), pork (for parasite control), and poultry, beef, lamb, and pork (for bacterial control)

5.4. Using Other Physical Methods to Remove or Destroy Microbes Radiation (continued…) Ultraviolet radiation destroys microbes directly Damages DNA Used to destroy microbes in air, water, and on surfaces Poor penetrating power Thin films or coverings can limit effect Cannot kill microbes in solids or turbid liquids Most glass and plastic block Must be carefully used since damaging to skin, eyes Microwaves kill by generated heat, not directly Microwave ovens heat food unevenly, so cells can survive

5.4. Using Other Physical Methods to Remove or Destroy Microbes High Pressure Used in pasteurization of commercial foods E.g., guacamole Avoids problems with high temperature pasteurization Employs high pressure up to 130,000 psi Destroys microbes by denaturing proteins and altering cell permeability Products maintain color, flavor associated with fresh food

5.4. Using Other Physical Methods to Remove or Destroy Microbes

5.5. Using Chemicals to Destroy Microorganisms and Viruses Potency of Germicidal Chemical Formulations Sterilants destroy all microorganisms Heat-sensitive critical instruments High-level disinfectants destroy viruses, vegetative cells Do not reliably kill endospores Semi-critical instruments Intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, fungi, and most viruses Disinfect non-critical instruments Low-level disinfectants destroy fungi, vegetative bacteria except mycobacteria, and enveloped viruses Do not kill endospores, naked viruses Disinfect furniture, floors, walls

5.5. Using Chemicals to Destroy Microorganisms and Viruses Selecting the Appropriate Germicidal Chemical Toxicity: benefits must be weighed against risk of use Activity in presence of organic material Many germicides inactivated Compatibility with material being treated Liquids cannot be used on electrical equipment Residues: can be toxic or corrosive Cost and availability Storage and stability Concentrated stock decreases storage space Environmental risk Agent may need to be neutralized before disposal

Classes of Germicidal Chemicals Alcohols 60–80% aqueous solutions of ethyl or isopropyl alcohol Kills vegetative bacteria and fungi Not reliable against endospores, some naked viruses Coagulates essential proteins (e.g., enzymes) More soluble in water; pure alcohol less effective Damage to lipid membranes Commonly used as antiseptic and disinfectant Limitations Evaporates quickly, limiting contact time Can damage rubber, some plastics, and others

Classes of Germicidal Chemicals Aldehydes Glutaraldehyde, formaldehyde, and orthophthalaldehyde Inactivates proteins and nucleic acids 2% alkaline glutaraldehyde common sterilant Immersion for 10–12 hours kills all microbial life Formaldehyde used as gas or as formalin (37% solution) Effective germicide that kills most microbes quickly Used to kill bacteria and inactivate viruses for vaccines Used to preserve specimens

Classes of Germicidal Chemicals Biguanides Chlorhexidine most effective Extensive in antiseptics Stays on skin, mucous membranes Relatively low toxicity Destroys vegetative bacteria, fungi, some enveloped viruses Common in many products: skin cream, mouthwash

Classes of Germicidal Chemicals Ethylene oxide Useful gaseous sterilant Destroys microbes including endospores and viruses Reacts with proteins Penetrates fabrics, equipment, implantable devices Pacemakers, artificial hips Useful in sterilizing heat- or moisture-sensitive items Many disposable laboratory items Petri dishes, pipettes Applied in special chamber resembling autoclave Limitations: mutagenic and potentially carcinogenic

Classes of Germicidal Chemicals Halogens: oxidize proteins, cellular components Chlorine: Destroys all microorganisms and viruses Used as disinfectant Caustic to skin and mucous membranes 1:100 dilution of household bleach effective Very low levels disinfect drinking water Cryptosporidium oocysts, Giardia cysts survive Presence of organic compounds a problem Chlorine dioxide used as disinfectant and sterilant Iodine: Kills vegetative cells, unreliable on endospores Commonly used as iodophore Iodine slowly released from carrier molecule Some Pseudomonas species can survive in stock solution

Classes of Germicidal Chemicals Metal Compounds Combine with sulfhydryl groups of enzymes, proteins High concentrations too toxic to be used medically Silver still used as disinfectant: creams, bandages Silver nitrate eyedrops were required to prevent Neisseria gonorrhoeae infections acquired during birth Antibiotics have largely replaced Compounds of mercury, tin, copper, and others once widely used as preservatives In industrial products To prevent microbial growth in recirculating cooling water Extensive use led to environmental pollution Now strictly regulated

Classes of Germicidal Chemicals Ozone O3: unstable form of oxygen Decomposes quickly, so generated on-site Powerful oxidizing agent Used as alternative to chlorine Disinfectant for drinking and wastewater

Classes of Germicidal Chemicals Peroxygens: powerful oxidizers used as sterilants Readily biodegradable, no residue Less toxic than ethylene oxide, glutaraldehyde Hydrogen peroxide: effectiveness depends on surface Aerobic cells produce enzyme catalase Breaks down H2O2 to O2, H2O More effective on inanimate object Doesn’t damage most materials Hot solutions used in food industry Vapor-phase can be used as sterilant Peracetic acid: more potent than H2O2 Effective on organic material Useful on wide range of material

Classes of Germicidal Chemicals Phenolic Compounds (Phenolics) Phenol one of earliest disinfectants Has unpleasant odor, irritates skin Phenolics kill most vegetative bacteria Mycobacterium at high concentrations Not reliable on all virus groups Destroy cytoplasmic membranes, denature proteins Wide activity range, reasonable cost, remain effective in presence of detergents and organic contaminants Leave antimicrobial residue Some sufficiently non-toxic; used in soaps, lotions Triclosan, hexachlorophene

Classes of Germicidal Chemicals Quaternary Ammonium Compounds (Quats) Cationic (positively charged) detergents Nontoxic, used to disinfect food preparation surfaces Charged hydrophilic and uncharged hydrophobic regions Reduces surface tension of liquids Aids in removal of dirt, organic matter, organisms Most household soaps, detergents are anionic But positive charge of quats attracts them to negative charge of cell surface Reacts with membrane Destroys vegetative bacteria and enveloped viruses Not effective on endospores, mycobacteria, naked viruses Pseudomonas resists, can grow in solutions

Classes of Germicidal Chemicals

5.6. Preservation of Perishable Products Chemical preservatives Food preservatives must be non-toxic for safe ingestion Weak organic acids (benzoic, sorbic, propionic) Inhibit metabolism, alter cell membrane function Control molds and bacteria in foods and cosmetics Nitrate and nitrite used in processed meats Inhibit endospore germination and vegetative cell growth Stops growth of Clostridium botulinum Higher concentrations give meats pink color Shown to be carcinogenic—form nitrosamines

5.6. Preservation of Perishable Products Low-Temperature Storage Refrigeration inhibits growth of pathogens and spoilage organisms by slowing or stopping enzyme reactions Psychrotrophs, psychrophilic organisms can still grow Freezing preserves by stopping all microbial growth Some microbial cells killed by ice crystal formation, but many survive and can grow once thawed

5.6. Preservation of Perishable Products Reducing Available Water Accomplished by salting, adding sugar, or drying food Addition of salt, sugar increases environmental solutes Causes cellular plasmolysis (water exits bacterial cells) Some bacteria grow in high salt environments Staphylococcus aureus Drying often supplemented by salting Lyophilization (freeze drying) foods Coffee, milk, meats, fruits, vegetables Drying stops microbial growth but does not reliably kill Numerous cases of salmonellosis from dried eggs

Contamination of an Operating Room Contamination occurs readily Cleaning afterwards critical

African Americans In Science: A Glimpse of History James Hildreth born in Camden, Arkansas. He was valedictorian of his high school class of 1975. He graduated in 1978 magna cum laude from Harvard University. In the fall of 1979 he enrolled at Oxford University in England. He graduated in 1982 with his Ph.D. in Immunology. He returned back to the U.S. and joined John Hopkins Medical School in Baltimore, MD. He took a one year leave of absence to do a post-doctoral fellowship in pharmacology from 1983 to 1984. He received his M.D. degree from John Hopkins in 1987 and joined the John Hopkins faculty. In 2002 he became the first African American in the 125 year history at John Hopkins School of Medicine to earn full professorship and tenure in the basic science field. His research focus is on the transmission of HIV and AIDS and how this disease enters cells via “lipid rafts.” He is currently working on a chemical based condom to help prevent the transmission of HIV to women. 46

African Americans In Science: A Glimpse of History A. Oveta Fuller born in Melbane, North Carolina on August 31, 1955 educated at the University of North Carolina, Chapel Hill were she received a Bachelor of Science in 1977, and a Ph.D. from the same school. She did postdoctoral study at the University of Chicago. She is a professor at the University of Michigan Medical School and is known for her study with herpes and other infectious diseases. She studies how they attach to the host cell and enter through the cell wall. 47