1. 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:

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

3. 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

4. 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. 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

6. 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

7. 5.1. Approaches to Control
Situational considerations: Microbial control methods depend upon situation and level of control required

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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

15. 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
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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)

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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)

26. 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

27. 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

28. 5.4. Using Other Physical Methods to Remove or Destroy Microbes

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. Classes of Germicidal Chemicals

42. 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

43. 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

44. 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

45. Contamination of an Operating Room
Contamination occurs readily
Cleaning afterwards critical

46. 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 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

47. 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