Bio 105 Lecture 6
A few definitions of microbial control: Sterilization
Sterilization: destruction of all microbes on an object
Aseptic Definition?
Aseptic: an environment free of contamination from pathogens
Disinfection Definition?
Disinfection: the use of physical or chemical agents to inhibit or destroy most microbes on inanimate objects
• Sanitization is disinfection of objects to meet public safety standards
Antisepsis Definition?
Antisepsis: chemical disinfection of living tissues with agents
Degerming Definition?
Degerming: removal of microbes from tissue by mechanical means
Cidal Definition?
-Cidal: an agent that kills microbes
Static Definition?
-Static: an agent that stops microbial growth
Mechanisms of Antimicrobial Agents Are?
– Cell wall damage: can cause osmotic pressure to burst cells or prevent viral attachment
– Cytoplasmic membrane damage: contents will leak out – Protein damage: denatured enzymes are non-
functional
– DNA/RNA damage: fatal mutants, non-functional mRNAs or ribosomes
Susceptibilities of microbes to antimicrobial agents Most Resistance?
Prions, Bacterial Endospores, Mycobacteria, Cysts of Protozoa, Active Stage Protozoas (Trophozoites)
Susceptibilities of microbes to antimicrobial agents Most Susceptible?
Most Gram-Negative Bacteria, Fungi, Nonenveloped Viruses, Most Gram-Positive Bacteria, Enveloped Viruses
The Microbial Death Curve is Also?
Logarithmic
– Decimal reduction time (D-value): time to kill 90% of microbes
Physical Control Methods Include
Extreme Heat
The thermal death point is the
lowest temperature required to kill all organisms in a sample in ten minutes
Thermal Death Time Does What?
measures time to kill all organisms at a set temperature
What Temperature is Thermal Death Point?
20 degrees Celcius
Physical control: moist heat
– Moist heat is more effective than dry heat!
– Method 1: boiling (intermediate disinfection)
• Kills vegetative bacteria, fungi, trophozoites, most viruses
– Excludes protozoan cysts, endospores, some viruses: not sterilization!
• 100° C maximum: useful for sanitizing tableware, baby bottles
Moist heat cont’d. Method 2
Method 2: autoclaving (sterilization)
• Uses pressure to increase effective temperature of steam:
15 psi = 121° C boiling temperature for 15 min. • Objects must be exposed to the steam!
Autoclaves Need?
Regular Maintenance and Checks to Ensure Functionality...
– Biological indicators include endospores that are autoclaved to ensure sterilization (spore test)
Yellow Medium Means?
Spores are viable autoclaved objects are not sterile
Red Medium Means?
Spores were killed autocalved objects are sterile
After Autoclaving, Flexible Vial Is?
Is squeezed to break ampule and release medium onto spore strip
Physical control methods: heat cont’d. Method 3
Method 3: pasteurization (low-level disinfection)
• Milk and other food products are heated to a point where
they destroy most spoiling bacteria
• Modern flash pasteurization heats milk to 72° C for 15 s
Physical control methods: heat cont’d.
Method 4
Method 4: dry heat (sterilization)
• Requires higher temperatures for sterilization (eg. 220° C
for 5 hours)
– Only useful for very heat resistant items
• Ovens for glass, ceramics and metals
• Incineration (bunsen burner) for metal tools
Physical Control Methods:cold
Refrigeration decreases metabolism of microbes, unless they’re psychrophiles like Listeria
• Slow freezing more effective than ‘flash’ freezing
Physical Control Methods: Drying
Drying (dessicating) foods removes water necessary for microbial growth
– Freeze-drying (lyophilization) is used to store cultures long term
Physical Control Methods Cold and Drying Are?
These methods are bacteriostatic (prevent microbial growth): not disinfecting or sterilizing
Physical Control Methods: Osmotic Pressure
– High concentrations of salt or sugar in foods to inhibit microbial growth
• Cells in hypertonic solution of salt or sugar lose water – Fungi have greater ability than bacteria to survive
hypertonic environments
Physical Control Methods:
Radiation Are?
Materials are sterilized with ionizing radiation, or disinfected with non- ionizing radiation
• Gamma rays or X-rays commonly used for ionizing radiation
• UV light used for non- ionizing
Physical control methods: membrane filtration (sterilization) Is?
– Useful for sterilizing larger volumes that can’t be autoclaved
– Filter must have appropriate pore size (≤ 0.22 μm)
Physical control methods:air filtration(sterilization) Is?
– Biological safety cabinets can keep a surface sterile protecting both sample and workers
• Requires HEPA filters (also dependent on pore size!)
What Are Germicides?
Germicides are chemical agents classified by their
relative abilities to disinfect objects
– High-level: kills almost all pathogens (excludes some
endospores)
• Typically can’t use on environmental surfaces: too reactive and/or toxic
– Intermediate-level: kills fungal spores, protozoan cysts, vegetative bacteria (excludes endospores)
– Low-level: kills vegetative bacteria, fungi, protozoa, some viruses (excludes naked viruses, mycobacteria, cysts, endospores)
What are we looking for in an ideal germicide?
– Cheap cost
– Odorless
– Easy to use
– ‘Residual effect’ on treated items/surfaces
– Non-damaging to treated items/surfaces
– Not affected by bioburden (number of microbes)
– Broad-spectrum (affects a large variety of microbes)
– Short contact time (chemical equivalent of TDP) • Pay attention to manufacturer's guidelines!
Chemical Methods: Surfactants (low) Are?
– Surfactant = ‘surface active’: main action is degerming by dissolving microbes in surfactant before washing away
• Mostly bacteriostatic
– Includes soaps and synthetic detergents
• ‘Quat’ detergents disrupt cellular membranes – Rule #1 of nursing club: wash your hands
• Contact time is important: 15-30s
Chemical Methods: Heavy Metals (low) Are?
Includes arsenic, zinc, mercury, copper, silver
• Major method of purifying water in ancient times • Still used in amalgam fillings, surgical dressings
• Main action is to denature proteins: bacteriostatic
Chemical Methods: Phenolics (intermediate) Are?
Denature proteins and disrupt membranes: bactericidal • Traditionally used for antisepsis during surgery
• Commonly found in antimicrobial soaps
• Remain active for prolonged time, but smell bad and irritate skin
Chemical Methods: Halogens (intermediate) Are?
Includes reactive elements that denature proteins: bacteriostatic or bactericidal
• Chlorine/bromine/fluoride used for water treatment
• Sodium hypochlorite (bleach) is a high-level germicide when
concentrated, but very corrosive/toxic
• Iodophors used to prepare skin for surgery
• All are relatively long lasting, but toxic at high concentrations
Chemical Methods: Alcohols (intermediate) Are?
Denature proteins and disrupts cytoplasmic membranes quickly (bactericidal), but evaporate quickly as well
• Only effective diluted in water (typically 70% alcohol) – Necessary for appropriate contact time
• Appropriate for hand disinfection
• Doesn’t deal with bioburden well, flammable
Chemical Methods: Aklylating Agents (High)
-Highly reactive, destroy proteins and DNA: bactericidal
– Non-corrosive to most materials
– 2% glutaraldehyde - short contact time (10 min):
disinfectant, long contact time (10 h): sterilizer • Toxic and carcinogenic!
Chemical Methods:Peroxygens (Intermediate to High) Are?
– These are ROS chemicals that oxidize microbial cell components: bactericidal
– Ozone (O3) is an intermediate-level water treatment
– Concentrated hydrogen peroxide (H2O2) and peracetic acid are effective high-level item disinfectants
• Peroxides are corrosive to metals!
• Peracetic acid is a very useful sporicide (endospore killer)
In-Use Tests Are Used To?
Gauge a disinfectant’s utility in the clinic
– After using a disinfectant on a surface, it’s incubated in media with an inactivator present