Sepsis

(2)

• Greek word for decay or putrid 
• Indicates bacterial contamination

Asepsis

(1)

• Absence of significant contamination

• are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery

• The process of freeing an article from microorganisms including their spores

• Reducing the number of pathogenic microorganisms to the point where they no longer cause disease(s)

Bacteriostatic Agent

(2)

• An agent that inhibits bacteria growth
• Most agents do not kill endospores

Bactericide

(2)

• An agent that kills bacteria
• Most do not kill endospores

Sporicide

(1)

• An agent that kills spores

• process where all microorganisms are destroyed in the final container

Aseptic technique

(4)

• process whereby personnel manipulate
• sterile preparations,
• sterile packaging components and
• sterile administration devices in a manner that excludes the introduction of microorganisms

Pyrogen

(6)

• “fever producing”
• Usually caused by bacterial organisms
• Endotoxins and Exotoxins
• e.g., LPS – Lipopolysaccharides
• Can also be caused by contaminants
• paper fibers, etc

Depyrogenation

(6)

• process whereby all pryogens are removed or inactivated from a product
• Many methods
• distillation,
• chromatography,
• chemical,
• heat

Pyrogen detection

Methods

(6)

• Two methods
• Rabbit Test – use just as it sounds
  rabbits have responses to endotoxins similar to humans
• LAL Test – Limulus Amebocyte Lysate (LAL)
• Amebocyte enzyme from horseshoe crab will form blood clots when exposed to endotoxins
• Commerical test kits available to perform LAL testing
• Negative LAL test indicates very low probability (but not zero) for endotoxins

Methods of Sterilization

(3)

• Physical sterilization
• Chemical sterilization
• Mechanical removal

Physical Sterilization

(2)

• Heat
• Radiation

Physical Sterilization

Heat

(9)

• Dry Heat
• Incineration
• Drying Oven
• Moist Heat
• Steam under pressure
• Sterilizing
• Boiling/Hot water
• Pasteurization
• Disinfecting

Physical Sterilization

Radiation

(7)

• Ionizing
• Xray,
• gamma ray
• sterilizing
• Non-ionizing
• Ultraviolet (UV)
• disinfecting

Physical Sterilization

Heat Sterilization

(5)

• Denaturation of cellular macromolecules -> death
• Death = exponential process
• Sterilization requires lower temps. for a longer period of time
• Moist heat is better than dry heat for sterilization
• Thermal death time (TDT): time to kill all cells of a certain pathogen

Physical Sterilization

Endospores

(3)

• Heat sterilization procedures are designed to destroy endospores
• A major factor in the resistance of endospores is the amount and state of water they contain (minimal amounts and becomes gel-like)
• Only 0.5 min and 65°C required at 15 psi to kill vegetative cells

Physical Sterilization

Steam Sterilization

(5)

• Steam is lighter than air
• Inlet should be on top of autoclave
• Newer autoclaves use a vacuum pump to remove air from the chamber – pulsed vacuum or dynamic vacuum
• Must be space between objects
• No air pockets

Physical Sterilization

Steam Under Pressure (Autoclave)

(7)

• Mechanism - coagulation of protein
• Moist heat coagulates cellular protein at a lower temperature as compared to dry heat
• Heat capacity
• 540 cal/gm at 1000C
• 524 cal/gm at 1210C
• Dry heat 1cal/gm
• Requires constant supply of steam

Physical Sterilization

Autoclave v. Pasteurization

(8)

• Autoclave
• Uses high temp and high pressure.
• 121°C, 15 psi, 15 min
• Pasteurization
• Reduces microbes in milk and other heat-sensitive foods
•  sterilization b/c not all org. are killed
• Prevents spread of pathogens and food spoilage
• 71°C, 15 sec

Physical Sterilization

Dry Heat Sterilization

(6)

• Kills by oxidation effects
• The oven utilizes dry heat to sterilize articles
• Operated between 50oC to 250/300oC
• A holding period of 160oC for 1 hr is desirable
• There is a thermostat controlling the temperature
• Double walled insulation keeps the heat in and conserves energy

Physical Sterilization

Dry Heat Sterilization

Advantage and Application

(11)

• Requires a longer exposure time @1210C it would take 84 hours!
• Major advantage -
• it can rid glassware of pyrogens.
• Requires a much higher temp.
• 180°C/45-60 minutes, 160°C/2 -3hours
• Application
• glassware used in compounding
• non-aqueous products –
• powders,
• oils,
• petrolatum

Physical Sterilization

Examples of dry heat sterilized products

(4)

• glycerin for injection
• petrolatum for use in eye ointments
• progesterone in oil injection
• talc

Physical Sterilization

Weakness

(3)

• Weakness is the aseptic final assembly
• rubber stoppers would burn up in oven
• must aseptically assemble in LAF

Physical Sterilization

Dry Heat Sterilization

MOA, Sterilization Times

(8)

• MOA is
• by oxidizing microorganisms
• Sterilization Times
• 1600 C for 120-180 minutes
• 1800 C for 45-60 minutes
• De-pyrogenation of glassware times
• 2300 C for 60-90 minutes
• 2500 C for 30-60 minutes

Physical Sterilization

Radiation Sterilization

(2)

• Ionizing radiation (ex. x-rays)
• Nonionizing radiation (UV light)

Physical Sterilization

Radiation Sterilization
Ionizing radiation (ex. x-rays)

(3)

• produces reactive molecules, which degrade DNA.
• Ionizing radiation also directly causes breaks in DNA.
• Used for sterilization and decontamination in the medical supplies and food industries

Physical Sterilization
Radiation Sterilization

Nonionizing Radiation (UV light)

(1)

• germicidal and used to sterilize exposed surfaces, but cannot penetrate deeper

Physical Sterilization

Ultrasonic and Sonic Vibration

(5)

• High frequency sound waves (>20k Hz)
• Killing mechanism is by heat generation
• Not very effective at heat/energy transmission
• Microorganisms vary in their sensitivity
• Not very reliable for pharmaceutical sterilization

Chemical Sterilization

How act and forms

(8)

• Chemical agents act by
• Protein coagulation
• Disruption of the cell membrane
• Removal of Sulfhydryl groups
• Substrate competition
• Two forms:
• Liquid chemicals
• Gas chemicals

Liquid Chemical Sterilization

Alcohols

• Ethanol /Isopropyl alcohol are frequently used
• No action on spores
• Concentration recommended 60-90% in water – per USP <797> … 70% IPA
• Uses
• Disinfection of thermometer/medical device
• Disinfection of the skin – Venipuncture

Liquid Chemical Sterilization

Aldehydes

(3)

• Formaldehyde & Glutaraldehyde are frequently used
• Formaldehyde is bactericidal, sporicidal & has a lethal effect on viruses.
• Glutaraldehyde is effective against Tubercle bacilli, fungi and viruses

Liquid Chemical Sterilization

Phenols

(4)

• Obtained by distillation of coal tar
• Phenols are powerful anti-microbial substances
• Phenolic derivatives have been widely used as disinfectants for various purposes in hospitals
• Examples: Lysol, cresol

Gas Chemical Sterilization

(2)

• Specially used for sterilizing heart-lung machines, respirators, sutures, dental equipment, books and clothing
• Also used to sterilize glass, metal and paper surfaces, plastics, oils, some foods and tobacco

Gas Chemical Sterilization

Ethylene Oxide

(2)

• Colorless, highly penetrating gas, has a sweet odor
• Effective against all types of micro-organisms including viruses and spores

Mechanical Removal

(9)

• Membrane Sterilization
• May be used for terminal sterilization
• May be used to reduce bioburden prior to
  terminal sterilization
• Eg – filter large volume parenterals prior to autoclaving
• Reduces endotoxin formation
• Uses filters
• Two types
• Depth filters
• Membrane (surface) filters

Filtration Sterilization

(2)

• Removes organisms rather than killing
• Risk of contamination is greater than with
  terminal sterilization but less than aseptic
  final assembly

Filter Sterilization

(3)

• Depth filter
• Membrane filter
• Nucleopore filter

Filter Sterilization

Depth filter

(2)

• fibrous mat that traps particles in paths created
  throughout the depth of the filter;
• often used as prefilters

Filter Sterilization

Membrane filter

(2)

• functions more like a sieve, trapping many particles on the filter surface, size of the holes and molecules
  passing through are precisely controlled,
• used to sterilize liquids

Filter Sterilization

Nucleopore filter

(2)

• sizes of the holes is precisely controlled,
• used in EM to remove and concentrate a sample from a liquid

Filter Sterilization

(5)

• Most filters are designed for pressure filtration.
• The membrane is not supported on the upstream side
• Bubble point test the filter when finished
• Fill syringe with air, re-attach, push down
• Large filters, put the distal end in a beaker of water and watch for bubbles

Filter Sterilization

Pore sizes

(7)

• Standard sizes for sterilization
• 1.2 micron, 0.8 micron, 0.45 micron, 0.22 micron
• 0.22 micron is considered antibacterial
• Does not remove virus or spores
• chances of the smaller pores being filled up first
• larger pores are left which increases the chance of contamination
• select products for sterility and pyrogen tests from the last ones filled

Aseptic Processing

(5)

• Certain pharmaceutical products must be sterile
• injections, ophthalmic preparations, irrigation solutions, hemodialysis solutions
• Two categories of sterile products
• those that can be sterilized in final container (terminally sterilized)
• those that cannot be terminally sterilized and must be aseptically prepared

Aseptic Processing

Objective

(2)

• Objective is to maintain the sterility of a product, assembled from sterile components
• Operating conditions so as to prevent microbial contamination