Section F P2 Flashcards
which wavelength is UV radiation most germicidal
UV radiation is most germicidial at 254 nm
. This is the wavelength at which the UV radiation is absorbed by DNA and RNA causing changes in their structure and rendering them unable to replicate.
Centrifugation separates the sample into:
pellet ( sedimented material)
supernatant (liquid that may be decanted from the tube.)
Sedimentation of a sample also depends on time and centrifugal force (or gravitational force.)
Factors affecting centrifugal force
- speed of rotation, expressed in rotation per minute (rpm).
- Radius of rotation (r), distance of the particle from the centre of rotation.
HORIZONTAL or SWING ROTOR (swinging or hanging bucket)
Mainly low speed centrifuges
In high speed ultra centrifuges
Tubes rotate from a vertical to horizontal position
Buckets return to vertical as centrifuge speed decreases (decelerates)
Advantages: produces well-formed pellets, versatile, rotor – buckets easy to change.
Disadvantages: longer path length, slower speed, so longer for separation; moving parts wear with extended use
Pelllet forms at the bottom tip
Fixed Angle Rotors
Advantages:short path length, so cells pellet faster
no moving parts so no major mechanical failures – wear only.
Disadvantage: cells may be damaged by being forced against wall of tube.
Pellet a little on the side
Centrifuge Maintenance Log
detail the number of runs age of rotors date of servicing rotation speed per run rotors send out for service name of person doing the maintenance or servicing comments
Maintenance and Quality Assurance of centrifuge
Clean inside of the centrifuge daily a 1/10 dilution of hypochlorite solution or an equivalent disinfectant
Clean bowl with a germicidal disinfectant.
Autoclave rotor heads and buckets
Remove all broken glass or plastic disposed of appropriately
Hazardous materials must be capped or in sealed buckets to prevent the release aerosols.
BROKEN TUBES in the CENTRIFUGE:
Turn off centrifuge.
If in sealed buckets, go to step 7; in unsealed cups, inform others in the vicinity and leave the immediate area for at least 30 minutes to allow aerosols to settle.
Slowly open centrifuge lid, remove all broken tubes, buckets, rotors, etc to a container of disinfectant which is non-corrosive; allow to stand for a time appropriate for the disinfectant used. Some items may be autoclavable.
Place any unbroken capped container in disinfectant for 60 minutes and then remove and rinse.
Wipe down the bowl of the centrifuge twice with disinfectant, rinse with water, then air dry.
Place towels or cloth used for the wipe down into autoclave bags in the biohazard disposal area.
Remove sealed bucket to the biological safety cabinet.
Leave broken tubes in the bucket, replace the lid loosely and autoclave the entire contents or place in disinfectant (see steps 3 & 4).
partial immersion thermometer
Has a line or ridge (called an immersion ring) on the stem (76 or 35 mm up from the bulb tip).
Designed for the bulb and a portion of the stem (up to the immersion ring) to be immersed
Waterbaths
Total immersion thermometer
Does not have an immersion line.
Is designed such that the bulb and the entire stem (liquid column-not the entire length of the thermometer) are immersed or exposed to the temperature being measured
Some manufacturers inscribing TOTAL or TOTAL IMMERSION on the reverse of the thermometer
Hot air ovens
Can be used in fridges , freezers and incubators
Complete Immersion Thermometers
Entire thermometer is exposed to the medium of the temperature is being measured
Designed for inside incubators, fridges, freezers
Calibration of liquid thermometers
Usually every three months - yearly
Traditional Method: of thermometer calibration
Measure the freezing point of water
Slushy mixture of ice and water-0º C (distilled water)
Wait least 3 minutes
Insert the thermometer, to a depth sufficient to cover the 0oC (32oF) graduation (total immersion), or to the immersion line (partial immersion).
Mark the point indicated when it comes to equilibrium
Take successive readings at least one minute apart. Readings should agree within one tenth of one graduation.
or do it with boiling water - 100D
Bi- Metallic Expansion
Principle- different metals expand at different rates when heated.
A bimetallic strip consists of two different metal strips fastened edge to edge. ( example: brass and steel)
Used in Refrigerator & Freezer, household thermostats
Electrical Continuous Reading Device:
Records the temperature inside the unit (freezer, fridge)
24 hours a day, on a sheet of paper
Some Immuno-Hematology (Blood bank) fridge. Built-in chart recorder or -70 Celsius freezer
Freezer and Refrigerator - Clean with disinfectant
Daily = highly used refrigerator and freezer, door handles
Weekly =less frequent used refrigerator and freezer, door handles
Monthly = (In addition to daily or weekly), frozen and refrigerated packs/gel packs ,thermometers and temperature monitors
Annually = Coils and motors(done by outside source) Thorough cleaning of : refrigerators, freezers, walk in cooler
water bath up keep
Drain, cleaned and re-filled water bath on a weekly basis to avoid build up of salts and contamination.
Cleaned with a mild laboratory detergent using a sponge or soft cloth.
Scale build up can be removed using a mild descaler and soft brush
Disinfectants like phenolic detergents are are added to the water bath to prevent bacterial contamination
Dont use bleach, Sodium azide- explosive, strong acid or salt
DISINFECTANT
chemical or physical agent that is applied to inanimate(non-living) objects to kill microbes, Example: Bench tops
ANTISEPTIC:
A non- toxic chemical agent that is applied to living tissue to kill microbes
May not be as effective at killing microbes as disinfectants
Can be used on human or animal tissue e.g. the skin
Not good for sterilization because of their slow action on some viruses, bacterial endospores and Mycobacterium tuberculosis
Bactericidal:
An antimicrobial that kills bacteria.
Bacteriostatic:
ntimicrobial that inhibits bacterial growth but does not kill all the bacteria.
Prolonged application of bacteriostatic agents can often result in bacterial death
The more organisms present the harder it is to disinfect.It als depends on the kinds of microorganism present-Endospores,M.tuberculosis,viruses etc.
Vegetative Bacteria
bacterial cells that are in the growing phase ie they can reproduce.
Endospore:
extremely resistant dormant cell structure produced by some bacterial species. If you break down the term endospore, ‘endo-‘ means ‘inside’ and ‘-spore’ refers to the ‘dormant structure,’ so the endospore is a structure formed inside the cell. It is a non-reproductive phase.
ECHANISMS OF ACTION OF CHEMICAL DISINFECTANT AND ANTISEPTIC AGENTS
Protein denature-disrupting the Hydrogen and Disulfide bonds
Membrane disruption
Nucleic acid damage-blocks metabolism
Inhibition of metabolism-e.g. lipids
Cell wall - chemical to use
Formaldehyde
Phenols
Chlorine releasing agents
Cytoplasmic coagulation
Chlorhexidine
Glutaraldehyde
Hexachlorophene
Cell membrane: membrane potential or electron transport
Hexachlorophene Phenols Parabens Weak acids used as food preservatives such as benzoic, sorbic and proprionic acids QACs (Quaternary ammonium compounds)
Leakage of cell components
Phenols
Chlorhexidine
Alcohols
Quats (Quaternary ammonium compounds)
Nucleic acids
Alkylating agents such as ethylene oxide gas
Spore core -Bacterial endospores:
Glutaraldehyde
Formaldehyde
Spore cortex -Bacterial endospores:
Chlorine releasing agents
Glutaraldehyde
Nitrous acid
Nitrates/nitrates act as food preservatives by preventing germination of endospores
Envelopes-surrounds the capsid
Chlorine releasing agents
Quats (Quaternary ammonium compounds)
Chlorhexidine
Viral nucleic acid
Chlorine releasing agents
Capsid-protein shell
Glutaraldehyde Quats (Quaternary ammonium compounds) Chlorine releasing agents Iodine Phenols Alcohols
Cell membrane of fungus
Chlorhexidine
Alcohols
Quats (Quaternary ammonium compounds)
Cell wall of fungus
Glutaraldehyde
Nucleic acid of fungus
Acridine dyes
Surfactants:
Mild microbicidal effect
Mechanically remove organisms by breaking the oily film on the skin (emulsification)
Water soluble but are able to dissolve lipids
Increases the solubility of lipids in water solutions
Examples of surfactants are soaps and detergents
Detergents
Cationic detergents are better at killing bacteria than anionic detergents-since there is negative charge on bacterial cell walls.
Mechanically remove microorganism, but are not microcidal (meaning they do not necessarily kill the microorganism)
Effective against vegetative bacteria , some spores and viruses
Ineffective against certain species of Pseudomonas, M. tuberculosis and endospores
Example laundry powder
Quaternary ammonium compounds (quats)
cationic detergent disinfectants are quaternary ammonium compounds (quats) Effective against: 1. Vegetative gram positive bacteria 2. Lipophilic viruses only 3. Fungi
Probs with Quats Their inactivation by soaps (organic material)
Their inhibition by calcium and magnesium ions
Their inability to kill certain species of Pseudomonas, spores, hydrophilic viruses, gram negative bacteria and M. tuberculosis
Quats are now often mixed with another agent to increase their detergent/disinfectant effectiveness.
Halogens
Two types:Iodine and Chloride
Chlorine gas reacts with water to form hypochlorite ions-do not heat
Acts by denaturing microbial enzymes by binding with protein
Iodine is often employed as a tincture(dissolved in alcohol)
A tincture is an alcohol solution of a substance, in this case iodine
Less effective than chlorine
Halogens Effective against:
- Vegetable bacteria-good
- Lipophilic and hydrophilic viruses
- Some bacterial spores-60% dilution
- M. tuberculosis-60% dilution
- Some fungi
- Effective against HIV
Uses of Halogen:
Work and equipment surfaces Glassware Contact time 10-30 minutes Working solution of bleach must be made fresh daily.A 1% solution of bleach is an effective disinfectant.It should be made daily because it gets inactivated in 24 hrs,in other words the shelf life of 1% bleach is 24 hours Examples: Wescodyne , Betadine,Bleach
ALCOHOLS
It Denatures protein
Need water to penetrate cell membrane.Best effective at 70%
Contact time 30 minutes
Use for skin and equipment disinfectant
Effective against : Vegetative bacteria, Virus and lipid envelopes, HIV, and TB
Physical properties of Alcohol
Non corrosive Slow acting..may evaporate from skin before disinfection is complete Not inactivated by organic material Flammable Shelf life -one week
PHENOL AND PHENOL DERIVATIVES (PHENOLICS)
Toxic to the cell .It alters membrane permeability and denatures protein
Proportional to concentration
Low concentration inhibits but do not kill
Some bacteria inactivate phenol compounds
2. 2% needed for activity
3. Contact time 30 minutes
4. Shelf life >1 week
5. Used on floors, equipment surface and glassware
Examples: Lysol, Phiso-Hex, and some throat lozenges
Vegetative bacteria,Lipophillic viruses, Rickettsiae, Fungi, M. tuberculosis
Physical properties of Phenol
Corrosive
Leaves residue and odour
Flammable
Halogenetic phenols inactivated by organic material
Skin and eye irritant-wear eye protection
Toxic
How do Aldehydes work?
They areStrong reducing agents -They Denature proteins formaldehyde - 0.2-8.0% Glutaraldehyde - 2% Contact time = 30 minutes Shelf life >1 week
Uses: for aldehydes
Fumigation of safety cabinets in a sealed area
Disinfecting surgical equipment/instruments
Embalming
Preserving biological specimens
Preparing vaccines
Nail polish hardeners (<5%)
Several home product contain formaldehyde
Effective against Vegetative bacteria, Bacterial spores, Lipophilic and hydrophilic viruses, Fungi
Non –corrosive
Non flammable
Active in the presence of organic material
Highly toxic (avoid use where possible)
GLUTARALDEHYDE
Use to sterilize equipment, may require many hours of exposure Kills vegetative bacteria in 30 minutes Spores - 4 hours Cold sterilization -10 hours Less toxic than formaldehyde Microcidal at 7.7 pH Working Solution good for 7-10days
Examples : Cidex and Sporocidin
Ethylene oxide
Ethylene oxide is a gas that has extraordinary penetrating power; this allows it to penetrate all sorts of nooks and corners
- Sterilize after 4-12 hours of exposure
- Explosive therefore mixed with an inert gas (Freon or CO2 )
- Used to sterilize heat sensitive items such as syringes, heart valve, textile, artificial heart and lung machines
- Denatures microbial protein
Vapours are toxic to the eyes, skin and mucous membrane
Carcinogenic
High Level disinfectant
Kills vegetative microorganisms and inactivates viruses, and bacterial spores.
Contact time 10-30minutes
Intermediate level disinfectant
Kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi and inactivate most viruses
Low level disinfectant
Kills most vegetative microorganisms except M. tuberculosis, some fungi and inactivate some pores
Cleaning Process (Steps)
Dissembling Soaking Washing Chemical energy –detergent Thermal energy Mechanical energy (friction) Rinsing Drying or draining Sterilizing or disinfectant
Body tissue, organs or parts disposal
Place in a biohazard bag or in durable, leak-proof containers, color-coded and labeled with the biohazard symbol
Arrange for pick up and incineration
Lab cultures disposal
Separate all glass from plastic
Place in separate autoclave bags
Autoclave prior to disposal.
Blood, body fluids disposal
Place in autoclave bags and autoclave prior to disposal
Sharp items disposal
Place in sealed, puncture resistant, leak-proof sharps containers
Do not bend or re-cap needles
Label or color-code container.
Sterilization:
• Freeing of an article from all living organisms, including bacteria and their spores.
Methods of Sterilization:
- Heat
a. Dry heat, e.g. hot air oven
b. Flaming or red heat (incineration), e.g. Inoculating loops, scalpels, quick flaming of mouths of tubes and bottles and glass slides
c. Moist heat, e.g. Autoclave
DRY HEAT
• Dry heat is produced by an oven usually heated by electricity - minimum one hour at 160°C
• To maintain sterility after the process, articles are either , wrapped in kraft paper, placed in sterilization pouches made of kraft paper or placed in metal containers.
• Dry heat is used to sterilize :
Empty glassware – flasks, pipettes, tubes;
Instruments like scissors and surgical forceps
Powders, oils and fats (are not penetrated by moist heat)
• Dry heat acts by oxidizing or burning up bacteria.
• Controls are performed by: spore testing
INCINERATION (RED HEAT) OR FLAME
• Produced by a microbiology loop incinerator or Bunsen burner.
MOIST HEAT
Steam under pressure of 15psi is hotter than steam produced at atmospheric pressure; therefore, destruction of bacteria takes less time
The autoclave is a chamber with a tightly fitted door into which steam can flow. -• At 15 psi = 121°C
Note that it is the temperature, not the pressure that kills organisms.
Minimum conditions for sterilization
• At 121°C, it requires 15 minutes to sterilize articles in the autoclave.
• Steam destroys bacteria and their spores by coagulating and denaturing their proteins and enzymes.
NOTE: Moist heat (100°C.-boiling water-steam) at atmospheric pressure can kill vegetative bacteria, viruses and fungi, but NOT bacterial endospores.
The Autoclave is used to sterilize:
Liquids
• culture media
Unwrapped items
• Instruments
• Tubing
Wrapped packages
• Wrapping prevents recontamination after removal from autoclave
• Laboratory biohazard garbage
The sterilizing cycle:
- Pre Cycle vacuum
• Removes air from the load - Heating time
• Air is removed from the chamber and it’s load and replaced by steam
• The chamber must be uncrowned for proper - Holding time
• The load is sterilized at 121°C for a minimum of 12minutes, plus 3 mins is added for “safety time” to ensure sterilization - Cooling Time
• Steam is exhausted from the chamber
• When the load includes liquids, steam must be exhaust slowly to avoid excess boil over
• The pressure must be released to normal air pressure
• The temperature must be at 100°C before the door can be safely opened
Autoclave Cycles
- Gravity (fast) Cycle: used for bagged tools
- Liquid (slow exhaust): Used for liquids and slow steam evacuation
- Dry Cycle: Used dry goods such as glassware or plastic. Can be used for waste -20mins
- Garbage Cycle: Biohazard waste -usually 50-60 mins to assure decontamination
clean the autoclave with mild detergent NO CHLORINE
clean things with a 0.1% bleach
Types of sterilization controls:
• Autoclave tape, glass tubes with pellets that melt at 121, paper and foil strips that change color (chemical)
Advantages: Immediate information
Disadvantages: No guarantee of sterility
• Bacterial Spores (biological) -Bacillus stearothermopilus or bacillus atrophaeus for dry heat or ethylene oxide
• Processed strip placed in TSB , with colour indicator for 24 hours@ 54-58°C (56°C)
• Examined for growth/no growth
– Colour stays the same –no growth of spores
– Colour changes sterilization not OK. Spores growing
• Growth control- non-autoclaved strip placed in TSB broth
• Sealed glass ampoules -• After autoclaving an internal glass ampoule is broken, releasing the spores into the growth medium. If the autoclave destroyed the spores, the medium will remain its original color.
-If autoclaving was unsuccessful the B. sterothermophilus will grow, causing a color change during the incubation.
disadvantage - time it takes
• Temperature recording chart (physical) -• PM performed on a quarterly
Material Preparation For Autoclaving
- Inspect glassware for cracks
- Wrap or bag loose material in steam-penetrable paper
- Cover container with a loosened lid
- Containers of liquid must be a maximum of 2/3 full, with lids loosened.
- Use heat -resistant borosilicate glassware or heat resistant Teflon plastic.
- Open when the pressure gauge is “0” wait 10 mins before removing items
- Add 1-2 inches of water (no more than half full) to secondary bin to prevent bottle bottom from breaking
LD50
lethal dose 50 - cause 50% death of animal population mg/kg or g/kg
very toxic if less than 50 milligrams per kilo of weight
lc50
Concentration of substance in air that when given over a specified period of time will cause death of 50% of animal population
ppm
Exposure limit
maximum limit of exposure to an air contaminant
TLV
Threshold limit value
PEL
legal permissible exposure limit
8-hour TWA - time weighted average
concentration in air average over 8 hour shift
STEL
Short term exposure limit exposure in 15 min periods
ceiling limit
concentration amounts that should never be exceeded
common routes of entry
inhalation and skin absorption
lipid soluble are more readily absorbed not water based
A1 carcinogen
benzene
ethylene oxide and formaldahyde is A2
benzoyl peroxide
oxidizer
increase speed and intensity of fire
provide both a source of fuel and oxygen
use solutions of oxidizers instead of dry forms or dilute them
other oxidizers are sulphuric, nitric and perchloric acid`
glacial acetic acid
store with flammables seperate from oxidizers
ethers q
insidious - creep up dead over time
oxidize and form peroxides which are shock sensitive do not remove old ethers call outside company
should be stored in the dark and away from heat
perchloric acid
can build up crystals in fume hood
explode when mixes with organic matter
sodium azide
dont pour down drain
explode in heat, friction and shock
forms metal azide which explodes on contact call disposal company
if 0.1% down drain put 100X more water
microscope clean
1:16 sporicidin solution
cryostat cleaning after CJD disease
sodium hydroxide
regular alcohol not bleach
picric acid
similar to TNT when dry
store in water
