LECTURE: Physical Methods of Control MICROBIOLOGY – ALCAMO LECTURE: Physical Methods of Control

Incident at the Pool It was July, 1988 in Los Angeles, Ca A person unintentionally defecated in a pool The pool was later used by a high school water polo team, a SCUBA diving class, and an elementary school field trip

Incident at the Pool In early August, pool users began to go to their doctor complaining of watery diarrhea, stomach cramps and fever 11 patients were tested for MO’s in their stool and it was found that they had the protozoan Cryptosporidium CDC investigators found that although chlorination was OK, the filters being used were not functioning

Incident at the Pool This shows how MO’s can spread if proper water purification techniques are not used Sterilization is the destruction or removal of all life forms Something can not be partially sterilized, it is all or nothing It is considered contaminated until sterilized Even if something is sterilized, it can still be harmful if the MO gave off a toxin

How Does Sterilization Kill Cells?? Denaturation Breaks H bonds in RNA, DNA, enzymes Coagulation of proteins (cooked eggs) Irreversible

How Does Sterilization Kill Cells?? Oxidation Large molecules break into small molecules, loss of electrons Cell walls, membranes hit first

How Does Sterilization Kill Cells?? Antimetabolite Interferes with normal cell reactions It is a molecule that is similar enough to a natural molecule used in an organism to interfere with the molecule’s function Antibiotics are antimetabolites as well as cold temperatures

Principal Methods of Sterilization Physical Agents: heat radiation filtration Aim of sterilization is destruction of bacterial spore most resistant forms of life carry on life processes at minimum rate possess enzymes to transform from dormant state

Control With Heat Heat has a killing effect on MO’s It is fast, reliable, inexpensive and does not introduce any chemicals MO’s have an optimal growth temperature Growth slows down both above and below this temperature

Effects of Heat Biochemical changes in organic molecules result in cell death Enzyme structure altered (can’t perform basic life functions) Structural molecule breakdown (cell membranes fall apart) Water loss can result in cell death Organisms depend on water Excessive heat dehydrates them

Heat Killing Rate Function of both time and temperature Each MO has: Thermal death time – time necessary for killing it at a specific temperature Thermal Death Point – the temperature at which it dies in a given time

How to Determine Time and Temperature Type of organism to be killed: To sterilize materials – direct method at bacterial spores To prepare milk – no need to sterilize, just heat to kill off vegetative cells Material to be treated Powders – use dry heat Solutions – use moist heat

Temperature High - 100˚C and above Direct flame: Loop in a Bunsen burner Incineration: Fast oxidation, kills all, +/- 1,000˚F long enough to burn up Example: disposable hospital gowns, cows that died of anthrax Cautions: Aerosols & Completeness Hot air oven: Slow oxidation, kills all, dry heat, 160˚C for 2 hours Example: powders, glass pipettes and syringes Non-burnables only

Temperature High - 100˚C and above Boiling Water: moist heat penetrates materials more quickly than dry heat lower temperatures and less time required works by denaturing proteins Not considered a sterilizing agent because can’t always be sure spores and viruses are inactivated Minimum exposure of 30 minutes

Autoclave Moist heat – pressurized steam Most dependable method for destruction of life forms When the pressure of a gas (steam) increases, the temperature of the gas increases proportionally (under 15 lbs pressure, the water temp goes up to 121-126˚C in 15 minutes) Reduces cooking time in a pressure cooker and sterilizing time in an autoclave Broadest professional application: Food, medical, labs, research

Autoclave Moist heat denatures proteins

Temperature Medium - Below 100˚C, selective kill, not sterile Pasteurization: Aimed at pathogens Reduces # of MO in a liquid and destroys organisms that could cause spoilage or disease Spores are not affected by pasteurization Fractional Sterilization (Tyndallization)- Repeated heating 3X with time for spore generation in between Holding Method - 63˚C/30 minutes Flash Pasteurization - 72˚C/15 seconds Ultrapasteurization - 82˚C/3 seconds

Heat sensitive liquids: Milk Wine Beer

. Low Temperature Refrigeration: Antimetabolite Static Measure Only – does not kill MO’s Warm up and growth recurs Food Spoilage implications 1-7˚C, Lowers MO’s enzyme reaction rates (watch out for psychrophiles) Freezing: 0˚C and Below, same as refrigeration

2. Water No water = no chemical reactions Antimetabolite Cidal or Static – Time & Organism Mycobacterium (TB) – Months Treponema (Syphilis) – Minutes Spores – Years, Decades

2. Water Desiccation = Water deprivation by drying out Osmosis = Water deprivation by chemical means

3. Radiation Electromagnetic waves that denature and kill Short: “X rays” & Gamma rays Also called ionizing radiation Penetrate objects well Human cells very sensitive Drugs, medical & dental supplies No apparent residual effects

3. Radiation

3. Radiation Long: Ultraviolet (100-400 nm) No penetration, surface only Can damage retina of eye Sterile culture rooms, O.R. No one around or special goggles

4. Sound Ultrasonic vibrations “Sonication” Limited use: High frequency sound waves When used in fluids – causes formation of microscopic bubbles or cavities and water appears to boil These cavities easily collapse and send out shock waves MO’s in fluid are disintegrated by external pressures Limited use: Dental – clean instruments Research – breaking open cells

5. Filtration Mechanical device for removing MO’s from a liquid MO’s get stuck in pores of filter and rest of fluid is decontaminated or sterile Size of unwanted particles must be known Used to purify IV solutions, Bacterial media, pharmaceuticals, beverages Types of Filters: Inorganic – porcelain or glass Organic – diatomaceous earth Membrane – cellulose HEPA – air filters

Terms Cidal – kills Static – controls Disinfectant – applied to inanimate object Antiseptic – applied to living tissue Sterilization – killing of “all” mo’s Toxin may exist