Micro Bio


Physical Methods for Controlling Microbial Growth includes exposure to….



o   Heat

o   Cold

o   Desiccation

o   Filtration

o   Osmotic pressure

o   Radiation

o   Ultrasonic waves



·         Most common method

·         Effective, economical, easy to do

·         Methods

o   Denatures proteins

o   Disrupting cell membrane and cell wall

o   Disrupting nucleic acids

·         Microbes vary in susceptibility to heat

o   Thermal death point – lowest temperature that will kill all microbes in a liquid suspension in 10 minutes

o   Thermal death time – minimum time to kill all microbes in a liquid suspension at a certain heat

o   Decimal reduction time – time to kill 90% at a set temperature

·         Heat can be moist or dry

o   Moist heat  (more efficient) kills at lower temperatures

§  Moisture better conductor of heat than air

§  Can disinfect, sanitize, or sterilize


Methods of moist heat


o   Boiling water

o   Autoclaving

o   Pasteurization

o   Ultrahigh-Temperature Sterilization

Boiling water



·         Kills bacteria, fungi, protozoa, and most viruses within 10 mins at sea level

·         Does not sterilize endospores, protozoan cysts, some viruses

·         100 deg Celsius is the highest temperature, boiling time only controllable variable

Longer times needed at higher elevations due to lower BP

Boiling water is commonly used to disinfect and sanitize



·         Application of moist heat in form of steam under pressure

·         Higher temperature under pressure than boiling

·         Reliable and recommended method for items that can withstand high temps, pressure, and moisture

·         121 degrees C  x 15 PSI x 15 mins à sterilization (KNOW!!)

·         Setup

o   Large pressurize chamber

o   Shelf

o   Sealable door

o   Pipes for introducing and evacuating steam

o   Pressure and Temperature gauges

·         All air in chamber must be replaced with steam

o   Without air evacuated à lower temps

o   Ejector valve crucial

§  Closes when 121 reached due to steam leaving

§  If prematurely closed, may not kill endospores

§  Temp and pressure must be monitored

·         Time dependent on volume of material or liquid

·         Steam must be able to come into contact

o   Permeable bags

o   Dry heat trapped in impermeable bags can produce lower temps

§  Aluminum foil example

·         Air heavier than steam

o   Steam will not displace air in upright containers with small openings

o   Tip them on side

·         PA State law requires med and dental practices to monitor with endospore sterilization indicator

Heat sensitive tape also used but doesn’t meet state requirements




·         Mild heating of beverages and food to kill pathogens and vegetative microbes causing spoilage

o   1860 – Louis Pasteur developed for wine

·         1930 milk pasteurization lowered TB in US

·         3 Methods

o   Heat to 63 for 30 min

o   High temp short time – 72 for 15 secs through tubes

§  Improves quality of milk without compromising pasteurization

o   Ultra-high temp – 134 for 1 sec through tubes

§  Affect taste and quality

§  Rarely used

·         Kills most vegetative bacteria, fungal spores, yeasts, enveloped and non- enveloped viruses, protozoan cysts and trophozoites, worm eggs & worm larvae

o   Can keep at 0-5 for days because it kills most except endospores, viruses, and thermophiles

Thermophiles don’t spoil food cause they don’t grow at or below room temp

   Ultrahigh-Temperature Sterilization


§  140 for 1-3 seconds with rapid cooling

§  Sterilized and kept indefinitely without spoiling

§  Degradation still occurs with time

Dry Heat Sterilization



·         Less efficient

·         Requires longer times and higher temps

·         Minimum

o   160 for 2 hours or 171 for 1 hour

§  Too high temp for paper, rubber, and plastics

·         Sterilizes powders, oils, and other materials not able to be sterilized by steam

Also direct flaming for wire loops or incineration

  Refrigeration and Freezing



·         Effects of cold depends on microbe and time

·         Refrigeration 0-5 degrees C inhibits growth of most microbes by slowing metabolism but doesn’t kill

o   Psychotrophs grow slowly at low temps and cause refrigerator spoilage

·         Most don’t grow

o   Exceptions

§  Listeria monocytogenes – food contaminant

§  Yersinia – blood contaminant

·         Freezing inhibits the growth of microbes by making water unavailable as ice and slowing metabolism

o   Freezing kills some cells by rupturing membrane

o   Many survive and resume when thawed

o   Freezing to -20 for 7 days kills tapeworms and roundworm larvae

o   Fish and squid

§  Infected by roundworm anasakids

§  Migrate to muscle at refrigerator temps

§  Killed by -20 for 7 days or -35 for 1 day

§  Raw fish must be frozen before sold because of sushi

o   Raw Pork

§  Contains Trichinella larvae and should never be eaten raw or undercooked

·         Killed by -20 for 7 days

Lunchmeats made with raw port like salami must be frozen first

Dessication and Lyophilization



·         Dessication inhibits microbial growth because metabolism requires water

·         Molds more tolerant of low moisture and spoil jerky, fruits, grains

·         Most microbes not killed by desiccation, some highly resistant

o   Mycobacterium tuberculosis can survive desiccation at room temp for months

o   Most viruses resistant to desiccation

·         Some microbes sensitive to dessication

o   Gonococci survive less than one hour on environmental surfaces

§  Hard to get gonorrhea from a toilet seat

·         Organic matter can protect microbes and enhance survival times

·         Lyophilization (freeze drying) is removal of water from frozen solution by sublimation under vacuum

Does not harm microbes and preserves them




§  Liquids and air sterilized by small pore filters

§  .2 um filter removes microbial cells from liquid media unstable to heat

·         Sterilizes media – all cells removed

·         Viruses do get through at times

·         .01 um remove viruses and prions but clog easily

§  HEPA used in hospitals for high risk patients and operating rooms


Osmotic pressure



§  Salt and sugar preserves foods (meat, fish, fruit, etc)

§  Creates hypertonic environment that draws out water

·         Plasmolysis – shrinkage of cytoplasm

·         Prevents cell growth, but doesn’t kill

§  Fungi grow more readily than bacteria in such environments and spoils jelly, ham, fruit, beef jerky





·         Release of high speed subatomic particles or waves of electromagnetic energy

o   Particulate radiation

o   Electromagnetic radiation – given off as waves by decomposing atoms and travels at speed of light

·         Depends on wavelength, intensity, duration

o   Some harmless (visible light)

o   Other kills

§  Ionizing

·         High energy radiation that creates radicals

o   Damage DNA and cell components

·         Produced by

o   Electromagnetic waves — Gamma rays and X-rays (less than 1nm wavelengths)

o   Particulate radiation — Electron beams

·         Electron beams have high energy and sterilize quickly but penetrate power is low

o   Sterilize disposable tissue culture and med supplies but not thick objects

·         Gamma rays and X-rays have high energy and penetrate deeply

o   Require hours to kill microbes

o   Can preserve food but public has been reluctant to embrace

§  Nonionizing

·         Longer than 1 nm

·         UV Light, visible light, infrared waves, microwaves, radiowaves

·         UV only non-ionizing electromagnetic radiation that is microcidal

o   Between 40 and 310 nm

o   Kills at 200-295nm by altering DNA bases

§  Thymine dimers

o   Maximal killing at 260 nm which is light absorption optimum of DNA

o   Disinfects things that come in direct contact with it

§  Disinfects air and uncovered nonporous surfaces in operating rooms, food cafeterias

o   Low penetrating power

§  Doesn’t penetrate plastics, paper, cellophane, cannot sterilize wrapped or covered materials

o   Harmful to humans

§  Sunburns, skin cancer, damage to retinas

§  Other Non-ionizing radiation

·         Sunlight has some antimicrobial effect

o   Forms singlet oxygen

o   Ozone screens out ionizing radiation

·         Microwaves

o   Don’t directly damage microbes

o   Kills vegetative cells by heating water in cytoplasm of cells

Too inefficient and unreliable for sterilization

  Ultrasonic Waves



·         Effective and recommended way to clean soiled instruments before sterilization

·         Instruments placed in detergent solution and ultrasonic bath that generates ultrasonic sound waves

The ultrasonic sound waves creates high energy cavitations that collapse on the surface and dislodge adherent material

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