Control of Microorganisms Bio3124 Lecture #6

Definitions sterilization destruction /removal of all viable organisms disinfection killing, inhibition, removal of pathogens disinfectants usually chemical used on inanimate objects sanitization reduction of microbial population to levels deemed safe antisepsis prevention of infection of living tissue by microorganisms antiseptics chemical agents, kill or inhibit growth of microorganisms when applied to tissues Chemotherapy: internal use chemicals to kill or inhibit microbes within host tissues

Definitions… Antimicrobials: -cidal agents: agent kills, commonly called germicides kills pathogens and many nonpathogens but not necessarily endospores include bactericides, fungicides, algicides, and virucides -static agents: agent inhibits growth include bacteriostatic and fungistatic

Microbial Death microorganisms are not killed instantly death curves are exponential Plot: log of survivors vs antimicrobial exposure time The slope: average death rate Bacterial Death Curve Survivorsx109 (CFU/ml) Survivors( log of CFU/ml) Time

Effectiveness of Antimicrobial Agent Activity Depends on: population size population composition vegetative vs dormant concentration duration of exposure longer exposure  more organisms killed temperature higher temperatures usually increase killing local environment e.g., pH, viscosity and concentration of organic matter organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents

Methods in controlling microorganisms Two major methods are used, Physical methods Heat Moist heat sterilization (autoclaves) Pasteurization Dry heat sterilization (ovens, incinerators) Low temperature (refrigeration, freezing) Filtration (for heat labile liquids) Irradiation (UV and ionizing radiation) Chemical methods Disinfectants and antiseptics (phenolics,alcohols, aldehydes, gases… etc) Chemotherapeutic agents (internal use)

Moist Heat Sterilization above 100oC , requires saturated steam under pressure (autoclave) effective against all types of microorganisms and spores degrades nucleic acids, denatures proteins, and disrupts membranes

The Autoclave or Steam Sterilizer 121°C, 15 psi (2 atm) for 20 minutes Kills all bacteria Kills endospores Clostridium botulinum Botulism Bacillus anthracis Anthrax

Pasteurization Louis Pasteur and Claude Bernard (1862) does not sterilize logarithmic reduction of germs rather than killing them all Most often ~5 log reduction; milk, beer, apple cider, fruit juice and other beverages Procedures High temperature short time: holding milk at 72 C for 15-30 seconds Ultra high temperature: exposure to ~130 C for a fraction of second Double pasteurization: 68C for 30 minutes followed by cooling and again heating at 68C for additional 30 minutes (spores germinate, killed upon entry to vegetative stage)

Dry Heat Sterilization less effective Clostridium botulinium spores killed in 2-3 hours Ovens: higher temperatures & longer exposure time (160-170oC for 2 to 3 hours) oxidizes cell constituents and denatures proteins Bench-top incinerators inoculating loops Institutional incineration

Measuring Heat-Killing Efficiency To develop standards for killing efficiency: specially important for industrial settings to develop SOPs decimal reduction time (D or D value) time required to kill 90% of microorganisms or spores in a sample at a specific temperature One log reduction

Kinetics of thermal reduction Time 106 105 104 103 100oC D100 1 log D is the time required for one log reduction (90% kill) Can be calculated using: DT= Δt log N1-logN2 Δt: total exposure time N1: initial population N2: population size after treatment # Bacteria T= applied Temperature

Example 1: calculate the D value for a bacterial suspension of 109 cfu/ml that was subjected to 85˚C for 15 minutes at which point its density was reduced to 106 cfu/ml. DT= Δt log N1-logN2 Δt: 15 minutes N1: 109 cfu/ml N2: 106 cfu/ml T= 85˚ C D85= 15 log 109-log106 D85= 15 9- 6 D85= 5 minutes

Example 2: the D90 value for a bacterium is 2 minutes. If starting culture has 108 cfu/ml, how long should this suspension be kept at 90C to kill the entire population? DT= Δt log N1-logN2 Δt: ? minutes N1: 108 cfu/ml N2: 1 cfu/ml T= 90˚ C 2= Δt log 108-log100 2= Δt 8- 0 Δt = 16 minutes

The D value: an index for sensitivity to thermal killing Which one is more sensitive to heat killing at 100˚C? Bacillus subtilis or E.coli? At 100C the time required to reduce Bacillus population is longer than that required for E.coli 106 105 104 103 100oC DE.coli DB.subtilis # Bacteria Time

The D value is temperature dependent D value decreases as the temperature increases ie. there is less time required to reduce the population by one log at higher temperatures Time 106 105 104 103 # Bacteria 120oC 110oC 100oC D110 D100 D120

Kinetics of thermal reduction: the Z value increase in temperature required to reduce D by 1/10 (one log reduction) 100 Z = ΔT log D1-logD2 10 D value (min) ΔT: Temperature change D1: initial D value D2: secondary D value 1 log 1 Z =10˚C 100 105 110 115 120 Temperature (T)

Kinetics of thermal reduction: the Z value by having D values for different temperatures one can seek for altering the sterilization protocol to fit to the industrial setting One question would be: how much the temperature can increase to reduce the D value to a given length This would provide a pragmatic approach in setting up SOPs in industrial settings

The use of Z value Example: A food processing company produces canned meat. Prevention of Clostridium botulinum spores from growing is important. The D121 for botulinum spores is 0.2 minutes and the Z value is 10˚C. the company wants to sterilize the canned food at 111˚C. what should be the length of sterilization if they consider to kill 1012 spores per can content. since every 10˚C decrease in treatment causes 10-fold increase in D value then: D111= D121x10 ie. D111= 0.2x10 = 2 minutes using, D111= Δt log1012-log100 2 = Δt 12- 0 Δt= 2x12= 24 minutes They should heat treat their product at 111˚C for 24 minutes.

Problem : try this on your own The Z value for a microorganism is 2oC. it takes 54 minutes at 75oC to reduce the population from 109 to 106. At what temperature should the microorganism be treated to achieve the same result in 10.8 sec. Answer=800C

Low Temperatures Freezing stops microbial reproduction due to lack of liquid water some microorganisms killed by ice crystal disruption of cell membranes Refrigeration slows microbial growth and reproduction Does not prevent psychrophilic microorganisms

Filtration Porous material with 0.1-0.45 um pore size reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms also used to reduce microbial populations in air

Filtration Bacillus megaterium Trapped on a nylon Membrane with 0.2 um pore size Enterococcus faecalis Trapped on a polycarbonate Membrane with 0.4 um pore size

Filtering air surgical masks cotton plugs on culture vessels high-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets laminar flow biological safety cabinet

Ultraviolet (UV) Radiation limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances has been used for water treatment Kills by inducing massive number of mutations How about escaping mutants?

Ionizing Radiation Gamma radiation from cobalt 60 is used penetrates deep into objects destroys bacterial endospores; not always effective against viruses used for sterilization of antibiotics, hormones, sutures, plastic disposable supplies, and food

Chemical Control Agents -Disinfectants and Antiseptics

Phenolics commonly used as laboratory and hospital disinfectants (2%) act by denaturing proteins and disrupting cell membranes tuberculocidal, effective in presence of organic material, and long lasting disagreeable odor and can cause skin irritation

Alcohols bactericidal, fungicidal, but not sporicidal Effective if diluted to 70% in water (95% is much less active) inactivate some enveloped viruses denature proteins and possibly dissolve membrane lipids

Halogens - Iodine skin antiseptic oxidizes cell constituents and iodinates proteins at high concentrations may kill spores skin damage, staining, and allergies can be a problem

Halogens - Chlorine oxidizes cell constituents important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant destroys vegetative bacteria and fungi, but not spores can react with organic matter to form carcinogenic compounds

Quaternary Ammonium Compounds detergents that have antimicrobial activity and are effective disinfectants organic molecules with hydrophilic and hydrophobic ends cationic detergents are effective disinfectants kill most bacteria, but not Mycobacterium tuberculosis or endospores safe and easy to use, but inactivated by hard water and soap

Aldehydes highly reactive molecules that cross link proteins sporicidal and can be used as chemical sterilants combine with and inactivate nucleic acids and proteins

Sterilizing Gases used to sterilize heat-sensitive materials EtO penetrates plastic packages Toxic, needs to be aerated microbicidal and sporicidal combine with and inactivate proteins BPL used for sterilizing vaccines Decomposes after use, but is carcinogenic

Evaluation of antimicrobial efficiency: Phenol coefficient 5 Minutes TEST 10 Minutes 5 Minutes Phenol 10 Minutes

Evaluation of antimicrobial efficiency calculation: The reciprocal of the lowest concentration of the test material that prevents the microorganism from growing over 10 minutes exposure but not at 5 minutes relative to that of phenol is considered as phenol coefficient of the test compound. In this example: PC= 320/160 PC= 2

Solution for take home problem: using formula the D75 would be, 54x60 = 54x60 9 - 6 = 54x60 3 D75= = 1080 sec log109-log106 With the same token to achieve same killing rate over 10.8 seconds the Dx should be equal to: Dx = 10.8 3 = 3.6 sec Using D1, Dx and T1 in Z expression, we can solve for T2: Z = T2 – T1 log D1-logD2 2(log 300) = T2 – 75 2x2.48 = T2 – 75 T2= 75+ 4.96 T2 = 79.96 or ~ 80 C 2 = T2 – 75 log 1080-log 3.6 NOTE: indeed this is what is done in so called ultra high temp short time pasteurization where a thin layer of milk is passed through hot plates over a few seconds 2(log 1080/3.6) = T2 – 75