1. Control of Microorganisms
Definitions
Conditions Influencing Antimicrobial Activity
Physical Methods
Chemical Agents
Preservation of Microbial Cultures

2. Definitions
Sterilization: A treatment that kills or removes all living cells, including viruses and spores, from a substance or object
Disinfection: A treatment that reduces the total number of microbes on an object or surface, but does not necessarily remove or kill all of the microbes

3. Definitions
Sanitation: Reduction of the microbial population to levels considered safe by public health standards
Antiseptic: A mild disinfectant agent suitable for use on skin surfaces
-cidal: A suffix meaning that “the agent kills.” For example, a bacteriocidal agent kills bacteria

4. Definitions
-static: A suffix that means “the agent inhibits growth.” For example, a fungistatic agent inhibits the growth of fungi, but doesn’t necessarily kill it.

5. Conditions Influencing Antimicrobial Activity
Under most circumstances, a microbial population is not killed instantly by an agent but instead over a period of time
The death of the population over time is exponential, similar to the growth during log phase

6. Conditions Influencing Antimicrobial Activity
Several critical factors play key roles in determining the effectiveness of an antimicrobial agent, including:
Population size
Types of organisms
Concentration of the antimicrobial agent
Duration of exposure
Temperature pH
Organic matter
Biofilm formation

7. Physical Methods Moist Heat Dry Heat Low Temperatures Filtration
Radiation

8. Physical Methods: Moist Heat
Mechanism of killing is a combinantion of protein/nucleic acid denaturation and membrane disruption
Effectiveness Heavily dependent on type of cells present as well as environmental conditions (type of medium or substrate)
Bacterial spores much more difficult to kill than vegetative cells

9. Physical Methods: Moist Heat
Measurements of killing by moist heat
Thermal death point (TDP): Lowest temperature at which a microbial suspension is killed in 10 minutes; misleading because it implies immediate lethality despite substrate conditions
Thermal death time (TDT): Shortest time needed to kill all organisms in a suspension at a specified temperature under specific conditions; misleading because it does not account for the logarithmic nature of the death curve (theoretically not possible to get down to zero)

10. Physical Methods: Moist Heat
Measurements of killing by moist heat (cont.)
Decimal reduction time (D value): The time required to reduced a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions
z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions
F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores

11. Physical Methods: Moist Heat
Calculations using D and z values
Given: For Clostridium botulinum spores suspended in phosphate buffer, D121 = min
How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 1012 spores to 100 spores (1 spore) at 121°C? Answer: Since 1012 to 100 is 12 decimal reductions, then the time required is 12 x min = 2.45 min

12. Physical Methods: Moist Heat
Methods of Moist Heat
Boiling at 100°C
Effective against most vegetative cells; ineffective against spores; unsuitable for heat sensitive chemicals & many foods
Autoclaving/pressure canning
Temperatures above 100°C achieved by steam pressure
Most procedures use 121.1°C, achieved at approx. 15 psi pressure, with min autoclave time to ensure sterilization
Sterilization in autoclave in biomedical or clinical laboratory must by periodically validated by testing with spores of Clostridium or Bacillus stearothermophilus

13. Autoclave

15. Physical Methods: Moist Heat
Methods of Moist Heat
Pasteurization
Used to reduce microbial numbers in milk and other beverages while retaining flavor and food quality of the beverage
Retards spoilage but does not sterilize
Traditional treatment of milk, 63°C for 30 min
Flash pasteurization (high-temperature short term pasteurization); quick heating to about 72°C for 15 sec, then rapid cooling

16. Pasteurization

17. Physical Methods: Moist Heat
Methods of Moist Heat
Ultrahigh-temperature (UHT) sterilization
Milk and similar products heated to °C for sec
Very quickly sterilizes the milk while keeping its flavor & quality
Used to produce the packaged “shelf milk” that does not require refrigeration

18. Physical Methods: Dry Heat
Incineration
Burner flames
Electric loop incinerators
Air incinerators used with ferementers; generally operated at 500°C
Oven sterilization
Used for dry glassware & heat-resistant metal equipment
Typically 2 hr at 160°C is required to kill bacterial spores by dry heat: this does not include the time for the glass to reach the required temp (penetration time) nor does it include the cooling time

20. Physical Methods: Low Temperatures
Refrigerator:
around 4°C
inhibits growth of mesophiles or thermophiles; psychrophiles will grow
Freezer:
“ordinary” freezer around -10 to -20°C
“ultracold” laboratory freezer typically -80°C
Generally inhibits all growth; many bacteria and other microbes may survive freezing temperatures

21. Physical Methods: Filtration
Used for physically removing microbes and dust particles from solutions and gasses; often used to sterilize heat-sensitive solutions or to provide a sterilized air flow
Depth filters: eg. Diatomaceous earth, unglazed porcelean
Membrane filters: eg. Nitrocellulose, nylon, polyvinylidene difluoride
HEPA filters: High efficiency particulate air filters used in laminar flow biological safety cabinets

22. Physical Methods: Radiation
Ultraviolet Radiation
DNA absorbs ultraviolet radiation at 260 nm wavelength
This causes damage to DNA in the form of thymine dimer mutations
Useful for continuous disinfection of work surfaces, e.g. in biological safety cabinets

23. Physical Methods: Radiation
Ionizing Radiation
Gamma radiation produced by Cobalt-60 source
Powerful sterilizing agent; penetrates and damages both DNA and protein; effective against both vegetative cells and spores
Often used for sterilizing disposable plastic labware, e.g. petri dishes; as well as antibiotics, hormones, sutures, and other heat-sensitive materials
Also can be used for sterilization of food; has been approved but has not been widely adopted by the food industry

24. Chemical Agents Phenolics Alcohols Halogens Heavy metals
Quaternary Ammonium Compounds
Aldehydes
Sterilizing Gases
Evaluating Effectiveness of Chemical Agents

25. Chemical Agents: Phenolics
Aromatic organic compounds with attached -OH
Denature protein & disrupt membranes
Phenol, orthocresol, orthophenylphenol, hexachlorophene
Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application
Disagreeable odor & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage

26. Chemical Agents: Alcohols
Ethanol; isopropanol; used at concentrations between 70 – 95%
Denature proteins; disrupt membranes
Kills vegetative cells of bacteria & fungi but not spores
Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench

27. Chemical Agents: Halogens
Act as oxidizing agents; oxidize proteins & other cellular components
Chlorine compounds
Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas)
Sodium Hypochlorite (Chlorine Bleach) used at % dilution as benchtop disinfectant
Halazone tablets (parasulfone dichloroamidobenzoic acid) used by campers to disinfect water for drinking

28. Chemical Agents: Halogens
Iodine Compounds
Tincture of iodine (iodine solution in alcohol)
Potassium iodide in aqueous solution
Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne
Used as antiseptics for cleansing skin surfaces and wounds

29. Chemical Agents: Heavy Metals
Mercury, silver, zinc, arsenic, copper ions
Form precipitates with cell proteins
At one time were frequently used medically as antiseptics but much of their use has been replaced by less toxic alternatives
Examples: 1% silver nitrate was used as opthalmic drops in newborn infants to prevent gonorrhea; has been replaced by erythromycin or other antibiotics; copper sulfate used as algicide in swimming pools

30. Chemical Agents: Quaternary Ammonium Compounds
Quaternary ammonium compounds are cationic detergents
Amphipathic molecules that act as emulsifying agents
Denature proteins and disrupt membranes
Used as disinfectants and skin antiseptics
Examples: cetylpyridinium chloride, benzalkonium chloride

31. Chemical Agents: Aldehydes
Formaldehyde and gluteraldehyde
React chemically with nucleic acid and protein, inactivating them
Aqueous solutions can be used as disinfectants

32. Chemical Agents: Sterilizing Gases
Ethylene oxide (EtO)
Used to sterilize heat-sensitive equipment and plasticware
Explosive; supplied as a 10 – 20% mixture with either CO2 or dichlorofluoromethane
Its use requires a special EtO sterilizer to carefully control sterilization conditions as well as extensive ventilation after sterilation because of toxicity of EtO
Much of the commercial use of EtO (for example, plastic petri dishes) has in recent years been replaced by gamma irradiation

33. Chemical Agents: Sterilizing Gases
Betapropiolactone (BPL)
In its liquid form has been used to sterilize vaccines and sera
Decomposes after several hours and is not as difficult to eliminate as EtO, but it doesn’t penetrate as well as EtO and may also be carcinogenic
Has not been used as extensively as EtO
Vapor-phase hydrogen peroxide
Has been used recently to decontaminate biological safety cabinets

34. Chemical Agents: Evaluating the Effectiveness
Phenol Coefficient Test
A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhi and Staphylococcus aureus and incubated at either 20°C or 37°C
Samples are removed at 5 min intervals and inoculated into fresh broth
The cultures are incubated at 37°C for 2 days
The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient

35. Chemical Agents: Evaluating the Effectiveness
Phenol Coefficient Test (cont.)
The reciprocal of the maximum effective dilution for the test disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient
For example: Suppose that, on the test with Salmonella typhi The maximum effective dilution for phenol is 1/90 The maximum effective dilution for “Disinfectant X” is 1/450 The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5

36. Chemical Agents: Evaluating the Effectiveness
Phenol Coefficient Test (cont.)
Phenol coefficients are useful as an initial screening and comparison, but can be misleading because they only compare two pure strains under specific controlled conditions
Use dilution tests and simulated in-use tests
Are tests designed to more closely approximate actual normal in-use conditions of a disinfectant

37. Preservation of Microbial Cultures
Periodic Transfer and Refrigeration
Mineral Oil Slant
Freezing in Growth Medium
Drying
Lyophilization
Ultracold Freezing

38. Preservation of Microbial Cultures: Periodic Transfer and Refrigeration
Stock cultures are aseptically transferred at appropriate intervals to fresh medium and incubated, then stored at 4°C until they are transferred again
Many labs use “agar slants;” care has to be taken to avoid contamination
Major problem with possible genetic changes in strains; most labs need a way to keep “long term” storage of original genetic stocks

39. Preservation of Microbial Cultures: Mineral Oil Slant
Sterile mineral oil placed over growth on agar slants to preserve cultures for longer period of time in the refrigerator
Contamination problems; messy; many organisms are sensitive to this; generally it is a poor technique and doesn’t work well

40. Preservation of Microbial Cultures: Freezing in Growth Medium
Used as a “long term” storage strategy
Broth cultures of the organisms are frozen at -20°C
Often, sterile glycerin (glycerol) is added at a 25 – 50% final concentration; this helps to prevent ice crystal formation and increases viability of many organisms

41. Preservation of Microbial Cultures: Drying
Suitable for some bacterial species
Samples are grown on sterile paper disks saturated with nutrient, then the disks are allowed to air dry and stored aseptically
Reconstituted by dropping disk into nutrient broth medium

42. Preservation of Microbial Cultures: Lyophilization
Suitable for many bacterial species as well as fungi and viruses
Broth cultures are placed in special ampules and attached to a vacuum pump; the vacuum removes all of the water from the cells leaving a “freeze-dried” powder
The culture is reconstituted by adding broth to the lyophilized powder and incubating it
Considered the best method of long-term storage for most bacterial species

43. Preservation of Microbial Cultures: Ultracold Freezing
Similar to freezing, but at very cold temperature
At about -70 to -80°C, in liquid nitrogen or in an ultracold freezer unit