1. The Control of Microbial Growth
Chapter 7
The Control of Microbial Growth
Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc. 1

3. The Terminology of Microbial Control
Learning Objective
7-1 Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis.

4. The Terminology of Microbial Control
Sepsis refers to microbial contamination
Asepsis is the absence of significant contamination
Aseptic surgery techniques prevent microbial contamination of wounds

5. The Terminology of Microbial Control
Sterilization: removing all microbial life
Commercial sterilization: killing C. botulinum endospores
Disinfection: removing pathogens
Antisepsis: removing pathogens from living tissue

6. The Terminology of Microbial Control
Degerming: removing microbes from a limited area
Sanitization: lowering microbial counts on eating utensils
Biocide/germicide: killing microbes
Bacteriostasis: inhibiting, not killing, microbes

7. The usual definition of sterilization is the removal or destruction of all forms of microbial life; how could there be practical exceptions to this simple definition? 7-1

8. The Rate of Microbial Death
Learning Objective
7-2 Describe the patterns of microbial death caused by treatments with microbial control agents.

9. Table 7.2 Microbial Exponential Death Rate: An Example

10. Figure 7.1a Understanding the Microbial Death Curve.
Plotting the typical microbial death curve logarithmically (red line) results in a straight line.
One log decrease =
90% of population
killed
log10 of number of surviving cells
Arithmetic number of surviving cells
Time (min)
(a) Plotting the typical microbial death curve arithmetically (blue line) is impractical: at 3 minutes the population of 1000 cells would only be a hundredth of the graphed distance between 100,000 and the baseline.

11. Effectiveness of Treatment
Depends on:
Number of microbes
Environment (organic matter, temperature, biofilms)
Time of exposure
Microbial characteristics

12. log10 of number of surviving cells
Figure 7.1b Understanding the Microbial Death Curve.
sterile surgical equipment
log10 of number of surviving cells
High population load
Low population load
Time (min)
(b) Logarithmic plotting (red) reveals that if the rate of killing is the same, it will take longer to kill all members of a larger population than a smaller one, whether using heat or chemical treatments.

13. How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria? 7-2

14. Actions of Microbial Control Agents
Learning Objective
7-3 Describe the effects of microbial control agents on cellular structures.

15. Actions of Microbial Control Agents
Alteration of membrane permeability
Damage to proteins
Damage to nucleic acids

16. Would a chemical microbial control agent that affects plasma membranes affect humans? 7-3

17. Physical Methods of Microbial Control
Learning Objectives
7-4 Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat.
7-5 Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth.
7-6 Explain how radiation kills cells.

18. Heat
Thermal death point (TDP): lowest temperature at which all cells in a culture are killed in 10 min
Thermal death time (TDT): time during which all cells in a culture are killed

19. Decimal Reduction Time (DRT)
Minutes to kill 90% of a population at a given temperature

20. Figure 7.10 A comparison of the effectiveness of various antiseptics.

21. Moist Heat Sterilization
Moist heat denatures proteins
Autoclave: steam under pressure

22. Figure 7.2 An autoclave.
Exhaust valve
(removes steam
after sterilization)
Operating valve
(controls steam from
jacket to chamber)
Steam to
chamber
Safety
valve
Pressure gauge
Steam
Door
Steam
chamber
Air
Perforated shelf
Sediment
screen
Steam jacket
Thermometer
Automatic ejector valve
(thermostatically controlled;
closes on contact with
pure steam when air is
exhausted)
Pressure regulator
for steam supply
To waste line
Steam supply

23. Steam Sterilization
Steam must contact item’s surface

24. Figure 7.3 Examples of sterilization indicators.

25. Pasteurization Reduces spoilage organisms and pathogens
Equivalent treatments
63°C for 30 min
High-temperature short-time: 72°C for 15 sec
Ultra-high-temperature: 140°C for <1 sec
Thermoduric organisms survive

26. Dry Heat Sterilization
Kills by oxidation
Dry heat
Flaming
Incineration
Hot-air sterilization
Hot-Air
Autoclave
Equivalent Treatments
170˚C, 2 hr
121˚C, 15 min

27. Filtration HEPA removes microbes >0.3 µm
Membrane filtration removes microbes >0.22 µm

28. Flask of sample Cap Membrane filter Cotton plug in vacuum line
Figure 7.4 Filter sterilization with a disposable, presterilized plastic unit.
Flask of
sample
Cap
Membrane filter
Cotton plug in
vacuum line
ensures sterility
Sterile
filtrate
Vacuum line

29. Physical Methods of Microbial Control
Low temperature inhibits microbial growth
Refrigeration
Deep-freezing
Lyophilization
High pressure denatures proteins
Desiccation prevents metabolism
Osmotic pressure causes plasmolysis

30. Figure 7.5 The radiant energy spectrum.

31. Radiation Ionizing radiation (X rays, gamma rays, electron beams)
Ionizes water to release OH•
Damages DNA
Nonionizing radiation (UV, 260 nm)
Microwaves kill by heat; not especially antimicrobial

32. How is microbial growth in canned foods prevented? 7-4
Why would a can of pork take longer to sterilize at a given temperature than a can of soup that also contained pieces of pork? 7-5
What is the connection between the killing effect of radiation and hydroxyl radical forms of oxygen? 7-6

33. Chemical Methods of Microbial Control
Learning Objectives
7-7 List the factors related to effective disinfection.
7-8 Interpret the results of use-dilution tests and the disk-diffusion method.

34. Principles of Effective Disinfection
Concentration of disinfectant
Organic matter pH
Time

35. Use-Dilution Test Metal rings dipped in test bacteria are dried
Dried cultures are placed in disinfectant for 10 min at 20°C
Rings are transferred to culture media to determine whether bacteria survived treatment

36. Staphylococcus aureus Pseudomonas aeruginosa
Figure 7.6 Evaluation of disinfectants by the disk-diffusion method.
Zone of inhibition
Chlorine
Chlorine
Chlorine
O-phenylphenol
O-phenylphenol
Hexachlorophene
O-phenylphenol
Hexachlorophene
Hexachlorophene
Quat
Quat
Quat
Staphylococcus aureus
(gram-positive)
Escherichia coli
(gram-negative)
Pseudomonas aeruginosa
(gram-negative)

37. Clinical Focus
Which preparation is more effective?

38. Disk-diffusion test of Zephiran against M. abscessus.
Clinical Focus 7.1 Infection Following Steroid Injection
Undiluted
1:10
Disk-diffusion test of Zephiran against M. abscessus.

39. If you wanted to disinfect a surface contaminated by vomit and a surface contaminated by a sneeze, why would your choice of disinfectant make a difference? 7-7
Which is more likely to be used in a medical clinic laboratory, a use-dilution test or a disk-diffusion test? 7-8

40. Chemical Methods of Microbial Control
Learning Objectives
7-9 Identify the methods of action and preferred uses of chemical disinfectants.
7-10 Differentiate halogens used as antiseptics from halogens used as disinfectants.
7-11 Identify the appropriate uses for surface-active agents.
7-12 List the advantages of glutaraldehyde over other chemical disinfectants.
7-13 Identify chemical sterilizers.

41. Phenol and Phenolics
Disrupt plasma membranes

42. (a) Phenol (b) O-phenylphenol
Figure 7.7ab The structure of phenolics and bisphenols.
(a) Phenol
(b) O-phenylphenol

43. Bisphenols
Hexachlorophene, triclosan
Disrupt plasma membranes

44. (c) Hexachlorophene (a bisphenol)
Figure 7.7cd The structure of phenolics and bisphenols.
(c) Hexachlorophene (a bisphenol)
(d) Triclosan (a bisphenol)

45. Biguanides
Chlorhexidine
Disrupts plasma membranes

46. Halogens Iodine Chlorine Tinctures: in aqueous alcohol
Iodophors: in organic molecules
Alter protein synthesis and membranes
Chlorine
Bleach: hypochlorous acid (HOCl)
Chloramine: chlorine + ammonia
Oxidizing agents

47. Alcohols Ethanol, isopropanol Denature proteins, dissolve lipids
Require water

48. Table 7.6 Biocidal Action of Various Concentrations of Ethanol in Aqueous Solution against Streptococcus pyogenes

49. Heavy Metals Ag, Hg, and Cu Oligodynamic action
Silver nitrate may be used to prevent gonorrheal ophthalmia neonatorum
Silver sulfadiazine used as a topical cream on burns
Copper sulfate is an algicide
Oligodynamic action
Denature proteins

50. Figure 7.8 Oligodynamic action of heavy metals.

51. Benzalkonium chloride
Figure 7.9 The ammonium ion and a quaternary ammonium compound, benzalkonium chloride (Zephiran).
Ammonium ion
Benzalkonium chloride

52. Surface-Active Agents, or Surfactants
Soap
Degerming
Acid-anionic detergents
Sanitizing
Quaternary ammonium compounds (cationic detergents)
Bactericidal, denature proteins, disrupt plasma membrane

53. Chemical Food Preservatives
Organic acids
Inhibit metabolism
Sorbic acid, benzoic acid, and calcium propionate
Control molds and bacteria in foods and cosmetics
Nitrite prevents endospore germination
Antibiotics
Nisin and natamycin prevent spoilage of cheese

54. Aldehydes
Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH)
Use: medical equipment
Glutaraldehyde, formaldehyde, and ortho-phthalaldehyde

55. Gaseous Sterilants Denature proteins Use: heat-sensitive material
Ethylene oxide

56. Plasma
Free radicals destroy microbes
Use: tubular instruments

57. Supercritical Fluids CO2 with gaseous and liquid properties
Use: medical implants

58. Peroxygens Oxidizing agents Use: contaminated surfaces
O3, H2O2, peracetic acid

59. Why is alcohol effective against some viruses and not others? 7-9
Is Betadine an antiseptic or a disinfectant when it is used on skin? 7-10
What characteristics make surface-active agents attractive to the dairy industry? 7-11
What chemical disinfectants can be considered sporicides? 7-12
What chemicals are used to sterilize? 7-13

60. Microbial Characteristics and Microbial Control
Learning Objective
7-14 Explain how the type of microbe affects the control of microbial growth.

61. Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.

62. Table 7.7 The Effectiveness of Chemical Antimicrobials against Endospores and Mycobacteria

63. The presence or absence of endospores has an obvious effect on microbial control, but why are gram-negative bacteria more resistant to chemical biocides than gram-positive bacteria? 7-14