1. The Control of Microbial Growth
Chapter 7
The Control of
Microbial Growth

2. The Terminology of Microbial Control
Sterilization: Removal of all microbial life
Commercial Sterilization: Killing Clostridium botulinum endospores
Disinfection: Removal of pathogens
Antisepsis: Removal of pathogens from living tissue
Degerming: Removal of microbes from a limited area (mechanical removal)

3. The Terminology of Microbial Control
Sanitization: Lower microbial counts on eating utensils
Biocide/Germicide: Kills microbes
Bacteriostasis: Inhibiting, not killing, microbes
Sepsis : refers to microbial contamination.

4. The Terminology of Microbial Control
Asepsis: the absence of significant contamination.
Aseptic surgery: prevent microbial contamination of wounds.
Aseptic technique: laboratory techniques used to minimize contamination

6. The Rate of Microbial Death
Bacterial populations die at a constant logarithmic rate when heated or treated with antimicrobial chemicals.
Figure 7.1a

7. The Rate of Microbial Death
Number of microbes
Environmental influences (organic matter, temperature, biofilms)
Time of exposure
Microbial characteristics
Figure 7.1b

8. Actions of Microbial Control Agents
Alternation of membrane permeability
Damage to the lipids or proteins of the plasma membrane causes cellular contents to leak and interferes with the growth of the cell
Damage to proteins
Denaturation of proteins by breaking H-bonds or covalent bonds (heat or certain chemicals)
Damage to nucleic acids
Cannot replicate or carry out normal metabolic functions (heat, radiation, or chemicals)

9. Physical Methods of Microbial Control
Need to consider effects on other things besides the microbes (e.g. vitamins, antibiotics, budget)
Heat
Usually used to sterilize laboratory media and glassware, and hospital instruments
Kill microbes by denaturing their enzymes
Heat resistance varies among different microbes
Need to consider suspending medium
presence of fats and proteins protects microbes; acidic condition more effective for heat sterilization

10. Physical Methods of Microbial Control: Heat
Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min.
Thermal death time (TDT): Time required to kill all cells in a culture
Decimal reduction time (DRT or D value): Minutes to kill 90% of a population at a given temperature
Related to bacterial heat resistance

11. Heat Sterilization: moist heat
Moist heat denatures (coagulates) proteins
Break H-bonds
Boiling
Kills vegetative forms of bacterial pathogens; almost all viruses, fungi and fungal spores
More reliable form of sterilization with moist heat requires higher temperature (>100 oC) e.g. Autoclave
Need to consider if heat or moisture can damage the material

12. Heat Sterilization: moist heat
Autoclave: Steam under pressure
121 oC, 20 psi, 15 min. at sea
level
Figure 7.2

13. Heat Sterilization: moist heat
Autoclave
Need direct contact with steam or contained in a small volume of aqueous solution
Used for culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items (have to be able to withstand high temperatures and pressure)
Solid materials and large containers requires extra time

14. Heat sterilization: Pasteurization
Pasteurization reduces spoilage organisms and pathogens
Used for milk, cream, and certain alcoholic beverages
Lowers microbial numbers, but thermoduric (heat resistant) bacteria survive
Thermoduric bacteria are non-pathogenic or do not cause spoilage
Equivalent treatments
63°C for 30 min
High-temperature short-time 72°C for 15 sec
Ultra-high-temperature: 140°C for <1 sec

15. Heat sterilization: Dry heat sterilization
Dry Heat Sterilization kills by oxidation
Flaming: sterilize inoculating loops
Incineration: sterilize and dispose contaminated paper cups, bags, and dressings
Hot-air sterilization: sterilize glassware, instruments, needles, and glass syringes
121˚C, 15 min
170˚C, 2 hr
Equivalent treatments
Autoclave
Hot-air

16. Physical Methods of Microbial Control
Filtration removes microbes
Virus passes through most bacteriological membrane filters (0.22 μm to 0.45 μm pore diameter)
Sterilize heat-sensitive materials (culture media, enzymes, vaccines, and antibiotic solutions)
High-efficiency particulate air (HEPA) filters to remove all microbes larger than 0.3 μm in diameter

17. Physical Methods of Microbial Control
Low temperature inhibits microbial growth
Decrease chemical reactions and possible changes in proteins
Used for food, drug, and culture preservation
Refrigeration: bacteriostatic effect
Deep freezing: preserve bacterial cultures (quick-frozen between -50oC to -95 oC)
Lyophilization: long-term preservation of microbial cultures

18. Physical Methods of Microbial Control
High pressure alter molecular structure of proteins and carbohydrates
Rapid inactivation of vegetative bacterial cells
Used for fruit juices (preserve flavors, colors, and nutrient values of the products)
Desiccation prevents metabolism
Used for food preservation
Microbes can remain viable for years

19. Physical Methods of Microbial Control
Osmotic pressure causes plasmolysis
Used for food preservation
Resembles preservation by desiccation (deny moisture needed for growth to microbial cells)
Radiation
Effects varies depending on its wavelength, intensity, and duration

20. Radiation
Figure 7.5

21. Physical Methods of Microbial Control
Ionizing radiation (X rays, gamma rays, electron beams): cause ionization of water to form highly reactive hydroxyl radicals that react with DNA
Used for sterilizing pharmaceuticals and disposable dental and medical supplies
Nonionizing radiation (UV): damages DNA by forming thymine dimers to inhibit correct replication of the DNA during reproduction
Used for disinfecting vaccines and other medical products; to control microbes in the air
(Microwaves kill by heat; not especially antimicrobial)

22. Chemical Methods of Microbial Control
Control the growth of microbes on both living tissues and inanimate objects
Most of them reduce microbial populations to safe levels or remove vegetative forms of pathogens from objects.
No single disinfectant or antiseptic is appropriate for all situation
Principles of effective disinfection
Concentration of disinfectant
Presence of organic matter
pH of the medium
Time of exposure

23. Chemical Methods of Microbial Control
Evaluating a disinfectant
Use-dilution test
1. Metal rings dipped in test bacteria are dried
2. Dried cultures placed in disinfectant for 10 min at 20°C
3. Rings transferred to culture media to determine whether bacteria survived treatment
Effectiveness determined by the number of cultures that grow

24. Chemical Methods of Microbial Control
Evaluating a disinfectant
Disk-diffusion method (in teaching laboratories)
Figure 7.6

25. Types of Disinfectants
Phenol
Used by Lister to control surgical infections; rarely used now
Cause skin irritation and disagreeable odor
Phenolics & bisphenols are derivative of phenol
Figure 7.7

26. Types of Disinfectants
Phenolics (e.g. Lysol, O-phenylphenol)
Used for disinfecting pus, saliva, and feces
Derivative of phenol; reduced skin irritation with increased antibacterial activity
Injure lipid-containing plasma membranes (cellular contents leak out); works on mycobacteria
Active even in the presence of organic compounds, stable, and persist for long periods after application

27. Types of Disinfectants
Bisphenols (e.g. Hexachlorophene, Triclosan)
Hexachlorophene (pHisoHex) used for surgical and hospital microbial control procedures (esp. staphylococci and streptococci)
Triclosan used in antibacterial soaps & many other products; works well against gram-positive and gram-negative bacteria as well as fungi
Derivative of phenol
Disrupt plasma membranes

28. Types of Disinfectants
Biguanides (e.g. Chlorhexidine)
Used for microbial control on skin and mucous membranes; for surgical hand scrubs and preoperative skin preparations in patients
Injure plasma membranes
Strong affinity for binding to the skin & mucous membranes
Biocidal against most vegetative bacteria, fungi, and certain enveloped virus

29. Types of Disinfectants
Halogens (e.g. Iodine, Chlorine)
Oxidizing agents; effective antimicrobial agents
Iodine, one of the oldest and most effective antiseptics; used mainly for skin disinfection and would treatment; or treat water (iodine tablet)
Effective against all kinds of bacteria, many endospores, various fungi, and some viruses
Available as a tincture and iodophor (e.g. Betadine and Isodine)

30. Types of Disinfectants
Bleach is hypochlorous acid (HOCl) formed by chlorine + water
Liquid form of compressed chlorine gas used for disinfecting municipal drinking water, water in swimming pool, & sewage
Chlorine compounds sued to disinfect dairy equipment, eating utensils, household items, and glassware
Gaseous form of chlorine, chlorine dioxide used for area disinfection to kill endospores of anthrax bacteria

31. Types of Disinfectants
Alcohols (e.g. Ethanol & isopropanol)
Used for swabbing skin and the surface of inanimate objects
Effectively kill bacteria, fungi, and some enveloped virus
Denature proteins, dissolve lipids
Leave no residue (act on a surface and then evaporates rapidly)
Optimum concentration of ethanol = 70%, but effective between 60 – 95%
Table 7.6

32. Types of Disinfectants
Heavy Metals and Their compounds. (e.g. sliver, mercury, and copper)
Oligodynamic action: the ability of small amounts of a heavy metal compound to exert antimicrobial activity
Denature proteins (metal ions combine with the sulfhydryl groups on cellular protein denature)
Silver nitrate used to prevent gonorrheal ophthalmia neonatorum

33. Types of Disinfectants
Mercuric chloride is bacteriostatic; use is limited; control mildew in paints
Copper sulfate sued chiefly to destroy green algae that grow in reservoirs, stock ponds, swimming pools, and fish tanks
Zinc chloride: common ingredient in mouth washes

34. Types of Disinfectants
Surface-Active Agents (Surfactants)
Decrease surface tension among molecules of a liquid
Bactericidal (gram-positive), fungicidal, amoebicidal, and virucidal (enveloped virus); Denature proteins, disrupt plasma membrane
Quaternary ammonium compounds Cationic detergents
Sanitizing (food industry, dairy utensils and equipment); anion reacts with plasma membrane; effective on thermoduric bacteria
Acid-anionic detergents
Degerming; mechanical removal of microbes by scrubbing
Soap

35. Types of Disinfectants
Chemical Food Preservatives
Organic Acids or salts of organic acids
Inhibit metabolism
Sorbic acid, benzoic acid, calcium propionate
Control molds and bacteria in foods and cosmetics
Nitrite prevents endospore germination & preserve the red color of the meat
Antibiotics (not for internal use)
Nisin (for endospore-forming spoilage bacteria) and natamycin (antifungal antibiotic) prevent spoilage of cheese

36. Types of Disinfectants
Aldehydes (e.g. Glutaraldehyde & formaldehyde)
Very effective antimicrobials
Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, —SH)
Glutaraldehyde used to disinfect hospital instruments including respiratory-therapy equipment
2% solution bactericidal, tuberculocidal, and virucidal (10 min.)

37. Types of Disinfectants
Formaldehyde (formalin) used to preserve biological specimens and inactivate bacteria and viruses in vaccines
Gaseous Chemosterilizers (e.g. ethylene oxide)
Chemicals that sterilize in a closed chamber
Denature proteins
Highly penetrating; widely used on medical supplies and equipment; suspected carcinogens
Propylene oxide and beta-propiolactone can also be used for sterilization

38. Types of Disinfectants
Peroxygens (e.g. O3, H2O2, peracetic acid)
Oxidizing cellular components
Ozone (O3) often used to supplement chlorine in the disinfection of water (neutralize taste and odor)
H2O2: effective disinfectant on inanimate objects; but not good for open wounds (may slow healing); neutralized by catalase (present in human cells)

39. Types of Disinfectants
Benzoyl peroxide: useful for treating wounds infected by anaerobic pathogen; medications for acne
Peracetic acid: one of the most effective liquid chemical sporicides available; considered a sterilant; effective on endospores and viruses
Also used in disinfection of food-processing and medical equipment

40. Microbial Characteristics and Microbial Control
Resistance due to:
Structural component
External LPS layer of gram-negative (porin)
Cell wall structure of mycobacteria
Endospore (bacterial)
Protozoan cysts and oocysts
Non-enveloped virus
No effective means to destroy (e.g. prions)
Figure 7.11

41. Microbial Characteristics and Microbial Control
Good
Fair
Glutaraldehyde
Poor
Alcohols
Chlorines
None
Quats
Phenolics
Mycobacteria
Endospores
Effectiveness against
Chemical agent