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

2. Levels & Types of Control
Sterilization - completely destroys all forms of microbial life
Commercial Sterilization - limited heat treatment, destroys pathogens but not all bacteria
Disinfection - destroys vegetative cells on a surface
reduces # of viable organisms in the material
disinfectant - chemical treatment used to disinfect inanimate objects

3. Sanitized for your protection.
Controls continued
Antisepsis - treatment of living skin or tissue to kill microorganisms
Degerming - physical removal of microbes (alcohol swab, soap)
Sanitize - systematic cleansing of inanimate objects to reduce the microbial count to a safe level (for public health, used in restrooms, kitchens)
Sanitized for your protection.

4. Effects of Agents Bacteriocidal (-cidal, to kill) - kill all bacteria
Bacteriostatic (-static, to stop) halt (inhibit) the growth of bacteria for as long as the inhibitory substance is present
if inhibitor is removed, growth begins again
Aseptic – prevention of (or absence of) sepsis
sepsis – bacterial contamination (decay, putrefaction)

5. Microbial Death Killing in a population is not instantaneous
death, like growth, occurs exponentially (if the log of the number of cells is plotted on the Y axis, then a straight line results, (see fig. 7.1)

6. In this example, 90% of survivors
are dying each minute
This figure indicates shorter time
required to kill a smaller population

7. Action of Antimicrobials
1. Alter membrane permeability
damage to lipids or proteins causes leaks and interferes with growth
2. Damage proteins or nucleic acids
Proteins - enzymes which cell needs for growth are damaged
damage to DNA or RNA prevents information from being transferred for protein synthesis

8. Physical Methods of Control
Heat (moist & dry)
Filtration
Cold
Desiccation
Osmotic Pressure
Radiation
each will be discussed in some detail in the following minutes…

9. Heat How it works - denatures (unwinds) proteins
Thermal Death Time (TDT) the time at a given temperature in which all the microorganisms in a liquid culture will be killed
Thermal Death Point (TDP) the lowest temperature at which all microorganisms in a liquid suspension are killed in 10 minutes
Heat sterilization high heat and high humidity together are the most effective in killing microorganisms, but dry heat can also be used

10. Moist Heat
Boiling - kills all vegetative cells, method called Tyndallization can be used with endospore-forming bacteria
Steam alone can reach 100oC, but under pressure (15 p.s.i.) can reach 121oC = Autoclave
to kill all microorganisms, all contents must be available for steam to reach on surface
kills endospores in 15 minutes at this temperature

12. More Moist Heat Techniques
Pasteurization uses temperature below boiling to kill pathogens and reduces total microorganism count (doesn’t kill all, some harmless microorganisms survive), does not alter food taste
Classical Pasteurization - 63oC for 30 minute
Flash Pasteurization - now use high-temperature short-time 72oC for 15 seconds. Sometimes called High Temperature Short Time (HTST) Pasteurization

13. Dry Heat Examples include:
Empty glassware can be sterilized in oven in 1-3 hours at 180oC
Aseptic techniques including flaming mouth of tubes, inoculating loops/needles
Incineration – (burning) a convenient method for destroying some disposable wastes

14. Cold low temperatures are bacteriostatic
home refrigerator is at 4oC, freezer at -10oC (commercial is -20oC)
some bacteria can grow at refrigerator temperatures, but most pathogens do not

15. Desiccation Desiccation = the absence of water
prevents growth (also used for bacterial storage, lypholization)
Lypholization = ‘freeze-drying’

16. Osmotic Pressure
hypertonic environments prevent growth of most organisms (dehydrates them)
Fungi are more resistant to osmotic pressure extremes than are bacteria

17. Ultraviolet Radiation
most lethal at wavelength of 260 nm
absorbed by DNA, leads to formation of thymine (pyrimidine) dimers
DNA has several methods of repair from this damage
UV used to sterilize air & surfaces (but cannot expose skin)

18. Ionizing Radiation example Gamma radiation from Cobalt 60
Advantage - penetrates deep into objects
can be used to sterilize plastic objects, even foods

19. Filtration physically remove bacteria from liquid or air
most bacterial filtration by .45 or .2 micron pore size

20. Review table 7.5 for a good summary of Physical control of microbes

21. Chemical Microbial Controls

22. Evaluating Effectiveness of Chemical Agents
Use-Dilution Testing – standardized metal rings dipped in bacterial cultures of Salmonella choleraesuis, Staphylococcus aureus & Pseudomonas aeruginosa, dried briefly then placed into disinfectants for 10 minutes at 20oC. These cultures are then allowed to grow and are later evaluated.
Disk-Diffusion Method – Filter paper soaked with chemical agent placed on inoculated agar plate containing a known organism, usually Staphylococcus aureus or Pseudomonas aeruginosa. The clear ‘death zone’ around the filter paper is measured hours later.

23. Phenol & Phenolics phenol and phenol derivatives
Phenol was the first chemical used to control microorganisms by Joseph Lister in 1867; used as disinfectants and antiseptics (example, Lysol)
Action of chemical: denature proteins and disrupt lipid containing cell membranes
Advantage: Stable & persist for long periods, good surface disinfectants

24. Bisphenols
bis = 2
A phenol derivative with TWO phenols linked by a bridge.
pHisoHex© used in surgical scrubbing & for hospital control of microbes.
particularly effective against G+ staphylococci & streptococci which affect newborns
Triclosan – another bisphenol that can be incorporated into plastics probably disrupts membranes – broad spectrum but particularly effective against G+ & fungi

25. Phenol
Bisphenols

26. Biguanide similar to phenolics, disrupts membranes
Used in some hospital surgical scrubs
Advantage: binds to skin & mucous membranes but has low toxicity to humans
Effective against vegetative bacterial cells (not spores), & fungi

27. Halogens
halogens are elements in the same group of the periodic table (fluorine, chlorine, bromine, iodine, etc.).
Iodine and chlorine are most important as antimicrobial agents
iodine often used in a tincture (alcohol solution)
chlorine may be used as Cl2 gas or in solution with other chemicals
Action: damage cellular materials (through oxidation)

28. Alcohols both disinfectants and antiseptics
ethanol and isopropanol used most (at 70-80% concentrations)
Actions: denature proteins, possibly dissolve membrane lipids

29. Heavy metals (fig. 7.8)
examples: mercury, silver, arsenic, zinc, copper
silver nitrate prevents neonatal ophthalmic gonorrhea
Actions: heavy metals inactivate cell proteins

30. Surfactants surface acting agents
Soaps & detergents.
Action: mechanical removal of microbes.
Some contain antibacterial agents such as triclocarban (effective against G+)

31. Acid-Anionic Surfactants
Important surface agents in dairy industry.
Action: Negative portion (anion) of molecule disrupts plasma membranes
Benefit: Acid-anionic sanitizers effective against broad spectrum of organisms including those resistant to heat (thermoduric). These agents are nontoxic, noncorrosive & quick acting

32. Quaternary Ammonium Compounds
Quats are composed of charged quaternary nitrogen and a long hydrophobic chain. A type of surfactant
The positively charged end is a version of the ammonium ion (NH4+) – this give the name (quat) and is the effective portion of the molecule.
Quats probably disrupt membranes.
stable and non-toxic, can be used on food utensils and as skin antiseptics.
Some bacteria actively grow in these compounds.

33. an example of a quat

34. Aldehydes examples: formaldehyde, glutaraldehyde
Action: cross-link proteins rendering them inactive
Benefit: sporicidal
aldehydes are highly reactive molecules

35. Chemical Food Preservatives
examples: sodium benzoate, sorbic acid, calcium propionate
Uually organic acids which are metabolized,
Action: keep food pH low to inhibit growth
these are biostatic and merely halt growth, they don’t kill organisms

36. Gases
useful for sterilizing heat-sensitive objects (example: the disposible Petri dishes in the lab)
Ethylene oxide, reacts with cell proteins
bactericidal and sporicidal

38. Peroxygens oxidizing agents – these oxidize cellular components.
Examples: ozone, hydrogen peroxiede, peracetic acid
Ozone often used to treat water
hydrogen peroxide often used to treat open wounds. Broken down to water & oxygen by they enzyme catalase

39. Chemical Methods & Microbial Resistance
Most Gram-positive organisms are generally more sensitive to chemical agents than are Gram-negatives
Endospores are less susceptible
Mycobacterium also resistant
(See Table 7.7 for examples)