Control of Microbial Growth Microbiology Chapter 7 Control of Microbial Growth

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

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.

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)

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)

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

Chemical Microbial Controls

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 24-48 hours later.

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

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

Phenol Bisphenols

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

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)

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

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

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

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

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.

an example of a quat

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

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

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

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

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)