Chemical & Physical Methods of Microbial Control [Disinfectants, Antiseptics, etc.]

Food Preservation summary

1. Temperature 2. pH 3. Drying 4. Chemicals 5. Carbon monoxide 6. Bacteriophages 7. Radiation

More Specifically on Food Preservation

1. Temperature - already discussed; factor most often used to preserve food; canning is the oldest method. two factors, time & temp, determine safe heat treatments for canning, 2. pH - already discussed 3. Drying - already discussed; drying & salting do not sterilize but preserve food by making it unable to support microbial growth for lack of water, an essential nutrient.

Alkylating Agents Formalin Mode of action - they alkylate (attach short chains of carbon atoms) to proteins and nucleic acids. Effect: sterilization Disadvantages: carcinogenic

37% solution of formaldehyde used to preserve tissues & to embalm; lower concentrations are used to inactivate microbes for killed vaccines.

These foods can become contaminated with Listeria during production, but unlike fresh meat and poultry, these foods are consumed without additional cooking that would kill the bacteria, thereby increasing the risk to Listeriosis, an infection caused by the bacteria. Furthermore, L. monocytogenes is capable of multiplying at low temperatures, so it can multiply in food over time even when the food is kept in the refrigerator. Persons with increased risk to Listeriosis are pregnant women, newborns, those with a compromised immune system, and the elderly.

Advantage: Kind of a form of biological control - not having to use chemicals, etc. Viruses are very specific for the cells they infect. Disadvantage: People freak out when they find out their food has been sprayed with viruses!

Halogens Chlorine Mode of Action - inactivates enzymes Effect - antiseptic/disinfectant

Advantage: kills endospores Disadvantages: activity reduced by the presence of organic materials (vomit, blood, etc.) & too irritating to use on skin/mucous membranes. Uses: disinfectant; ingredient in household bleach; added to swimming pools;

Radiation - Radiation is classified by wavelength with ionizing radiation (gamma rays) at the short-wavelength end, visible light in the middle, & radio waves at the long-wavelength end. The shorter the wavelength, the greater its energy, & the more lethal it is to microorganisms.

Advantages: does not add chemicals to the product, environmentally friendly, fast acting, does not changes taste if used on food Disadvantages: can also be harmful to humans doing the irradiating; ionizing radiation is also technically complex

Low pH Mode of Action - denatures proteins Effect - Acidity (low pH) prevents the growth of most microbes, especially in an anaerobic environment Advantages: Low pH also increases the effectiveness of heat treatments (ex. acidic foods like tomatoes can be canned merely by boiling).

Disadvantages: can change appearance or taste of foods Examples of uses: sorbic acid, benzoic acid, propionic acid adding vinegar (acetic acid) to foods (pickles, peppers, etc.) dipping meat into 8OoC solution of lactic acid kills 90% of bacteria; disadvantage - changes color of meat

Nanotechnology Mode of Action - Nanospheres of oil droplets are suspended in water to create a nanoemulsion requiring only miniscule amounts of EcoTru's active ingredient, PCMX. The nanospheres carry surface charges that efficiently penetrate the surface charges on microorganisms' membranes, much like breaking through an electric fence.

Effect - Equal in efficacy to Quats, and bleach-based disinfecting products Advantages: Non-corrosive, non-irritating and does not require U.S. Environmental Protection Agency handling, warning or precautionary statements. Kills TB in 5 minutes, HIV, Staphylococcus, E. coli, and other bacteria, viruses, and fungi. No toxic fumes, non-flammable, noncorrosive, does not dry out skin, pleasant odor. No harmful dermal, ocular, inhalation, or ingestion effects.

More Specifically on Food Preservation Carbon monoxide

Effect - NOT microbiocidal or microbiostatic, rather it is a pigment fixative; it gives meat a bright pink color that lasts weeks Disadvantage - may deceive shoppers who depend on color to help them avoid spoiled meat Note: levels of carbon monoxide used are not toxic to consumers

Alcohols Ethanol & Isopropanol Structure - compounds with a hydroxyl group (-OH); a 50 to 70% solution in water is the most effective concentration (one of the few exceptions to the rule: increase effectiveness by increasing concentration) Mode of Action - when mixed with water it disrupts lipids in cell membranes & denatures proteins

Effect - disinfectant/antiseptic Advantages - less drying than soaps, no bacterial resistance, quicker to use (don't need to dry with papertowel as with handwashing) Disadvantages - evaporates quickly from surfaces; does not kill endospores; not good for wound treatment (cause coagulation of patients proteins producing a layer under which microbes continue to grow). Uses: alcohol gels to disinfect hands

Phenol & Phenolics Hexachlorophene Structure - compounds with hydroxyl groups (-OH) attached to a benzene ring. Mode of Action - denature proteins, disrupt cell membranes. Effect - disinfectant; kills many microorganisms

Effect: antiseptic; Uses: once widely used as an ingredient in soaps & lotions (ex. in Physohex); Disadvantage: in 1970's was found to increase risk of brain damage in babies; prescription only for adults now.

Types of Germicides Quaternary ammonium salts ["quats" - ex. benzalkonium chloride] - Uses: indicated for the antisepsis of skin, mucous membranes, and wounds; used for preoperative preparation of the skin, surgeons' hand and arm soaks, treatment of wounds, preservation of ophthalmic solutions, irrigations of the eye, body cavities, bladder, urethra, and vaginal douching.

Effect: most effective against G(+); do not kill spores. Disadvantage: effectiveness is decreased in the presence of soap - can actually support Pseudomonas growth; now being mixed with other agents to overcome some of these problems. Ex. Zephiran, Cepacol, Bactine, Mercurochrome (used to be mercury based - see under heavy metals)

Ionizing Radiation: X-rays, Gamma rays Mode of Action: cause a chain of ionizations by stripping electrons from atoms - causes free radicals to form; free radicals damage other molecules in the cell, such as DNA.

Effect: sterilization Uses: sterilize foods (meat, produce, grains, spices, poultry); in this process no radioactive material is added to the food

Nonionizing Radiation: UV Light Mode of Action - excites electrons in molecules - when this happens in DNA molecules, it results in formation of extra bonds between adjacent bases (called dimers) - this changes the shape of the DNA and causes problems during replication and transcription; bacteria actually have special enzymes that can fix these dimers

Effects: results in sterilization, but kills only on surfaces Uses: UVc (UVa & b too long) used in surgery wards, hospital/clinics air conditioning units, water treatment facilities Note: microwaves do not have much effect on microbes - too low energy (heat generated will denature proteins)

Testing Germicides: Phenol coefficients

Lister introduced carbolic acid (phenol) as a disinfectant in 1867. Germicides can be tested by comparing their effectiveness to phenol, a traditional germicide. The procedure involves preparing several dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen) or Staphylococcus aureus, incubating the tubes, and then checking for cloudiness (turbidity) in the tubes, indicating growth.

Dyes Ex. Crystal violet [gentian violet]

MOA: blocks cell wall synthesis. Effect: It effectively inhibits growth of G(+) bacteria & yeast Uses: G(+) bacteria and yeast infections Disadvantage: It stains everything purple!

Osmotic Strength Effect - kills most bacteria with exception of halophiles and endospore-formers. Mode of action - crenation or shrinkage can occur (you're placing the microbes in a hypertonic environment).

Methods/Uses - use high concentrations of salt or sugar (corn syrup) in food industry (beef jerky, fruits, etc.) Advantages - inexpensive Disadvantage - once added, salt or sugar cannot be easily removed; changes taste of foods

Drying [Desiccation] - the removal of water Effect - does not sterilize Mode of Action - depends on method; drying limits growth (bacteria don't like dry environments); if using heat, also denatures proteins

Methods: 1. Evaporation involving heat - Effect: kills many microbes, but temps are not high enough to result in sterilization; Uses: in food industry (dried fruit, etc.). 2. Lyophilization [freeze drying] - Method: removes water directly by converting water from a solid state (ice) to a gaseous state; materials are frozen & placed in a chamber to which a partial vacuum is applied Uses: in the microbiology lab to preserve perishable materials such as proteins, blood products, & reference cultures of microbes; used in food industry to make instant coffee, dried fruit, etc.; water can be added later to resume original state Advantage - avoids chemical changes caused by heat drying Disadvantage - requires special equipment & expensive!

Cold Effect - depends on temp (see below); microbiostatic; does not sterilize; don't forget about psychrophiles! Mode of Action - slows down chemical reactions Advantages - increases shelflife of foods and other materials Disadvantage - does not sterilize; some products harmed by freezing over time (freezer burn)

Methods: 1. Refrigeration (5o C) - stops the growth of most species of microbes; most disease-causing microbes are mesophiles, not psychrophiles; one exception is Listeria, which causes listeriosis (food poisoning). Uses: Preserving food 2. Freezing (-10o C) - kills most bacteria, but survivors can remain alive for long periods in frozen state MOA: ice crystals disrupt structure of bacteria Uses: Preserving food; bacterial cultures can be preserved by rapid freezing, sometimes with the addition of a compound called DMSO, milk, or glycerol to protect proteins.

Heat Advantages - simple, inexpensive; best method if material being treated is not damaged by heat Disadvantage - high heat may change flavor of some foods and some materials may be damaged Mode of Action - denatures proteins Effect - depends on temperature (see below) Thermal death point (TDP) - lowest temp at which all microbes in a liquid will be killed in 10 minutes Thermal death time (TDT) - time is takes for all microbes to be killed at a given temperature.

Methods: Dry Heat Sterilization (171oC, 1hr., 160oC for 2 hr., 121oC for 16 hrs.); Uses: sterilize materials that can withstand high temps; ex. flaming your loop; incineration in hot air ovens is used to sterilize and dispose of paper dressings, paper cups, etc. Moist Heat - ex. boiling or autoclaves; Advantage: effective at a lower temperature than dry heat & it penetrates more quickly because of water. Disadvantage of boiling - does not kill thermophiles or endospores and can change flavor of some foods. Autoclave achieves sterilization - it uses pressure to raise the temperature above that of boiling (121oC, 15psi, for 20 min.); Autoclave uses: sterilize liquids, glassware & media, instruments, syringes, transfusion equipment. Pasteurization - Effect: limits growth, but does not sterilize (disadvantage); Uses: slow spoilage of milk & dairy products, wine, beer, "egg beaters"; Advantage: causes minimal damage to the product; developed by Louis Pasteur; standard treatment: heat to 63oC for 30 min. or 72oC for 15 sec (the latter is called HTST - high temp, short time pasteurization).

Heavy Metals

Mode of Action - heavy metals (mercury, copper, silver) react with the sulfhydryl groups of proteins to cause denaturation Effect - antibacterial/antifungal (disinfectant/antiseptic) Disadvantages - some are very toxic, especially the mercury based preparations

Membrane Filtration Effect - depends on pore size of filter; filters out most microbes; exceptions: Mycoplasma (no cell wall so they can squeeze through small spaces), spirochetes (corkscrew their way through small holes), & viruses (too small)

Mode of Action - physically removes cellular organisms Disadvantages: can't filter out all microorganisms (see above for examples) Advantages: causes less damage to materials than heat; can be used on heat sensitive materials (see below) Methods/Uses: used with media, antibiotics, & other heat sensitive materials; replacing pasteurization in some causes, because filtration causes even less damage; you may have heard of "cold filtered" beers.

Types of Germicides Surfactants/Detergents Structure - compounds with hydrophilic & hydrophobic parts. Wetting agents are surfactants that are often used with other chemical agents to help the agent penetrate fatty substances. Surfactants are not germicidal by themselves!

Mode of action - Penetrate oily substances in water & break them into small droplets that become coated with surfactant molecules. The hydrophobic end of the surfactant sticks into the droplets & the hydrophilic end is attracted to the water. The result is an emulsion, a fine suspension of oily droplets in water, which can now be rinsed away. Effect of soaps & detergents - wash away microbes, but do not kill them.

Phenol & Phenolics

Structure - compounds with hydroxyl groups (-OH) attached to a benzene ring. Mode of Action - denature proteins, disrupt cell membranes. Effect - disinfectant; kills many microorganisms

Halogens Chloramines Mode of Action - inactivates enzymes Effect - antiseptic/disinfectant

Structure: chlorine + ammonia Effect: disinfectant Advantage: weaker but more stable than chlorine - does not react with organic material like chlorine Uses: now used by water treatment facilities instead of chlorine for disinfection of drinking water

Testing Germicides: Phenol coefficient

Testing Germicides: The ratio of the effective dilution of the chemical agent to the dilution of phenol that has the same effect. A disinfectant with a p.c. of 1.0 has the same effectiveness as phenol. A p.c. of less than 1.0 means the disinfectant is less effective than phenol. Greater than 1.0 means it's more effective than phenol. Example: Lysol has a p.c. of 5 against S. aureus and a p.c. of 3.2 against S. typhi.

Chemical Control

The effectiveness of a chemical antimicrobial agent is affected by time, temperature, pH, and concentration.

Bacteriophages (phages) are viruses that infect only bacteria and do not infect mammalian or plant cells. Phages are ubiquitous in the environment, and humans are routinely exposed to them at high levels through food and water without adverse effect.

The phage preparation will be used in meat and poultry processing plants for spray application to the surface of RTE meat and poultry products, such as lunch meats and hot dogs, to kill Listeria. FDA approved is a mixture of equal proportions of six phages specific against Listeria monocytogenes. The petitioner's rationale for incorporating six phages in one formulation is to minimize the possibility of L. monocytogenes developing resistance to the additive. The approved phage preparation is reported to be effective against 170 strains of L. monocytogenes.

Why would you want to determine the lowest concentration of a germicide that kills the most bacteria?

To prevent resistance (is what I believe)

Halogens Mode of Action - inactivates enzymes Effect - antiseptic/disinfectant Iodine - antiseptic; Advantage: broad spectrum against bacteria, viruses, fungi Tincture - Structure: iodine in a dilute alcohol solution; Use: one of first skin antiseptics Iodophor - Structure: mixture of iodine and surfactants

Uses: Betadine and Isodine (used for surgical scrubs and to prepare skin for surgery); Disadvantage: contamination of Betadine with Pseudomonas bacteria has been reported; Povidone-Iodine (PVP iodine) - surgical scrubs; antiseptic before punctures or injections; ointment used to prevent infection in cuts, scrapes, etc.; also used in antidandruff shampoo.

Phenol & Phenolics - Lysol Structure - compounds with hydroxyl groups (-OH) attached to a benzene ring. Mode of Action - denature proteins, disrupt cell membranes. Effect - disinfectant; kills many microorganisms

Uses: common household and hospital disinfectant; Advantage: action is not impaired by organic materials as is Chlorox (remain active even in the presence of blood, feces, etc.)

Phenol & Phenolics Triclosan Structure - compounds with hydroxyl groups (-OH) attached to a benzene ring. Mode of Action - denature proteins, disrupt cell membranes. Effect - disinfectant; kills many microorganisms

Uses: found in many antibacterial soaps; impregnated into cutting boards Disadvantages: overuse has been found to lead to resistant strains; not a good idea to use on a regular basis in a normal household (by killing the normal bacteria, it creates an environment where mutated bacteria that are resistant to triclosan are more likely to survive and reproduce). Studies have raised concerns that it can disrupt hormones critical for reproduction and development in humans. Now a call to ban triclosan in the U.S. - Minnesota is the only state to enact the ban so far.

Biguanides Structure - salt with lots of nitrogen Mode of Action - disrupts cell membrane Effect - antiseptic Example: Chlorhexidine gluconate

Uses: mouthwashes for gingivitis (Peridex or Periogaurd), impregnated on tiny chips and inserted into gums to treat gum disease (Periochip); also impregnated into catheters, combined with soap or alcohol for surgical hand scrubs, and used in contact lens solutions Disadvantages: tooth staining is a common side effect when taking iron supplements; may interfere with vitamin K absorption so supplements should be taken; deactivated by surfactants in toothpaste Advantages: low toxicity and strong affinity to skin and mucous membranes

Hydrogen peroxide (H2O2) Mode of Action - oxidizing agent (denatures proteins & DNA) Effect - antiseptic Note: when H2O2 comes into contact with tissue, it bubbles producing oxygen gas; this is because all aerobes (incl. eukaryotes) produce the enzymes catalase & peroxidase which decompose H2O2 into oxygen & H2O. It bubbles when oxygen is produced.

You can differentiate between Staphylococcus & Streptococcus using H2O2; Staphylococcus is catalase positive, Streptococcus is catalase negative, so Staphylococcus would bubble and Streptococcus would not. Staphylococcus is more resistant to H2O2 because of the catalase it produces. Disadvantage - not a great antiseptic, but doesn't sting, so is widely popular; can cause tissue damage if used in high concentrations (>3%). Uses: better as a disinfectant; found in soft contact lens solutions

More Specifically on Food Preservation Chemicals - Various chemical preservatives are added to commercially prepared foods.

calcium propionate - antifungal agent added to bread. sorbic acid - antifungal agent added to soft drinks, salad dressings, cheeses. sodium benzoate - antifungal agent added to soft drinks, salad dressings, cheeses. sodium nitrate (nitrite) - antibacterial agent that prevents germination of Clostridium botulinum spores when added to bacon, ham, hot dogs. chlorine - disinfection of raw chicken

Alkylating Agents Glutaraldehyde [Cidex, Glutarex, Sonacide] Mode of action - they alkylate (attach short chains of carbon atoms) to proteins and nucleic acids. Effect: sterilization Disadvantages: carcinogenic

colorless liquid; used to sterilize medical/dental surgical instruments, disinfect lab table tops, tissue fixation in histology labs, development of x-rays.

sterilization

destroy all microbial life -even bacterial endospores and fungal spores; there are no degrees of sterility!

Commercial sterilization

food is subjected only to enough heat to destroy Clostridium botulinum spores; there are some thermophiles that are spore-producers and could spoil food, but do not cause human disease. disinfection (sanitation) - reduce the number of pathogens to a level at which they pose no danger of disease; disinfectants are used to kill microbes on inanimate objects like bathroom surfaces, etc. (most are too harsh for use on delicate tissue); most disinfectants do not kill spores.

Phenol & Phenolics Cresol Structure - compounds with hydroxyl groups (-OH) attached to a benzene ring. Mode of Action - denature proteins, disrupt cell membranes. Effect - disinfectant; kills many microorganisms

from the creosote or greasewood bush. Uses: used to prevent the rotting of wooden posts, fences, railroad ties (coal tar creosote is the most widely used wood preservative in the US); has been used to treat skin diseases, in insecticides, and fungicides; creosote leaves have been used as an herbal remedy for various illnesses. Disadvantages: taking large amounts may cause kidney/liver damage); coal tar creosote is extremely toxic and can result in death.

Heavy Metals Copper sulfate

fungicide used to control fungal diseases of fruit, vegetable, nut and field crops; also used as an algaecide in pools and fish tanks and as a molluscicide against slugs and snails.

antisepsis

kill microbes or inhibit their growth on skin or other living tissue; antiseptics are applied to living tissue.

Heavy Metals Selenium sulfide

kills fungi, including spores; commonly used to treat fungal skin infections; included in dandruff shampoos (dandruff is often caused by a fungus).

Alkylating Agents Ethylene oxide Mode of action - they alkylate (attach short chains of carbon atoms) to proteins and nucleic acids. Effect: sterilization Disadvantages: carcinogenic

odorless gas; advantages: disappears from the object after treatment; disadvantage: extremely toxic to humans so must be used in a sealed chamber (expensive equipment); used to sterilize materials destroyed by heat (medical supplies, plastic, rubber gloves, animal feed, mattresses, pillows, telephones).

Heavy Metals Silver Nitrate

once applied to eyes of newborns to prevent gonorrhea; the trend for a while was toward using antibiotics instead, but the development of antibiotic-resistant strains has necessitated the use of silver nitrate again.

Heavy Metals Thimerosal Merthiolate (tincture); banned?; when you google it, there are lots of Spanish sites, so still made in Mexico?; once used in basic first aid kit supplies for antiseptic use

once used widely as a vaccine preservative; thimerosol has been reduced or eliminated from many childhoold vaccines; not found in any live vaccines; multidose vials of vaccine are more likely to have thimerosal than single dose higher chance of contamination with a multidose vial, so that's why the mercury is used. found in some cosmetics (Loreal Miracle Wear Mascara), some contact lens solutions (most are thimerosal free), nasal sprays (Neo-Synephrine)

Paper disc method

paper discs are saturated with the chemical agent and placed on the surface of an agar plate inoculated with a test organism. Clear "zones of inhibition" appear around the discs if the chemical agent is effective.

"-static"

treatments that inhibit rather than kill; ex. refrigeration. (microbiostatic, bacteriostatic, fungistatic)

"-cidal"

treatments that kill. (germicidal, microbiocidal, bactericidal, fungicidal, viricidal)

sanitizer

typically used on food-handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards (may mean just washing with soap in some cases).