Microbial Growth Control: Chapter 5

• Lecture 6 focuses on controlling microbial growth, corresponding to Chapter 5 in the textbook

Important Terms

• Sterilization is the complete removal/destruction of all microbial life, including the elimination of all microorganisms and endospores
• Disinfection involves the destruction of pathogenic microorganisms, killing most but not all microbes; for example, chemical disinfectants like alcohol and bleach
• Antisepsis refers to disinfecting living tissues, requiring a gentler chemical treatment
• Degerming is the removal of most microbes from a limited area, such as swabbing skin with alcohol (mechanical removal)
• Sanitization lowers microbial counts on a surface to a safe level

Bacteriocidal vs Bacteriostatic

• Agents ending in 'cide' kill microbes; a bacteriocidal agent kills bacteria
• Agents ending in 'static' inhibit microbial growth; a bacteriostatic agent prevents bacteria from reproducing, maintaining a constant bacterial number
• Asepsis (aseptic) is when an area is free of significant contamination; surgical techniques are performed aseptically to minimize infection risks

Physical Methods of Microbial Control

• Heat kills microbes by destroying their fundamental enzymes and is bacteriocidal

Moist Heat

• Boiling disrupts protein structure by breaking hydrogen bonds, killing most pathogens and viruses and accomplishing disinfection in 5 minutes; it does not kill endospores
• Autoclaving uses 15psi pressure and 121°C steam to kill all endospores in about 15 minutes, sterilizing all organisms and best used when heat damage is not a concern
• Autoclaving is also used for sterilizing culture media, hospital instruments and materials able to withstand heat/moisture; all liquids must reach 121°C and all physical surfaces must be exposed to steam

Pasteurization

• Pasteurization does not result in sterilization but eliminates pathogens to slow spoilage
• Pasteurization involves mild heating of a liquid
• Classic treatment is 63°C for 30 minutes
• Alternative treatments include:

HTST Pasteurization

• High Temperature Short-Time Pasteurization is 72°C for 15 seconds and is the most common today

UHT Pasteurization

• Ultra-High Temperature Pasteurization is 140°C for 3 seconds, and the product can be stored without refrigeration

Dry Heat Sterilization

• Several methods exist that kill via oxidation
• Direct flaming is used in microbiology labs to sterilize inoculation needles/loops, where metallic instruments must be red hot to be sterile
• Incineration is an effective way to sterilize and dispose of contaminated materials such as paper cups and dressings
• Hot air sterilization utilizes a drying oven at 170°C for 2 hours to ensure sterilization
• Hot air sterilization is used for dry materials that are not temperature-sensitive, ie glassware and dry chemicals sensitive to moisture

Filtration

• Vacuum is used to force liquid through a filter with very small pores: 0.10µm, 0.22μm, or 0.45 μm
• Pores are too small for bacteria to pass through
• It is used to sterilize/filter temperature-sensitive liquids such as antibiotics, enzymes, some vaccines, and culture media
• Viruses are much smaller than bacteria and can pass through the pores

Air Filtration

• High efficiency particulate air (HEPA) filters filter air and are used in operating rooms to remove microbes larger than 0.30μm in diameter, as well as in rooms for burn patients

Low Temperatures

• Refrigeration slows/stops microbial growth and is bacteriostatic
• Freezing stops microbial growth; reduces metabolic rate, with no reproduction or toxin synthesis, killing only a small portion of microbes

High Pressure

• Liquid suspensions are treated with high pressure which disturbs protein/molecular structure and kills most bacterial cells, but does not kill endospores or sterilize; it is a bacteriocidal method

Dessication

• Dessication controls microbial growth and is bacteriostatic in action when performed
• The processes of removal of water (drying) slows/stops microbial growth but does not kill microbes; microbes can remain viable for years in a dehydrated state

Osmotic Pressure

• High concentrations of salts and sugars creates a hypertonic environment in which bacterial cells will dehydrate and shrivel
• Osmotic Pressure can kill some bacteria, therefore it is both bacteriocidal and bacteriostatic
• Molds and yeasts are more resistant

Radiation

• Radiation destroys DNA
• X-rays and gamma rays penetrate materials
• UV and high-energy electron beams are used on surfaces

Microwaves

• Microwaves use longer wavelengths that do not have high enough energy to directly kill microorganisms
• Instead, microwaves heat the water, which then kills the microorganisms, and/or the high temperatures created by the microwaves disinfect materials

Chemical Methods of Microbial Control

• Most methods can disinfect but may not sterilize; they reduce microbial counts to safe levels
• Assessing effectiveness is done using the disk diffusion Method: A disk of filter paper is soaked in the chemical disinfectant; the disk is placed on the agar plate already been inoculated with bacteria
• The chemical diffuses from the paper to the plate
• The plate is incubated until the inoculated bacteria grow, and the zone of clearance around the bacteria is used to determine disinfectant effectiveness

Types of Disinfectants and Antiseptics

• Phenolics and Bisphenols disrupt plasma membranes and denature proteins
• They are bacteriocidal, very effective, remain active a long time and are stable with other organic compounds
• They are suitable for the disinfection of surfaces contaminated by saliva, pus, and feces and are mixed with other compounds to decrease their toxicity
• Examples include Phenolics and Bisphenols:

Phenolics

• Lysol is used for household disinfection

Bisphenols

• Hexachlorophene is useful against bacteria that cause skin infections in newborns and disinfects nurseries; caution since it may cause neurological disorders
• Triclosan is used in some personal hygiene products and cosmetics such as deodorant soaps

Biguanides

• Chlorhexidine attacks the plasma membrane with a broad spectrum of activity
• It's used to control microbes in surgical hand scrubs and is combined with alcohol/detergents and Bacteriocidal
• Only kills enveloped viruses and does not destroy endospores; mycobacteria species are also resistant
• Chlorhexidene can persist for 6 hours

Halogens

• Chlorine and iodine impair protein synthesis and alter membranes
• They are used as household bleach and iodine, which is a very effective antiseptic
• Chlorine, used as a gas/combined with other chemicals, is a strong oxidizing agent that destroys enzymes
• Iodine, one of the oldest and most effective antibiotics, is available as a tincture (in solution in aqueous alcohol) or an iodophore combined with iodine and an organic molecule that slowly releases the iodine, i.e. betadine

Alcohols

• Alcohols denature proteins and disrupt lipid membranes, killing bacteria and fungi
• They do not kill endospores and non-enveloped viruses
• Acts very quickly and evaporates to leave no residue
• It is used to wipe skin surfaces before injections and is unsatisfactory applied directly to wounds but combined with water for required alcohol for denaturation
• Types: Ethanol (70% most effective) and isopropanol (rubbing alcohol) are two commonly used types

Heavy Metals

• Metals like copper, silver, zinc, and mercury can inhibit microbes
• Silver nitrate (1%) is used as an antiseptic where eye drops are given to newborn infants help protect against gonorrhea of the eye
• Silver sulfadiazine, a combination of silver and sulfadiazine drug, is used as a topical cream for burns
• Silver ions can also be incorporated into dressings and catheters to prevent hospital-acquired infections
• Copper sulfate can destroy green algae in swimming pools/fish tanks
• Zinc chloride has been used in mouthwashes

Soaps and Detergents

• Soaps and detergents do not work as antiseptics but as surface agents
• They are most important in the mechanical removal of microbes
• Soap breaks up oil on the skin, which is then washed away along with debris by water
• Soap and water are therefore good degerming agents

Quaternary Ammonium Compounds (Quats)

• Quats are cationic detergents that kill most bacteria, fungi, and some viruses, but not endospores or mycobacteria
• They are less effective against gram-negative than gram-positive organisms, affecting the plasma membrane
• They are strongly antimicrobial, colorless, tasteless, odorless, and non-toxic at low concentrations: Cepacol as mouthwash
• Pseudomonas not only survives in these compounds but actively grows in them

Gaseous Chemosterilizers

• Chemical gases used for sterilization, such as ethylene oxide, denature protein and are bacteriocidal
• They kill all microbes, including endospores and viruses, because exposure requires lasting 4-18 hours, are penetrating and, can be used to sterilize disposable hospital equipment and lab supplies ie. plastic tubing and disposable swabs

Oxidizing Agents

• Oxidizing agents oxidize and denature proteins and are useful against anaerobic organisms
• Hydrogen peroxide is a common antiseptic for wounds
• Ozone is used with chlorine to disinfect water
• Benzoyl peroxide is used as antiseptic on skin, kills anaerobic bacteria in tissues, and is an example for bacteria that cause acne

Gradient of Resistance

• Most resistant: Prions
• Endospores of bacteria
• Mycobacteria
• Cysts of protozoa
• Vegetative protozoa
• Gram-negative bacteria
• Fungi, including most fungal spores
• Viruses without envelopes
• Gram-positive bacteria
• Viruses with lipid envelopes
• Least resistant