Microbial Growth Control

Control of Microbial Growth in the Environment

• Microbial growth control is important in public health, health care, laboratories, and at home

Terminology of Microbial Control

• Sterilization involves removing/destroying all microbes, including endospores and viruses
• Commercial sterilization of canned food targets Clostridium botulinum endospores
• Aseptic environments or procedures are free of contamination, specifically pathogens
• Aseptic surgery techniques are used to prevent microbial contamination of wounds
• Disinfection uses chemicals or physical agents to kill/inhibit microbe growth, especially pathogens
• Disinfectants are used to treat inanimate objects/surfaces
• Antisepsis uses chemicals on skin or tissue, chemicals are called antiseptics
• Degerming removes microbes by scrubbing/swabbing, using soaps or alcohol
• Washing hands and preparing an area of skin for an injection is degerming
• Sanitization disinfects places/things used by the public to meet public health standards
• Steam and hot water are used to sanitize restaurant utensils, chemicals for public toilets
• Pasteurization applies heat to kill pathogens and reduce spoilage microbes in food/beverages

Activity of Chemical and Physical Agents

• Chemical/physical agents have -static (inhibiting) or -cidal (killing) activity
• -stasis or -static agents inhibit microbial metabolism and growth without killing microbes
• -cide or -cidal agents kill target microbes
• Bacteriostatic agents inhibit bacterial growth, while bactericidal agents kill bacteria
• Fungistatic agents inhibit fungal growth, while fungicidal agents kill fungi
• Virustatic agents inhibit viral growth, while virucidal agents kill viruses
• Germicides are antimicrobial chemicals that kill pathogens

Microbial Death Rates

• Microbial death is the permanent loss of reproductive capability
• Antimicrobial agent effectiveness is determined by microbial death rate, which is constant
• Microbicidal agents do not kill all cells simultaneously, but kill a constant percentage over time
• Death rate example: 90% of cells die after each minute of treatment

Ideal Antimicrobial Agent Characteristics

• Inexpensive
• Fast-acting
• Stable during storage
• Controls growth/reproduction of microbes
• Harmless to humans, animals, and objects
• However, all antimicrobial agents possess limitations, advantages and disadvantages

Considerations When Selecting Antimicrobial Methods

• Nature of the site/object to be treated
• Sensitivity to heat or chemicals must be considered
• Potential risk of infection with medical instruments
• Invasive vs non-invasive equipment
• Number and susceptibility of microbes
• Environmental conditions like temperature, pH, and organic material should be considered

Antimicrobial Chemicals & Medical Instruments

• Germicide effectiveness is classified as high, intermediate, or low
• Classification relies on their ability to destroy microbes on non-sterilizable instruments
• High-level germicides kill all microbes, including endospores and are used on invasive instruments like catheters and implants
• Intermediate-level germicides do not kill endospores and are used on non-invasive instruments like endoscopes that contact mucous membranes
• Low-level germicides are not effective against endospores/all viruses, used on instruments that contact skin, e.g., stethoscopes and electrodes

Biosafety Levels

• CDC provides guidelines for four safety levels in microbiology labs
• Each level increases personnel/environmental safety through strict lab techniques, safety equipment, and facility design
• Pathogens are classified by risk groups (BSL1-4) based on pathogenicity, transmission, and treatment availability
• BSL1: Non-pathogenic E. coli
• BSL2: Staphylococcus aureus (MRSA)
• BSL3: M. tuberculosis, B. anthracis
• BSL4: Ebola virus, Smallpox

Heat Treatment for Microbial Control

• Microbes vary in their susceptibility to heat, which can be used for disinfection/sterilization
• Thermal death point is the lowest temperature that kills all cells in a broth in 10 minutes
• Thermal death time is the time needed to sterilize a specific liquid volume at a set temperature
• Decimal reduction time (D) is the time required to kill 90% of microbes in a sample
• The canning industry uses D value to kill 90% of Clostridium botulinum endospores per treatment at 121°C

Commercial Canning Process

• Industrial-sized autoclaves (retorts) are used
• Aimed to reduce 10^12 Clostridium botulinum endospores to only 1, which is a 12 D process
• A D value at 121°C is 0.204 min
• Endospores in canned foods can germinate and vegetative cells produce botulinum toxin
• This neurotoxin causes botulism from contaminated food

Additional Heat Treatment Information

• Heat treatment is reliable, safe, fast, inexpensive, and non-toxic
• Methods include moist and dry heat
• Boiling kills most microorganisms and viruses, but does not sterilize due to endospore survival
• Pasteurization kills pathogens like Brucella melitensis and Mycobacterium bovis
• Pasteurization decreases the number of heat-sensitive microbes, reducing spoilage

Sterilization with Pressurized Steam (Autoclave)

• Sterilization typically occurs at 121°C and 15 psi for 15-20 minutes
• Autoclaving destroys endospores, but not prions
• Dry heat is less effective than moist heat
• Dry heat requires longer times and higher temperatures (ovens)
• Incineration destroys medical waste, diseased animal carcasses, and sterilizes inoculating loops

Refrigeration and Freezing

• Refrigeration and freezing, desiccation, lyophilization, and osmotic pressure are used to store microbial cultures
• Refrigeration is short-term storage because it inhibits the growth of many pathogens and spoilage microbes
• Some pathogens, like Listeria and Yersinia, reproduce in refrigerated food and blood products
• Freezing stops microbial growth by limiting water availability
• Survivors will grow and spoil food once thawed

Additional Storage Methods

• Desiccation is also used, supplemented by addition of salt/sugars for preservation
• Lyophilization (freeze-drying) is used for coffee, milk, meats, fruits, and vegetables
• Salting and adding sugars increase environmental solute concentration, resulting in cellular plasmolysis

Mechanical Methods for Controlling Microbial Growth

• Filtration is useful for counting microbes and/or sterilizing heat-sensitive materials
• Filtration is used for culture media, drugs, vitamins, enzymes, and antibiotic solutions/vaccines
• HEPA filters are placed in safety cabinets or air ducts to protect immunocompromised individuals and to help TB containment

Ionizing Radiation

• Gamma and X-rays are used
• Radiation removes electrons from atoms, thus produces ROS (reactive oxygen species)
• ROS damages cell structures-DNA
• Used to sterilize medical equipment and drugs
• Approved by FDA/WHO/UNFAO on food to eliminate pathogens and decrease the amount of spoilage organisms in spices, fruit, vegetables, poultry, beef, lamb, pork

Non-Ionizing Radiation

• Ultraviolet radiation forms thymine dimers, causing DNA mutations
• UVGI (Ultraviolet Germicidal Irradiation) uses the UVC range for disinfection, as well as food preservation, air purification, or wastewater treatment
• UV lamps are utilized in hospitals (nurseries and operating rooms) and inside of biological safety cabinets
• Has poor penetrating power because turbid liquids, solids, glass and plastic block UV radiation

Effectiveness of Chemical Agents: Factors to Consider

• Concentration of the chemical
• Temperature, pH, and exposure time
• Presence of contaminating organic material
• Numbers and types of microbes present
• The object or site being treated

Phenol and Phenolics

• Phenol was originally used by Lister to prevent surgical wound infections
• Triclosan, a bisphenolic, is in diapers, garbage bags and cutting boards
• Phenolic compounds are still used in healthcare since they work in the presence of organic material

Alcohol

• Includes Isopropanol and ethyl alcohol
• Alcohols are used as disinfectants and antiseptics
• Pure alcohol is ineffective, meaning that 70-90% alcohol solutions are used
• They evaporate quickly and may not contact microbes long enough

Halogens

• Iodine, chlorine, fluorine, and bromine fall into this category
• Iodine is an antiseptic
• Tinctures are iodine solutions in alcohol
• Iodophors are organic compounds containing iodine that slowly releases it
• Betadine (Iodophor) is used for surgery preparation on a hand
• Chlorine disinfects drinking water and swimming pools, and waste-water
• Sodium hypochlorite (household chlorine bleach) and calcium hypochlorite are effective disinfectants
• Chlorine dioxide (gas) was employed in 2001 for federal facilities to remove anthrax spores
• Chloramine is used as a disinfectant and antiseptic in wound dressings

Oxidizing Agents

• Oxidizing agents (e.g. H2O2) are high-level disinfectants and antiseptics
• H2O2 is used as a disinfectant and antiseptic (not on open wounds because catalase inactivates it), it is used also for sterilization
• Ozone disinfects drinking water as an expensive but safer alternative to chlorine
• Peracetic acid is used for equipment sterilization and by food processing/medical personnel which is unaffected by contaminants

Surfactants

• This includes Soaps and detergents as a method of microbial control
• Soaps are good degerming agents but have poor antimicrobial activity
• Detergents have quaternary ammonium compounds (quats), found as antimicrobials in several products
• Benzalkonium chloride (Zephiran) / Cetylpyridinium is in Cepacol mouthwash
• Their action is retarded by organic contaminants
• Notably, Pseudomonas aeruginosa thrives in quats

Heavy Metals as Disinfectants

• These act as low-level disinfectants, they are bacteriostatic and fungistatic
• Including silver, mercury, copper, zinc, and arsenic
• Silver nitrate was used prevent newborn blindness caused by Neisseria gonorrhoeae
• Moreover, silver is still used as an antimicrobial in burn creams, surgical dressings and catheters
• Heavy metals denature proteins

Formaldehyde

• A 2% glutaraldehyde solution is used for disinfection or sterilization, depending on length of contact
• A 37% formaldehyde solution (formalin) disinfects rooms and instruments

Alternative methods of Microbial Control

• Includes Enzymatic Methods
• To control microbes in food (like cheese) and or to prevent wine from spoiling, lysozyme is used
• Lysozyme is produced by the human body, working as antimicrobial by breaking the NAM/NAG link in peptidoglycans
• In a second example, prionzyme is enzymatic against prions, it removes them from surgical instruments

Evaluating Antimicrobial Agents

• Multiple methods exist
• Including disk-diffusion