Control of Microbial Growth

1
Control of Microbial Growth

• Chapter 5

2
Approaches to Control

• Control mechanisms either physical or chemical
• May be a combination of both
• Physical methods
• Heat
• Irradiation
• Filtration
• Mechanical removal
• Chemical methods
• Use a variety of antimicrobial chemicals
• Chemical depends on circumstances and degree of
  control required

3
Approaches to Control

• Principles of control
• Sterilization
• Removal of all microorganisms
• Sterile item is absolutely free of microbes,
  endospores and viruses
• Can be achieved through filtration, heat,
  chemicals and irradiation
• Disinfection
• Eliminates most pathogens
• Some viable microbes may exist
• Disinfectants used on inanimate objects and
  surfaces
• Antiseptics used on living tissues
• Pasteurization
• Brief heat treatment used to reduce organisms
  that cause food spoilage
• Surfaces can also be pasteurized

4
Approaches to Control

• Principles of control
• Decontamination
• Treatment to reduce pathogens to level considered
  safe
• Degerming
• Mechanism uses to decrease number of microbes in
  an area
• Particularly the skin
• Sanitized
• Implies a substantially reduced microbial
  population
• This is not a specific level of control
• Preservation
• Process used to delay spoilage of perishable
  items
• Often includes the addition of growth-inhibiting
  ingredients

5
Approaches to Control

• Situational considerations
• Microbial control methods are highly variable
• Depends on situation and degree of control
  required
• Daily life
• Hospital
• Microbiology laboratories
• Food and food production facilities
• Water treatment

6
Approaches to Control

• Daily life
• Washing a scrubbing with soaps and detergents
  achieves routing control
• Hand washing single most important step to
  achieving control
• Soap acts as wetting agent
• Aids in mechanical removal of microorganisms
• Removes numerous organism from outer layer of
  skin
• Normal flora usually unaffected because it
  resides in deeper layers

7
Approaches to Control

• Hospitals
• Minimizing microbial population very important
• Due to danger of nosocomial infections
• Patients are more susceptible to infection
• Pathogens more likely found in hospital setting
• Numerous organisms develop antimicrobial
  resistance due to high concentrations of
  antibiotics
• Instruments must be sterilized to avoid
  introducing infection to deep tissues

8
Approaches to Control

• Microbiology laboratories
• Use rigorous methods of control
• To eliminate microbial contamination to
  experimental samples and environment
• Aseptic technique and sterile media used for
  growth
• Eliminates unwanted organisms
• Contaminated material treated for disposal
• Eliminate contamination of environment

9
Approaches to Control

• Food and food production facilities
• Retention of quality enhanced through prevention
  of microbial growth and contamination
• Achieved through physical removal and chemical
  destroying organisms
• Heat treatment most common and most reliable
  mechanism
• Irradiation approved to treat certain foods
• Chemicals prevent spoilage
• Risk of toxicity

10
Approaches to Control

• Water treatment facilities
• Ensures drinking water is safe
• Chlorine generally used to disinfect water
• Can react with naturally occurring chemicals
• Form disinfection by-products (DBP)
• Some DBP linked to long-term health risks
• Some organism resistant to chemical disinfectants

11
Selection of Antimicrobial Procedure

• Selection of effective procedure is complicated
• Ideal method does not exist
• Each has drawbacks and procedural parameters
• Choice of procedure depends on numerous factors
• Type of microbe
• Extent of contamination
• Number of organisms
• Environment
• Risk of infection
• Composition of infected item

12
Selection of Antimicrobial Procedure

• Type of microorganism
• Most critical consideration
• Is organism resistant or susceptible to generally
  accepted methods?
• Resistant microbes include
• Bacterial endospores
• Resistant to heat, drying and numerous chemicals
• Protozoan cysts and oocysts
• Generally excreted in feces and cause diarrheal
  disease
• Mycobacterium species
• Cell wall structure initiates resistance
• Pseudomonas species
• Can grow in presence of many chemical
  disinfectants
• Naked viruses
• Lake envelope and are more resistant to chemical
  killing

13
Selection of Antimicrobial Procedure

• Number of organisms initially present
• Time it takes to kill it directly affected by
  population size
• Large population more time
• Commercial effectiveness is gauged by decimal
  reduction time
• A.k.a D value
• Time required to kill 90 of population under
  specific conditions
• Washing reduces time required to reach
  disinfection or sterilization

14
Selection of Antimicrobial Procedure

• Environmental conditions
• Environmental conditions strongly influence
  effectiveness
• pH, temperature and presence of organic materials
  can increase or decrease effectiveness
• Most chemicals are more effective at higher
  temperatures and lower pH
• Effectiveness can be hampered by the presence of
  organism molecules
• Can interfere with penetration of antimicrobial
  agent

15
Selection of Antimicrobial Procedure

• Potential risk of infection
• Medical items categorized according to potential
  risk of disease transmission
• Critical items come in contact with body
  tissues
• Needles and scalpels
• Semicritical instruments contact mucous
  membranes but do not penetrate body tissues
• Endoscope
• Non-critical instruments contact unbroken skin
  only
• Show little risk of transmission
• Stethoscope

16
Selection of Antimicrobial Procedure

• Composition of the item
• Some sterilization and disinfection methods
  inappropriate for certain items
• Heat inappropriate for plastics and other heat
  sensitive items

17
Heat as Control

• Heat treatment most useful for microbial control
• Relatively fast, reliable, safe and inexpensive
• Heat can be used to sterilize or disinfect
• Methods include
• Moist heat
• Dry heat

18
Heat as Control

• Moist heat
• Destroys through irreversible coagulation of
  proteins
• Moist heat includes
• Boiling
• Pasteurization
• Pressurized steam

19
Heat as Control

• Boiling (100 C)
• Destroys most microorganisms and viruses
• Not effective means of sterilization
• Does not destroy endospores
• Pasteurization
• Pasteur developed to avoid spoilage of wine
• Does not sterilize but significantly reduces
  organisms
• Used to increase shelf life of food
• Most protocols employ HTST method
• Heated to 72C and held for 15 seconds
• Other protocol UHT
• Heated to 140C - 150C, held for several seconds
  then rapidly cooled

20
Heat as Control

• Pressurized steam
• Autoclave used to sterilize using pressurized
  steam
• Heated water ? steam ? increased pressure
• Preferred method of sterilization
• Achieves sterilization at 121C and 15psi in 15
  minutes
• Effective against endospores
• Flash autoclaving sterilizes at 135C and 15psi
  in 3 minutes
• Prions destroyed at 132C and 15psi for 4.5 hours

21
Heat as Control

• Dry heat
• Not as effective as moist heat
• Sterilization requires longer times and higher
  temperatures
• 200C for 1.5 hours vs. 121C for 15 minutes
• Incineration method of dry heat sterilization
• Oxidizes cell to ashes
• Used to destroy medical waste and animal
  carcasses
• Flaming laboratory inoculation loop incinerates
  organism
• Results in sterile loop

22
Other Physical Methods of Control

• Heat sensitive materials require other methods of
  microbial control
• Filtration
• Irradiation
• High-pressure treatment

23
Other Physical Methods of Control

• Filtration
• Membrane filtration used to remove microbes from
  fluids and air
• Liquid filtration
• Used for heat sensitive fluids
• Membrane filters allow liquids to flow through
• Traps microbes on filter
• Depth filters trap microbes using electrical
  charge

• Filtration of air
• High efficiency particulate air (HEPA) filter
  remove nearly all microbes from air
• Filter has 0.3µm pores to trap organisms

24
Other Physical Methods of Control

• Radiation
• Electromagnetic radiation
• Energy released from waves
• Based on wavelength and frequency
• Shorter wavelength, higher frequency more
  energy
• Range of wavelength is electromagnetic spectrum
• Radiation can be ionizing or non-ionizing

25
Other Physical Methods of Control

• Ionizing radiation
• Radiation able to strip electrons from atoms
• Three sources
• Gamma radiation
• X-rays
• Electron accelerators
• Causes damage to DNA and potentially to plasma
  membrane
• Used to sterilize heat resistant materials
• Medical equipment, surgical supplies, medications
• Some endospores can be resistant

26
Other Physical Methods of Control

• Ultraviolet radiation
• Non-ionizing radiation
• Only type to destroy microbes directly
• Damages DNA
• Causes thymine dimers
• Used to destroy microbes in air, drinking water
  and surfaces
• Limitation
• Poor penetrating power
• Thin films or coverings can limit effect

27
Other Physical Methods of Control

• High pressure processing
• Used in pasteurization of commercial foods
• Does not use high temperatures
• Employs high pressure
• Up to 130,000 psi
• Destroys microbes by denaturing proteins and
  altering cell membrane permeability

28
Chemicals as Control

• Chemicals can be used to disinfect and sterilize
• Called germicidal chemicals
• Reacts with vital cell sites
• Proteins
• DNA
• Cell membrane

29
Chemicals as Control

• Potency of chemicals
• Formulations generally contain more than one
  antimicrobial agent
• Regulated by
• FDA
• Antiseptics
• EPA
• Disinfectants
• Germicidal agents grouped according to potency

• Sterilants
• Destroy all microorganisms
• High-level disinfectants
• Destroys viruses and vegetative cells,
• Not endospores
• Intermediate-level disinfectants
• Kills vegetative cells fungi, most viruses,
• Not endospores
• Low-level disinfectants
• Removes fungi, vegetative bacteria and enveloped
  viruses
• Not mycobacteria, naked viruses or endospores

30
Chemicals as Control

• Selecting appropriate chemical
• Points to consider
• Toxicity
• Benefits must be weighed against risk of use
• Activity in presence of organic material
• Many germicides inactivated in presence of
  organic matter
• Compatibility with material being treated
• Liquids cannot be used on electrical equipment
• Residue
• Residues can be toxic or corrosive
• Cost and availability
• Storage and stability
• Concentrated stock relieves some storage issues
• Environmental risk
• Is germicidal agent harmful to environment

31
Chemicals as Control

• Classes of chemicals
• Germicides represent a number or chemical
  families
• Alcohols
• Aldehydes
• Biguanides
• Ethylene oxide
• Halogens
• Metals
• Ozone
• Peroxides
• Phenolics
• Quaternary ammonium compounds

32
Chemicals as Control

• Alcohols
• Solutions of 60 - 80 isopropyl or ethyl alcohol
  kill vegetative bacteria and fungi
• Not effective against endospores and some naked
  viruses
• Mode of action
• Coagulation of proteins and essential enzymes
• Damage to lipid membranes
• Commonly used as antiseptic and disinfectant
• Limitations
• Evaporates quickly limiting contact time
• May damage material such as rubber and some
  plastics

33
Chemicals as Control

• Aldehydes
• Destroy organisms by inactivating proteins and
  DNA
• 2 glutaraldehyde solution most widely used
  liquid sterilant
• Orthophthalaldehyde studied as alternative
• Formalin used to kill bacteria and inactivate
  viruses
• Also used for specimen preservation
• Formalin is solution made from formaldehyde

34
Chemicals as Control

• Biguanides
• Most effective member of group is chlorhexidine
• Extensively used in antiseptics
• Relative low toxicity
• Destroys wide range of organisms

35
Chemicals as Control

• Ethylene oxide
• Useful gaseous sterilant
• Destroys microbes including endospores and
  viruses
• Mode of action
• Reacts with proteins
• Useful in sterilizing heat or moisture sensitive
  items
• Limitations
• Mutagenic and potentially carcinogenic

36
Chemicals as Control

• Halogens
• Common disinfectants
• Mode of action
• Oxidizing proteins and other cell components
• Includes chlorine and iodine
• Chlorine
• Destroys all types of organisms and viruses
• Used as disinfectant
• Caustic to skin and mucous membranes
• Chlorine dioxide replacing chlorine in many
  applications
• Iodine
• Kills vegetative cells
• Not reliable with endospores
• Used in tincture or iodophore on skin

37
Chemicals as Control

• Metal compounds
• Compounds combine with enzymes and proteins
• Interfering with function
• High concentrations of many metals toxic to human
  tissue
• Silver still used as disinfectant
• Creams containing silver sulfadiazine used to
  prevent secondary infections
• Also available on bandages for wound care

38
Chemicals as Control

• Ozone
• O3
• Unstable form of oxygen
• Powerful oxidizing agent
• Used as alternative to chlorine
• As disinfectant for drinking and waste water

39
Chemicals as Control

• Peroxygens
• Includes hydrogen peroxide and peracetic acid
• Powerful oxidizing agents
• Readily biodegradable
• Less toxic than ethylene oxide and glutaraldehyde

40
Chemicals as Control

• Hydrogen peroxide
• Effectiveness depends on surface being treated
• Living tissue produce catalase enzyme
• Breaks down hydrogen peroxide to oxygen and water
• More effective on inanimate object
• Useful as disinfectant
• Leaves no residue
• Doesnt damage most materials
• Hot solutions used in food industry
• Vapor-phase can be used as sterilant
• Peracetic acid
• More potent then hydrogen peroxide
• Effective on organic material
• Can be used on wide range of material

41
Chemicals as Control

• Phenolics
• A.k.a carbolic acid
• One of the earliest disinfectants
• Now has limited use
• Active ingredient in Lysol
• Mode of action
• Destroy plasma membrane
• Denature proteins
• Kills most vegetative cells
• Can kill mycobacterium at high concentrations
• Not reliable on all groups of viruses
• Triclosan and hexachlorophene phenols used in
  soaps and lotions

42
Chemicals as Control

• Quaternary ammonium compounds
• A.k.a Quats
• Cationic detergents
• Nontoxic
• Used to disinfect food preparation surfaces
• Mode of action
• Reduces surface tension
• Aids in removal of dirt and organic matter
• Facilitates mechanical removal of organisms
• Positive charge attracts Quats to negative charge
  of cell surface
• Reacts with membrane
• Destroys vegetative bacteria and enveloped
  viruses
• Not effective on endospores, mycobacteria and
  naked viruses

43
Preservation of Perishable Products

• Preservation extends shelf-life of many products
• Chemicals are often added to prevent or slow
  growth of microbes
• Other methods include
• Low temperature storage
• Freezing
• Reducing available water

44
Chemicals as Control

• Chemical preservatives
• Numerous chemicals are used as preservatives
• Formaldehyde, Quats, and phenols
• Weak organic acids often used as food
  preservatives
• Benzoic, ascorbic and propionic acids
• Used in bread, cheese and juice
• Mode of action
• Alter cell membrane function
• Interfere with energy transformation
• Nitrates and nitrites used in processed meats
• Inhibits germination of endospores and growth of
  vegetative cells
• Have been shown to be potent carcinogen

45
Chemicals as Control

• Low temperature storage
• Microbial growth is temperature dependent
• Low temperatures slow down or stop enzymatic
  reactions of mesophiles and thermophiles
• Some psychrophiles still able to grow
• Freezing means of food preservation
• Essentially stops microbial growth
• Irreversibly damages cell
• Kills up to 50 of microbes
• Reaming cells still pose potential threat

46
Chemicals as Control

• Reducing water availability
• Decreasing water availability accomplished by
  salting or drying food
• Addition of salt increases environmental solutes
• Causes cellular plasmolysis
• Numerous bacteria can continue to grow in high
  salt environments
• Staphylococcus aureus can survive in high salt
  concentrations
• Desiccation or drying is often supplemented by
  other methods
• Salting
• Lyophilization (freeze drying)
• Widely used to preserve foods like coffee, milk
  and meats