Transcript Clostridium botulinum

Chapter 7
The Control of
Microbial Growth
The Terminology of Microbial
Control
• Sterilization: Removal of all microbial life
• Commercial Sterilization: Killing
Clostridium botulinum endospores
• Disinfection: Removal of pathogens
• Antisepsis: Removal of pathogens from
living tissue
• Degerming: Removal of microbes from a
limited area (mechanical removal)
The Terminology of Microbial
Control
• Sanitization: Lower microbial counts on
eating utensils
• Biocide/Germicide: Kills microbes
• Bacteriostasis: Inhibiting, not killing,
microbes
• Sepsis : refers to microbial contamination.
The Terminology of Microbial
Control
• Asepsis: the absence of significant
contamination.
• Aseptic surgery: prevent microbial
contamination of wounds.
• Aseptic technique: laboratory techniques
used to minimize contamination
The Rate of Microbial Death
• Bacterial populations die at a constant logarithmic rate
when heated or treated with antimicrobial chemicals.
Figure 7.1a
The Rate of Microbial Death
• Number of
microbes
• Environmental
influences
(organic matter,
temperature,
biofilms)
• Time of exposure
• Microbial
characteristics
Figure 7.1b
Actions of Microbial Control Agents
• Alternation of membrane permeability
– Damage to the lipids or proteins of the plasma
membrane causes cellular contents to leak and
interferes with the growth of the cell
• Damage to proteins
– Denaturation of proteins by breaking H-bonds or
covalent bonds (heat or certain chemicals)
• Damage to nucleic acids
– Cannot replicate or carry out normal metabolic
functions (heat, radiation, or chemicals)
Physical Methods of Microbial
Control
• Need to consider effects on other things besides
the microbes (e.g. vitamins, antibiotics, budget)
• Heat
– Usually used to sterilize laboratory media and
glassware, and hospital instruments
– Kill microbes by denaturing their enzymes
– Heat resistance varies among different microbes
– Need to consider suspending medium
• presence of fats and proteins protects microbes; acidic
condition more effective for heat sterilization
Physical Methods of Microbial
Control: Heat
– Thermal death point (TDP): Lowest
temperature at which all cells in a culture are
killed in 10 min.
– Thermal death time (TDT): Time required to
kill all cells in a culture
– Decimal reduction time (DRT or D value):
Minutes to kill 90% of a population at a given
temperature
• Related to bacterial heat resistance
Heat Sterilization: moist heat
• Moist heat denatures (coagulates) proteins
– Break H-bonds
• Boiling
– Kills vegetative forms of bacterial pathogens;
almost all viruses, fungi and fungal spores
• More reliable form of sterilization with
moist heat requires higher temperature
(>100 oC) e.g. Autoclave
– Need to consider if heat or moisture can
damage the material
Heat Sterilization: moist heat
• Autoclave:
Steam under
pressure
– 121 oC, 20 psi,
15 min. at sea
level
Figure 7.2
Heat Sterilization: moist heat
• Autoclave
– Need direct contact with steam or contained in
a small volume of aqueous solution
– Used for culture media, instruments, dressings,
intravenous equipment, applicators, solutions,
syringes, transfusion equipment, and numerous
other items (have to be able to withstand high
temperatures and pressure)
– Solid materials and large containers requires
extra time
Heat sterilization: Pasteurization
• Pasteurization reduces spoilage organisms and
pathogens
– Used for milk, cream, and certain alcoholic beverages
– Lowers microbial numbers, but thermoduric (heat
resistant) bacteria survive
• Thermoduric bacteria are non-pathogenic or do not cause
spoilage
• Equivalent treatments
– 63°C for 30 min
– High-temperature short-time 72°C for 15 sec
– Ultra-high-temperature: 140°C for <1 sec
Heat sterilization: Dry heat
sterilization
• Dry Heat Sterilization kills by oxidation
– Flaming: sterilize inoculating loops
– Incineration: sterilize and dispose contaminated
paper cups, bags, and dressings
– Hot-air sterilization: sterilize glassware, instruments,
needles, and glass syringes
Hot-air
Equivalent treatments 170˚C, 2 hr
Autoclave
121˚C, 15 min
Physical Methods of Microbial
Control
• Filtration removes microbes
– Virus passes through most bacteriological membrane
filters (0.22 μm to 0.45 μm pore diameter)
– Sterilize heat-sensitive materials (culture media,
enzymes, vaccines, and antibiotic solutions)
– High-efficiency particulate air (HEPA) filters to
remove all microbes larger than 0.3 μm in diameter
Physical Methods of Microbial
Control
• Low temperature inhibits microbial growth
– Decrease chemical reactions and possible
changes in proteins
– Used for food, drug, and culture preservation
– Refrigeration: bacteriostatic effect
– Deep freezing: preserve bacterial cultures
(quick-frozen between -50oC to -95 oC)
– Lyophilization: long-term preservation of
microbial cultures
Physical Methods of Microbial
Control
• High pressure alter molecular structure of
proteins and carbohydrates
– Rapid inactivation of vegetative bacterial cells
– Used for fruit juices (preserve flavors, colors,
and nutrient values of the products)
• Desiccation prevents metabolism
– Used for food preservation
– Microbes can remain viable for years
Physical Methods of Microbial
Control
• Osmotic pressure causes plasmolysis
– Used for food preservation
– Resembles preservation by desiccation (deny
moisture needed for growth to microbial cells)
• Radiation
– Effects varies depending on its wavelength,
intensity, and duration
Radiation
Figure 7.5
Physical Methods of Microbial
Control
– Ionizing radiation (X rays, gamma rays, electron
beams): cause ionization of water to form highly
reactive hydroxyl radicals that react with DNA
• Used for sterilizing pharmaceuticals and disposable dental
and medical supplies
– Nonionizing radiation (UV): damages DNA by
forming thymine dimers to inhibit correct replication
of the DNA during reproduction
• Used for disinfecting vaccines and other medical products;
to control microbes in the air
– (Microwaves kill by heat; not especially
antimicrobial)
Chemical Methods of Microbial
Control
• Control the growth of microbes on both living tissues
and inanimate objects
• Most of them reduce microbial populations to safe
levels or remove vegetative forms of pathogens from
objects.
• No single disinfectant or antiseptic is appropriate for all
situation
• Principles of effective disinfection
–
–
–
–
Concentration of disinfectant
Presence of organic matter
pH of the medium
Time of exposure
Chemical Methods of Microbial
Control
• Evaluating a disinfectant
– Use-dilution test
1.
2.
Metal rings dipped in test bacteria are dried
Dried cultures placed in disinfectant for 10
min at 20°C
3.
Rings transferred to culture media to
determine whether bacteria survived
treatment
• Effectiveness determined by the number of cultures that
grow
Chemical Methods of Microbial
Control
• Evaluating a disinfectant
– Disk-diffusion method (in teaching laboratories)
Figure 7.6
Types of Disinfectants
• Phenol
– Used by Lister to
control surgical
infections; rarely used
now
– Cause skin irritation
and disagreeable odor
– Phenolics & bisphenols
are derivative of phenol
Figure 7.7
Types of Disinfectants
• Phenolics (e.g. Lysol, O-phenylphenol)
– Used for disinfecting pus, saliva, and feces
– Derivative of phenol; reduced skin irritation
with increased antibacterial activity
– Injure lipid-containing plasma membranes
(cellular contents leak out); works on
mycobacteria
– Active even in the presence of organic
compounds, stable, and persist for long periods
after application
Types of Disinfectants
• Bisphenols (e.g. Hexachlorophene, Triclosan)
– Hexachlorophene (pHisoHex) used for surgical
and hospital microbial control procedures (esp.
staphylococci and streptococci)
– Triclosan used in antibacterial soaps & many
other products; works well against gram-positive
and gram-negative bacteria as well as fungi
– Derivative of phenol
– Disrupt plasma membranes
Types of Disinfectants
• Biguanides (e.g. Chlorhexidine)
– Used for microbial control on skin and mucous
membranes; for surgical hand scrubs and
preoperative skin preparations in patients
– Injure plasma membranes
– Strong affinity for binding to the skin &
mucous membranes
– Biocidal against most vegetative bacteria,
fungi, and certain enveloped virus
Types of Disinfectants
• Halogens (e.g. Iodine, Chlorine)
– Oxidizing agents; effective antimicrobial agents
– Iodine, one of the oldest and most effective
antiseptics; used mainly for skin disinfection and
would treatment; or treat water (iodine tablet)
• Effective against all kinds of bacteria, many
endospores, various fungi, and some viruses
• Available as a tincture and iodophor (e.g. Betadine and
Isodine)
Types of Disinfectants
– Bleach is hypochlorous acid (HOCl) formed by
chlorine + water
• Liquid form of compressed chlorine gas used for
disinfecting municipal drinking water, water in
swimming pool, & sewage
• Chlorine compounds sued to disinfect dairy
equipment, eating utensils, household items, and
glassware
• Gaseous form of chlorine, chlorine dioxide used for
area disinfection to kill endospores of anthrax
bacteria
Types of Disinfectants
• Alcohols (e.g. Ethanol & isopropanol)
– Used for swabbing skin and the surface of
inanimate objects
– Effectively kill bacteria, fungi, and some
enveloped virus
– Denature proteins, dissolve lipids
– Leave no residue (act on a surface and then
evaporates rapidly)
– Optimum concentration of ethanol = 70%, but
effective between 60 – 95%
Table 7.6
Types of Disinfectants
• Heavy Metals and Their compounds. (e.g.
sliver, mercury, and copper)
– Oligodynamic action: the ability of small
amounts of a heavy metal compound to exert
antimicrobial activity
– Denature proteins (metal ions combine with the
sulfhydryl groups on cellular protein denature)
– Silver nitrate used to prevent gonorrheal
ophthalmia neonatorum
Types of Disinfectants
• Mercuric chloride is bacteriostatic; use is
limited; control mildew in paints
• Copper sulfate sued chiefly to destroy green
algae that grow in reservoirs, stock ponds,
swimming pools, and fish tanks
• Zinc chloride: common ingredient in mouth
washes
Types of Disinfectants
• Surface-Active Agents (Surfactants)
– Decrease surface tension among molecules of a liquid
Soap
Acid-anionic detergents
Quaternary ammonium
compounds
Cationic detergents
Degerming; mechanical removal of
microbes by scrubbing
Sanitizing (food industry, dairy
utensils and equipment); anion reacts
with plasma membrane; effective on
thermoduric bacteria
Bactericidal (gram-positive),
fungicidal, amoebicidal, and virucidal
(enveloped virus); Denature proteins,
disrupt plasma membrane
Types of Disinfectants
• Chemical Food Preservatives
– Organic Acids or salts of organic acids
• Inhibit metabolism
• Sorbic acid, benzoic acid, calcium propionate
• Control molds and bacteria in foods and cosmetics
– Nitrite prevents endospore germination & preserve
the red color of the meat
– Antibiotics (not for internal use)
• Nisin (for endospore-forming spoilage bacteria) and
natamycin (antifungal antibiotic) prevent spoilage of
cheese
Types of Disinfectants
• Aldehydes (e.g. Glutaraldehyde &
formaldehyde)
– Very effective antimicrobials
– Inactivate proteins by cross-linking with
functional groups (–NH2, –OH, –COOH, —
SH)
– Glutaraldehyde used to disinfect hospital
instruments including respiratory-therapy
equipment
• 2% solution bactericidal, tuberculocidal, and
virucidal (10 min.)
Types of Disinfectants
– Formaldehyde (formalin) used to preserve
biological specimens and inactivate bacteria and
viruses in vaccines
• Gaseous Chemosterilizers (e.g. ethylene
oxide)
– Chemicals that sterilize in a closed chamber
– Denature proteins
– Highly penetrating; widely used on medical
supplies and equipment; suspected carcinogens
• Propylene oxide and beta-propiolactone can also be
used for sterilization
Types of Disinfectants
• Peroxygens (e.g. O3, H2O2, peracetic acid)
– Oxidizing cellular components
– Ozone (O3) often used to supplement chlorine
in the disinfection of water (neutralize taste and
odor)
– H2O2: effective disinfectant on inanimate
objects; but not good for open wounds (may
slow healing); neutralized by catalase (present
in human cells)
Types of Disinfectants
– Benzoyl peroxide: useful for treating wounds
infected by anaerobic pathogen; medications
for acne
– Peracetic acid: one of the most effective liquid
chemical sporicides available; considered a
sterilant; effective on endospores and viruses
• Also used in disinfection of food-processing and
medical equipment
Microbial Characteristics and
Microbial Control
• Resistance due to:
– Structural component
• External LPS layer of
gram-negative (porin)
• Cell wall structure of
mycobacteria
• Endospore (bacterial)
• Protozoan cysts and
oocysts
• Non-enveloped virus
– No effective means to
destroy (e.g. prions)
Figure 7.11
Microbial Characteristics and
Microbial Control
Chemical agent
Phenolics
Quats
Chlorines
Alcohols
Glutaraldehyde
Effectiveness against
Endospores
Mycobacteria
Poor
Good
None
None
Fair
Fair
Poor
Good
Fair
Good