Transcript Lesson 3Control of Microbial Growth

Lesson 3
Control of Microbial Growth
January 22, 2015
Reproduction in Prokaryotes
• Binary fission—asexual reproduction by a
separation of the body into two new bodies
– DNA replication
– Cytokinesis—splitting of the cell
• Budding—asexual reproduction in which a
new individual develops from some generative
anatomical point of the parent organism
– Yeasts (Candida albicans and Cryptococcus
neoformans)
Figure 6.12a Binary fission in bacteria.
Cell elongates and DNA is
replicated.
Cell wall and plasma membrane
begin to constrict.
Cell wall
Plasma
membrane
DNA
(nucleoid)
Cross-wall forms, completely
separating the two DNA
copies.
FtsZ protein
Cells separate.
(a) A diagram of the sequence of cell division
Budding
YEAST AND FUNGI
Phases of Bacterial Growth
• Bacteria have four distinctive growth phases
– Lag—intense activity preparing for population
growth but has little to no increase in population
– Log—(logarithmic growth) period of growth where
cellular reproduction is most active
– Stationary—growth rate slows. Number of
bacterial death balances the number of new cells.
Population stabilizes
– Death—number of deaths exceeds the number of
new cells formed. Population can die out entirely
Figure 6.15 Understanding the Bacterial Growth Curve.
Lag Phase
Log Phase
Stationary Phase
Intense activity
preparing for
population growth,
but no increase in
population.
Logarithmic, or
exponential,
increase in
population.
Period of equilibrium;
microbial deaths balance
production of new cells.
Death Phase
Population Is
decreasing at a
logarithmic rate.
The logarithmic growth in the
log phase is due to
reproduction by binary fission
(bacteria) or mitosis (yeast).
Staphylococcus spp.
Question
• If a culture of mesophilic bacteria were placed
in a refrigerator, what phase of growth would
it be in? Incubator (set at 37 degrees)?
Terminology of Microbial Control
• Sepsis refers to microbial contamination of the body
– Septic shock occurs when the body responds to
pathogen/toxin contaminating the blood
– SIRS—Systemic Inflammatory Response Syndrome
• Not specific to infection (burns, pancreatitis, trauma)
• Asepsis is the absence of significant contamination
• Aseptic surgery techniques prevent microbial
contamination of wounds
The Terminology of Microbial Control
• Sterilization: removing ALL microbial life
– Eradicates microbes AND their endospores
– Any agent used to sterilized is a sterilant
– Steam and pressure (autoclave)
• Commercial sterilization:
– How do you think can goods are sterilized?
– Heated enough to kill C. botulinum endospores
• Other thermophilic endospores still persist
The Terminology of Microbial Control
• Disinfection: destruction of vegetative (growing)
pathogens
–
–
–
–
Endospores still persist
Can be mechanical or chemical
Chemicals, UV radiation, boiling water, and steam
Usually refers to inanimate objects.
• Antisepsis: the removal of pathogens from living
tissue
The Terminology of Microbial Control
• Degerming: refers to the removal of microbes
from a limited area
– Alcohol swabbing before an injection
• Mechanical and chemical
– Server wiping down table at a restaurant
• Sanitization is the practice of lowering microbial
counts on public eateries
– High-temperature dishwashing and dipping drinking
glasses in chemical disinfectant
• Hotel Room Sanitation?!?!
• http://www.youtube.com/watch?v=kLssaWxip
N8
• Names of treatments that cause death of a
microbe end in the suffix –cide
– Biocide/germicide is the generic name for killing
microbes
• Bactericide
• Fungicide
• Virucide
• Names of treatments that only stop microbial
growth end in the suffix –static/stasis
– Once the bacteriostatic agent is removed, growth
resumes
Factors that affect the efficacy of
Antimicrobial Treatments
1. Number of microbes. More microbes; longer it
takes to kill them
2. Environmental influences.
3. Time of exposure. Chemical antimicrobials
require extended time to function to affect more
resistant microbes
4. Microbial characteristics. Features of the
microbe itself determine the effectiveness of
antimicrobial agent (constituents of membrane,
enzymes, endospores, etc.)
Rate of Microbial Death
• Bacterial populations usually die at a constant
rate.
• Different antimicrobial agents have varying microbial
death rates
• If an antimicrobial agent “x” kills 90% of a
microbial population in 1 minute. It will
subsequently kill the same amount each
additional minute
Table 7.2 Microbial Exponential Death Rate: An Example
Actions of Microbial Control Agents
on Microbes
• Microbial agents primarily affect microorganisms via
three mechanisms
1. Alteration of membrane permeability
–
Plasma membrane is the target of many microbial agents
2. Damage to ribosomes and/or proteins
–
Denaturing proteins or abrogating its production results
in the inability to carry out metabolic reactions
3. Damage to nucleic acids
–
•
DNA/RNA carry information for replication/metabolism
http://www.ncbi.nlm.nih.gov/books/NBK7986/
Control of Microbial Growth
• There are two means of controlling microbial
growth
1. Physical
2. Chemical
Physical Methods
1.
2.
3.
4.
5.
6.
7.
Heat
Filtration
Low Temperatures
High Pressure
Dessication
Osmotic Pressure
Radiation
Heat
• Heat kills microorganisms by inactivating their
enzymes
– Heat resistance varies among different microbes
• Thermal death point (TDP): lowest
temperature at which all cells in a culture are
killed in 10 min
• Thermal death time (TDT): time during which
all cells in a culture are killed
Moist Heat Sterilization
• Moist heat kills microbes by denaturing
proteins
• Boiling kills vegetative forms of bacteria, most
viruses, fungi and their spores in 10’
– Some viruses and endospores may survive
• Autoclave: steam under pressure destroys
endospores
– Most effective use of moist-heat sterilization
– Some materials can be damaged by autoclaving
Moist Heat Sterilization
• Pasteurization—reduces spoilage organisms and
pathogens in milk/juices
– Raises the temp right below boiling
– DOES NOT ERADICATE ALL MICROBES!!!!
– High-temperature short-time: 72°C (161°F) for 15 sec
• Milk in U.S. Must be refrigerated. Short storage life.
– Ultra-high-temperature: 140°C (284°F) for 1-2 sec
• Milk became popular in Europe. Can be stored for several
months without refrigeration.
– Equivalent treatments
• As temperature increases, length of time to kill microbes
decreases
Dry Heat Sterilization
• Kills microbes by oxidation effects
– Dry heat
– Flaming
– Incineration
– Hot-air sterilization (oven)
Equivalent Treatments
Hot-Air
Autoclave
170˚C, 2 hr
121˚C, 15 min
Dry Heat
Moist Heat
Filtration
• Blocks the passage of microorganisms
• HEPA (high-efficiency particulate air) removes
microbes >0.3 µm.
• Membrane filtration (liquid) removes
microbes >0.22 µm
Figure 7.4 Filter sterilization with a disposable, presterilized plastic unit.
Flask of
sample
Cap
Membrane filter
Cotton plug in
vacuum line
ensures sterility
Sterile
filtrate
Vacuum line
Physical Methods of Microbial Control
• Low temperature inhibits microbial growth
– Refrigeration (4°C)
– Deep-freezing (-20°)
– Lyophilization (freeze-drying) (-80°)
• Uses liquid nitrogen
• High pressure denatures proteins
– Some endospores are resistant. Combined with
elevating temps and alternating pressure cycles
• Desiccation is the removal of water and prevents
metabolism
• Osmotic pressure causes plasmolysis
Radiation
1. Ionizing radiation (X rays, gamma rays,
electron beams)
– Ionizes water to release OH•
– Damages DNA, proteins, membranes
2. Non-ionizing radiation (UV, 260 nm)
– Damages DNA by causing thymine dimers
– Inhibit DNA replication
Figure 7.5 The radiant energy spectrum.
Chemical Methods of Microbial
Control
• Chemical agents are used to control microbial
growth on inanimate objects and living tissue
• Only a few chemicals achieve “sterility” due to
1. Endospores
2. Characteristics of some bacteria resist destruction
• NOTE: No single disinfectant is appropriate for all
circumstances
Principles of Effective Disinfection
1. Concentration of disinfectant
– Greater the concentration, more effective
2. Organic matter that is being disinfected
3. pH
– Can affect the action of the chemicals
4. Time
– Longer the exposure; more microbes are killed
Evaluating a Disinfectant
• Use-Dilution Test
• Metal/glass cylinders are dipped in test
bacteria (broth) are dried at 37°C for a brief
period of time
• Dried cultures are placed in disinfectant of
varying concentrations for 10 min at 20°C
• Bacteria from cylinders are transferred to
culture media to determine whether bacteria
survived treatment
Disk Diffusion Test
• Disk of filter paper is soaked in a chemical agent
• Filters are placed on a “lawn” of bacteria to
evaluate growth inhibition
• The area where growth is inhibited is called zone
of inhibition
– Larger the zone, the more effective the chemical
agent is at controlling microbes
– Antibiotic discs
Figure 7.6 Evaluation of disinfectants by the disk-diffusion method.
Zone of inhibition
Chlorine
O-phenylphenol
Hexachlorophene
Quat
Staphylococcus aureus
(gram-positive)
Chlorine
O-phenylphenol
Hexachlorophene
Quat
Escherichia coli
(gram-negative)
Disk Diffusion Method
Chlorine
O-phenylphenol
Hexachlorophene
Quat
Pseudomonas aeruginosa
(gram-negative)
Chemicals in Disinfectants
Phenol and Phenolics
• Also referred to as carbolic acid
• Disrupt plasma membranes and results in cellular
leakage
• Found in some disinfectants, mouth washes, and
throat sprays (1% concentration)
– Over the counter throat lozenges are lower
concentration
• Therefore are not antimicrobial
Bisphenols
• Derivative of phenol
– Two phenols bridge together
• Hexachlorophene, triclosan
– Disrupt plasma membranes and inhibits enzyme needed
for synthesis of fatty acids (lipids)
• Used in nurseries
– Affects the growth of gram positive staph and strep in
newborns
• Over use results in resistance
Figure 7.7cd The structure of phenolics and bisphenols.
(c) Hexachlorophene (a bisphenol)
(d) Triclosan (a bisphenol)
Biguanides
• Broad spectrum of activity
– Taken into the plasma membrane and abrogates permeability
– Binds to DNA thus affecting transcription
• Especially effective against Gram positive bacteria
– Not as effective against Gram negative bacteria and viruses
• Chlorhexidine disrupts plasma membranes
• Used for surgical hand scrubs and pre-operative skin prep
in patients
Halogens
• Iodine
– Active against all bacteria
– Tinctures: Iodine in aqueous alcohol
– Iodophors: Iodine in solubilizing agent such as a
surfactant
– Alter protein synthesis and membranes
• Chlorine
– Bleach: hypochlorous acid (HOCl)
– Chloramine: chlorine + ammonia
• Water treatment facilities
– Mode of action is widely debated
Alcohols
• Kills bacteria and fungi
– Ineffective at killing endospores and non-enveloped
viruses
• Ethanol, isopropanol
– Denature proteins, dissolve lipids, disrupts
membranes
• Requires water to facilitate movement across membrane
• 70% more effective than 100%
– Certain pathogens that lack a lipid envelope are
resistant to alcohols
• Clostridium difficile
Heavy Metals
• Ag (silver), Hg (mercury), and Cu (copper)
– Silver sulfadiazine used as a topical cream on
burns
– Copper sulfate is an algicide (kills algae)
• Used in pools
• Oligodynamic action—refers to the
antimicrobial effect of heavy metals
– Method of action is unknown
• Silverware (100%) self-sanitize!!!!
Figure 7.8 Oligodynamic action of heavy metals.
Surface-Active Agents (Surfactants)
Surfactants decrease surface tension among molecules of a liquid
Soap (emulsifies oil) “little
antiseptic value” (Axe, Dove)
Degerming
Acid-anionic detergents (attacks
membrane) (dishwashing liquid)
Sanitizing
Quaternary ammonium compounds
(cationic detergents) “Quats”
Bactericidal, denature proteins,
More effective on gram(+)
disrupt plasma membrane
Food Preservatives
• Organic acids
– Control molds and bacteria in foods and cosmetics
– Inhibit metabolism
– Sorbic acid (canned goods), benzoic acid (facial
cleaners), and calcium propionate (bread)
• Sodium nitrite is added to meats to prevent
endospore germination
• Antibiotics
– Nisin (bacteriocin) and natamycin prevent spoilage of
cheese
Aldehydes
• Inactivate proteins by cross-linking with the
functional groups (–NH2, –OH, –COOH, –SH)
• Use: Sterilization of medical equipment
– Glutaraldehyde and formaldehyde
Peroxygens
• Oxidizing agents
• Use: contaminated surfaces
– O3, H2O2, peracetic acid
• Especially effective against anaerobic bacteria
Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.