Microbiology #3
lulsab
Chapter5.pptx5.7 Physical, Chemical, and Biological Control of Microbes
A variety of terms are used to describe antimicrobial control measures:
Sterilization: killing of all living organisms from inanimate objects
Disinfection: reduction of pathogens from inanimate objects (10 minutes)
Antisepsis: killing or removal of pathogens from the surface of living tissues
Sanitation: reducing the microbial population to safe levels (30 seconds)
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Cells Die at a Logarithmic Rate
Microbes die according to a negative exponential curve, where cell numbers are reduced in equal fractions at constant intervals.
Decimal reduction time (D-value) is the length of time it takes an agent or a condition to kill 90% of the population.
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FIGURE 5.25 ■ The death curve and the determination of D-values.
Physical Agents That Kill Microbes – 1
High temperature
Moist heat is more effective than dry heat.
Boiling water (100oC) kills most cells.
Killing spores (resistant to adverse conditions) and thermophiles usually requires a combination of high pressure and temperature.
At high pressure, the boiling point of water rises to a temperature rarely experienced by microbes.
Even endospores quickly die under these conditions.
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Physical Agents That Kill Microbes – 2
Steam autoclave
121oC (250oF) at 15 psi for 20 minutes
Conditions produced in pressure cookers when canning vegetables
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FIGURE 5.26 ■ Steam autoclave.
Physical Agents That Kill Microbes – 3
Pasteurization
Many different time and temperature combinations can be used.
LTLT (low temperature/long time)
63oC for 30 minutes
HTST (high temperature/short time)
72oC for 15 seconds
Both processes kill Coxiella burnetii, the causative agent of Q fever.
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Physical Agents That Kill Microbes – 4
Cold
Low temperatures slow growth and preserve strains.
Refrigeration temperatures (4oC–8oC) are used for food preservation.
For long-term storage of cultures
Placing solutions in glycerol at –70oC
Lyophilization or freeze-drying
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Physical Agents That Kill Microbes – 5
Filtration
Micropore filters with pore sizes of 0.2 mm can remove microbial cells, but not viruses, from solutions.
Samples from 1 ml to several liters can be drawn through
a membrane filter by vacuum or can be forced through it using a syringe.
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FIGURE 5.27 ■ Membrane filtration devices.
Physical Agents That Kill Microbes – 6
Air can also be sterilized by filtration.
This process forms the basis of several personal protective devices.
Laminar flow biological safety cabinets force air through HEPA filters, which remove > 99.9% of airborne particulate material 0.3 μm in size or larger.
Biosafety cabinets are critical to protect individuals working with highly pathogenic material.
Newer technologies embed antimicrobial agents or enzymes directly into the fibers of the filter.
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Physical Agents That Kill Microbes – 7
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FIGURE 5.28 ■ Biological safety cabinet. CREDITS: A: A: Labconco Corporation; C: Nikki-Universal Co., Ltd., 2000
Physical Agents That Kill Microbes – 8
Irradiation
Ultraviolet light
Has poor penetrating power
Used only for surface sterilization
Gamma rays, electron beams, and X-rays
Have high penetrating power
Used to irradiate foods and other heat-sensitive items
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Some Bacteria Are Highly Resistant to Physical Control Measures
Deinococcus radiodurans
Nicknamed “Conan the bacterium”
Has the greatest ability to survive radiation of any known organism
Has exceptional capabilities
for repairing DNA damaged
by radiation
Was genetically engineered
for use in bioremediation
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FIGURE 5.29 ■ Deinococcus radiodurans. CREDITS: A: John R. Battista, Louisiana State University
Chemical Agents – 1
A number of factors influence the efficacy of a given chemical agent, including:
The presence of organic matter
The kinds of organisms present
Corrosiveness
Stability, odor, and surface tension
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Chemical Agents – 2
The phenol coefficient test compares the effectiveness of disinfectants.
| Chemical agent | Staphylococcus aureus | Salmonella enterica |
| Phenol | 1.0 | 1.0 |
| Chloramine | 133.0 | 100.0 |
| Cresols | 2.3 | 2.3 |
| Ethanol | 6.3 | 6.3 |
| Formalin | 0.3 | 0.7 |
| Hydrogen peroxide | Empty cell | 0.001 |
| Lysol | 5.0 | 3.2 |
| Mercury chloride | 100.0 | 143.0 |
| Tincture of iodine | 6.3 | 5.8 |
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Table 5.3, Phenol Coefficients for Various Disinfectants.
Commercial Disinfectants and Antiseptics – 1
These include:
Ethanol
Iodine (Wescodyne and Betadine)
Chlorine
Ethylene oxide (a gas sterilant)
These damage proteins, lipids, and/or DNA.
Are used to reduce or eliminate microbial content from objects
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Commercial Disinfectants and Antiseptics – 2
| Phenolics | Alcohols | Aldehydes | Quaternary ammonium compounds | Gases |
| The 2 dimensional structure of phenol. An alcohol group is attached to one of the carbons on a benzene ring. | The 2 dimensional structure of ethanol. The carbon of a methyl group is bonded to another carbon, which is also bonded to two hydrogens and an alcohol group. | The 2 dimensional structure for formaldehyde. A carbon atom is double bonded to an oxygen atom and single bonded to two hydrogen atoms. | The 2 dimensional structure for cetylpyridinium chloride. The cetylpyridinium ion has a ring of 5 carbon atoms and 1 nitrogen atom. Every other bond in this ring is a double bond, totaling to 3 double bonds in the ring. The nitrogen atom is also bonded to a left parenthesis upper C upper H 2 right parenthesis 15 upper C upper H 3 group. This ion has a positive 1 charge. The chloride ion has a negative 1 charge. | The 2 dimensional structure for ethylene oxide. The geometry for this structure is trigonal planar between the carbon atoms of two methyl groups and an oxygen atom. |
| The 2 dimensional structure of hexachlorophene. There are two benzene rings connected by an additional carbon atom. The carbon atom is also bonded to 2 hydrogens. The structure is symmetric with the plane of symmetry at the connecting carbon atom. The top carbon of each benzene ring is connected to an alcohol group. Three of the other four carbons not attached to groups are bonded to chlorine atoms. | The 2 dimensional structure of isopropanol or also known as rubbing alcohol. There is a chain of three carbons. The central carbon is bonded above by a hydrogen and below by an alcohol group. The other two carbons are a part of methyl groups. | The 2 dimensional structure for glutaraldehyde. There is a 5 carbon chain. The three carbon atoms in the middle are each bonded to 2 hydrogens. The carbon atoms on the end are double bonded to an oxygen and single bonded to a hydrogen. The left end carbon atom has the oxygen bonded above the hydrogen and the right end carbon atom has the oxygen bonded below the hydrogen. | The 2 dimensional structure for benzalconium chloride or also known as mixture. This is an ionic complex whose cation has the following structure. A benzene ring on the left is attached to a carbon atom that is also bonded to two hydrogen atoms. This carbon atom is bonded to the right by a nitrogen atom. This nitrogen atom is bonded from the top and bottom by methyl groups and to the right by an R group. Below the structure, R equals an alkyl that is upper C 8 upper H 17 to upper C 18 to upper H 37. The anion for this compound is the chloride ion. | The 2 dimensional structure for betapropiolactone. There is a rectangular structure between the carbon atoms of two methyl groups, an oxygen atom, and another carbon atom. The carbon atom is also double bonded to another oxygen atom. |
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FIGURE 5.30 ■ Structures of some common disinfectants and antiseptics.
Bacteria Can Develop Resistance to Disinfectants
This has been achieved via several mechanisms:
Altering the fatty acid synthesis protein normally targeted by triclosan
Producing membrane-
spanning, multidrug
efflux pumps
Forming multispecies
biofilms, which offer
collaborative protection
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FIGURE 5.31 ■ Mixed biofilm. CREDITS: A. Bridier et al. 2011. Biofouling 27:1017–1032
Antibiotics – 1
Antibiotics are chemical compounds synthesized by one microbe that kill or inhibit the growth of other microbial species.
Penicillin mimics part of the bacterial cell wall.
Prevents cell wall formation and is bactericidal
Other antibiotics target:
Protein synthesis
DNA replication
Cell membranes
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Antibiotics – 2
Penicillin
Produced by Penicillium notatum
Mimics a part of the cell wall
Prevents peptidoglycan synthesis and prevents cell wall formation
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FIGURE 5.32 ■ Penicillin. CREDITS: B: USPS; C: Christine L. Case/Skyline College
Antibiotics – 3
Penicillin
Is bactericidal because actively growing cells lyse without the support of the cell wall
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FIGURE 5.33 ■ Effect of ampicillin (a penicillin derivative) on E. coli. CREDITS: ART GIRARD, ANNE KLEIN, & A.J. MILICI, PFIZER GLOBAL RESEARCH AND DEVELOPMENT
Biological Control of Microbes
Biocontrol is the use of one microbe to control the growth of another.
Probiotics contain certain microbes that, when ingested, aim to restore balance to intestinal flora.
Lactobacillus and Bifidobacterium
Phage therapy aims to treat infectious diseases with a virus targeted to the pathogen.
Bacteriophages are a possible alternative to antibiotics in the face of rising antibiotic resistance.
Commercial phage products are now available to target a few food-borne pathogens.
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