Microbial Control - Vidyamandira

Microbial Control

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Source : Prescot et al Microbiology,

Microbiology by Pelczar, Brock et

al Microbiology

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Microbial deathThe permanent loss of reproductive capability, even under

optimum growth conditions is the accepted microbial definition of death

Factors affecting microbial death rate

a) Number of microorganisms

b) Nature of the microorganisms in the target population

c) Temperature and pH of the environment

d) Concentration of the agent

e) Mode of action of the agent

f) Presence of solvents, interfering organic matter, and inhibitors

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Practical Concerns in Microbial

Control

Selection of method of control depends on circumstances:

• Does the application require sterilization?

• Is the item to be reused?

• Can the item withstand heat, pressure, radiation, or chemicals?

• Is the method suitable?

• Will the agent penetrate to the necessary extent?

• Is the method cost- and labor-efficient and is it safe?

Basic Principles of

Microbial Control

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The Agents Versus the Processes• –cide: to kill

– Bactericide: chemical that destroys bacteria (not endospores)

– Fungicide: a chemical that can kill fungal spores, hyphae, and yeasts

– Virucide: a chemical that inactivates viruses

– Sporicide: can destroy bacterial endospores

– Germicide and microbicide: chemical agents that kill microorganisms

• Stasis and static: to stand still

– Bacteristatic: prevent the growth of bacteria

– Fungistatic: inhibit fungal growth

– Microbistatic: materials used to control microorganisms in the body, for example

Relative susceptibilities of microbes to antimicrobial agents

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Targets to Control Microbial Presence

• Injure cell wall

• Injure cell membranes

• Interfere with nucleic acid synthesis

• Interfere with protein synthesis

• Interfere with protein function

Which of the above would effect our cells too?

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Bacterial populations die at a constant logarithmic rate

The Selection of Microbial Control Methods

• Factors Affecting the Efficacy of Antimicrobial Methods

▫ Site to be treated

Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects

Method of microbial control based on site of medical procedure

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The Selection of Microbial Control Methods

• Factors Affecting the Efficacy of Antimicrobial Methods

▫ Relative susceptibility of microorganisms

Germicides classified as high, intermediate, or low effectiveness

High-level kill all pathogens, including endospores

Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria

Low-level kill vegetative bacteria, fungi, protozoa, and some viruses

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Physical Methods of Microbial Control

• Heat-Related Methods▫ Effects of high temperatures

Denature proteins

Interfere with integrity of cytoplasmic membrane and cell wall

Disrupt structure and function of nucleic acids

• Heat▫ Thermal death point (TDP): Lowest temperature at which all cells

in a culture are killed in 10 min.

▫ Thermal death time (TDT): Time to kill all cells in a culture

▫ Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature

▫ z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions

▫ F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores

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Physical Methods: Moist Heat

• Measurements of killing by moist heat (cont.)

▫ Decimal reduction time (D value): The time required to reduced a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions

▫ z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions

▫ F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores


• Heat-Related Methods▫ Moist heat

Used to disinfect (kill organisms and remove spores), sanitize (kill organisms but not necessarily their spores), and sterilize (kill all organisms and spores)

Denatures proteins and destroys cytoplasmic membranes

More effective than dry heat Methods of microbial control using moist heat

Boiling

Autoclaving

Pasteurization

Ultrahigh-temperature sterilization

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• Heat-Related Methods

▫ Moist heat

Boiling

Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses

Boiling time is critical

▫ Different elevations require different boiling times

Endospores, protozoan cysts, and some viruses can survive boiling

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• Heat-Related Methods

▫ Moist heat

Autoclaving

Pressure applied to boiling water prevents steam from escaping

Boiling temperature increases as pressure increases

Autoclave conditions – 121ºC, 15 psi, 15 min

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The relationship between temperature and pressure

Figure 9.5

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• Moist heat denatures proteins

• Autoclave: Steam under pressure

• 15 min at 121oC at 15 psi

Autoclaving

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Nonpressurized Steam

• Tyndallization – intermittent sterilization for substances that cannot withstand autoclaving

• Items exposed to free-flowing steam for 30 –60 minutes, incubated for 23-24 hours and then subjected to steam again

• Repeat cycle for 3 days.

• Used for some canned foods and laboratory media

• Disinfectant


• Heat-Related Methods▫ Moist heat

Pasteurization Used for milk, ice cream, yogurt, and fruit juices

Not sterilization▫ Heat-tolerant microbes survive

Pasteurization of milk▫ Batch method

▫ Flash pasteurization (High temp, short time)

▫ Ultrahigh-temperature pasteurization (very high temp, very short time)

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Pasteurization

63oC for 30 minutes

72oC for 15 seconds

140oC for 1 second

Pasteurization reduces spoilage organisms and pathogens

Pasteurization of milk

Batch method • The batch method uses a vat pasteurizer which consists

of a jacketed vat surrounded by either circulating water, steam or heating coils of water or steam.

• Batch method: Expose to 63°C to 66°C for 30 minutes (LTLT)

In the vat the milk is heated and held throughout the holding period while being agitated. The milk may be cooled in the vat or removed hot after the holding time is completed for every particle.

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Flash method• High Temperature Short Time (HTST)

• Milk is heated to 72°C (161.6°F) for at least 15 seconds.

• Used for perishable beverages like fruit and vegetable juices, beer, and some dairy products. Compared to other pasteurization processes, it maintains color and flavor better.

• It is done prior to filling into containers in order to kill spoilage microorganisms, to make the products safer and extend their shelf life. Flash pasteurization must be used in conjunction with sterile fill technology.

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Ultrahigh-temperature method

• Heating for 1-2 seconds at a temperature exceeding135°C (275°F), which is the temperature required to killspores in milk and then rapid cooling.

• Treated liquids can be stored at room temperature.

• The most common UHT product is milk, but the processis also used for fruit juices, cream, soy milk, yogurt,wine, soups, and stews.

• Can cause browning and change the taste and smell ofdairy products.

• UHT canned milk has a typical shelf life of six to ninemonths, until opened.

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• Heat-Related Methods▫ Dry heat

Used for materials that cannot be sterilized with moist heat

Denatures proteins and oxidizes metabolic and structural chemicals

Requires higher temperatures for longer time than moist heat

Incineration is ultimate means of sterilization

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• Dry Heat Sterilization kills by

oxidation

▫ Incineration

▫ Hot-air sterilization


Hot-air Autoclave

Equivalent treatments 170˚C, 2 hr 121˚C, 15 min

Dry Heat: Hot Air and Incineration

• Incineration

▫ Ignites and reduces microbes to ashes and gas

▫ Common practice in microbiology lab-incineration on inoculating loops and needles using a Bunsen burner

▫ Can also use tabletop infrared incinerators

Dry Oven

• Usually an electric oven

• Coils radiate heat within an enclosed compartment

• Exposure to 150°C to 180°C for 2 to 4 hours

• Used for heat-resistant items that do not sterilize well with moist heat


• Refrigeration and Freezing▫ Decrease microbial metabolism, growth, and

reproduction

Chemical reactions occur slower at low temperatures

▫ Psychrophilic microbes can multiply in refrigerated foods

▫ Refrigeration halts growth of most pathogens▫ Slow freezing more effective than quick freezing▫ Organisms vary in susceptibility to freezing

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• Dessication and Lyophilization

▫ Dessication is drying (98% of the water is removed) inhibits growth due to removal of water

▫ Lyophilization (freeze-drying)

Substance is rapidly frozen and sealed in a vacuum

Substance may also be turned into a powder

▫ Used for long-term preservation of microbial cultures

Prevents formation of damaging ice crystals

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The use of dessication as a means of preserving

apricots

Figure 9.8

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• Effective for removing microbes from air and liquids

• Fluid strained through a filter with openings large enough for fluid but too small for microorganisms

• Filters are usually thin membranes of cellulose acetate, polycarbonate, and a variety of plastic materials

• Pore size can be controlled and standardizes

Decontamination by Filtration:

Techniques for Removing Microbes

Filtration equipment used for microbial control

Figure 9.9

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The role of HEPA filters in biological safety cabinets

Figure 9.10

High-Efficiency Particulate Arresting (HEPA) air filters are used in medical facilities, automobiles, aircraft, and homes. The filter must remove 99.97% of all particles greater than 0.3 micrometer from the air that passes through.

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• Prepare liquids that can’t withstand heat

• Can decontaminate beverages without altering their flavor

• Water purification

• Removing airborne contaminants (HEPA filters)

Applications of Filtration


• Osmotic Pressure

▫ High concentrations of salt or sugar in foods to inhibit growth

▫ Cells in hypertonic solution of salt or sugar lose water

▫ Fungi have greater ability than bacteria to survive hypertonic environments

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• Radiation damages DNA

▫ Ionizing radiation (X rays, gamma rays, electron beams)

▫ Nonionizing radiation (UV)- surface sterilization only

▫ (Microwaves kill by heat; not especially antimicrobial)



• Radiation: Cold sterilization▫ Ionizing radiation

Wavelengths shorter than 1 nm Electron beams, gamma rays

Excites the electrons to the point that they are ejected from the molecule entirely causing the formation of ions

Ions disrupt hydrogen bonding, cause oxidation, and create hydroxide ions Hydroxide ions denature other molecules (DNA) Also causes lethal chemical changes in organelles

and the production of toxins Electron beams – effective at killing but do not

penetrate well Gamma rays – penetrate well but require hours to kill

microbes

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• Radiation

▫ Nonionizing radiation

Wavelengths greater than 1 nm

Excites electrons, causing them to make new covalent bonds

Leads to abnormal linkages and bonds within molecules

Affects 3-D structure of proteins and nucleic acids

UV light causes pyrimidine dimers in DNA

UV light does not penetrate well

Suitable for disinfecting air, transparent fluids, and surfaces of objects

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Ionizing Radiation• Food products

• Medical products (drugs and tissues)Potential problems include changing flavor and nutritional

value, and introducing undesirable chemical reactions

Potential danger to machine operators and possible damage to some materials

Nonionizing Radiation• Usually disinfection rather than sterilization

• Hospital rooms, operating rooms, schools, food prep areas, dental offices

• Treat drinking water or purify liquidsPoses threat to human tissue if overexposure occurs

Applications of Radiation

Increased shelf life of food achieved by ionizing

radiation

Figure 9.11

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Irradiation food Radura

• The word "Radura" is derived from radurization, combining "radiation" with the stem of "durus", the Latin word for hard, lasting.


Sound Waves (Sonication)a) Used high-frequency sound waves to disrupt cell structure.

b) Sonicator–water-filled chamber through which the sound waves become vibrations that can disrupt cell structure.

c) Application of ultrasound waves causes rapid changes in pressure within the intracellular liquid; this leads to cavitation, the formation of bubbles inside the cell, which can disrupt cell structures and eventually cause the cell to lyse or collapse.

d) Gram-negative bacteria are most susceptible.

e)Often used to clean debris from instruments before sterilization.

Not a reliable form of disinfection or sterilization.

Useful in the laboratory for efficiently lysing cells to release their contents for further research.

Outside the laboratory, sonication is used for cleaning surgical instruments, lenses, and a variety of other objects such as coins, tools, and musical instruments.


• Biosafety Levels▫ Four levels of safety in labs dealing with

pathogens Biosafety Level 1 (BSL-1)

Handling pathogens that do not cause disease in healthy humans

Biosafety Level 2 (BSL-2) Handling of moderately hazardous agents

Biosafety Level 3 (BSL-3) Handling of microbes in safety cabinets

Biosafety Level 4 (BSL-4) Handling of microbes that cause severe or fatal disease

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A BSL-4 worker carries

Ebola virus cultures

Figure 9.12

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Chemical Methods of Microbial Control

• Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA

• Effect varies with differing environmental conditions

• Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

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Acids and Alkalis

• Very low or high pH can destroy or inhibit microbial cells

• Limited in applications due to their corrosive, caustic, and hazardous nature



• Phenol and PhenolicsPhenol (carbolic acid) was first used by Lister as a disinfectant.

Rarely used today because it is a skin irritant and has strong odor.

Phenolics are chemical derivatives of phenol

Cresols (Lysol): Derived from coal tar.Biphenols: Effective against gram-positive staphylococci

and streptococci. Excessive use in infants may cause neurological damage.

Destroy plasma membranes and denature proteins.Advantages: Stable, persist for long times after applied, and

remain active in the presence of organic compounds.

▫ Intermediate- to low-level disinfectants

▫ Remain active for prolonged time

• Commonly used in health care settings, labs, and homes

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• Alcohols

▫ Intermediate-level disinfectantsKill bacteria, fungi, but not endospores or naked viruses.

Act by denaturing proteins and disrupting cell membranes.

Used to mechanically wipe microbes off skin before injections or blood drawing.

Not good for open wounds, because cause proteins to coagulate.

Ethanol: Drinking alcohol. Optimum concentration is 70%.

Isopropanol: Rubbing alcohol. Better disinfectant than

ethanol. Also cheaper and less volatile.

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70% vs 100% Ethanol

Pure alcohol (100%) coagulates protein in contact. Suppose the purealcohol is poured over a single celled organism. The alcohol will gothrough the cell wall of the organism in all direction, coagulating theprotein just inside the cell wall. The ring of the coagulated protein wouldthen stop the alcohol from penetrating farther from the cell, and nomore coagulation would take place. At this time the cell would becomeinactive but not dead. Under the favorable conditions the cell wouldthen begin to function. If 70 % of alcohol is poured to a single celledorganism, the diluted alcohol also coagulates the protein, but at aslower rate, so that it penetrates all the way through the cell beforecoagulation can block it. Then the entire cell is coagulated and theorganism dies.


Halogens• Chlorine – Cl2, hypochlorites (chlorine bleach), chloramines

▫ Denaturate proteins by disrupting disulfide bonds

When mixed in water forms hypochlorous acid:

Cl2 + H2O ------> H+ + Cl- + HOCl

▫ Intermediate level▫ Unstable in sunlight, inactivated by organic matter ▫ Water, sewage, wastewater, inanimate objects

• Iodine - I2, iodophors (betadine) Iodine tincture (alcohol solution) was one of first antiseptics used.

▫ Denature proteins▫ Intermediate level▫ Milder medical & dental degerming agents, disinfectants,

ointments

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Oxidizing Agents

Oxidize cellular components of treated microbes.

Disrupt membranes and proteins.

A. Ozone:

Used along with chlorine to disinfect water.

Helps neutralize unpleasant tastes and odors.

More effective killing agent than chlorine, but less stable and more expensive.

Highly reactive form of oxygen.

Made by exposing oxygen to electricity or UV light

B. Hydrogen Peroxide:

Not useful for treating open wounds due to catalase activity: the tissues convert it into H2O and 0xygen bubbles.

Effective in disinfection of inanimate objects

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• Surfactants▫ “Surface active” chemicals

Reduce surface tension of solvents

▫ Soaps and detergents Soaps have hydrophilic and hydrophobic ends

Good degerming agents but not antimicrobial

Detergents are positively charged organic surfactants

▫ Quats (Quaternary ammonium cations) Low-level disinfectants; disrupts cell membranes Ideal for many medical and industrial applications Good against fungi, amoeba, and enveloped viruses,

but not endospores, Mycobacterium tuberculosis and non-enveloped viruses.

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• Heavy Metals▫ Heavy-metal ions denature proteins

▫ Low-level bacteriostatic and fungistatic agents

▫ Include copper, selenium, mercury, silver, and zinc.

▫ Very tiny amounts are effective.

A. Silver:

1% silver nitrate used to protect infants against gonorrheal eye infections, now has been replaced by erythromycin.

B. Mercury

Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds.

C. Copper

Copper sulfate is used to kill algae in pools and fish tanks.

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• AldehydesInclude some of the most effective antimicrobials.Inactivate proteins by forming covalent crosslinks with

several functional groups (–NH2, –OH, –COOH, —SH).

A. Formaldehyde:Excellent disinfectant, 2% aqueous solution.

Commonly used as formalin, a 37% aqueous solution.Formalin was used extensively to preserve biological

specimens and inactivate viruses and bacteria in vaccines.

Irritates mucous membranes, strong odor.

B. Glutaraldehyde:

Less irritating and more effective than formaldehyde.

Commonly used to disinfect hospital instruments.

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Chemical Methods of Microbial Control• Gaseous Agents

▫ Microbicidal and sporicidal gases used in closed chambers to sterilize items

▫ Denature proteins and DNA by cross-linking functional groups▫ Used in hospitals and dental offices Disadvantages

Can be hazardous to people Often highly explosive Extremely poisonous Potentially carcinogenic

Ethylene Oxide: Kills all microbes and endospores, but requires exposure of 4 to 18

hours. ETO is a colorless gas that is flammable and explosive but it is liquid at

temperatures below 10.8oC. Alkylation reactions with organic compounds such as enzymes (eg

inactivation of enzymes having sulfhydryl group) and other proteins. It has high penetrating power and passes through and sterilizes

large packages of materials, bundles of cloth, and even certain plastics.

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• Enzymes▫ Antimicrobial enzymes act against microorganisms▫ Human tears contain lysozyme

Digests peptidoglycan cell wall of bacteria

▫ Enzymes to control microbes in the environment Lysozyme used to reduce the number of bacteria in

cheese Prionzyme can remove prions on medical

instruments

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Evaluating the Effectiveness

• Phenol Coefficient Test

▫ A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhiand Staphylococcus aureus and incubated at either 20°C or 37°C

▫ Samples are removed at 5 min intervals and inoculated into fresh broth

▫ The cultures are incubated at 37°C for 2 days

▫ The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient


Evaluating the Effectiveness

• Phenol Coefficient Test (cont.)▫ The reciprocal of the maximum effective dilution for the test

disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient

▫ For example:Suppose that, on the test with Salmonella typhiThe maximum effective dilution for phenol is 1/90The maximum effective dilution for “Disinfectant X” is 1/450The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5

▫ Phenol coefficients are useful as an initial screening and comparison, but can be misleading because they only compare two pure strains under specific controlled conditions


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Microbial Control - Vidyamandira