SEM-III_CC6-CONTROL OF MICROORGANISMS.ppt

Microbial death
The 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

4
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
5

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
15

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?
17

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
19

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
20

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
22

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

▫ 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
24

▫ 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
26

▫ 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
27

The relationship between temperature and pressure
Figure 9.5
28

• Moist
heat
denatures
proteins
• Autoclave
: Steam
under
pressure
• 15 min at
121oC at
15 psi
Autoclaving

30
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

▫ 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)
31

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.
33

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.
34

Ultrahigh-temperature method
• Heating for 1-2 seconds at a temperature exceeding
135°C (275°F), which is the temperature required to kill
spores in milk and then rapid cooling.
• Treated liquids can be stored at room temperature.
• The most common UHT product is milk, but the process
is also used for fruit juices, cream, soy milk, yogurt,
wine, soups, and stews.
• Can cause browning and change the taste and smell of
dairy products.
• UHT canned milk has a typical shelf life of six to nine
months, until opened.
35

▫ 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
36

• 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
40

• 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
41

The use of dessication as a means of preserving
apricots
Figure 9.8
42

• 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
46

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.
47

• 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
49

• 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
51

• 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
53

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 liquids
Poses threat to human tissue if overexposure occurs
Applications of Radiation

Increased shelf life of food achieved by ionizing
radiation
Figure 9.11
56

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
 Handling of moderately hazardous agents
 Handling of microbes in safety cabinets
 Handling of microbes that cause severe or fatal disease
59

A BSL-4 worker carries
Ebola virus cultures
Figure 9.12
60

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
61

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 Phenolics
Phenol (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
64

• Alcohols
▫ Intermediate-level disinfectants
Kill 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.
65

66
70% vs 100% Ethanol
Pure alcohol (100%) coagulates protein in contact. Suppose the pure
alcohol is poured over a single celled organism. The alcohol will go
through the cell wall of the organism in all direction, coagulating the
protein just inside the cell wall. The ring of the coagulated protein would
then stop the alcohol from penetrating farther from the cell, and no
more coagulation would take place. At this time the cell would become
inactive but not dead. Under the favorable conditions the cell would
then begin to function. If 70 % of alcohol is poured to a single celled
organism, the diluted alcohol also coagulates the protein, but at a
slower rate, so that it penetrates all the way through the cell before
coagulation can block it. Then the entire cell is coagulated and the
organism 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
67

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
68

• 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.
69

• 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.
70

• Aldehydes
Include 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.
72

• 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.
74

• 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
75

Evaluating the Effectiveness
• Phenol Coefficient Test
▫ A series of dilutions of phenol and the experimental
disinfectant are inoculated with Salmonella typhi
and 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 typhi
The maximum effective dilution for phenol is 1/90
The maximum effective dilution for “Disinfectant X” is
1/450
The 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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