Control of Control of MicroorganismsMicroorganisms
Microbiology
Control of Microbial GrowthControl of Microbial Growth
Effected in two basic ways:1. Biocidal action: Killing Microorganisms 2. Biostatic action :By inhibiting the Growth of
MicroorganismsUsually involves the use of:1. Physical Agents that Kill or Prevent Growth2. Chemical Agents that Kill or Prevent Growth3. Biological Agents: Bacteria, Fungi, Phage
etc.
Level of ResistanceLevel of Resistance1. Endospores (Most)2. Parasites3. Mycobacteria4. Fungal Spores5. Small Non-enveloped Viruses
- Polio, Rotavirus, Rabies5. Vegetative Fungal Cells6. Enveloped Viruses
- Herpes, Hepatitis B & C, HIV5. Vegetative Bacteria (Least)
Principles of Microbial Principles of Microbial ControlControlPrevention / Control of Growth to Prevent
Infection and Spoilage, AsepsisSterilization / Complete Destruction
- Real sterilization is All or Nothing) Commercial Sterilization
The best we can doReduce numbers to safe level
- Utilizes a. Heatb. Radiationc. Chemicalsd. Physical Removale. Hyperbaric (High pressure)
Principles of Microbial ControlPrinciples of Microbial Control
Commercial Sterilization / Heat Treatment of Canned Foods
Disinfection / Reducing Growth Nonliving Surfaces
Antisepsis / Reducing Growth Living TissueAntibiotics ( “magic bullets”) Internal usePreservativesSanitize – Subject to High Temperature
(70oC) Washing (Dishwashers) or Hypo
Principles of Microbial ControlPrinciples of Microbial Control
Cide – Suffix Meaning to KillStat – Suffix Meaning to InhibitSepsis – Bacterial ContaminationAsepsis – Lack of Bacterial Contamination
Rate of Microbial DeathRate of Microbial DeathBacteria Usually Die At a Constant RatePlotted Logarithmically This Will Give a Straight
Line
Time to Kill in Proportion to Time to Kill in Proportion to the Population Sizethe Population Size
Large Numbers Require Greater TimeSmall Numbers Require Less Time
Susceptibilities VarySusceptibilities VaryEndospores are Difficult to KillOrganic Matter May Interfere with
Heat Treatments and Chemical Control Agents
Must clean first
Control Agents Act By:Control Agents Act By:
Alteration of Membrane Permeability- Susceptibility of membrane is due to
its lipid and protein composition- Control Agents can alter permeability
Damage to Proteins and Nucleic Acids- Break hydrogen and covalent bonds in
proteins- Interfere with DNA, RNA, Protein Synthesis
Physical Methods of Physical Methods of Microbial ControlMicrobial Control
Physical MethodsPhysical Methods
HeatFiltrationLow TemperaturesDesiccationOsmotic PressureRadiation
HeatHeat
Most Frequent and Widely Used.Always Consider
1. Type of Heat2. Time of Application3. Temperature
Endospores are the most heat resistant of all cells.
Moist Heat / Denatures EnzymesThermal Death Point (TDP) / Lowest
Temp to Kill All the Bacteria in a Broth in 10 Minutes
Thermal Death Time (TDT) / Time Span Required to Kill All the Bacteria in a Broth at a Given Temperature
Decimal Reduction Time (DRT) / Length of Time in Which 90% of a Bacterial Population will be Killed at a given Temperature
Boiling / Kills Many Vegetative Cells and Inactivates Viruses Within 10 Minutes
Moist Heat SterilizationMoist Heat SterilizationBoilingAutoclaving
BoilingBoiling
• 100° C for 30 Minutes
• Kills Everything Except Some Endospores
• Intermittent boiling can kills spores
Boil, incubate, boil, incubate, boil
Does not always work
AutoclavingAutoclavingSteam Under
Pressure121° C for 15
Minutes at 15 lb/in2
Heat-labile Substances will be Denatured
Steam Must Contact the Material
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Measuring heat-killing efficiencyMeasuring heat-killing efficiency
thermal death time (TDT)◦shortest time needed to kill all microorganisms in a suspension at a specific temperature and under defined conditions
decimal reduction time (D or D value)◦time required to kill 90% of microorganisms or spores in a sample at a specific temperature
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Other measures…Other measures…Z value
◦increase in temperature required to reduce D by 1/10
F value◦time in minutes
at a specific temperature needed to kill a population of cells or spores
Figure 7.2Figure 7.2
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Moist heatMoist heat
Autoclaves Autoclaves
– – used to kill endospores efficientlyused to kill endospores efficiently
– – use saturated steam under pressure to reach temperaturesuse saturated steam under pressure to reach temperatures
above boilingabove boiling
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Moist heat…Moist heat…
pasteurization◦controlled heating at temperatures well below
boiling◦reduces total microbial population and thereby
increases shelf life of treated material
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Pasteurization of milkPasteurization of milk
flash pasteurization (high temperature short-term – HTST)◦72°C for 15 seconds then rapid cooling
ultrahigh-temperature (UHT) sterilization◦140 to 150°C for 1 to 3 seconds
Cook Chill F values Cook Chill F values Copyright © The McGraw-Hill Copyright © The McGraw-Hill
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Cook ChillCook ChillCopyright © The McGraw-Hill Copyright © The McGraw-Hill
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Dry Heat SterilizationDry Heat SterilizationDirect FlamingIncinerationHot-Air Sterilization (Oven)
Flaming the Loop
Flaming the loop helps to prevent contamination of the bacteria.
When flaming the loop, make sure that all of the wire has been heated to redness.
IncinerationIncineration
• Burns and Physically Destroys Organisms
• Used for
a. Needles
b. Inoculating Wires
c. Glassware
d. Body Parts?
Dry Heat (Hot Air Oven)Dry Heat (Hot Air Oven)
• 160° C for 2 Hours or 170° C for 1 hour
• Used for
a. Glassware
b. Metal
c. Objects That Won’t Melt
PasteurizationPasteurizationA High Temperature
Is Used For a Short Time
Batch Method (LTLT)63 °C for 30 Minutes
Flash Method (HTST)72 °C for 15 Seconds
Ultra-High-Temperature is 140 °C for 3 seconds
FiltrationFiltrationThe passage of a liquid or gas through a filter
with pores small enough to retain microbes.Especially important to sterilize solutions
which would be denatured by heat (antibiotics, injectable drugs, amino acids, vitamins.)
HEPA FiltersHEPA Filters
High-Efficiency Particulate Air Filters1. Operating Rooms2. Burn Units3. Fume Hoods
Low TemperaturesLow Temperatures
Decreasing Temperature Decreases Chemical ActivityLow Temps are Not BactericidalRefrigeration <50 C
◦Listeria still grows but slow◦Spoilage organisms still grow
Freezing -180 C◦Spoilage due to enzymes
DesiccationDesiccation
Disrupts Microbial MetabolismStops Growth / Still ViableFreeze-drying / Dehydration
◦Used to preserve bacteria
Viruses and Endospores Can Resist Desiccation
Osmotic PressureOsmotic Pressure
PlasmolysisSugar Curing / SaltingMay Still Get Some Mold or Yeast Growth
◦Staphylococcus and Listeria grows in 10% salt
RadiationRadiationActs By Destroying DNA or Damaging It.Dependent on the Wavelength,
Intensity, and Duration
RadiationRadiation
Ionizing Radiation1. High Degree of Penetration2. Examples
- Gamma Rays- X-rays- High Energy
Electron Beams
Ultraviolet Radiation1. Nonionizing2. Low Degree of Penetration3. Low Penetration4. Harmful / Skin / Eyes5. Cell Damage / Thymine6. Germicidal / 260 nm
MicrowavesMicrowaves
Kill Microbes Indirectly with Heat
Chemical Control MethodsChemical Control Methods
Phenols and PhenolicsHalogensAlcohols (70% ethanol) (iso-propanal)Heavy Metals and Their CompoundsSurface-Active AgentsQuaternary Ammonium CompoundsChemical Food PreservativesAldehydesAntibiotics
Two Conditions Influence the Two Conditions Influence the Effectiveness of Chemical Effectiveness of Chemical DisinfectantsDisinfectants
Type of Microbe- G+ More Susceptible to Disinfectants- Pseudomonands Can Grow in
Disinfectants and Antiseptics- M. tuberculosis is Resistant to Many Disinfectants- Endospores Most Resistant
Environment- Organic Matter and Increased Temp
Evaluating a DisinfectantEvaluating a DisinfectantOld Standard is the Phenol Coefficient
Test(FYI -- The phenol coefficient is the value obtained by dividing the highest dilution of the test solution by the highest dilution of phenol that sterilizes the given culture of bacteria under standard conditions of time and temperature.)
In Lab We Use Soaked Filter In Lab We Use Soaked Filter PapersPapers
Antibiotic SensitivitiesAntibiotic Sensitivities
Types of DisinfectantsTypes of DisinfectantsPhenol and Phenolics
- Another Name for Carbolic Acid / Lysol
- Joseph Lister- Exert Influence By
1. Injuring Plasma membranes2. Inactivating Enzymes3. Denaturing Proteins
- Long Lasting, Good for Blood and Body
Fluids, No Effect on Spores
Types of DisinfectantsTypes of DisinfectantsHalogens
- Can be Used Alone or in Solution- Chlorine -- Purifies Drinking Water
a. 2-4 Drops of Chlorine per Liter / 30 Min
b. Forms an Acid Which is Bactericidalc. Disinfectant in Gaseous Form or in Solution as Calcium Hypochlorite
- Iodine – combines with Amino Acidsa. Inactivates Enzymesb. Tincture / Alcoholc. Iodophor / Organic Molecule /
Betadine
Types of DisinfectantsTypes of Disinfectants
Alcohols- Denature Proteins- Dissolve Lipids- Tinctures- Wet Disinfectants
a. Aqueous Ethanol (60% - 95%)
b. Isopropal Alcohol
Types of DisinfectantsTypes of Disinfectants
Heavy Metals and Their Compounds- Used for Burn Treatment- Prevents Neonatal Gonorrheal
OpthalmiaSilver nitrate
- Denature Proteins
Types of DisinfectantsTypes of Disinfectants
Surface-Active Agents- Decrease Molecular Surface
Tension- Include Soaps and Detergents- Soaps Have Limited Germicidal
Action but Assist in the Removal of
Organisms by Scrubbing- Acid-Anionic Detergents / Dairy
Types of DisinfectantsTypes of Disinfectants
Quaternary Ammonium Compounds (QUATS)
- Cationic Detergents Attached to NH4
+1
- Disrupt Plasma Membranes- Most Effective on Gram-Positive
Bacteria- Mouthwashes and Sore Throat
Remedies
Types of DisinfectantsTypes of Disinfectants
Chemical Food Preservatives- Sorbic Acid- Benzoic Acid
InhibitFungus- Propionic Acid- Nitrate and Nitrite Salts / Meats / To Prevent Germination of
Clostridium botulinum endospores
Types of DisinfectantsTypes of Disinfectants
Aldehydes- Formaldehyde- Glutaraldehyde- Most Effective of all Chemical Disinfectants- Carcinogenic- Oxidize Molecules Inside Cells
Types of DisinfectantsTypes of DisinfectantsGases
◦Ethylene oxide◦Ozone◦Sulfur dioxide, ◦ chlorine, ◦ glutaraldehyde, ◦methylene chloride, ◦formaldehyde, ◦ammonia
Types of DisinfectantsTypes of Disinfectants
Antibiotics- Used to Preserve
Cheese- Tomato products pH
> 4.5- Nisin
Stock feeds Promote faster growth
Antibiotic ResistanceAntibiotic Resistance
Growing ProblemIndiscriminant and Inappropriate UseSuper Bugs
1. Methicillin Resistant S. aureus2. Vancomycin Resistant
Enterococcus3. Multidrug Resistant M.
tuberculosisImportant to test Sensitivities