Control of Microorganisms Microbiology Control of Microbial Growth Effected in two basic ways: 1....

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


reproduction or display.reproduction or display. 1919Figure 7.1Figure 7.1

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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



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



Moist heat…Moist heat…

pasteurization◦controlled heating at temperatures well below

boiling◦reduces total microbial population and thereby

increases shelf life of treated material



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


Heavy Metals and Their Compounds- Used for Burn Treatment- Prevents Neonatal Gonorrheal

OpthalmiaSilver nitrate

- Denature Proteins


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


Quaternary Ammonium Compounds (QUATS)

- Cationic Detergents Attached to NH4

+1

- Disrupt Plasma Membranes- Most Effective on Gram-Positive

Bacteria- Mouthwashes and Sore Throat

Remedies


Chemical Food Preservatives- Sorbic Acid- Benzoic Acid

InhibitFungus- Propionic Acid- Nitrate and Nitrite Salts / Meats / To Prevent Germination of

Clostridium botulinum endospores


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


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

Control of Microorganisms Microbiology Control of Microbial Growth Effected in two basic ways: 1....

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