Control of Microorganisms Microbiology

Control of Microbial Growth Effected in two basic ways: 1. Biocidal action: Killing Microorganisms 2. Biostatic action :By inhibiting the Growth of Microorganisms Usually involves the use of: 1. Physical Agents that Kill or Prevent Growth 2. Chemical Agents that Kill or Prevent Growth 3. Biological Agents: Bacteria, Fungi, Phage etc.

Level of Resistance 1. Endospores (Most) 2. Parasites 3. Mycobacteria 4. Fungal Spores 5. Small Non-enveloped Viruses - Polio, Rotavirus, Rabies 5. Vegetative Fungal Cells 6. Enveloped Viruses - Herpes, Hepatitis B & C, HIV 5. Vegetative Bacteria (Least)

Principles of Microbial Control Prevention / Control of Growth to Prevent Infection and Spoilage, Asepsis Sterilization / Complete Destruction - Real sterilization is All or Nothing)  Commercial Sterilization The best we can do Reduce numbers to safe level - Utilizes a. Heat b. Radiation c. Chemicals d. Physical Removal e. Hyperbaric (High pressure)

Principles of Microbial Control Commercial Sterilization / Heat Treatment of Canned Foods Disinfection / Reducing Growth Nonliving Surfaces Antisepsis / Reducing Growth Living Tissue Antibiotics ( “magic bullets”) Internal use Preservatives Sanitize – Subject to High Temperature (70 o C) Washing (Dishwashers) or Hypo

Principles of Microbial Control Cide – Suffix Meaning to Kill Stat – Suffix Meaning to Inhibit Sepsis – Bacterial Contamination Asepsis – Lack of Bacterial Contamination

Rate of Microbial Death Bacteria Usually Die At a Constant Rate Plotted Logarithmically This Will Give a Straight Line

Time to Kill in Proportion to the Population Size Large Numbers Require Greater Time Small Numbers Require Less Time

Susceptibilities Vary Endospores are Difficult to Kill Organic Matter May Interfere with Heat Treatments and Chemical Control Agents Must clean first

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 Microbial Control

Physical Methods Heat Filtration Low Temperatures Desiccation Osmotic Pressure Radiation

Heat Most Frequent and Widely Used. Always Consider 1. Type of Heat 2. Time of Application 3. Temperature Endospores are the most heat resistant of all cells.

Moist Heat / Denatures Enzymes Thermal 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 Sterilization Boiling Autoclaving

Boiling 100° C for 30 Minutes Kills Everything Except Some Endospores Intermittent boiling can kills spores   Boil, incubate, boil, incubate, boil   Does not always work

Autoclaving Steam Under Pressure 121° C for 15 Minutes at 15 lb/in 2 Heat-labile Substances will be Denatured Steam Must Contact the Material

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.18 Measuring 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

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.19 Figure 7.1

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.20 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 7.2

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.21

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.22 Moist heat Autoclaves – used to kill endospores efficiently – use saturated steam under pressure to reach temperatures above boiling

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.23 Moist heat… pasteurization ◦controlled heating at temperatures well below boiling ◦reduces total microbial population and thereby increases shelf life of treated material

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.24 Pasteurization 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.25

Cook Chill Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.26

Dry Heat Sterilization Direct Flaming Incineration Hot-Air Sterilization (Oven)

Incineration Burns and Physically Destroys Organisms Used for a. Needles b. Inoculating Wires c. Glassware d. Body Parts?

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

Pasteurization A 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

Filtration The 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 Filters High-Efficiency Particulate Air Filters 1. Operating Rooms 2. Burn Units 3. Fume Hoods

Low Temperatures Decreasing Temperature Decreases Chemical Activity Low Temps are Not Bactericidal Refrigeration <5 0 C ◦Listeria still grows but slow ◦Spoilage organisms still grow Freezing C ◦Spoilage due to enzymes

Desiccation Disrupts Microbial Metabolism Stops Growth / Still Viable Freeze-drying / Dehydration ◦Used to preserve bacteria Viruses and Endospores Can Resist Desiccation

Osmotic Pressure Plasmolysis Sugar Curing / Salting May Still Get Some Mold or Yeast Growth ◦Staphylococcus and Listeria grows in 10% salt

Radiation Acts By Destroying DNA or Damaging It. Dependent on the Wavelength, Intensity, and Duration

Radiation Ionizing Radiation 1. High Degree of Penetration 2. Examples - Gamma Rays - X-rays - High Energy Electron Beams Ultraviolet Radiation 1. Nonionizing 2. Low Degree of Penetration 3. Low Penetration 4. Harmful / Skin / Eyes 5. Cell Damage / Thymine 6. Germicidal / 260 nm

Microwaves Kill Microbes Indirectly with Heat

Chemical Control Methods Phenols and Phenolics Halogens Alcohols (70% ethanol) (iso-propanal) Heavy Metals and Their Compounds Surface-Active Agents Quaternary Ammonium Compounds Chemical Food Preservatives Aldehydes Antibiotics

Two Conditions Influence the Effectiveness of Chemical Disinfectants 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 Disinfectant Old 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 Papers

Antibiotic Sensitivities

Types of Disinfectants Phenol and Phenolics - Another Name for Carbolic Acid / Lysol - Joseph Lister - Exert Influence By 1. Injuring Plasma membranes 2. Inactivating Enzymes 3. Denaturing Proteins - Long Lasting, Good for Blood and Body Fluids, No Effect on Spores

Types of Disinfectants Halogens - 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 Bactericidal c. Disinfectant in Gaseous Form or in Solution as Calcium Hypochlorite - Iodine – combines with Amino Acids a. Inactivates Enzymes b. Tincture / Alcohol c. Iodophor / Organic Molecule / Betadine

Types of Disinfectants Alcohols - Denature Proteins - Dissolve Lipids - Tinctures - Wet Disinfectants a. Aqueous Ethanol (60% - 95%) b. Isopropal Alcohol

Types of Disinfectants Heavy Metals and Their Compounds - Used for Burn Treatment - Prevents Neonatal Gonorrheal Opthalmia Silver nitrate - Denature Proteins

Types 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 Disinfectants Quaternary Ammonium Compounds (QUATS) - Cationic Detergents Attached to NH Disrupt Plasma Membranes - Most Effective on Gram-Positive Bacteria - Mouthwashes and Sore Throat Remedies

Types of Disinfectants Chemical Food Preservatives - Sorbic Acid - Benzoic AcidInhibitFungus - Propionic Acid - Nitrate and Nitrite Salts / Meats / To Prevent Germination of Clostridium botulinum endospores

Types of Disinfectants Aldehydes - Formaldehyde - Glutaraldehyde - Most Effective of all Chemical Disinfectants - Carcinogenic - Oxidize Molecules Inside Cells

Types of Disinfectants Gases ◦Ethylene oxide ◦Ozone ◦Sulfur dioxide, ◦ chlorine, ◦ glutaraldehyde, ◦methylene chloride, ◦formaldehyde, ◦ammonia

Types of Disinfectants Antibiotics - Used to Preserve Cheese - Tomato products pH > Nisin Stock feeds  Promote faster growth

Antibiotic Resistance Growing Problem Indiscriminant and Inappropriate Use Super Bugs 1. Methicillin Resistant S. aureus 2. Vancomycin Resistant Enterococcus 3. Multidrug Resistant M. tuberculosis Important to test Sensitivities