CH. 7 | The Control of Microbial Growth

Overview

Microbial control agents are physical or chemical methods used to inhibit, kill, or remove microorganisms to prevent infection, spoilage, and contamination. Key types include chemical disinfectants (alcohols, phenolics, halogens), antiseptics, antibiotics, and physical methods like heat, radiation, and filtration. These agents target bacteria, viruses, and fungi on living tissue or surfaces.

Microbial Growth

• The increase in the number of cells or total population mass of microorganisms

7 - 1 Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis.

Bactericidal

• Kills bacteria

Bacteriostatic

• Inhibits/Prevents the growth of bacteria

Sterilization

• Complete removal or destruction of all living microorganisms, including endospores

• Methods include physical and chemical ways

• The most common is autoclaving, or using aseptic technique (heating an inoculating loop/needle) — flaming the instruments / tubes through a bunsen burner flame

• Complete sterilization is often not required in other settings
  
  

• Commercial sterilization
  → A process of treating canned goods aimed at destroying microbes and endospores of C. botulinum
  → Limited heat treatment
  
  

Disinfection

• Any treatment used on inanimate objects to kill or inhibit the growth of microorganisms

• Destruction of vegetative pathogens (Non-endospore forming)

• Makes use of chemicals, ultraviolet radiation, boiling water, or steam

• Commonly applied as chemical agents to treat an inert surface or substance
  → i.e., alcohol, bleach, hydrogen peroxide
  
  

• Antisepsis
  → A chemical method for disinfectation that is directed at living tissue and or skin
  
  

Degerming

• The removal of microorganisms in an area / mechanical removal rather than the killing of microbes in a limited area

• Usual target is on living tissue and or skin
  → i.e., when someone is about to receive an injection, the skin is swabbed with alcohol
  
  

Sanitization

• Intended to lower microbial counts to safe public health levels and minimize the chances of disease transmission from one user to another

• Accomplished by high-temperature washing, etc
  
  

Biocide + Germicide

• A substance capable of killing microorganisms

• Kills microorganisms (with certain exceptions)

• i.e., a fungicide kills fungi, a virucide inactivates viruses, etc
  
  

Bacteriostasis

• A treatment capable of inhibiting bacterial growth
  
  

Asepsis

• Absence of significant contamination by unwanted organisms

• Clean and sterile techniques

• Involves washing hands
  → i.e., hand sanitizer, using alcohol wipes before injections

7 - 2 Describe the patterns of microbial death caused by treatments with microbial control agents.

Pattern: Exponential (Constant Rate) Death

• Microbes don’t die all at once, they die at a constant proportional rate. Each unit of time, the same percentage of whatever is left gets killed

• Constant rate: The proportion being killed each minute, and it stays the same throughout the entire treatment
  → i.e., 90%

• The number of deaths decreases by one each minute, but the rate (90% per minute) remains constant. This is known as exponential death
  
  

What affects this pattern?

• Number of microbes — Bigger starting population = longer it takes to eliminate completely

• Environmental conditions — Warmer temperatures and acidic conditions make treatments more effective

• Organic matter — Blood, feces, and biofilms can shield microbes and slow killing (Usually protective of microorganisms)

• Time of exposure — Longer exposure is needed for tough organisms or endospores

• Microbial characteristics — Cell wall composition and endospore formation affect resistance
  
  

In other words…

Microbes die at a constant proportional rate (exponential death), meaning the same percentage dies each time interval, which appears as a straight line on a logarithmic graph. This rate is influenced by factors such as population size, environment, organic matter, exposure time, and microbial traits.

7 - 3 Describe the effects of microbial control agents on cellular structures.

3 Primary Ways —in which various agents can kill or inhibit microbes—

• Plasma membrane — Actively regulates the passage of nutrients into the cell, and deals with the elimination of waste from it
  → Damage to the lipids or proteins by microbial control agents can cause cellular contents to leak into the surrounding medium and interfere with cell growth
  
  

• Enzymes — Vital to all cellular activities = Destroying its enzymes destroys the cell
  → Damage / Breakage of the hydrogen bonds results in the denaturation of the protein
  
  

• Nucleic Acids — Carrier of the cell’s genetic information
  → Damage to these nucleic acids is lethal for the cell, as it can no longer replicate or carry out metabolic functions (such as the synthesis of enzymes)

7 - 4 Compare the effectiveness of moist heat and dry heat.

Bacterial Heat Resistance

• Thermal Death Point (TDP)
  → Lowest temperature at which all the microorganisms in a particular liquid suspension will be killed in 10 minutes
  → i.e., Salmonella, if you heated it to 59°C for 10 minutes, some cells might survive. But at 60°C for 10 minutes, every single cell is dead — that's the thermal death point

• Thermal Death Time (TDT)
  → Minimal length of time for all bacteria in a particular liquid culture to be killed at a given temperature

• Decimal Reduction Time (DRT)
  → Time in minutes, in which 90% (one log) of a population of bacteria at a given temperature will be killed
  
  

Moist Heat Sterilization

• Boiling
  → Usual temperature: 100^o c
  → Kills vegetative forms of bacterial pathogens
  → Endospores are not killed/destroyed by this

• Autoclaving
  → Preferred method of sterilization (unless the material can get damaged by heat or moisture)
  → Higher the pressure, higher the temperature
  → 121^o c for 15 minutes will kill all organisms and endospores

• Pasteurization
  → Developed by Louie Pasteur
  → Mild heating — sufficient to kill organisms that cause spoilage, without damaging the taste of the product
  → Kills harmful/pathogenic microorganisms in food and beverages without completely sterilizing them
  → High-temperature short time pasteurization (HTST)
  → Ultra-high temperature treatments (UHT)
  
  

Dry Heat Sterilization

• Flaming
  → The process of sterilizing an inoculating loop or needle by holding it in an open flame
  → Incineration

• Hot Air Sterilization
  → Sterilization by the use of an oven at 170^o c for approximately 2 hours

7 - 5 Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth.

Filtration

• Works by passing a liquid or gas through a screen-like material with pores small enough to trap and retain microorganisms physically — it doesn't kill them, it simply removes them from the substance

• In other words, filtration physically removes microorganisms rather than suppressing or killing them
  → i.e., strainer
  
  

Low Temperatures

• Refrigeration
  → Works by slowing down the metabolic rate of microorganisms, so much so that they can barely function (cannot reproduce or synthesize toxins)
  → Bacteriostatic effect — Inhibits / Prevents bacterial growth

• Freezing
  → Rapid Freezing — Microbes are dormant, but not dead
  → Slow Freezing — Most harmful, ice crystals form and physically disrupt the cellular and molecular structure
  → Thawing — Most damaging, slow, but causes more ice crystal damage

• Lyophilization / Freeze-drying
  → Laboratory process for preserving microbes by freezing, reducing pressure, and removing water
  
  

High Pressure

• If the pressure is high enough, it can alter the molecular structures of proteins and carbohydrates, resulting in the rapid inactivation of vegetative bacterial cells

• Endospores are highly resistant to high pressure
  
  

Desiccation (Dry)

• Absence of water → microorganisms cannot grow or reproduce, but can remain viable

• Prevents metabolism
  
  

Osmotic Pressure

• High concentrations of salt or sugar create a hypertonic environment outside the microbial cell, causing water to rush out of the cell through osmosis

• Dehydrates the cell, which starves the cell of moisture (which is needed for it to survive and reproduce)

• Mostly bacteriostatic, but can become bactericidal if dehydration is severe enough

7 - 6 Explain how radiation kills cells.

Ionizing Radiation

• Gamma rays

• X-rays

• High-energy electron beams
  → Shorter wavelength = carries more energy
  → Ionization of water makes it so that highly reactive hydroxyl radicals form, which can kill organisms by reacting with their organic cellular components (especially DNA), and damage them
  → Destruction of DNA
  
  

Nonionizing Radiation

• UV light
  → Longer wavelength
  → Does not have enough energy to ionize atoms
  → UV light damages cells by being absorbed into their DNA. This absorption causes bonds to form between adjacent pyrimidine bases (usually thymines) in the chain, creating thymine dimers. These dimers block proper DNA replication during cell reproduction, ultimately preventing the cell from reproducing correctly and eventually leading to cell death
  → Damage to DNA

Physical Methods used to control Microbial Growth

7 - 7 List the factors related to effective disinfection.

Concentration of the Disinfectant
→ Directly affects how well a disinfectant will work
→ Must always be diluted exactly as specified by the manufacturer
→ Too weak = Ineffective
→ Too strong = Potentially wasteful/harmful

Temperature

→ The activity of most disinfectants increases as the temperature rises
→ Too high a temperature can end up inactivating the disinfectant itself

pH of the Medium

• Significantly affects pH activity

• Chlorine is a good example; it is most effective at pH 6, but ineffective at pH > 8
  
  

Nature / Organic matter

• Organic materials present on a surface can interfere with disinfectant action

• This is why surfaces need to be scrubbed and rinsed before applying the disinfectant
  
  

Time

7 - 8 Interpret the results of use-dilution tests and the disk-diffusion method.

Use-Dilution Tests

• A method of determining the effectiveness of a disinfectant using serial dilutions

• Measures the presence and or absence of bacterial growth to determine the effectiveness of a disinfectant
  
  How it works:

• Metal/glass cylinders are dipped into standardized bacterial cultures. Thereafter, they are removed and dried at 37^o c for a short time. Afterwards, they are placed into the disinfectant solution at the recommended concentration and left for 10 minutes at 20^o c, and then transferred over to a growth medium

• To interpret results:
  → No bacterial growth = Disinfectant effectively killed all bacteria
  → Some bacterial growth = Disinfectant was partially effective, some survived, some died
  → Heavy bacterial growth = Disinfectant was ineffective
  
  

Disk-Diffusion Method (Kirby-Bauer Test)

• An agar-diffusion test to determine microbial susceptibility to chemotherapeutic agents

• Measures the diameter of the zone of inhibition surrounding the antimicrobial-soaked disk to determine the susceptibility of bacteria to drugs
  
  How it works:

• A filter paper disk is soaked with the chemical agent, which is then placed on an agar plate that has been inoculated with the test organism. Afterwards, the plate gets incubated, and the results are up for discussion

• To interpret results:
  → Clear zone of inhibition around disk = Chemical is effective, meaning that bacteria cannot grow in the zone
  → No clear zone = Chemical is ineffective, meaning that bacteria can grow

7 - 9 Identify the methods of action and preferred uses of chemical disinfectants.

Antimicrobial action/activity

• Inhibits the growth of or killing of microorganisms through mechanisms such as damaging their cell walls, disrupting membranes, inhibiting protein synthesis, or blocking DNA replication

• The following examples are chemical antimicrobial agents and their specific mechanisms of antimicrobial activity
  
  

Phenolics

• Suitable agents for disinfecting pus, saliva, and feces

• Exerts antimicrobial activity by injuring plasma membranes, which results in leakage of cellular contents

• Remain active in the presence of organic compounds → stable → persist for long periods of time
  
  

Bisphenols

• Used to control infections in nurseries, and is an ingredient in soaps, toothpastes, and mouthwashes
  
  

• Triclosan (Bisphenol used in s, t, and m)
  → Was eventually banned because it was not considered safe in healthcare settings. It would inhibit an enzyme needed for the biosynthesis of fatty acids, which affects the integrity of the plasma membrane
  → Effective against gram + bacteria
  
  

Biguanides

• Affects bacterial cell membranes

• Effective against gram + and gram - bacteria
  
  

• Chlorhexidine
  → Common in microbial control on skin and mucous membranes
  → Often used for surgical hand scrubs & skin preparation in patients
  
  

Essential Oils

• Mixture of hydrocarbons extracted from plants

• Similar to phenolics, they can be used to disinfect hard surfaces like countertops, and can be used on skin
  
  

Halogens

• Iodine and Chlorine are effective antimicrobial agents
  → Iodine: Impairs protein synthesis and alters membranes (their structure and ability to function)
  → Chlorine: Oxidizing agent, shuts down cellular enzyme systems
  
  

Alcohols

• Effectively kill bacteria and fungi but not endospores and non-enveloped viruses

• Denatures proteins and can disrupt membranes

• Ethanol and Isopropanol
  
  

Heavy Metals

• Can be biocidal or antiseptic
  
  

• Oligodynamic action
  → The ability of small amounts of a heavy metal compound to exert antimicrobial activity

7 - 12 List the advantages of glutaraldehyde over other chemical disinfectants.

Glutaraldehyde

• An Aldehyde (effective antimicrobial)

• Less irritating, more effective than formaldehyde

• Effective against bacteria, viruses, fungi, spores, etc

• Used in hospital settings as a disinfectant

• Considered a sterilizing agent

7 - 13 Identify chemical sterilizers.

Plasmas

• Consists of an electrically excited gas

• Free radicals destroy microbes

• Advantage: Requires only low temperatures
  
  

Supercritical Fluids

• CO2 with gaseous and liquid properties

• Organisms exposed to this supercritical CO2 are inactivated (including most vegetative organisms that cause spoilage and foodborne illnesses)
  
  

Peroxygens

• Oxidizing agents

• Used for contaminated surfaces and food packaging

7 - 14 Explain how the type of microbe affects the control of microbial growth.

Ethylene Oxide

• Kills all microbes and endospores, but requires a lengthy exposure time for several hours

1. Prions — MOST RESISTANT

• Infectious proteins (not even living cells!)

• Cause neurological diseases like mad cow disease

• Normal autoclaving is inadequate

• To destroy: incineration of infected carcasses

• For surgical instruments: WHO/CDC recommend sodium hydroxide + autoclaving at 121°C for 1 hour OR 134°C for 18 minutes

• Disposable instruments must be incinerated

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2. Bacterial Endospores

• Affected by relatively few biocides

• Extremely tough protective coating makes them highly resistant

• Require very harsh conditions to destroy (e.g. autoclaving at 121°C)

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3. Mycobacteria

• Non-endospore-forming but still greater-than-normal resistance

• Example: Mycobacterium tuberculosis

• Resistance due to waxy, lipid-rich cell wall

• Special tuberculocidal tests developed specifically to test biocides against them

• Disinfectant labels often indicate if they are tuberculocidal

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4. Cysts & Oocysts of Protozoa

• Relatively resistant to chemical disinfection

• The cyst form is a protective stage making them harder to kill than vegetative forms

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5. Vegetative Protozoa

• Active, non-cyst form

• More susceptible than cysts but still moderately resistant

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6. Gram-Negative Bacteria

• More resistant than Gram-positive due to:

  • External lipopolysaccharide (LPS) layer

  • Porins — highly selective structural openings that control what enters the cell, blocking many biocides

• Pseudomonas and Burkholderia are especially resistant — can even grow actively in some disinfectants (e.g. quaternary ammonium compounds)

• Also resistant to many antibiotics

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7. Fungi (including most fungal spores)

• Moderately resistant

• More tolerant of osmotic pressure and acidic conditions than bacteria

• Reason molds cause spoilage in fruits and grains

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8. Viruses Without Envelopes (Nonenveloped)

• Have only a protein coat

• More resistant — fewer biocides are effective against them

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9. Gram-Positive Bacteria

• Generally more susceptible than Gram-negative

• Lack the external LPS layer and selective porins

• Most biocides work well against them

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10. Viruses With Lipid Envelopes — LEAST RESISTANT

• Most susceptible because their lipid envelope is easily disrupted

• Lipid-soluble antimicrobials are very effective against them

• Labels indicate effectiveness against lipophilic viruses