1. Microbial Control

2. Terminology for Microbial Control
Sterilization- removal or destruction of all forms of microbial life
Commercial sterilization- subjects canned food to only enough heat to destroy the endospores of Clostridium botulinum
Disinfection- is the destruction of vegetative pathogens on a surface, usually with chemicals
Spores and viruses are not necessarily destroyed
Antisepsis- is the chemical disinfection of living tissue, such as skin or mucous membranes

3. Terminology for Microbial Control
Asepsis- is the absence of pathogens on an object or area, as in antiseptic surgery
Degerming (degermation)- is the removal of transient microbes from the skin by mechanical cleansing or by antiseptic
Sanitation- is the reduction of microbial populations on objects to safe public health levels
A biocide or germicide- kills microorganisms
Fungicides kill fungi, virucides kill viruses
Suffix – cide means the killer of a specified microorganism
Suffix- stat used in this way indicates only that the substance inhibits – for example bacteriostasis
Example for asepsis is handwashing Example for bacteriostatic is a refrigerator

4. Rate of Microbial Death
Bacterial populations killed by heat or chemicals tend to die at constant rates—for example, 90% every 10 minutes. Plotted logarithmically, these figures form straight descending lines.

5. Factors that influence effectiveness of an antimicrobial treatment:
1. Number of microbes
-more cells, more time needed to kill all
2. Environmental influences
-organics often inhibit chemical agents (blood, feces, vomit)
-temperature (disinfectants work better in warm temperatures)
-pH (heat is more effective in an acid pH)
Environmental- any medium containing fats and protein will tend to protect bacteria

6. Factors that influence effectiveness of an antimicrobial treatment:
3. Time of exposure
-Chemical antimicrobials require a certain amount of exposure; same agent may need longer on resistant organisms or spores
-with heat, lower temps require longer to kill
4. Microbial characteristics
-resistance genes, protective structures
(e.g.capsules) etc. can inhibit action
-bioflims prevent penetration
susceptibility to different agents varies among microbes

7. Type and Age of Microbe
Bacteria- susceptible to protein denaturing BUT mycobacteria is not because of its hydrophobic coat
Growth cycles- physiologically young bacteria(early in growth cycle) susceptible to heat
Endospores more resistant to heat the older they get

8. Properties of Materials:
Plastic and rubber- not heat tolerant
Cutting edges of surgical instruments- no moisture
or corrosive chemicals
Edges can become pitted due to rust and corrosion, microbes can hide there
Fabrics- no chemical disinfection; some too fragile

9. Actions of Microbial Control Agents
1. Alteration of membrane permeability
The plasma membrane controls the passage of nutrients and wastes into and out of the cell.
Damage to the plasma membrane causes:
leakage of cellular contents
interferes with cell growth.
= Leak lysis, death
Cell lysis is a process in which a cell is broken down or destroyed

10. Actions of Microbial Control Agents
2. Damage to proteins and Nucleic Acids
Enzymes and other proteins are essential for cell function
Denatures proteins
Enzymes (no reactions)
Proteins necessary for bacteria metabolism
Hydrogen bonds are broken
Covalent bonds are also broken
3. Damage to nucleic acids
Prevent replication, transcription, or translation
Hydrogen bonds hold proteins in the characteristic 3-dimensional shape required for their functions. Heat and certain chemicals break these bonds and the shape is lost. This is called denaturation.
DNA and RNA carry the cell’s genetic information and function in protein synthesis. Damage to these by heat, radiation, or chemicals usually kills the cell.

11. Physical Methods of Microbial Control
-to disinfect objects, food, and solutions common methods:
-Temperature: kill or inhibit growth
Heat
Low Temps.
-Filtration: physical removal
-Desiccation: inhibit growth
-Osmotic pressure
-Radiation: kill

12. Methods involving Heat:
Heat: Common food preservation
Denatures protein (changes shape)
Thermal Death Point (TDP) = lowest temp at which all microbes in liquid suspension will be killed in 10 min
Thermal Death Time (TDT)= minimal length of time for all microbes in liquid suspension to be killed at given temp
*Both are different for different species due to microbial variation in heat tolerance
Decimal Reduction Time (DRT) = Time in minutes in which 90% of bacteria at a given temperature will be killed
Concept of equivalent treatments:
With any heat treatment, the higher the temperature used the shorter the exposure time needed to achieve the same effect

13. Methods involving Heat:
1. Moist heat will always kill faster than dry heat at the same temperature
Kills microbes by coagulation of cell proteins
A. Boiling (100°C) kills vegetative forms of bacterial pathogens, many viruses, and fungi within 10 minutes
Some mo’s and viruses are resistant
Endospores (up to 20 hrs.) and some viruses (30 minutes) survive boiling for longer times.
This occurs faster in the presence of water, so moist heat requires lower temperatures and less time of exposure than dry heat.
Edospores-dormant stage of some bacteria that allow them to survive harsh conditions-ex. Extreme heat

14. Methods involving Heat:
B. Autoclaves
Moist Heat (steam) and pressure for sterilization
Achieves higher temps. than boiling
Preferred method (sealed chamber, air is exhausted, and steam under pressure is injected) for all materials that can withstand it
Kills all organisms and their endospores in about minutes
An autoclave is shown in Figure 1.1 and 1.2
Here we use an autoclave to sterilize culture media. In hospitals, doctors’ offices, dentists’ offices, etc. this methods is used to sterilize medical equipment.
Materials being autoclaved are often wrapped in paper, so that after sterilization the outside of the package can be handled without contaminating the sterile item inside.
The size of the container, the volume of a liquid, and the type of wrapping can influence the time and temperature required for sterilization.

15. Autoclave Figure 1.1 Figure 1.2 Autoclave tape
Autoclave tape works by changing color after exposure to temperatures commonly used in sterilization processes, typically 121°C in a steam autoclave. It is important to note that the presence of autoclave tape that has changed color on an item does not ensure that the product is sterile, as the tape will change color upon exposure only. For steam sterilization to occur, the entire item must completely reach and maintain 121°C for minutes with proper steam exposure to ensure sterilization. Diagonal shaped more leat, words Autoclaved lead free
Figure 1.2
Autoclave tape

16. Methods involving Heat:
C. Pasteurization
Louis Pasteur
Mild Heating
Kills most pathogens
Kills bacteria that cause spoilage
Preserves taste in product
Lowers bacterial numbers
Pasteurization or pasteurisation is a process of heating a food, which is usually a liquid, to a specific temperature for a predefined length of time and then immediately cooling it after it is removed from the heat. This process slows spoilage caused by microbial growth in the food.
Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food. Instead, it aims to reduce the number of viable pathogens so they are unlikely to cause disease (assuming the pasteurized product is stored as indicated and is consumed before its expiration date).

17. Methods involving Heat:
2. Dry Heat
Kills by burning to ashes or by oxidation
Flaming- we use this on loops in labs
Incineration- burning of contaminated paper
Hot air sterilization
Hot ovens
Mainly used for items not suitable for autoclaving
Oily substances, powders
Large amounts of glassware

18. Low Temperature
Lower temp inhibits growth, rapid freezing limits moisture (bacteriostatic)
Refrigerator temperatures (0° to 7°C) slow the metabolic rate of microbes; however
Psychrotrophic species still grow slowly.
Some organisms grow at temperatures slightly below freezing, but microbes at the usual temperatures of freezer compartments are completely dormant.

19. Filtration Liquids Heat- sensitive materials
Small pores prevent passage of bacteria
High-efficiency particulate air filters (HEPA)
Operating rooms
Special clean rooms
Masks
Used with solutions for: renal dialysis, heart bypass machines, I.V.’s
Catch particles autoclaves miss

20. Desiccation
Microbes require water for growth, and adequately dried (desiccated) foods will not support their growth, therefore inhibiting growth
Absence of Water
Lyophilization- rapid freeze drying
Used for blood products , serum products, enzymes, cultures
Avoids ice crystal formation; cells burst when water expands
Species vary in their susceptibility. Dry surroundings kill some vegetative cells in one hour or less. Others can survive for months or even years. Bacteria that produce endospores are extremely resistant to drying. Many viruses are also resistant to drying.

21. Osmotic Pressure
High salt or sugar concentrations cause water to leave the cell; this is an example of osmosis.
Generally, molds and yeasts resist osmotic pressures better than bacteria.

22. Radiation Ionization Radiation
includes X rays, gamma rays, and high-energy electron beams
very short wavelengths and high levels of energy
Penetrate deeply
Ionizes water to form hydroxyl radicals
These destroy cell components, especially DNA
Kills: vegetative cells, viruses, most endospores with adequate exposure
Applications: food preservation, sterilization of pharmaceuticals, medical supplies, mail
Recently, approval has been granted for use of low level radiation of fruits and meats. The post office is now using this method to sterilize some mail. This process does involve the use of dangerous radiation and can only be used in a properly shielded room, so it is mostly used in factories where widescale use of the setup makes it economical.

23. Radiation Nonionizing radiation
Have a longer wavelength and less energy
Ultraviolet (UV) light is the common example
Causes the formation of thymine dimers, which interferes with DNA replication and formation of mRNA.
UV lamps are used in hospitals and in food service
This method does not sterilize, but it does reduce bacterial growth
Penetrating power is very low, so any type of covering protects microbes.
Sunlight has some weak antimicrobial effects (biocidal), but the wavelengths of sunlight are too long to work well.

24. Types of Chemical Agents
1. Phenols and Phenolics
A. Phenol (carbolic acid)
Irritating to skin and mucous membranes
Bad odor
Rarely used today
B. Phenolics
Chemicals derived from phenol
Chemically altered to make it less irritating and more effective
Damages plama mbs., inactivates enzymes, denature proteins
Often used as disinfectants as they remain active in the presence of organic matter
Its main use now is in throat lozenges and sprays, but the concentration is so low that there is little antimicrobial effect, although there is some local anesthetic action. Some throat sprays may have a concentration above 1% and these may show antibacterial action.

25. Types of Chemical Agents
C. Bisphenols
contain 2 phenolic groups connected by a bridge
Hexachlorophene
pHisoHex is an example
Prescription antibacterial lotion
Used in nurseries to control gram + bacteria
Skin bacteria: Staphylococcus and Streptococcus
Triclosan
Found in antibacterial soap
Effective against G+ and G- bacteria

26. Types of Chemical Agents
2. Biguanides
Chlorhexidine is an example
Hibiclens soap
Used on skin and mucous membranes
Similar to phenolics but less toxic
Disrupt plasma mb.
Broad spectrum
Effective against most vegetative bacteria and fungi, but not against endospores and many viruses.
Damaging to eyes

27. Types of Chemical Agents
3. Halogens- Effective alone or in compounds
A. Iodine (I2)
One of the oldest and most effective
Very effective on: all bacteria, many endospores, fungi and some viruses
Combines with amino acids in proteins and denatures proteins
Alters plasma mb.
Negative Aspects
Staining
Sometimes irritating to the skin
May trigger allergies
Applications
Skin disinfection, wound treatment, water treatment

28. Types of Chemical Agents
B. Chlorine (Cl2)
Action: forms hypochlorus acid with water → oxidizing agent, denatures proteins
Broad spectrum: bacteria, fungi, some endospores, some viruses
Positive aspects: -effective against all vegetative cells including Mycobacterium
-cost effective
Negative aspects: -action inhibited by organics
-can form carcinogenic compounds
Applications: water and sewage treatment, surface and instrument disinfection
Sodium hypochlorite (NaClO): Is active ingredient of bleach
The U.S. military uses tablets of sodium dichloroisocyanurate for fielddisinfection of water

29. Types of Chemical Agents
C. Alcohols- frequently used for skin degerming
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, instrument disinfection
Not good for open wounds, because cause proteins to coagulate and leave bacteria unharmed
Ethanol (ethyl alcohol): Optimum concentration is 70%.
Isopropanol: Rubbing alcohol
Better disinfectant than ethanol
Usual concentration is 90%
Also cheaper and less volatile.

30. Types of Chemical Agents
5. Heavy metals and their compounds
Oligodynamic action- Very tiny amounts are effective
Includes- silver, mercury, copper, zinc
Bind sulfur groups causing inactivation or denaturing of proteins
A. Silver:
1% silver nitrate used to protect infants against gonorrheal eye infections until recently.
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 (fungicidal, algicidal)
D. Zinc
Used in mouthwashes
Superficial fungal and bacterial infections
However, because these drops often cause the baby's eyes to be irritated, most hospitals now use erythromycin ointment instead

31. Types of Chemical Agents
6. Surface-Acting Agents
Disrupt plasma membrane & denature proteins
Decrease surface tension
Include soaps and detergents
Washing with soap breaks up the oily film that covers skin and allows microbes and dirt to be washed away
7. Quaternary Ammonium Compounds
Widely used surface active agents
Denature proteins & disrupt cell membranes
Cationic (positively charge) detergents
Effective against gram positive bacteria, less effective against gram-negative bacteria.
Also destroy fungi, amoebas, and enveloped viruses.

32. Types of Chemical Agents
8. Aldehydes
These can act very effectively against microbes
Inactivate proteins
Action: cross-link (thus inactivate) nucleic acids and proteins
High activity (sterilization)
biocidal including endospores
Positive aspects
achieves sterilization
Negative aspects
unstable
-toxic
-volatile with noxious fumes
Applications: specimen preservation (embalming), vaccine sterilization

33. Types of Chemical Agents
9. Gaseous Sterilizers
Chemicals that sterilize in a chamber similar to an autoclave.
Denature proteins, by replacing functional groups with alkyl groups
A. Ethylene Oxide
Kills all microbes and endospores , but requires exposure of 4 to 18 hours.
Toxic and explosive in pure form.
Highly penetrating.
Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.

34. Types of Chemical Agents
10. Peroxygens
Oxidize cellular components
A. Hydrogen Peroxide
Common household antiseptic
Not good for open wounds because quickly broken down by catalase present in human cells
used in deep wounds because it releases oxygen as it breaks down, which makes conditions unfavorable for anaerobic bacteria
Effective in disinfection of inanimate objects (kills endospores)
Sporicidal at higher temperatures
Used by food industry and to disinfect contact lenses

35. Types of Chemical Agents
B. Benzoyl Peroxide
Main ingredient in many acne treatments
May be used in treating wound infections caused by anaerobes
C. Peracetic Acid
One of the most effective liquid sporicides available
Sterilant :
Kills bacteria and fungi in less than 5 minutes
Kills endosporesand viruses within 30 minutes
Used widely in disinfection of food and medical instrument because it does not leave toxic residues