How to minimize the microbial growth by different methods.

1. The Control of Microbial Growth

2. Sterilization
Commercial sterilization - for canned food -
• Sterilization is the removal or destruction of all living microorganisms.
• Heating is the most common method used for killing microbes, including the
most resistant forms, such as endospores.
• A sterilizing agent is called a sterilant.
• Liquids or gases can be sterilized by filtration.
Complete sterilization is often not required in somesettings.
For example, the body’s normal defences can cope with a few microbes entering a surgical wound.
A drinking glass or a fork in a restaurant requires only enough microbial control to prevent the
transmission of possibly pathogenic microbes from one person to another
endospores of Clostridium botulinum

3. Term Definition Comments
Sterilization Destruction or removal of all forms of microbial
life, including endospores
Usually done by steam under pressure ora
sterilizing gas, such as ethyleneoxide.
Disinfection Destruction of vegetative pathogens on
inanimate objects.
May make use of physical or chemical methods.
Antisepsis Destruction of vegetative pathogens onliving
tissue.
Treatment is almost always by chemical
antimicrobials.
Degerming Removal of microbes from a limited area, such as
the skin around an injection site.
Mostly a mechanical removal by an alcohol-
soaked swab.
Sanitization Treatment is intended to lower microbialcounts
on eating and drinking utensils to safe public
health levels.
May be done with high-temperature washing or
by dipping into a chemical disinfectant.
Terminology
Sepsis, indicates bacterial contamination, as in septic tanks for sewage treatment or a disease condition.
Aseptic means that an object or area is free of pathogens.
Bacteriostatic: Inhibits bacterial reproduction. Bactericidal: Kills bacteria

4. Rate of Microbial Death
Bacterial populations subjected to heat/antimicrobial chemicals die at a constant rate.
Rate: 90% / min
1. Alteration of Membrane Permeability 2. Damage to Proteins and Nucleic Acids

5. Several factors influence the effectiveness of antimicrobial treatments:
1.The number of microbes
The more microbes there are to begin with, the longer it takes to eliminate the entire population.
2. Environmental influences
- Warm conditions/solutions
- Presence of organic matter e.g, blood, vomitus, or feces
- Microbes in surface biofilms, are difficult for biocides to reach effectively
- Suspending medium, especially protective medium (fat, protein) protects microbes
- Heat is also measurably more effective under acidic conditions
3. Time of exposure
Chemical antimicrobials often require extended exposure to affect more-resistant microbes or
endospores.
4. Microbial characteristics
Microbial characteristics affect the choice of chemical and physical control methods.

7. Physical Methods of Microbial Control
Drying (desiccation) and salting (osmotic pressure) were probably among the earliest techniques
Heat is very effective (fast and cheap).
Heat appears to kill microorganisms by denaturing their enzymes; the resultant changes to the three-
dimensional shapes of these proteins inactivate them
 Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10min.
 Thermal death time (TDT): Time to kill all cells in a culture.
 Decimal Reduction Time (DRT): Minutes to kill 90% of a population at a giventemperature.
These are useful guidelines that indicate the severity of treatment required to kill a given population
of bacteria.

8. Moist Heat Sterilization
Moist heat kills microorganisms primarily by coagulating proteins (denaturation) – breakage of the
hydrogen bonds that hold the proteins in their three-dimensional structure.Example?
Boiling kills vegetative forms of bacteria, almost all viruses, fungi and their spores about 10 minutes.
Free-flowing (unpressurized) steam is same temp as boiling water but some bacterial endospores can
resist boiling for more than 20 hours. Not always a reliable sterilization procedure. However, brief
boiling will kill most pathogens. Examples ?
Steam under pressure in an autoclave - The higher the pressure in the autoclave, the higher the temp.
Autoclaving is the preferred method of sterilization in health care environments, unless the material
to be sterilized can be damaged by heat or moisture.

9. Autoclaving is used to sterilize culture media,
instruments, dressings, intravenous equipment,
solutions, syringes, transfusion equipment etc.
Sterilization in an autoclave is most effective
when the organisms either are contacted by
the steam directly or are contained in a small
volume of aqueous (primarily water) liquid.
Under these conditions, steam at a pressure of
about 15 psi (121°C) will kill all organisms and
their endospores in about 15 minutes.
Sterilizing the surface of a solid requires that
steam actually contact it.

10. Pasteurization
Pasteur used mild heating, which was sufficient to kill the organisms that caused the particular
spoilage problem without seriously damaging the taste of the product.
Significant number reduction (esp. spoilage and pathogenic organisms)  does not sterilize!
Classic holding method: 63C for 30 min
Flash pasteurization (HTST): 72C for 15 sec. Thermo-tolerant organisms survive
Ultra High Temperature (UHT): 140C for 4 sec then rapidly cooled in vacuumchamber.
Technically not pasteurization because it sterilizes.
Products other than milk, such as ice cream, yogurt, and beer, all have their own
pasteurization times and temperatures, which often differ considerably.
The phosphatase test (phosphatase is an enzyme naturally present in milk). If the
product has been pasteurized, phosphatase will have been inactivated

11. Dry Heat Sterilization
It kills by oxidation effects.
• Flaming of loop
• Incineration of carcasses
• Anthrax
• Foot and mouth disease
• Bird flu
• Hot-air sterilization
• Hot-air oven 170˚C, 2 hr

12. Filtration
Filtration is the passage of a liquid or gas through a screen-
like material with pores small enough to retain
microorganisms.
A vacuum is created in the receiving flask; air pressure then
forces the liquid through the filter. Filtration is used to
sterilize heat-sensitive materials, such as some culture
media, enzymes, vaccines, and antibiotic solutions.
High-efficiency particulate air (HEPA) filters remove almost
all microorganisms larger than about 0.3 μm in diameter.
Membrane Filters
Industrial Filters = 1 mm
Filters for bacteria = 0.25 to 0.45 μm
Filters for viruses = 0.01 μm

14. Low Temperatures
• Slows enzymatic reactions  inhibits microbial growth
• Freezing forms ice crystals that damage microbial cells
• Refrigeration, deep freezing, lyophilization
Other Methods
 High pressure in liquids denatures bacterial proteins and preserves flavor Example ?
 Desiccation prevents metabolism – Example ?
 Osmotic pressure causes plasmolysis – Example ?

15. Radiation
Ionizing Radiation: Shorter Wavelength < 1nm, so more energy
X-rays, Gamma-rays, electron beams  dislodge e- from atoms  production of free radicals and
other highly reactive molecules. eg, Cobalt - 60 radioisotope
The principal effect of ionizing radiation is the ionization of water, which forms highly reactive
hydroxyl radicals. These radicals kill organisms by reacting with organic cellular components,
especially DNA, and damaging them.
Non-ionizing Radiation: Wavelength > 1nm
Ultraviolet (UV) light damages DNA of exposed cells by causing bond formation between adjacent
pyrimidine bases, usually thymines. The thymines-dimers inhibits correct DNA replication.
UV is used to control microbes in the air. A UV “germicidal,” lamp is commonly found in hospital
rooms, nurseries, operating rooms, and cafeterias. UV light is also used to disinfect vaccines and
other medical products.
Disadvantage of UV, not very penetrating, so the organisms to be killed must be directly exposed.
Also damage human eyes, and prolonged exposure can cause burns and skin cancer

16. • Wavelength: 1 mm – 1m
• H2O quickly absorbs energy  release as heat to environment
• Do not have much direct effect on microorganisms
• Moisture-containing foods are heated by microwave action, and the heat
will kill most vegetative pathogens.
• Solid foods heat unevenly because of the uneven distribution of moisture.
Microwave

18. Chemical Methods of Microbial Control
• Few chemical agents achieve sterility.
• Consider presence of organic matter, degree of contact with
microorganisms, and temperature
• Disinfectants regulated by EPA, Antiseptics regulated by FDA
Use-dilution test
1. Metal rings dipped in test bacteria are dried.
2. Dried cultures of S. aureus and P. aeruginosa are placed in
disinfectant for 10 min at 20C.
3. Rings are transferred to culture media to determine whether
bacteria survived treatment.

19. Types of Disinfectants
• Phenol = carbolic acid
(historic importance)
• Phenolics:
Cresols (Lysol) - disinfectant
• Bisphenols
• Hexachlorophene
(pHisoHex, prescription), hospitals, surgeries, nurseries
• Triclosan (toothpaste, antibacerial soaps, etc.)
Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls
Remain active in presence of organic compounds

20. Chlorine
• Oxidizing agent
• Widely used as disinfectant
• Forms bleach (hypochlorous acid) when added to water.
• Broad spectrum, not sporicidal (pools, drinking water)
Iodine
More reactive, more germicidal. Alters protein synthesis and membranes.
Tincture of iodine (solution with alcohol)  wound antiseptic
Iodophors combined with an organic molecule  iodine detergentcomplex
(e.g. Betadine®).
Halogens

21. • Ethyl (60 – 80% solutions) and isopropyl alcohol
Denature proteins, dissolve lipids
• No activity against spores and poorly effective
against viruses and fungi
• Easily inactivated by organic debris also used in
hand sanitizers and cosmetics
Alcohols

22. • Soaps and Detergents
• Major purpose of soap: Mechanical removal and use as wetting agent
• Definition of detergents
Acidic-Anionic detergents Anion reacts with plasma membrane.
Nontoxic, non-corrosive, and fast acting. Laundry soap, dairy industry.
Cationic detergents  Quarternary ammonium compounds (Quats).
Strongly bactericidal against wide range, denature proteins, disrupt plasma
membrane esp. Gram+ bacteria
Surface Acting Ingredients / Surfactants
Soap Degerming
Acid-anionic detergents Sanitizing
Quarternary ammonium compounds
(cationic detergents)
Strongly bactericidal,

23. Aldehydes (alkylating agents)
• Inactivate proteins by cross-linking with
functional groups: (–NH2, –OH, –COOH, –SH)
• Glutaraldehyde: Sterilant for delicate surgical
instruments (Kills S. aureus in 5, M. tb in 10 min)
• Formaldehyde: Virus inactivation for vaccines
Aldehydes and Chemical Sterilants

24. Plasma
• Luminous gas with free radicals that destroy microbes
• Use: Tubular instruments, hands, etc.

25. Hydrogen Peroxide: Oxidizing agent
Inactivated by catalase  Not good for open wounds
Good for inanimate objects; packaging for food industry
(containers etc.)
3% solution (higher conc. available)
Esp. effective against anaerobic bacteria: Effervescent
action, may be useful for wound cleansing through removal
of tissue debris