1. Control of Microbial growth
Dr. Hala Al Daghistani

2. Terminology
Sepsis: Characterized by the presence of pathogenic microbes in
living tissues or associated fluids.
Asepsis: absence of significant contamination.
Aseptic surgery techniques prevent microbial contamination of wounds.
Antimicrobial chemicals, expected to destroy pathogens but not to achieve sterilization
Disinfectant: used on objects (reduce the number of viable microorganisms)
Antiseptic: used on living tissue, destroys or inhibits the growth of microorganisms
Nosocomial Infection(Hospital Acquired Infection) an infection that is contracted from the environment or staff of a healthcare facility.

3. Bacteriostatic: Inhibits bacterial reproduction
Sterilization: A defined process used to render a surface or product free from viable organisms, including bacterial spores.
Biocide: A chemical or physical agent, usually broad spectrum, that inactivates microorganisms.
Chemical biocides include hydrogen peroxide, alcohols, bleach, cycloheximide, and phenols
physical biocides include heat and radiation.
Fungicide, Sporicide, Germicide
Sanitization: reduces microbial numbers to safe levels (e.g.: eating utensils)
Bacteriostatic: Inhibits bacterial reproduction
Bactericidal: Kills bacteria

4. Preservation: The prevention of multiplication of M. O
Preservation: The prevention of multiplication of M.O. in formulated products, including pharmaceuticals and foods.
Antibiotics: Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations.

5. Effectiveness of Antimicrobial Treatment Depends on
Time it takes to kill a microbial population is proportional to number of microbes.
Microbial species and life cycle phases (e.g.: endospores) have different susceptibilities to physical and chemical controls.
Organic matter may interfere with heat treatments and chemical control agents.
Exposure time: Longer exposure to lower heat produces same effect as shorter time at higher heat.

6. Actions of Microbial Control Agents
Disruption of the Cell Membrane or cell Wall
Damage to proteins (disruption of the tertiary structure
of a protein or protein denaturation)
Damage to nucleic acids (include ionizing radiations, ultraviolet light, and DNA-reactive chemicals( e.g. alkylating agents and other compounds that react covalently with purine and pyrimidine bases). Ultraviolet light, induces cross-linking between adjacent pyrimidines on one or the other of the two polynucleotide strands, forming pyrimidine dimers
The susceptibility of the plasma membrane is due to its lipid and protein components.
2. Certain chemical control agents damage the plasma membrane by altering its permeability.
Some microbial control agents damage cellular proteins by breaking hydrogen bonds and covalent bonds.
4. Other agents interfere with DNA and RNA replication and protein synthesis.

7. Disruption of Free Sulfhydryl Groups
Enzymes and coenzyme containing cysteine have side chains terminating in sulfhydryl groups. Such enzymes and coenzymes cannot function unless the sulfhydryl groups remain free and reduced. Oxidizing agents and heavy metals do widespread damage.
Chemical Antagonism
The interference by a chemical agent with the normal reaction between a specific enzyme and its substrate is known as chemical antagonism.
The antagonist acts by combining with some part of the holoenzyme (the protein apoenzyme, the mineral activator, or the coenzyme), thereby preventing attachment of the normal substrate.

8. (e.g. carbon monoxide and cyanide combine with the iron atom in heme-containing enzymes and prevent their function in respiration).

9. Physical Methods of Microbial Control
Heat is very effective (fast and cheap).
Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min.
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 given T.
Heat is frequently used to kill microorganisms.
2. Moist heat kills microbes by denaturing enzymes.

10. A temperature of 100°C will kill all but not spore forms of bacteria within 2–3 minutes in laboratory-scale cultures.
a temperature of 121°C, pressure of 15 lb/sq inches for 15 minutes is used to kill spores. Steam is generally used, both because bacteria are more quickly killed when moist and because steam provides a means for distributing heat to all parts of the sterilizing vessel.

11. For sterilizing materials that must remain dry, circulating hot air electric ovens are available. because heat is less effective on dry material, it is
customary to apply a temperature of 160–170°C for 1 hour or more.
Under these conditions ( excessive temperatures
applied for long periods of time), heat acts by denaturing cell proteins and nucleic acids and by disrupting cell membranes.

12. Moist Heat Sterilization
Denatures proteins
Autoclave: Steam under pressure, Most dependable sterilization method
Steam must directly contact material to be sterilized.
All microorganisms even spore forming bacteria are killed at 121.5C for 15 min.
Prion destruction: 132C for 4.5 hours
Cl. Perfringens and botulinum (food poisoning) survive hours of boiling. Also very resistant Large numbers of hepatitis A virus, fungal spores and protozoal cysts.

13. Pasteurization
Significant number reduction (esp. spoilage and pathogenic organisms)  does not sterilize!
Historical goal: destruction of M. tuberculosis
Classic holding method: 63C for 30 min
Flash pasteurization (HTST): 72C for 15 sec.
-Most common method. - Thermoduric organisms survive
Ultra High Temperature (UHT): 140C for < 1 sec. Technically not pasteurization because it sterilizes.
UHT-pasteurized milk that is packaged aseptically results in a "shelf stable" product that does not require refrigeration until opened

14. Dry heat sterilization kills by oxidation
Flaming of loop
Incineration of carcasses
Anthrax
Foot and mouth disease
Bird flu
Hot-air sterilization
Hot air sterilization: Glassware is heated for 2-3 hours at 320º-360ºF (160º-180ºC)
Also used for powder, water free oils
Advantage: no dulling and corrosion
Hot-air
Autoclave
Equivalent treatments
170˚C, 2 hr
121˚C, 15 min

15. Filtration
Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 m
Membrane filters for fluids.
Pore size for bacteria: 0.2 – 0.4 m
Pore size for viruses: m
Fig 7.4

16. Various Other Methods Low Temperature
Slows enzymatic reactions  inhibits microbial growth
Freezing forms ice crystals that damage microbial cells
lyophilization
Various Other Methods
High pressure in liquids denatures bacterial proteins and preserves flavor
Desiccation prevents metabolism
Osmotic pressure causes plasmolysis

17. Ionizing Radiation
X-rays, -rays, electron beams  production of free radicals and other highly reactive molecules
Commonly used Cobalt-60 radioisotope
Salmonella and Pseudomonas are particularly sensitive
Sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material

18. Nonionizing Radiation: UV light
Most effective wave legnth ~ 260 nm
Effect: thymine dimers
Actively dividing organisms are more sensitive because thymine dimers cause ?
Used to limit air and surface contamination. Use at close range to directly exposed microorganisms
E.g.: germicidal lamps in our lab

19. Chemical Methods of Microbial Control
Few chemical agents achieve sterility.
Consider presence of organic matter, degree of contact with microorganisms, and temperature

20. Disk-diffusion Method
Disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a zone of inhibition indicates effectiveness.
Fig 7.6

21. Types of Disinfectants
Phenol = carbolic acid (historic importance)
Phenolics: Cresols (Lysol) - disinfectant
Bisphenols
Hexachlorophene (pHisoHex, prescription), hospitals, surgeries, nurseries
Triclosan (toothpaste, antibacerial soaps, etc.)
Fig 7.7
Pseudomonas very resistant to triclosan
Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls (??)
Remain active in presence of organic compoundsP

22. Halogens Chlorine Iodine 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 detergent complex (e.g. Betadine®). Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity.

23. Alcohols 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
Table 7.6

24. Heavy Metals
Oligodynamic action: toxic effect due to metal ions combining with sulfhydryl (—SH) and other groups  proteins are denatured.
Mercury (HgCl2, Greeks & Romans for skin lesions); Thimerosal
Copper against chlorophyll containing organisms  Algicides
Silver (AgNO3): Antiseptic for eyes of newborns
Zinc (ZnCl2) in mouthwashes, ZnO in antifungal in paint
The oligodynamic effect (Greek oligos = few, dynamis = force) was discovered in 1893 by the Swiss KW Nägeli as a toxic effect of metal-ions on living cells, algae, molds, spores, fungus, virus, prokaryotic and eukaryotic microorganisms, even in relatively low concentrations. This antimicrobial effect is shown by ions of: mercury, silver, copper, iron, lead, zinc, bismuth, gold, aluminium and other metals.

25. Surface Acting Ingredients / Surfactants
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 against wide range, but esp. Gram+ bacteria
Soap
Degerming
Acid-anionic detergents
Sanitizing
Quarternary ammonium compounds (cationic detergents)
Strongly bactericidal, denature proteins, disrupt plasma membrane

26. Chemical Food Preservatives
Sulfur dioxide
wine
Organic acids
Inhibit metabolism
Sorbic acid, benzoic acid, and calcium propionate
Control molds and bacteria in foods and cosmetics
Sodium nitrate and nitrite prevents endospore germination. In meats. Conversion to nitrosamine (carcinogenic)

27. Aldehydes and Chemical Sterilants
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. tuberculosis in 10 min)
Formaldehyde: Virus inactivation for vaccines
Chemical Sterilants for heat sensive material
Denature proteins
Ethylene oxide

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

29. 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 (e.g.:
Effervescent action, may be useful for wound cleansing through removal of tissue debris

30. Microbial Characteristics and Microbial Control
Fig 7.11
the end