Chapter 7

The Control of Microbial Growth

SLOs

Define sterilization, disinfection, antisepsis, sanitization, biocide, germicide, bacteriostasis, and asepsis.

Describe the microbial death curve.

Describe the effects of microbial control agents on cellular structures.

Compare effectiveness of moist heat (autoclaving, pasteurization) vs .dry heat.

Describe how filtration, low temperature, high pressure, desiccation, and osmotic pressure suppress microbial growth.

Explain how radiation kills cells.

List the factors related to effective disinfection.

Interpret results the disk-diffusion test.

Identify some methods of action and preferred uses of chemical disinfectant.

Differentiate between two halogens used as antiseptics and disinfectants.

List the advantage of glutaraldehyde and ethylene oxide over other chemical disinfectants.

Identify the method of sterilizing plastic labware.

Explain how microbial control is affected by the type of microbe.

How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria?

Would a chemical microbial control agent that affected plasma membranes affect humans?

How is microbial growth in canned foods prevented?

What is the connection between the killing effect of radiation and hydroxyl radical forms of oxygen?

If you wanted to disinfect a surface contaminated by vomit and a surface contaminated by a sneeze, why would your choice of disinfectant make a difference?

Why is alcohol effective against some viruses and not others?

Is Betadine an antiseptic or a disinfectant when it is used on skin?

What chemicals are used to sterilize?

The presence or absence of endospores has an obvious effect on microbial control, but why are gram-negative bacteria more resistant to chemical biocides than gram-positive bacteria?

SLOs cont.: Check Your Understanding

Terminology

Sepsis: microbial contamination.

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

Antiseptic: used on living tissue

Nosocomial

see

. . . More Terminology

Sterilization: Removal of all microbial life (heat, filtration)

For food: Commercial sterilization to kill C. botulinum endospores

Sanitization: reduces microbial numbers to safe levels (e.g.: eating utensils)

Bacteriostatic: Inhibits bacterial reproduction

Bactericidal: Kills bacteria

Fungicide, sporicide, germicide, biocide

Single most effective measure!

Remember Semmelweis,

Pasteur, and Lister from

Ch 1

Rate of Microbial Death

Microbial Death Curve, plotted logarithmically, shows this constant death rate as a straight line.

Bacterial populations subjected to heat or antimicrobial chemicals die at a constant rate.

Rate: 90% / min

Foundation Fig 7.1

How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria?

Foundation Fig 7.1 cont.

Effectiveness of Antimicrobial Treatment

Time it takes to kill a microbial population is to number of microbes.

Different microbial species and life cycle phases (e.g.:_____________) have different susceptibilities to physical and chemical controls.

Organic matter may interfere.

Temperature determines exposure time: Longer exposure to lower heat produces same effect as shorter time at higher heat.

Actions of Microbial Control Agents

Alteration of membrane permeability

Damage to proteins

Damage to nucleic acids

Check your understanding:

Would a chemical microbial agent that affects

plasma membranes affect humans?

Moist Heat Sterilization

___________proteins

Autoclave: Steam under pressure

Most dependable sterilization method

Steam must directly contact material to be sterilized.

Pressurized steam reaches higher temperatures.

Normal autoclave conditions: _____C for ___ min.

Prion destruction: 132C for 4.5 hours

Limitations of the autoclave Fig 7.2

Physical Methods of Microbial Control

Pasteurization

Significant number reduction (esp. spoilage and pathogenic organisms) does not sterilize!

Historical goal: destruction of M. tuberculosis

Classic holding method: 63C for 30 min

Flash pasteurization (HTST): 72C for 15 sec. Most common in US. Thermoduric organisms survive

Ultra High Temperature (UHT): 140C for < 1 sec. Technically not pasteurization because it sterilizes.

Hot-air Autoclave

Equivalent treatments

170˚C, 2 hr 121˚C, 15 min

Dry Heat Sterilization Kills by Oxidation

Flaming of loop

Incineration of carcasses Anthrax

Foot and mouth disease

Bird flu

Hot-air sterilization

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: 0.01 m

Compare to Fig 7.4

Low Temperature

Slows enzymatic reactions inhibits microbial growth Refrigeration (watch out for _______________!

Freezing forms ice crystals that damage microbial cells

Deep freezing and lyophilization

Various Other Methods

Desiccation prevents metabolism

Osmotic pressure causes plasmolysis

Ionizing Radiation

X-rays, -rays have short wave length dislodge e- from atoms production of free radicals and other highly reactive molecules

Used for sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material

Also as “cold pasteurization” of food Consumer fears!?

Effect: thymine dimers

Actively dividing organisms are more sensitive because thymine dimers cause ______________?

Used to fight air and surface contamination. Only kills at close range and directly exposed microbial agents

E.g.: germicidal lamps in OR, cafeteria, and our lab ??

Nonionizing Radiation: UV light

Heats H2O

Indirectly kills bacteria. How ?

Solid food heats unevenly. Why?

Nonionizing Radiation: Microwave

Fig 7.5

Few chemical agents achieve sterility.

Disinfectants regulated by EPA

Antiseptics regulated by FDA

Evaluating Disinfectants:

Use-dilution test

Disk-diffusion method

Chemical Methods of Microbial Control

Fig 7.6

Types of Antibacterial Chemicals

Phenol = carbolic acid (historic importance)

Who used first?

Many derivatives today:

Phenolics, e.g.: Lysol

Bisphenols, e.g.:

Hexachlorophene (in pHisoHex used in hospitals)

Triclosan (toothpaste, antibacerial soaps, etc.)

Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls (??)

Remain active in presence of organic compounds

Fig 7.7

Halogens

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: Iodine plus organic molecule. E.g.: complexed with detergent: Betadine®. Occasional skin sensitivity.

Cl I

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

Heavy Metals

Oligodynamic action: toxic effect due to metal ions combining with sulfhydryl (—SH) and other functinal groups proteins are denatured.

Silver (1% AgNO3): Antiseptic for eyes of newborns

Copper against chlorophyll containing organisms Algicides; also X-gel hand sanitizer

Zinc (ZnCl2) in mouthwashes, ZnO as antifungal in paint

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, but esp. Gram+ bacteria

Surface Acting Ingredients / Surfactants

Soap Degerming

Acid-anionic detergents Sanitizing

Quarternary ammonium compounds (cationic detergents)

Strongly bactericidal, denature proteins, disrupt plasma membrane

Sodium nitrate and nitrite prevent endospore Prevents ES germination. Used in meats. Conversion to nitrosamines: Carcinogenic!

Organic acids Inhibit metabolism E.g.: Sorbic acid, benzoic acid, etc. In foods and cosmetics

Sulfur dioxide wine

Chemical Food Preservatives

Aldehydes (alkylating agents)

Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH)

Formaldehyde: Embalming Formalin

Virus inactivation for vaccines

Glutaraldehyde: Liquid Sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tuberculosis in 10 min, ES in 3 – 10h)

Ethylene oxide: Gaseous Sterilant

Aldehydes and Chemical Sterilants

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)

Especially effective against anaerobic bacteria (e.g.:

Effervescent action, may be useful for wound cleansing through removal of tissue debris

Microbial Characteristics and Microbial Control

Fig 7.11