chapter 7 the control of microbial growth. the terminology of microbial control sterilization:...

Chapter 7

The Control of Microbial Growth

The Terminology of Microbial Control

• Sterilization: Removal of all microbial life• Commercial Sterilization: Killing

Clostridium botulinum endospores• Disinfection: Removal of pathogens• Antisepsis: Removal of pathogens from

living tissue• Degerming: Removal of microbes from a

limited area (mechanical removal)


• Sanitization: Lower microbial counts on eating utensils

• Biocide/Germicide: Kills microbes

• Bacteriostasis: Inhibiting, not killing, microbes

• Sepsis : refers to microbial contamination.

• Asepsis: the absence of significant contamination.

• Aseptic surgery: prevent microbial contamination of wounds.

• Aseptic technique: laboratory techniques used to minimize contamination


• Bacterial populations die at a constant logarithmic rate when heated or treated with antimicrobial chemicals.

Figure 7.1a

The Rate of Microbial Death

• Number of microbes

• Environmental influences (organic matter, temperature, biofilms)

• Time of exposure

• Microbial characteristics

The Rate of Microbial Death

Figure 7.1b

• Alternation of membrane permeability– Damage to the lipids or proteins of the plasma

membrane causes cellular contents to leak and interferes with the growth of the cell

• Damage to proteins– Denaturation of proteins by breaking H-bonds or

covalent bonds (heat or certain chemicals)

• Damage to nucleic acids– Cannot replicate or carry out normal metabolic

functions (heat, radiation, or chemicals)

Actions of Microbial Control Agents

• Need to consider effects on other things besides the microbes (e.g. vitamins, antibiotics, budget)

• Heat– Usually used to sterilize laboratory media and

glassware, and hospital instruments– Kill microbes by denaturing their enzymes– Heat resistance varies among different microbes– Need to consider suspending medium

• presence of fats and proteins protects microbes; acidic condition more effective for heat sterilization

Physical Methods of Microbial Control

Physical Methods of Microbial Control: Heat

– Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min.

– Thermal death time (TDT): Time required to kill all cells in a culture

– Decimal reduction time (DRT or D value): Minutes to kill 90% of a population at a given temperature

• Related to bacterial heat resistance

Heat Sterilization: moist heat

• Moist heat denatures (coagulates) proteins– Break H-bonds

• Boiling– Kills vegetative forms of bacterial pathogens;

almost all viruses, fungi and fungal spores

• More reliable form of sterilization with moist heat requires higher temperature (>100 oC) e.g. Autoclave– Need to consider if heat or moisture can

damage the material

• Autoclave: Steam under pressure– 121 oC, 20 psi,

15 min. at sea

level


Figure 7.2


• Autoclave– Need direct contact with steam or contained in

a small volume of aqueous solution– Used for culture media, instruments, dressings,

intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items (have to be able to withstand high temperatures and pressure)

– Solid materials and large containers requires extra time

• Pasteurization reduces spoilage organisms and pathogens– Used for milk, cream, and certain alcoholic beverages– Lowers microbial numbers, but thermoduric (heat

resistant) bacteria survive• Thermoduric bacteria are non-pathogenic or do not cause

spoilage

• Equivalent treatments– 63°C for 30 min– High-temperature short-time 72°C for 15 sec– Ultra-high-temperature: 140°C for <1 sec

Heat sterilization: Pasteurization

• Dry Heat Sterilization kills by oxidation– Flaming: sterilize inoculating loops– Incineration: sterilize and dispose contaminated

paper cups, bags, and dressings – Hot-air sterilization: sterilize glassware, instruments,

needles, and glass syringes

Heat sterilization: Dry heat sterilization

121˚C, 15 min170˚C, 2 hrEquivalent treatments

AutoclaveHot-air

• Filtration removes microbes– Virus passes through most bacteriological membrane

filters (0.22 μm to 0.45 μm pore diameter)– Sterilize heat-sensitive materials (culture media,

enzymes, vaccines, and antibiotic solutions)– High-efficiency particulate air (HEPA) filters to

remove all microbes larger than 0.3 μm in diameter



• Low temperature inhibits microbial growth– Decrease chemical reactions and possible

changes in proteins– Used for food, drug, and culture preservation– Refrigeration: bacteriostatic effect– Deep freezing: preserve bacterial cultures

(quick-frozen between -50oC to -95 oC)– Lyophilization: long-term preservation of

microbial cultures


• High pressure alter molecular structure of proteins and carbohydrates– Rapid inactivation of vegetative bacterial cells– Used for fruit juices (preserve flavors, colors,

and nutrient values of the products)

• Desiccation prevents metabolism– Used for food preservation– Microbes can remain viable for years


• Osmotic pressure causes plasmolysis– Used for food preservation– Resembles preservation by desiccation (deny

moisture needed for growth to microbial cells)

• Radiation– Effects varies depending on its wavelength,

intensity, and duration

Figure 7.5

Radiation

– Ionizing radiation (X rays, gamma rays, electron beams): cause ionization of water to form highly reactive hydroxyl radicals that react with DNA

• Used for sterilizing pharmaceuticals and disposable dental and medical supplies

– Nonionizing radiation (UV): damages DNA by forming thymine dimers to inhibit correct replication of the DNA during reproduction

• Used for disinfecting vaccines and other medical products; to control microbes in the air

– (Microwaves kill by heat; not especially antimicrobial)


• Control the growth of microbes on both living tissues and inanimate objects

• Most of them reduce microbial populations to safe levels or remove vegetative forms of pathogens from objects.

• No single disinfectant or antiseptic is appropriate for all situation

• Principles of effective disinfection– Concentration of disinfectant– Presence of organic matter– pH of the medium– Time of exposure

Chemical Methods of Microbial Control

• Evaluating a disinfectant– Use-dilution test

1. Metal rings dipped in test bacteria are dried

2. Dried cultures placed in disinfectant for 10 min at 20°C

3. Rings transferred to culture media to determine whether bacteria survived

treatment

• Effectiveness determined by the number of cultures that grow



Figure 7.6

• Evaluating a disinfectant– Disk-diffusion method (in teaching laboratories)

Types of Disinfectants

Figure 7.7

• Phenol– Used by Lister to

control surgical infections; rarely used now

– Cause skin irritation and disagreeable odor

– Phenolics & bisphenols are derivative of phenol


• Phenolics (e.g. Lysol, O-phenylphenol)– Used for disinfecting pus, saliva, and feces– Derivative of phenol; reduced skin irritation

with increased antibacterial activity– Injure lipid-containing plasma membranes

(cellular contents leak out); works on mycobacteria

– Active even in the presence of organic compounds, stable, and persist for long periods after application

• Bisphenols (e.g. Hexachlorophene, Triclosan)– Hexachlorophene (pHisoHex) used for surgical

and hospital microbial control procedures (esp. staphylococci and streptococci)

– Triclosan used in antibacterial soaps & many other products; works well against gram-positive and gram-negative bacteria as well as fungi

– Derivative of phenol – Disrupt plasma membranes



• Biguanides (e.g. Chlorhexidine)– Used for microbial control on skin and mucous

membranes; for surgical hand scrubs and preoperative skin preparations in patients

– Injure plasma membranes– Strong affinity for binding to the skin &

mucous membranes– Biocidal against most vegetative bacteria,

fungi, and certain enveloped virus

• Halogens (e.g. Iodine, Chlorine)– Oxidizing agents; effective antimicrobial agents– Iodine, one of the oldest and most effective

antiseptics; used mainly for skin disinfection and would treatment; or treat water (iodine tablet)

• Effective against all kinds of bacteria, many endospores, various fungi, and some viruses

• Available as a tincture and iodophor (e.g. Betadine and Isodine)



– Bleach is hypochlorous acid (HOCl) formed by chlorine + water

• Liquid form of compressed chlorine gas used for disinfecting municipal drinking water, water in swimming pool, & sewage

• Chlorine compounds sued to disinfect dairy equipment, eating utensils, household items, and glassware

• Gaseous form of chlorine, chlorine dioxide used for area disinfection to kill endospores of anthrax bacteria


Table 7.6

• Alcohols (e.g. Ethanol & isopropanol)– Used for swabbing skin and the surface of

inanimate objects– Effectively kill bacteria, fungi, and some

enveloped virus– Denature proteins, dissolve lipids– Leave no residue (act on a surface and then

evaporates rapidly)– Optimum concentration of ethanol = 70%, but

effective between 60 – 95%

• Heavy Metals and Their compounds. (e.g. sliver, mercury, and copper)– Oligodynamic action: the ability of small

amounts of a heavy metal compound to exert antimicrobial activity

– Denature proteins (metal ions combine with the sulfhydryl groups on cellular protein denature)

– Silver nitrate used to prevent gonorrheal ophthalmia neonatorum



• Mercuric chloride is bacteriostatic; use is limited; control mildew in paints

• Copper sulfate sued chiefly to destroy green algae that grow in reservoirs, stock ponds, swimming pools, and fish tanks

• Zinc chloride: common ingredient in mouth washes

• Surface-Active Agents (Surfactants)– Decrease surface tension among molecules of a liquid


Bactericidal (gram-positive), fungicidal, amoebicidal, and virucidal (enveloped virus); Denature proteins, disrupt plasma membrane

Quaternary ammonium compoundsCationic detergents

Sanitizing (food industry, dairy utensils and equipment); anion reacts with plasma membrane; effective on thermoduric bacteria

Acid-anionic detergents

Degerming; mechanical removal of microbes by scrubbing

Soap

• Chemical Food Preservatives– Organic Acids or salts of organic acids

• Inhibit metabolism

• Sorbic acid, benzoic acid, calcium propionate

• Control molds and bacteria in foods and cosmetics

– Nitrite prevents endospore germination & preserve the red color of the meat

– Antibiotics (not for internal use)• Nisin (for endospore-forming spoilage bacteria) and

natamycin (antifungal antibiotic) prevent spoilage of cheese


• Aldehydes (e.g. Glutaraldehyde & formaldehyde)– Very effective antimicrobials– Inactivate proteins by cross-linking with

functional groups (–NH2, –OH, –COOH, —SH)

– Glutaraldehyde used to disinfect hospital instruments including respiratory-therapy equipment

• 2% solution bactericidal, tuberculocidal, and virucidal (10 min.)



– Formaldehyde (formalin) used to preserve biological specimens and inactivate bacteria and viruses in vaccines

• Gaseous Chemosterilizers (e.g. ethylene oxide)– Chemicals that sterilize in a closed chamber– Denature proteins– Highly penetrating; widely used on medical

supplies and equipment; suspected carcinogens• Propylene oxide and beta-propiolactone can also be

used for sterilization

• Peroxygens (e.g. O3, H2O2, peracetic acid)

– Oxidizing cellular components

– Ozone (O3) often used to supplement chlorine in the disinfection of water (neutralize taste and odor)

– H2O2: effective disinfectant on inanimate objects; but not good for open wounds (may slow healing); neutralized by catalase (present in human cells)



– Benzoyl peroxide: useful for treating wounds infected by anaerobic pathogen; medications for acne

– Peracetic acid: one of the most effective liquid chemical sporicides available; considered a sterilant; effective on endospores and viruses

• Also used in disinfection of food-processing and medical equipment

Microbial Characteristics and Microbial Control

Figure 7.11

• Resistance due to: – Structural component

• External LPS layer of gram-negative (porin)

• Cell wall structure of mycobacteria

• Endospore (bacterial)• Protozoan cysts and

oocysts• Non-enveloped virus

– No effective means to destroy (e.g. prions)

Microbial Characteristics and Microbial Control

GoodFairGlutaraldehyde

GoodPoorAlcohols

FairFairChlorines

NoneNoneQuats

GoodPoorPhenolics

MycobacteriaEndospores

Effectiveness againstChemical agent

chapter 7 the control of microbial growth. the terminology of microbial control sterilization:...

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