Chapter 7 - Control of Microbial Growth



Chapter 11 – Physical and Chemical Control of Microorganisms

I. Controlling Microorganisms

History – See Insight 11.1

General Considerations

Relative Resistance of Microbial Forms – See Table 11.1

Highest resistance – prions, bacterial endospores

Moderate resistance – naked viruses, protozoan cysts, M. tuberculosis, Pseudomonas, S. aureus

Lowest resistance – most bacteria, enveloped viruses, protozoan trophs, most fungal spores

Terminology and Methods – know definitions and examples

Sterilization - destruction of all forms of microbial life (including endospores)

Microbicidal Agents vs Bacteriostatic Agents

cidal" - "has a killing action" - e.g., a "bactericide" kills bacteria

"static" - "limiting or inhibiting the growth" - "bacteriostatic " agents limit growth, but don't kill

Germicides, Disinfection, Antisepsis

Germicide – any chemical agent that kills pathogenic microorganisms

Disinfection / disinfectant - destruction of vegetative pathogens (non-spore-formers) on inert

surfaces

Sepsis - bacterial contamination; growth of bacteria in blood or other tissues

Asepsis - the absence of bacterial contamination ; any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection

Antisepsis / antiseptic - destruction of vegetative pathogens on living tissue

Methods that reduce the Numbers of Microbes

Degerming - the mechanical removal of most of the microbes in a limited area

Sanitization - treatment that lowers microbial counts to safe public health levels

Microbial Death

Factors that influence the effectiveness of antimicrobial treatments see Fig 11.2

• Time of exposure

• The number of microbes

• Microbial characteristics

-- gram positive vs. gram negative

-- type of organism (bacteria, fungus, protozoa)

-- endospores

-- viruses - with or without envelopes

-- Prions – see box – p.318

• Environmental influences

• Temperature ( usually more effective under warm conditions)

• pH- usually more effective under acid conditions

• Using the proper concentration of the agent or the proper diluting medium

• Presence of organic matter ( blood, vomit, feces, fats)

• The mode of action of the agent

II How do Antimicrobial Agents work – Modes or Mechanisms of Action

1. Effects on the Cell Wall

• Block synthesis, break it down or digest cell wall – cells will lyse

• Antibiotics (penicillins), detergents, alcohols

2. Alter the permeability of the plasma membrane –

• cell contents leak out; damaging substances can enter

• surfactants ( detergents)

3. Damage nucleic acids - DNA or RNA damage - the cell can't replicate or make proteins

• Antibiotics that bind to bacterial ribosome (chloramphenicol)

• Block protein synthesis

• Some bind to DNA – block replication, transcription or translation

• Some are mutagens (chemical or physical agents)

4. Damage proteins - denatured proteins lose shape - cannot function

• Coagulation by moist heat or solvents

III. Physical Means of Microbial Control - HEAT

• Denatures enzymes

• Heat resistance - Microbes vary in their ability to resist heat

• Spores – very resistant – require temperatures above boiling – Table 11.1

• Vegetative bacteria, fungi, viruses – More sensitive – table 11.

Thermal Death Time (TDT)

The shortest length of time required to kill all test microbes at a specified temperature

e.g., 121°C - 30 mins – commercial preparation – prevents botulism ( e.g. canning green beans)

Thermal Death Point (TDP)

The lowest temperature required to kill all microbes in a sample in 10 minutes

Moist Heat vs. Dry Heat

Moist Heat - Uses lower temperature and shorter times than dry heat ; Coagulates protein ( e.g., when you boil an egg) or denatures proteins

Dry Heat – need higher temperatures and longer times ; dehydrates or oxidizes (burns) cells

Common Methods of Moist Heat Control

1. Steam under pressure

2. Pasteurization

3. Boiling water

Steam Under Pressure (Autoclaving) – Fig 11.5

1. temperatures above that of boiling water

2. steam at 15 psi will reach temperature of 1210C

3. Most autoclaving - 1210C ; 15 p.s.i.; 10-40 minutes

4. will kill ALL endospores and organisms

5. conditions - solids -- direct surface contact with steam is needed

can't wrap in aluminum foil ( steam won't penetrate)

liquids - small amounts of aqueous solutions

moisture-proof material ( petroleum jelly, mineral oil) - need special

procedures

6. Sterilization Indicators

pressure / temperature sensitive tapes or labels

paper strips containing bacterial endospores - The BEST way to ensure that

sterilization has occurred

7. Commercial canning & Home canning – kills Clostridium botulinum spores

Pasteurization

1. first done by Pasteur - beer, wine, milk ( wanted to eliminate organisms that cause TB from milk)

2. milk-borne diseases caused by – Salmonella, Campylobacter, Listeria, Brucella, Coxiella, Mycobacterium tuberculosis

3. mild heating

4. eliminates most pathogens; lowers bacterial numbers; Thermodurics ( e.g., lactobacillus, Micrococcus, yeast)

5. doesn't sterilize - product still needs to be refrigerated, but keeps for a longer time

BoilingWater : Disinfection

1. 100°C – 30 min

2. kills most pathogens; endospores and some viruses are not killed by boiling

3. Steam - same as boiling

4. drinking water; utensils, bedding, clothing, etc.

Dry Heat Sterilization

• Needs a higher temperature and longer exposure time than moist heat.

• Dehydrates the cell

• Denatures proteins

• Oxidizes ( burns) – Incineration

• Flaming – Bunsen Burner ( 1870°C)

• Hot-air sterilization ( oven ) - requires higher temperature and more time than autoclaving

• Glassware, metal instruments, powders, oils, gels etc.

IV. Physical Means of Microbial Control - Low Temperatures

Retards activity of most microbes; Some survive – endospores of Clostridium and Bacillus,

Staphylococcus, Listeria, Clostridium, Streptococcus, yeasts, molds, viruses, Salmonella

A. Refrigeration

• Usually bacteriostatic

• Psychrotrophs - will grow at refrigerator temps, but not usually pathogenic

• Exception: Listeria p577-578

B. Freezing

• Rapid freezing - bacteria become dormant, but not killed

• Slow Freezing - ice crystal formation kills bacteria

• Important way to kill parasitic roundworms

• Not routinely reliable

V. Physical Means of Microbial Control - Dessication ( Drying)

A. Organisms cannot grow or reproduce but can remain viable for years

B. Lyophilization – quick freeze combined with dessication

Freeze-dried samples of bacteria are used to maintain laboratory stocks

C. organisms vary in their ability to withstand drying

M. tuberculosis is very resistant to drying

Neisseria gonorrhoeae is very sensitive to drying

Endospores are very resistant to drying ( have survived centuries)

Bacteria can persist in dried mucus, urine, pus, clothing, dust, bedding, wound dressings,

VI. Physical Means of Microbial Control - Osmotic Pressure

A. High concentrations of salt or sugar will preserve foods

B. hypertonic environment - water leaves the bacterial cell

C. salted meats, salted fish, jams, preserves, sugared fruits

D. molds and yeasts (fungi) are more resistant to high salt or high sugar solutions

E. Halophiles - like to grow in high salt concentrations

VII. Physical Means of Microbial Control – Radiation – Fig 11.7

A. Ionizing - gamma rays, X-rays, electron beams ( cathode rays)

• Short wavelength = high energy

• Ejects electrons from atoms – forms ions

• Ionize water - form oxidizing particles that react with and damage DNA, cause chemical changes in cell or produce toxic substances

• Good for materials that are heat or chemical sensitive

• used for foods ( spices, meats, vegetables)

• Protects against E.coli, Salmonella, Trichinella

• used for pharmaceuticals, plastic syringes, surgical gloves

B. Nonionizing radiation

• Longer wavelength than ionizing radiation ( e.g., UV light )

• Damages DNA - causes formation of pyrimidine dimmers- Fig 11.9

• Most effective UV wavelength for killing microorganisms is 260 nm. -- This wavelength is strongly absorbed by DNA

• Germicidal lamps used to disinfect room areas, surfaces ; must be close to the surface

• Good for airborne microbes – can reduce about 99%

• Not penetrating -- UV light can be blocked by plastic, cardboard, paper, cloth

• Object must be directly exposed

• UV light can cause – retinal damage, cancer, burns, wrinkling of skin

• Sunlight contains UV radiation - can damage DNA in cells

• Some bacteria have evolved mechanisms of resistance to UV light - endospores, protective pigments

VIII. Physical Means of Microbial Control – Filtration – Fig 11.11

• filters - liquid passes through, but pores are small enough to retain the bacteria

• used for heat-sensitive materials ( e.g., culture media, enzymes, vaccines, antibiotics, other

Pharmaceuticals, serum, blood products, milk, beer)

• Membrane filters(cellulose acetate) or plastic ( teflon) , nylon, charcoal particles

• HEPA filters - remove almost all microorganisms larger than 0.3 um

• Historically - viruses were discovered as filterable infectious agents - pathogens small enough

to pass through bacterial filters - would cause disease even after the material was filtered

Today there are filters with pores small enough to filter out viruses

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