Chapter 9 Control of Microbial Growth

• Ignaz Semmelweis and Joseph Lister developed the first microbial control methods.– Mid 1800s

• Before aseptic surgical techniques ~10% of all deaths were due to nosocomial infections

• Delivering mothers as high as 25% death rate• During the American Civil War, a surgeon may

have cleaned his scalpel by wiping it on his boot sole between incisions.

• Sepsis refers to microbial contamination• Asepsis is the absence of significant

contamination• Aseptic surgery techniques prevent microbial

contamination of wounds

The Terminology of Microbial Control

The Terminology of Microbial Control

• Sterilization: removing all microbial life– Prions are highly resistant to all forms of

sterilization– The definition of sterilization implies the absence

of prions.• Disinfection: removing pathogens

– Surfaces or substances• Antisepsis: removing pathogens from living

tissue

Effectiveness of Microbial Treatment

• Depends on:– Number of microbes

• The more you start with the longer it takes to eliminate

– Environment • Organic matter (vomit or blood) inhibit certain chemicals• Biofilms cells are protected by polysaccharide matrix• Temperature disinfectants work better under warm conditions

– Time of exposure• Chemical often requires extended exposure to microbes

– Spray, wipe, re-spray

– Microbial characteristics• Endospores and Gram negatives are more resistant

Microbial Characteristics and Microbial Control

• Gram negative more resistant than Gram positive– Pseudomonas highly resistant to biocides

• Outer membrane provides protection • Porins are highly selective of molecules that they permit

into the cell

• Mycobacterium tuberculosis– Mycolic acid– Waxy lipid component in cell wall resists effect of

chemicals

Microbial Characteristics and Microbial Control

• Prions: infectious proteins known to be heat and protease resistant– Autoclave in sodium hydroxide to destroy– Or incineration

Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.

Effectiveness of Microbial Treatment

• Bacterial populations when treated with heat or chemicals die at a constant rate.

Table 7.2 Microbial Exponential Death Rate: An Example

Actions of Microbial Control Agents

• Alteration of membrane permeability– Damage results in cell contents to leak from cell

• Damage to proteins– Little bags of enzymes- enzymes are folded

proteins unfolding due to chemical treatments makes enzymes not function

• Damage to nucleic acids– Heat, radiation, or chemicals are lethal to cells

Physical Methods of Microbial Control

Temperature: Heat Sterilization

• Moist heat denatures proteins• Heat preserved canned foods, most common

method of food preservation• Autoclave: steam under pressure

– 15 pounds of pressure per square inch – 121 degrees C– Kills all organisms and endospores but not prions!– Use: glassware, media, intravenous equipment….

Examples of sterilization indicators. Why do we need to use sterility indicators?

Figure 9.7 Sterility indicators

Cap that allowssteam to penetrate

Flexible plasticvial

Crushable glassampule

Nutrient mediumcontaining pHcolor indicator

Endospore strip

Incubation

After autoclaving, flexiblevial is squeezed to breakampule and releasemedium onto spore strip.

Yellow mediummeans spores areviable; autoclavedobjects are notsterile.

Red mediummeans spores werekilled; autoclavedobjects aresterile.

Bacillus stearothermophilus

• Kills by oxidation– Dry heat– Flaming– Incineration– Hot-air sterilization

Hot-Air Autoclave

Equivalent Treatments 170˚C, 2 hr 121˚C, 15 min

Temperature: Dry Heat Sterilization

Temperature: Pasteurization

• Reduces spoilage organisms and pathogens– High-temperature short-time: 72°C for 15 sec

• Milk

– Ultra-high-temperature: 140°C for <1 sec• Organic Milks are commonly pasteurized with this

method• Coffee creamers

Temperature: Low

• Low temperature inhibits microbial growth– Refrigeration– Deep-freezing

• Sub optimal growth temperatures

Filtration

• HEPA removes microbes >0.3 µm =300 nm• Membrane filtration (liquids) removes

microbes >0.22 µm• Viruses 5-300nm• Influenza virus: ~ 125-150 nm in diameter• Rhinovirus common cold: 18-28 nm in

diameter

Figure 7.4 Filter sterilization with a disposable, presterilized plastic unit.

Flask ofsample

Cap

Membrane filter

Cotton plug invacuum lineensures sterility

Vacuum lineSterilefiltrate

Radiation• Ionizing radiation (X rays, gamma rays)

– Damages DNA– Low level radiation approved for use in US on spices,

meats, and vegetables– Disposable dental and medical supplies such as plastic

syringes and surgical gloves– Postal service uses electron beams to sterilize certain

classes of mail• Nonionizing radiation (Ultra Violet)

– Damages DNA– Does not penetrate only good for surface applications

• Microwaves kill by heat; not especially antimicrobial

Figure 9.11 Irradiated and non-irradiated food

Non-irradiated Irradiated

Chemical methods of microbial control

Chemical Disinfection

• No single disinfectant is appropriate for all circumstances!

• See Lab 26 for clinically important Disinfectants

• Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA

• The chemical compounds are not to be used to treat disease only for controlling growth and preventing disease

Chemical Methods of Microbial Control

• Antimicrobial Soap: To much of a good thing? Page 281

• Development of Resistant Microbes– Little evidence that products containing antiseptic and

disinfecting chemicals add to human or animal health– Use of such products promotes development of

resistant microbes– Use of antimicrobial soaps should be limited to food

handlers, caring for newborns, and caring for high-risk patients (immunocompromised)

– CDC recommends using regular soap and washing with warm water for at least 10-15 seconds

Chemical Methods of Microbial Control

• Development of Resistant Microbes– Little evidence that products containing

antiseptic and disinfecting chemicals add to human or animal health

– Use of such products promotes development of resistant microbes

• Triclosan

• Triclosan– Disrupt plasma membranes– Active ingredient in antimicrobial soaps,

toothpaste, deodorants – Incorporated into cutting boards and knife handles– Widespread use has resulted in reports of

resistance

Bisphenols