Chapter 7

Control of Microbial Growth

• Boiling kills microbes by coagulating the proteins – endospores are not destroyed.

• Autoclaving - Temperature 121oC, 15 PSI, 15 min – even the endospores are destroyed.

• Dry heat – direct flaming is used to sterilize the inoculating loop.

• Refrigeration – slows down the growth of microbes. It does not kill the microbes.

• Osmotic environment – high concentration of salt – plasmolysis.

• UV light – mutation – inducing the formation of thymine dimers.

• Iodine - inactivates enzymes – skin infection, wound infection.

• 1% silver nitrate – ophthalmea neonatorum – eye infection of the newborn.

• Gas sterilization – ethylene oxide – mattresses, petri plates.

• Quaternary ammonium compounds – damage to the plasma membrane – mouthwash.

• Sodium nitrite – preserve meat products – prevents the germination of botulism endospores – used to preserve hot dogs.

• 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

• Commercial sterilization: Killing C. botulinum endospores

• Disinfection: Removing pathogens

• Antisepsis: Removing pathogens from living tissue

The Terminology of Microbial Control

• Degerming: Removing microbes from a limited area

• Sanitization: Lowering microbial counts on eating utensils

• Biocide/germicide: Kills microbes

• Bacteriostasis: Inhibiting, not killing, microbes

• The usual definition of sterilization is the removal or destruction of all forms of microbial life;

Effectiveness of Treatment

• Depends on– Number of microbes– Environment (organic matter, temperature,

biofilms)– Time of exposure– Microbial characteristics

Actions of Microbial Control Agents

• Alteration of membrane permeability

• Damage to proteins

• Damage to nucleic acids

Moist Heat Sterilization

• Moist heat denatures proteins

• Autoclave: Steam under pressure

Figure 7.2

An Autoclave

Pasteurization

• Reduces spoilage organisms and pathogens

• Equivalent treatments– 63°C for 30 min

Hot-Air Autoclave

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

Dry Heat Sterilization

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

Figure 7.4

Filtration

• Membrane filtration removes microbes >0.22 µm

Physical Methods of Microbial Control

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

• High pressure denatures proteins

• Desiccation prevents metabolism

• Osmotic pressure causes plasmolysis

Figure 7.5

Radiation

Principles of Effective Disinfection

• Concentration of disinfectant

• Organic matter

• pH

• Time

Figure 7.7c, d

Bisphenols

• Hexacholorphene, triclosan– Disrupt plasma membranes

Biguanides

• Chlorhexidine– Disrupt plasma membranes

Halogens

• Iodine– Tinctures: In aqueous alcohol– Iodophors: In organic molecules– Alter protein synthesis and membranes

• Chlorine– Bleach: Hypochlorous acid (HOCl)– Chloramine: Chlorine + ammonia– Oxidizing agents

Heavy Metals

• Ag, Hg, and Cu– Silver nitrate may be used to prevent

gonorrheal ophthalmia neonatorum– Silver sulfadiazine used as a topical cream on

burns– Copper sulfate is an algicide

• Oligodynamic action– Denature proteins

Chemical Food Preservatives

• Organic acids– Inhibit metabolism– Sorbic acid, benzoic acid, and calcium

propionate– Control molds and bacteria in foods and

cosmetics

• Nitrite prevents endospore germination

• Antibiotics– Nisin prevent spoilage of cheese

Gaseous Sterilants

• Denature proteins

• Use: Heat-sensitive material– Ethylene oxide

Chapter 18

Practical Applications of Immunology

History of Vaccines

• Variolation: Inoculation of smallpox into skin

(18th century)

• Vaccination: – Inoculation of cowpox virus into skin (Jenner) -

1790

Types of Vaccines

• Attenuated whole-agent vaccines– MMR

• Inactivated whole-agent vaccines Salk polio

• Toxoids– Tetanus

Types of Vaccines

• Subunit vaccines– Acellular pertussis– Recombinant hepatitis B

• Nucleic acid (DNA) vaccines– West Nile (for horses)– DNA injected into skin cells, protein made –

stimulates immune system.

The Development of New Vaccines

• Culture pathogen

• rDNA techniques– In plants

• Adjuvants

• Deliver in combination

Diagnostic Immunology

• Sensitivity: Probability that the test is reactive if the specimen is a true positive

• Specificity: Probability that a positive test will not be reactive if a specimen is a true negative

Monoclonal Antibodies (Mabs)

• Hybridoma: “Immortal” cancerous B cell fused with an antibody-producing normal B cell, produces

• Monoclonal antibodies

Monoclonal Antibodies

Figure 18.2

Monoclonal Antibodies

Figure 18.2

Monoclonal Antibodies

Figure 18.2

Monoclonal Antibodies (Mabs)

• Infliximab: For Crohn’s disease, blocks cytokine

• Ibritumomab + rituximab: For non-Hodgkin’s lymphoma, destroy cancer cells

• Trastuzumab: Herceptin for breast cancer

• Spread of cancer

Figure 18.5

Agglutination Reactions

• Particulate antigens and antibodies

• Antigens on a cell (direct agglutination)

Figure 18.7

Indirect Agglutination

Complement Fixation Test

Figure 18.10

Complement Fixation Test

Figure 18.10

Figure 18.11a

Fluorescent-Antibody (FA) Techniques

• Direct FA

Figure 18.11b

Indirect Fluorescent-Antibody Test

Enzyme-Linked Immunosorbent Assay

• Also called ELISA

• Enzyme linked to Ab is the indicator

Direct ELISA

Figure 18.14a

Indirect ELISA

Figure 18.14b

Serological Tests

Figure 18.13

Serological Tests

• Direct tests detect antigens (from patient sample)

• Indirect tests detect antibodies (in patient’s serum)

ELISA

• Enzyme liked immunosorbent assay

• Plastic plates with wells in them are used

• Direct ELISA – presence of antigen in a sample

• Indirect ELISA – presence of antibodies against a specific antigen in a sample.

Neisseriagonorrhoeae

Antibodies against HIV

Secondary antibodies

Against human Antibodies.

Enzyme linked antibodies

Substrate – colorlessProduct – blue