Chapter 7 Control of Microbial Growth

Boiling kills microbes by coagulating the proteins – endospores are not destroyed. Autoclaving - Temperature 121 o C, 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

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-AirAutoclave Equivalent Treatments170˚C, 2 hr121˚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)

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.

Neisseria gonorrhoeae Antibodies against HIV Secondary antibodies Against human Antibodies. Enzyme linked antibodies Substrate – colorless Product – blue