Microbial Control and Detection (Module 8 – Fish 154) PDF

Microbial Control and Detection Module 8 – Fish 154 Rhoda Mae C. Simora, PhD.  What is microbial control? Control of microbial growth means to inhibit or prevent growth of microorganisms. This control is affected in two basic ways: (1) by killing microorganisms o...

Microbial Control and Detection Module 8 – Fish 154 Rhoda Mae C. Simora, PhD.  What is microbial control? Control of microbial growth means to inhibit or prevent growth of microorganisms. This control is affected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of microorganisms.  Let’s learn some important terminologies… ❑ Agents which kill cells are called -cidal ❑ Agents which inhibit the growth of cells (without killing them) are referred to as -static agents Can you now define bactericidal and bacteriostatic? ✓Pathogens – can affect food Safety Can result to illness, disease or death ✓Spoilage microorganisms– can affect food quality poor quality, off-flavors short shelf-life, customer dissatisfaction  Methods of Microbial Control Physical Chemical Biological Mechanical Methods Methods Methods Removal Methods Disinfectants Filtration Heat Antibiotics Antiseptics Radiation Preservatives A. Moist heat Methods include boiling, pasteurization, and autoclaving. Very inexpensive and readily available; usually 100oC for 15 minutes – many vegetative cells and viruses are killed/inactivated within 10 minutes at 100oC. Pasteurization Primarily used to decrease the number of pathogenic organisms in food without adversely affecting the flavor; a) ultra pasteurization of milk is 130oC - 140oC for 1 - 2 seconds b) flash pasteurization is 72oC for 15 seconds c) normal pasteurization at 63oC for 30 minutes. Autoclaving/Sterilization Steam under pressure - the most effective moist heat method; usually 121.5oC at 15 psi for 15 minutes Digital autoclave B. Dry heat Direct flaming or incineration and hot air (160oC-170oC) -160oC for 1.5 to 2 hours ; 170oC for 1 hour C. Freezing Temperatures between 0o and 7oC may inhibit the reproduction of Direct flaming certain organisms or the production of toxins. Rarely bactericidal Not be an effective method of disinfection a. Quick freezing - is often used to store microorganisms for long periods of time b. Slow freezing - causes severe damage to cellular constituents and may be bactericidal. D. Desiccation Freezing fish fillets Drying or Freeze-drying can be used to inhibit growth (via inhibition of enzymes); organisms remain viable. E. Radiation Ionizing radiation (alpha, beta, gamma, and x-rays, cathode rays, high-energy protons and neutrons) exhibits a high degree of penetration. ▪ It creates free radicals in the medium, leading to the denaturation of proteins and nucleic acids. It can result in mutations. 1) Used in food preservation processes 2) Viruses and spores are somewhat resistant. 3) Gram-negative bacteria are more sensitive to ionizing radiation than are gram-positive organisms. Ultraviolet radiation is a form of non-ionizing radiation. -Low degree of penetration -Results in thymine dimers (cross-linkages) in DNA that interfere with replication. Microwave - usually do not directly kill organisms Laminar flow hood with UV light - indirectly from heat generated in microwave materials  Chemical Agents Disinfectants-antimicrobial agent used on inanimate objects Antiseptics-antimicrobial agents used on living tissues Preservatives – added to products to prevent decomposition by microbial growth  Disinfectants: Bleach (Chlorine)– generic term for the sodium hypochlorite, which imparts whitening properties. Used to disinfect water and for cleaning surfaces (e.g. floors, counters) and has proven effective in destroying HIV. Mode of Action Kills microbes by inhibiting enzyme activity and oxidizing cellular contents so that they no longer perform normal metabolic functions. Chlorine reacts with organic materials in the cell and is used up. Therefore, to be effective, chlorine concentrations must be high enough to allow the chlorine to attach to all the organic material present and still have some residual.  Disinfectants: Non-Bleach – utilize quaternary ammonium compounds as the primary disinfecting agent ex. Lysol brand -often as effective as bleach but these compounds are also much gentler especially on fabrics. Mode of Action exert their germicidal effect by denaturing proteins and destroying the selective permeability of the cell membrane which permits "leakage" of cellular contents. Antiseptics Alcohols: Ethanol, isopropanol •Alcohols: – Denature proteins, Ethanol, dissolve lipids isopropanol – Optimum •Denature proteins, concentration is 70% dissolve lipids •Optimum concentration is 70%. Table 7.6 Antiseptics •Surface-active agents or surfactants Surface-active ingredients or surfactants Soap Degerming Acid-anionic detergents Sanitizing Quarternary ammonium Bactericidal, Denature compounds proteins, disrupt plasma (Quats) membrane Quarternary ammonium compounds Antiseptics Heavy Metals Oligodynamic action: toxic effect due to metal ions combining with sulfhydryl (—SH) and other groups of proteins are denatured. Mercury (HgCl2), Greeks & Romans for skin lesions Copper against chlorophyll containing organisms. ex. Algicides Types of Disinfectants Silver (AgNO3):Biguanides: Antiseptic for eyes of Chlorhexidine newborns • Disrupt plasma membranes Zinc (ZnCl2) in mouthwashes, ZnO in Halogens: Iodine, chlorine antifungal in paint • Oxidizing agents • Bleach is hypochlorous acid (HOCl) • Surgical area, water, household disinfectant Heavy metals: Ag, Hg, and Cu • Oligodynamic action • Denature proteins  Biological Agents Antimicrobial agents o Antibiotics - also known as antibacterials, are medications that destroy or slow down the growth of bacteria. o They include a range of powerful drugs and are used to treat diseases caused by bacteria. o Antibiotics cannot treat viral infections, such as cold, flu, and most coughs. Modes of action fall into two basic categories: 1. Alteration of cell walls or cytoplasmic membranes 2. Interference with protein and nucleic acid structure  Cell wall - maintains integrity of cell When disrupted, cannot prevent cell from bursting due to osmotic effects Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell When damaged, cellular contents leak out Viral envelope responsible for attachment of virus to target cell Damage to envelope interrupts viral replication Non-enveloped viruses have greater tolerance of harsh conditions  Protein function depends on 3-D shape Extreme heat or certain chemicals denature proteins Nucleic Acids can be damaged or Protein structure destroyed by chemicals, radiation, and heat. Can produce fatal mutants Can halt protein synthesis through action on RNA Nucleic acid structure  Mechanical Agents A.Filtration Mechanical means of removing microorganisms. The liquid or gas is passed through a filter with pores small enough to prevent passage of microbes. This method can be used for substances that are sensitive to heat. a. HEPA (high efficiency particulate air) filter: removes at least 99.97% HEPA filter of airborne particles 0.3 micrometers (µm) in diameter. b. ULPA (ultra-low particulate air) filter: remove from the air at least 99.999% of dust, pollen, mold, bacteria and any airborne particles with a size of 120 nanometers or larger. B. Osmotic pressure Extremely hypertonic conditions can cause plasmolysis (i.e., contraction of all the cell membrane away from the cell wall). The basic principle for testing any control agent, (whether temperature, chemical or antibiotics) is always the same: 1. Expose the organism to the agent. 2. Remove the agent. 3. Put the organisms in favorable growth media. 4. Look for reproduction of organisms.  Minimum inhibitory concentration (MIC)- smallest amount of agent needed to inhibit the growth of a test organism Antimicrobial agent susceptibility assay using dilution methods. A series of increasing concentrations of antimicrobial agent is prepared in the culture medium. Each tube is inoculated with a specific concentration of a test organism, followed by a defined incubation period. Growth, measured as turbidity, occurs in those tubes with antimicrobial agent concentrations below the MIC.  Disc diffusion method- disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a zone of inhibition indicates effectiveness. Relative Susceptibility of Microorganisms  Detecting Microbial Contamination in Food/Fish The range of laboratory methods available to identify microbial hazards in food include: conventional culturing methods immunological protocols molecular methods recently the introduction of spectroscopic methods (e.g.,MALDI- TOF and Raman) The choice of method used depends on the technology platforms available, the specific risks to be identified, the complexity of the samples, the required speed of test response, and the expected microbial load.  Common Foodborne Pathogens detected in Food ❑Salmonella species ❑Campylobacter spp. ❑Listeria monocytogenes ❑Escherichia coli O157:H7 Salmonella spp. Campylobacter spp. Listeria monocytogenes E. coli O157:H7  Conventional Culturing Methods Typical workflow of microbial ID using conventional methods  Immunological Methods Antibody-based detection systems: o ELISA (enzyme-linked immunosorbent assay) – can detect antigens present on a cell surface or toxins produced by a food pathogen o Serological testing – blood tests that look for antibodies in your blood o Microarrays (antibody microarray) -a collection of capture antibodies are spotted and fixed on a solid surface such as glass, plastic, membrane, or silicon chip, and the interaction between the antibody and its target antigen is detected. These detection systems are usually very sensitive and specific, with detection limit that can be as low as a few CFU/g.  Molecular Methods ✓ FISH (fluorescent in-situ hybridization) ✓ Amplification methods – PCR, qPCR, rt-PCR ✓ DNA microarrays ✓ Whole genome sequencing These methods involve the analysis of certain genomic markers which allow for microbial ID at genus level, and can provide information on microbial susceptibility and viability.  Spectroscopic Methods ✓ MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry)- identify microorganisms on the basis of a protein profile or “fingerprint” ✓ Raman spectroscopy – based on inelastic MALDI-TOF workflow scattering of monochromatic light, usually from a laser source; can rapidly detect foodborne pathogens - End of Module 8 -