Conventional & Rapid Methods for Assessing Micorbial Loads PDF

Republic of the Philippines CENTRAL BICOL STATE UNIVERSITY OF AGRICULTURE Calabanga | Pasacao | Pili | Sipocot Department of Food Science FST 101 – Food Microbiology CONVENTIONAL AND RAPID METHODS...

Republic of the Philippines CENTRAL BICOL STATE UNIVERSITY OF AGRICULTURE Calabanga | Pasacao | Pili | Sipocot Department of Food Science FST 101 – Food Microbiology CONVENTIONAL AND RAPID METHODS OF ASSESSING MICROBIAL LOADS IN VARIOUS FOOD COMMODITY ALESSANDRA M. DOMANACO Assistant Professor IV  Lesson IV: Utilization of Conventional and Rapid Method of Assessing Microbial Loads in Various Food Commodity 1. Classify spoilage microorganisms according to the food or foods they are most likely to affect based on intrinsic and environmental characteristics of the food and compare to various storage methods to determine which method would be most appropriate for the given product; 2. Carry out a series of laboratory experiments aimed at isolating and identifying common food borne microbial pathogens; and 3. Design and carry out laboratory experiment to evaluate the microbial safety of a common food product.  INTRODUCTION ❖ Food serves as an excellent substrate for MICROBIAL GROWTH. ❖ Microbiological characteristics are assessed in terms of the microorganisms – molds, yeasts, bacteria and virus – present in food, their characters, ability to change the quality, their influence on the health of the consumers. ❖ The examination of foods for the presence, types, and numbers of microorganisms and/or their products is basic to food microbiology.  Definition of Terms Colony - is defined as a visible mass of microorganisms all originating from a single mother cell. Culture medium - is a mixture of substances that promotes and supports the growth and differentiation of microorganisms. Viable organism - metabolically or physiologically active. Serial dilution - used to decrease a bacterial concentration to a required concentration for a specific test method, or to a concentration which is easier to count when plated to an agar plate. Isolation - refers to the separation of a strain from a natural, mixed population of living microbes. Fastidious microorganisms - microorganisms that require special conditions and substances for growth. Pure culture - a laboratory culture containing a single species of organism. Mixed culture - one that contains more than one type of organism growing in a sterile medium  CULTURE MEDIA On Consistency On Nutritional Component Liquid Media Routine Lab Synthetic Media Media Solid Media Basal Media Semi - Solid Media Enriched Media Biphasic Media Selective Media Differential Media Transport Media CULTURE MEDIA BASED ON CONSISTENCY  Liquid Media - is a liquid medium used for the cultivation of a wide variety of organisms. - sometimes referred as “broths” (e.g. nutrient broth). - bacteria grow uniformly producing general turbidity - it is used when a large number of bacteria have to be grown and the numbers in the inoculum is suspected to be low. - can be enriched with other ingredients such as blood, serum, sugars, etc., for special purposes. Example: Vibrio & Bacillus are known to grow as a thin film called ‘surface pellicle’, Bacillus anthracis is know to produce stalactite growth flocculent  Solid Media - Liquid medium that is added with certain solidifying agent (such as Agar) - Agar – is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium. It is composed of two long- chain polysaccharides (70% agarose and 30% agaropectin). - It melts at 95°C and solidifies at 42 °C. Semi-Solid Media - Agar medium that has reduced amount of agar (0.2 – 0.5%) - Fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains. - e.g. Stuart’s & Amies media, Hugh & Leifson’s Oxidation Fermentation Test Medium  Biphasic Media - A culture system that comprises of both liquid and solid medium in the same bottle. - Other solidifying agent such as egg yolk and serum can be used to solidify culture media. - e.g. Loeffler’s Serum Slope, Lowenstein Jensen Medium and Dorset Egg Medium CULTURE MEDIA BASED ON NUTRITIONAL COMPONENT  Basal Media - are simple media that supports most non-fastidious bacteria. - e.g. Peptone water, nutrient broth and nutrient agar  Enriched Media - Addition of extra nutrients in the form of blood, serum, egg yolk, etc. to basal medium. - Used to grow nutritionally exacting (fastidious) bacteria. - e.g. Blood agar, chocolate agar, Leoffler’s serum slope  Selective Media - Designed to inhibit unwanted or contaminating bacteria and help recover pathogen from a mixture of bacteria. - Addition of certain inhibitory agents that don’t affect the pathogen. - Example: addition of antibiotics, dyes, chemicals alteration of pH or a combination of these Differential Media - Designed in such a way that different bacteria can be recognized on the basis of their colony colour. - Incorporation of dyes, metabolic substrates, etc. - e.g. MacConkey’s Agar, CLED agar, TCBS agar, XLD agar  Transport Media - Used when specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms. - Prevents drying (desiccation) of the colony, maintain the pathogen to commensal ration. - e.g. Stuart’s & Amie’s agar, Cary Blair medium, Venkatraman Ramakrishnan medium, Sach’s buffered glycerol saline  CULTURE METHODS PURPOSE ✓To isolate bacteria in pure cultures ✓To demonstrate their properties ✓To determine sensitivity to antibiotics ✓To estimate viable counts ✓Maintain stock cultures  CULTURE METHODS Streak culture Lawn culture Stroke culture Stab culture Pour Plate and Spread Plate Method  Spread Plate Method ❖ is the method of isolation and enumeration of microorganisms in a mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in a solution.  The spread plate technique involves using a sterilized spreader with a smooth surface made of metal or glass to apply a small amount of bacteria suspended in a solution over a plate. The plate needs to be dry and at room temperature so that the agar can absorb the bacteria more readily. A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate.  Pour Plate Method  pour plate method, a fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of a sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well. After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours.  Streak Culture - The streak plate technique is used to isolate the organisms (mostly bacteria) from a mixed population into a pure culture. - The inoculum is streaked over the agar surface to “thin out” the bacteria to produce pure culture - Wire loop (Platinum wire or Nichrome wire) is used. Streak plate method patterns Colony growth shapes in agar plates  Lawn Culture - Provides a uniform surface growth of the bacterium - Used for: ❖Bacteriophage typing ❖Antibiotic sensitivity testing ❖In the preparation of bacterial antigens and vaccines - Prepared by flooding the surface of the plate with a liquid suspension of the bacterium.  Stroke Culture Made in tubes containing agar slopes (slant). Inoculation is made in zig-zag pattern by the help of inoculating wire loop. Employed for providing a pure culture of the bacterium for slide agglutination. Colony growth shapes in agar slants  Stab Culture - Prepared by puncturing suitable medium – gelatin or glucose agar with a long, straight, charged wire. - Uses: ❖Demonstration of gelatin liquefaction ❖Oxygen requirements of the bacterium under study ❖Maintenance of stock culture  MICROBIOLOGICAL EXAMINATION STANDARD PLATE COUNTS (SPC) OR AEROBIC PLATE COUNTS (APC) CONVENTIONAL MOST PROBABLE NUMBER (MPN) METHODS DYE REDUCTION TECHNIQUES DIRECT MICROSCOPIC COUNTS (DMC) GENETIC METHOD (Nucleic Acid Probe) IMMUNOLOGICAL BASED METHOD (Enzyme RAPID METHODS Linked Immunosorbent Assay) BIOSENSOR METHOD  MICROBIOLOGICAL EXAMINATION Conventional Methods Rapid Methods CONVENTIONAL METHODS STANDARD PLATE COUNTS (SPC) OR AEROBIC PLATE COUNTS (APC) MOST PROBABLE NUMBER (MPN) DYE REDUCTION TECHNIQUES DIRECT MICROSCOPIC COUNTS (DMC) STANDARD PLATE COUNT (SPC) It is useful in determining the viable number of microbes in food and food products. From dilution blanks, measured amounts of diluted microorganisms are transferred by spread plating/pour plating over the plate count agar plates and incubated for 24 – 48 hours in inverted position. STANDARD PLATE COUNT (SPC) Food samples are blended or homogenized Serially diluted in an appropriate diluent Plated in or onto a suitable agar medium Incubated at an appropriate temperature for a given time All visible colonies are counted  Advantages Disadvantages Simple Plating procedures for selected Can determine viable groups are limited by the number of microbes degree of inhibition and effectiveness of the selective and/or differential agents employed MOST PROBABLE NUMBER (MPN) The method was introduced by McCrady in 1915 and widely used in food microbiology for determination of the presence of coliforms in potable water. The selective medium used is MacConkey’s medium which contains a bile salt inhibitory for growth of non-intestinal lactose fermenting bacteria. As the method is statistical in nature, MPN results re generally higher than SPC.  Water to be tested is diluted serially and inoculated in lactose broth coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is determined by counting the number of tubes giving positive reaction (i.e both color change and gas production) and comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables. ✓ MPN test is performed in 3 steps 1.Presumptive test 2.Confirmatory test 3.Completed test  Advantages Disadvantages Relatively simple Large volume of glassware Results from one laboratory required are more likely than SPC results to agree with those The lack of opportunity to from another laboratory observe the colonial Specific groups of organisms morphology of the organisms can be determined by use of Lack of precision appropriate selective and differential media It is the method of choice for determining fecal coliform densities  DYE REDUCTION TEST This test involves the use of redox dyes like methylene blue or resazurin to determine the quality of milk. Dyes (methylene blue from blue to white upon reduction and resazurin from slate blue to pink or white upon reduction) reduced and lose its color in the presence of actively growing bacteria. Assumed that the larger the number of bacteria in milk, the faster will the oxygen be consumed, and in turn the quicker will the color disappear. The time taken for the reduction of methylene blue or resazurin is inversely proportional to the number of viable bacteria present in sample and the shorter the dye reduction time poorer is the quality of the milk.  Advantages Disadvantages Simple Not all organisms reduce the Rapid dyes equally Inexpensive They are not applicable to food specimens that contain Only viable cells actively reductive enzymes reduce the dyes  DIRECT MICROSCOPIC COUNT The basis of a direct count is the actual counting of every organism present in a food sample. Identification through staining and observation of morphology is possible. This method is very quick and precise for determination of microorganisms in liquid foods like milk by the use of haemocytometer or Neubauer counting chamber. The method is quick and required practical experience.  Advantages Disadvantages Rapid It is a microscopic method (fatiguing to the analysts) Simple Both viable and nonviable cells are enumerated Cell morphology can be Food particles are not always assessed distinguishable from microorganisms Lends itself to fluorescent Microbial cells are not uniformly probes for improved distributed relative to single cells efficiency and clumps Some cells do not take the stains well and may not be counted DMC counts are invariably higher than counts by SPC.  RAPID METHODS GENETIC METHOD (Nucleic Acid Probe) IMMUNOLOGICAL BASED METHOD (Enzyme Linked Immunosorbent Assay) BIOSENSOR METHOD GENETIC METHOD: NUCLEIC ACID PROBE Probe-based tests is used by immobilizing probes to inorganic supports (dipsticks) that allow the user to easily manipulate the probe (e.g., wash off unhybridized DNA) without damaging or losing it. This is referred to as solid-phase hybridization systems (e.g. solution- based) are also possible. The principle advantage of this system is the reduced time required foe positive identification of Salmonella. Examples of some kits based on gene probes are AccuProbe for Campylobacter, GENE-TRAK for Campylobacter, E. coli, Listeria, Salmonella, Staphylococcus aureus and Yersinina enterocolitica.  1. Short single strands of DNA that are complementary to genes present in a pathogenic microbe are synthesized. 2. The food sample must then be treated so that any microbial cells are lysed, releasing their DNA. Reference: DNA Probe DNA hybridization HD Animation (youtube.com) 3. Microbial DNA is then treated to convert it from double strands to single strands, and the probe is added. Hybridization (annealing of complementary strands) then occurs between the single-stranded DNA probe and single-stranded DNA released from pathogenic microbes present in the food. 4. Probe DNA that has not hybridized must then be removed, usually by washing the sample, and the presence of hybridized DNA probes can then be detected. IMMUNOLOGICAL METHOD: ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) This is the most popular rapid method for the detection of food borne pathogens. This is also based on the specificity of antigen-antibody reaction. A few examples of some ELISA based kits for the detection of pathogens include EHEC-TEK, Assurance, HECO157, TECRA, Premier O157, E. coli Rapitest, Transia Card E. coli O157 for E. coli O157:H7. Several such kits are also available for other pathogens.  1. In this format antibody is coated on the surface of microtiter well. This antibody is known as primary antibody. Reference: Enzyme-linked immunosorbent assay (ELISA) - Animation (youtube.com) 2. Bacterial antigen obtained from the food sample is added to the antibody coated wells. Antigen-antibody reaction is allowed to occur followed by washing the wells to remove the unbound antigen. 3. After this, a secondary antibody tagged with an enzyme is added to the microtitre plate. This antibody is also specific for the antigen to be detected but it recognizes a different epitope on the surface of antigen. 4. Followed by the addition of substrate for the enzyme. This substrate is usually colourless and a coloured product is formed by the action of antibody bound enzyme. The colour development is taken as a positive reaction and even the intensity of the colour can be measured using ELISA reader. BIOSENSOR METHOD Biosensors have a biological sensor that is connected to a transducer. A transducer is a device capable of converting signals from the biological sensor into signals (usually electrical) that can be easily recorded and stored. For example, a number of biosensors use the specificity of antibody-antigen binding to detect pathogens in food samples. When the pathogen is present, it binds to the antibody. The key event follows: binding of antigen to antibody produces an electrical signal that can be detected and recorded. Biosensors, then, are an example of what science fiction authors describe as: ‘cybernetics’– the fusion of organic matter to electronic circuitry. THANK YOU!