FOS 4222 FOOD MICROBIOLOGY LABORATORY MANNUAL

SPRING SEMESTER, 2008 M-W 1:00-3:50 Dr. ANITA WRIGHT

Room 214 Aquatic Food Products Building Phone: 392-1991, Ext. 311 Lab Ext. 312 Email: acw@ufl.edu Website: http://fshn.ifas.ufl.edu/faculty/ACWright/FOS4222.html

Teaching Assistants: Dr. Venata Vedum-Mai and Melissa Jones

FOOD

TABLE OF CONTENTS

LAB EXERCISES Section 1: LAB EXERCISE 1: INTRODUCTION LAB EXERCISE 2: MEDIA LAB EXERCISE 3: MICROSCOPY LAB EXERCISE 4: ENUMERATION LAB EXERCISE 5: MPN LAB EXERCISE 6: E. COLI LAB EXERCISE 7: FUNGI LAB EXERCISE 8: SALMONELLA

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3 6 11 17 21 24 27 29

Section 2: LAB EXERCISE 9: IMMUNOASSAYS LAB EXERCISE 10: DNA PROBE LAB EXERCISE 11: PCR LAB EXERCISE 12: MOLECULAR TYPING 34 38 41 46

LAB EXERCISE 1: INTRODUCTION TO THE LABORATORY OVERVIEW: These lab exercises in this mannual are designed to supplement your course work and will chronologically follow (for the most part) the sections you are studying in class. They should provide practical hands on experience with standard microbiology, as well as recently developed rapid methods and molecular techniques. Basic principles of experimental controls, data presentation, and interpretation will be explored. Two very important issues in microbial science are safety and data handling. We will be working with appropriate precautions and equipment for pathogens, but remember ALL bacteria are potential pathogens for some people or at some doses. If you are not sure about a procedure: ALWAYS ASK!

Lab Safety Rules: 1. Absolutely no food or drink 2. No mouth pipetting 3. Always wear gloves and lab coat 4. Report any spills/contamination 5. Disinfect area before and after 6. Dispose of gloves and all contaminated materials in biohazard 7. Breakable or sharp or sharp objects are disposed to sharps container 8. WASH HANDS BEFORE LEAVING! Failure to respect safety rules will result in expulsion from lab.

Lab Safety Assignment: 1. Fill out Safety form 2. Locate fire extinguisher

Grading: Lab notebook with answers to study questions provided for each lab (20%) Lab report (20%) Quizzes on current lab (10%) Note: You are required to read lab assignments before class. Two Exams (25% each = 50% total) Be Prepared! Lab quiz will be given prior to each lab based on the material that will be covered that day.

Lab notebook: Laboratory data are the backbone of science, and your records should be accurate and precise. Scientific papers are generally composed of a number of experiments. Your notes should be sufficient such that you can later examine them and comprehend what, why and how you did something. Data should be recorded such that you (or anyone else) will be able to repeat the experiment from the description in your notebook. Some laboratory work, especially where data may be patentable or subject to government regulation, will require bound notebooks to be dated, signed and witnessed daily. All pages must be written in ink on sequentially numbered pages, and copies made on a regular basis. In practice, raw data is generally recorded outside of the notebook and then entered later (but not too much later). Pictures, tables, and other tabulations are permanently attached and are not removed from the notebook. Multiple notebooks are used when working on independent projects. The following elements should be present the recording of all experiments: 1. Date and Title of experiment. 2. Statement of purpose: Brief description of rational for experiment 3. Methods: Detailed step by step outline of experiment to include all variables and controls. Include anything that might be relevant: source of reagents, strains, temperatures, phase of the moon, etc. The object of this section is to provide a history of what you have done so that you or anybody else should be able to read and follow the protocol and reproduce the data. This section should be written before beginning any experiment. If you are following the protocol from the lab manual, you can cut and paste this from the lab manual. If there are deviations to the protocol that occur, such as a tube that was omitted or the wrong incubation temp., etc., record those in your results. Once a standard protocol has been established it can be referred to by a reference name or number 4. Results: Record everything! Including negative data, screw ups, changes in protocol, contamination. You do not know what might become important later. For example if you are doing plate counts, include the actual number of colonies at each dilution. Making tables ahead of time in your notebook is useful so you do not forget to record anything. 5. Data analysis: This section should be well-labeled so that it is easy to comprehend. For example, if you have been using a code for your strains (#1-10), your table or chart should be converted back to the strain names so it does not have to be deciphered every time you go back to it. Publishable data requires statistics, minimally mean, Standard deviation, p values. 6. Discuss you interpretation of results. This section should record any thoughts you have about the meaning of the data. Discuss how the data compares to expected results based on prior data or your own or in the literature. Are comparisons of strains statistically valid. Include implications for future experiments. 7. Provide answers to study question in your notebook.

Lab report: Lab report will cover the last two laboratory sections on PCR and molecular tying. A 4-6 page (double space) lab report will discuss the methods and results of these experiments. Send in word format to Maria (chatz@ufl.edu). 1. Statement of Purpose (1-2 pages): brief description of the purpose of the experiment. Should include some background on the topic and a rationale for the particular variables you are investigating. State hypothesis if possible. Direct quotes use quotation marks and any outside supporting material must be referenced (use ASM format). 2. Methods Description (1 page): Describe protocol in your own words so that someone else could do the experiment. Divide in to sections such as the following: Bacteria, culture conditions, biofilm assay. Include reagents and manufacturer if known. 3. Results (1-2 pages): Describe results in writing without interpreting but do not draw any conclusions. Prepare tables or graphs to incorporate raw data and analysis as needed. 4. Discussion. Conclusions (1 page): Discuss what the results and draw conclusions. Did you prove your hypothesis? Why or why not? How do your results compare to other data in the literature? What future experiments would you recommend? 5. References: Two required

LAB EXERCISE 2: MEDIA Isolation and cultivation of specific microorganisms is essential to the study of microbiology. In the environmental food, bacteria exist in complex communities that are extremely diverse and are rapidly altered with changing conditions. Robert Koch is credited with the concept of a pure culture, which allowed microbiologists to characterize a particular organism independently of related flora. This development was essential to the study of infectious disease and to establishing Kochs Postulates. His first observation of bacterial colonies on solid medium was on a potato. Later agar was incorporated into broth medium to provide solid support and the addition of a variety of components into the medium. The method for inoculation of bacteria varies with the target organism and the matrix from which the organism is isolated. Several methods are listed below: Agar Inoculation: 1. Spread plating: Liquid sample (usually 100 l) on agar plate is spread evenly across the surface using a sterile hockey stick. 2. Pour plating: Liquid sample (usually 1 ml) is pipetted into an empty sterile petri dish, and liquid agar (at 50C) is pored into dish and swirled to distribute the sample evenly throughout the agar. 3. Streak plating: Method for isolation of individual colonies. Using sterile loop (flamed) streaking sample across one edge of the plate two or three times. Re-flame and make another two or three parallel streaks by drawing the loop across the initial streaks once at a right angle. Flame the loop and repeat as before, this time pulling the loop through the second set of streaks. Stabbing: Inoculate by stabbing strait down the center of the media in slant tube. 4. Ensure that the stab is as straight as possible to help prevent misinterpretation of bacteria spreading Selective/differential media were developed to provide the ability to differentiate among different genera or even species of bacteria. These agars are generally used for presumptive identification and require further confirmatory assays for positive identification. The use of these media requires application of appropriate microbial controls to ensure proper media reaction. Both a positive control and a negative are required. Most bacterial culture media resemble the natural substrate on which the microorganisms usually grow. The content of a particular medium will depend upon then nutrient requirements of the microorganisms of interest. There are several different forms of media, which include liquid or broth medium and solid or semisolid agars. Broth medium contains nutrients dissolved in water and is used often as enrichment for more robust growth of bacteria. Solid medium contains nutrients in water and a solidifying agent. The most common solidifying agent is agar, which is a complex polysaccharide composed of galactose and galacturonic acid. It is found in many marine plants and is extracted commercially from certain marine algae. Agar is resistant to enzymatic hydrolysis by most microorganisms; however, a few species, mainly in the marine environment, do hydrolyze agar. Silica gel (from inorganic chemical silicic acid) can be used in place of agar as a solidifying agent and is also used when microorganisms need to be cultivated on media that are inorganic matter free. Bacteria generally grow in colonies on solid media, but slant tubes can be inoculated by stabbing

bacteria into the agar in order to observe chemical reactions, such as for the production of acid or H2S. Semisolid media have a lower concentration of solidifying agent, which results in a more jellylike consistency, and is used for motility or gelatin-hydrolysis testing of bacteria. The nutritional requirements of bacteria vary widely. For example, autotrophs may require only simple inorganic substrates while more fastidious bacteria (including most human pathogens) require complex vitamins and growth factors just to survive and multiply. Necessary nutrients to support bacterial growth always include the following: a) Water, b) Nitrogen (inorganic salts, peptides, proteins, amino acids); c) Energy or carbon source (carbohydrates, peptides, proteins, amino acids) ; d) Accessory growth factors (i.e., blood for pathogens, milk for milk microorganisms). Food microbiologist use media to detect and differentiate microorganisms from the food we consume. The types of media are as varied as the organisms that they target. General categories of media include the following: 1. Non-Selective Media. Multipurpose media that incorporate a wide variety of nutrients and growth factors for general cultivation of most bacteria. 2. Enrichment Media. Microorganisms to be analyzed are often outnumbered or outcompeted by other microbes (especially in food, water, and soil). Enrichment media increase the number of target microorganisms while suppressing the growth of competing microflora. 3. Elective Media. Satisfy the minimum nutritional requirements and are particularly useful if the microorganism has unusual nutritional requirements. Wild yeast cells can be isolated by using lysine agar on which other microorganisms can not grow unless they utilize lysine as their sole source of nutrition. 4. Selective Media. Generally support the growth of several species, but also include inhibitory agents which restrict the growth of undesired microorganisms. 5. Differential Media. Contains reagents (usually dyes) that react with pH changes or other metabolic consequences of growth to produce reactions that are indicative of particular species. Colonies will have species-specific characteristics which are easily recognized. For example, haemolytic and non-haemolytic microorganisms can be easily distinguished by examining for zones of lysis formed on blood agar plates. Unfortunately these reactions are not always 100% reliable and generally further tests are required for identification. Note: Media for food microbiology are commonly both selective and differential. Some media can be purchased already prepared, but proper preparation of most media is an essential skill in the food laboratory. The following criteria apply to all reagents and media used in microbial analysis: Preparation criteria: 1. Reagents and chemicals must be of suitable grade and composition. 2. Purchase smaller 500g quantities to ensure minimal exposure to atmosphere. 3. Store media in a clean, dry, cool place away from sun (no longer than two years). 4. List dates of receipt and first opened on each container. 5. Solutions should be prepared using laboratory pure (distilled or deionized) water. 6. Use borosilicate glassware or other non-reactive vessels. 7. Weigh media to the nearest 0.1 gram. 8. Stir continuously with heat to prevent scorching and provide an even mixture. 9. Check pH before and after sterilization. 7

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Clearly label all media preparations accordingly before sterilization.

Sterilization criteria: 1. Media with sugars should be filtered or autoclaved for < 10 minutes. 2. Total heat exposure should not exceed 45 minutes for any medium. 3. Adequately space items for proper sterilization to take place. 4. Indicator strips are run with each autoclave operation to ensure proper sterilization. 5. Media should be removed immediately. 6. Sterilize rehydrated media within 2 hours (30 minutes optimal) of preparation. 7. Media is never re-autoclaved. 8. Maintained sterilization records (batch number, date, etc). 9. Cool in water bath between 46-50oC for not more than 4 hours. 10. Discard Durham fermentation tubes with any air bubbles. Storage of Reagents and Media: 1. Store prepared media in clean, dry areas without light exposure. 2. Store media containing sugars at room temperature for not more than 7 days. 3. Store prepared media under refrigeration (generally not to exceed 4 weeks). 4. Refrigerated media brought to room temperature 4 hours prior to use. 5. Tubes with water loss should be discarded. Media should be checked for contamination prior to use. Cultures can fail to grow or produce unexpected or invalid results when reagents are prepared incorrectly. Common errors include following: 1. Incorrect weighing of reagents or measuring of water volume. 2. Deterioration of media because of exposure to heat, moisture, or oxidation. 3. Water impurities such as heavy metals, pesticides or chlorine. 4. Contamination of glassware with detergents or chemicals. 5. Incomplete mixing. 6. Overheating of some media can produce caramelization of sugars or breakdown of indicator dyes. 7. Improper pH determination leading to non-ideal growth conditions. Media are often sterilized by autoclaving. An autoclave is a chamber in which steam sterilization is used to add heat, humidity, and pressure to the chamber. By increasing the pressure within the chamber the boiling point of liquids is increased and sterilization time is decreased. Autoclave sterilization requires 121C and 15 psi for 15 to 60 min. Media are autoclaved in a container that will accommodate at least twice the volume to be prepared in order to prevent boiling over. Procedure: Day 1: Media Pouring Contest and Autoclave Field Trip. Media preparation: 1. Measure 5 grams 0.1 grams tryptone, 2.5 grams 0.1 grams yeast extract, and 5 grams 0.1 grams NaCl, using the weigh boats and scales provided in the back room. Carefully poor it into 1 liter media bottle or flask. 2. Add 500 ml of distilled water. 8

3. Tilt the bottle at a 45 angle and drop a stir bar into it. 4. Place the bottle onto the center of the hotplate and allow it to stir sat a slow speed (do not use the heat) until reagents are in solution. 5. Add 7.5 grams of agar. 6. Briefly stir the mixture and bring to autoclave. Agar will go into solution during autoclaving. (Some media require heating until agar is dissolved before autoclaving.) Plate pouring: 1. After autoclaving media is generally cooled to 45-50C to prevent condensation of plates. We will use pre-autoclaved and pre-cooled media that will be provided in the waterbath. 2. Separate plates into stacks of four (save the plastic sleeves for storage). 3. Pore the warm media (about 50C) into the plate until the bottom is completely covered. Pouring media that is too hot will produce excess condensation and water on the plates. 4. Stack up to 20 plates on top of each other and allow to solidify. This helps to prevent condensation from building up on the lids. 5. Once solid, invert plates so that the agar is on the top and allow to sit overnight. Sitting overnight allows some of the excess water to evaporate. 6. The next morning place the plates back into the plastic selves, tape shut and label with the date and name of the media. 7. Incubate 10 uninoculated plates at room temperature to check for sterility at 24 and 48 hours. 8. Refrigerate to store. Days 1-3: Isolation and selective media. Materials: A. L. agarThis is a non-selective agar that will support growth of most aerobic heterotrophic bacteria and will serve as a positive control for the viability of the organisms assayed. B. TCBS agar Thiosulfate-citrate-bile salts-sucrose agar is a combination selective and differential media. Bile salts provide the selective reagent by preventing the growth of most gram positive strains of bacteria. The fermentation of sucrose by Vibrio cholerae produces yellow colonies due to a change in pH, while most other Vibrios produce green colonies since they do not ferment sucrose. C. EMB agar Eosin-methylene blue agar (Levine): Lactose utilization and eosin and methylene blue dyes permit differentiation among enteric lactose fermentors and nonfermentors as well as the identification of Escherichia coli. E. coli colonies are blue-black with a metallic green sheen caused by the large quantity of acid that is produced and the precipitates of dyes onto the growths surface. D. TSI/LIA agar slants Triple sugar-iron (TSI) is designed to differentiate among the different groups of Enterbacteriaceae, which are capable of fermenting glucose with the production of acid. TSI slants contain lactose, sucrose, glucose and the acid base indicator phenol red. Phenol red indicates the production of acid by a change in color of the medium

from orange-red to yellow. Typical salmonella will have a red top with a yellow butt. In most cases H2S is produced which will appear as a black precipitate. Lysine iron agar- Salmonellae will produce an alkaline reaction from the decarboxylation of lysine to cadaverine. LIA contains brom thymol blue, which turns yellow with the production of acid. Typical salmonella will have a purple top with a yellow butt. In most cases, there is also production of H2S, which will appear as a black precipitate. E. Motility Agar. Motility can be observed with the microscope, but observations can be difficult because there is very little contrast between the bacterial cell and its environment in live, unstained cells. The MOT Agar Motility Test determines motility when culture growth radiates out from the line of inoculation in semi-solid medium. Non-motile organisms will grow only on the inoculation line. It is important to insert and remove the needle in a single, straight line. 1. Streak each of the control strains and unknown bacteria to the different media listed above and incubate at the 35C. Label all plates on the bottom and include date, your initials, organism, and type of media. Record the observed reactions for each species at 24 and 48h. 2. Using the same organisms from above, transfer the bacteria via sterile flamed needle by inoculating the MOT agar with a single straight line into the tube and incubate at room temperature. Observe the tubes at 24 and 48 hours. Check the tube for growth (motility) that radiates out from the inoculation line. STUDY QUESTIONS: 1. Why is it necessary to avoid condensation on solid medium? 2. What are the advantages of spread plating? Stabbing? 3. What are sources of contamination of microbial media? 4. Why are bile salts used in selective media? 5. What would be the expected results for these strains for media incubated at room temp? at 4 C?

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LAB EXERCISE 3: THE MICROSCOPE AND STAINING TECHNIQUES Compound microscope: The compound microscope (Figure 1) consists of two separate lens systems and is the most widely used in the field of microbiology. The first lens system in the compound microscope, called the objective, is nearest to the specimen being viewed. The objective magnifies the Figure 1. A. Compound Microscope specimen and produces a real image within the body tube. The objective lens system is comprised of both convex and concave lenses, which correct various chromatic and spherical aberrations inherent to a simple convex lens. Typically there are at least three objectives: the lowpower objective (16mm), the high-dry objective (4mm), and the oilimmersion objective (1.8mm). Focal length of the objective is determined by the distance (mm) used to produce the real image. The shorter the focal length of the objective, the shorter will be the working distance between the objective and specimen. The ocular lens consists of the eyepiece that you look through. The ocular magnifies the real image in the body tube to produce a virtual image that is seen by the eye while viewing the specimen. The total magnification can be calculated by multiplying the magnification of the objective by the magnification of the ocular. The objective magnification is usually found engraved on the side of the objective mount and the ocular magnification is usually found on the eyepiece. Resolving power is the ability to show two closely adjacent points as both distinct and separate. Resolving power is a function of both the wavelength of the light used to view the specimen and the numerical aperture of the lens system. Therefore, the shorter the wavelength of light, the smaller will be the structure that is clearly visible. However, decreasing wavelength to increase resolution is limited due to the narrow spectrum of visible light (400 to 750nm). The greatest increase in resolution can be achieved by increasing the numerical aperture, which is defined as the function of the effective diameter of the objective in relation to its focal length and the refractive index of the space between specimen and the objective. To decrease the refraction between the slide and objective, immersion oil is used because it has roughly the same refractive index as the glass slide. This decrease in refraction increases the light rays entering the objective and results in a greater resolution and clearer image. Fluorescence microscope: Fluorescence microscopy requires a special type of powerful illumination source, usually a mercury lamp. The light from the lamp passes through special colored filters, which only allow light with distinct wavelengths to pass. This narrow band of light hits the bacterial specimen. Certain compounds in the specimen (either natural compounds 11

Figure 2. Fluorescence Microscope

or fluorescent stains) capture the light and reflect it back up as light with a lower energy. This reflected light is detected either with the viewer's eye, or with sensitive detectors. Because this type of microscopy uses reflected light on a dark background, very small amounts of light (and of your sample) can be seen. Fluorescent compounds include natural compounds such as chlorophyll, as well as certain DNA-binding dyes such as ethidium bromide and DAPI. Sometimes fluorescent stains are attached to the constant region of antibodies, to generate very specific, very sensitive bacterial tags.

STAINING TECHNIQUES Bacteria are almost colorless and require staining to increase the contrast in their color with their surroundings. Stains generally react with the cell wall, not the background. Three advantages to staining include: 1) Providing contrast between the bacteria and the background to determine cell morphology; 2) Permitting the study of internal structures, such as the cell wall, vacuoles, or nuclear bodies; and 3) Allowing higher magnification as a result of the increased contrast. Simple stain. The Simple Stain is the easiest method, but lacks the power of discrimination seen with Gram and other stains. Simple staining is routinely used to determine size, shape, and arrangement of bacterial cells, as well as for direct microscopic cell counts. Typical dyes are crystal violet, safranin, carbol fuchsin, and methylene blue. These dyes are generally salts that include a colored ion or chromophore. For example, methylene blue is actually methylene blue chloride, and the color is derived from the positively charged (cationic) methylene blue ion. The interior of a bacterial cell has a slight negative charge in medium at neutral pH combines with the positively charged methylene blue ion. Basic dyes have a positively charged color-bearing ion, while an acidic dye will be negatively charged. Gram stain: Hans Christian Joachim Gram, a Danish physician invented the Gram Stain method in 1884. The Gram Stain is a differential stain that permits the division of bacterial species into two distinct groups: gram positive and gram negative. The two cell groups differ in permeability of the cell surface layers, and in cell wall composition. Gram positive cell walls have much more extensive peptidoglycan layers than do gram negative organisms, while gram positives lack the double membrane seen with gram negatives. Both gram-positive and gram-negative bacteria take up crystal violet and iodine, but the crystal violet-iodine complex is trapped inside the grampositive cell by the dehydration and reduced porosity of the thick peptidoglycan layer. Cell walls

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of gram-negative bacteria contain more lipids than gram-positive cell walls, and are more soluble in alcohol and acetone. It is theorized that these lipids are removed from the cell walls during the decolorizing step or that due to their higher lipid content, the gram-negative cells may contain fewer acidic reactive sites for binding with the basic dye. Four different solutions are used in a Gram stain: 1. The basic dye (crystal violet) stains bacterial cells with a purple blue color. 2. The mordant (Iodine) increases the affinity between the bacterial cell and the dye. Acids, bases, and metallic salts are examples of a mordant. A mordant complex is required to prevent certain cells from losing the stain during the decolorization step. 3. The decolorizing agent (95% ethanol) removes the dye from some of the stained cell. Cells with thick peptidoglycan layers will resist destaining and remain blue. 4. The counterstain (safranin) is another basic dye but of a different color from the initial stain and is used to stain the decolorized cells. The counterstained cells will appear red or pink. Stained cells that retain the basic dye following decolorization are termed gram-positive, while those that are decolorized are termed gram-negative. Spore stain: Species of the genera Bacillus and Clostridium produce a highly resistant body called an endospore (a cell with a tough spore coat). The endospore provides the bacteria with the capability to survive long periods of time in high temperatures or toxic chemicals. Endospores are also resistant to standard staining techniques, and heat must be applied along with a suitable stain in order to stain the endospore. Malachite green dye used in conjunction with steam is a common spore stain and is not removed from the endospore by washing. Safranin counterstain stain is then applied to stain the cell interior light red and contrast with spore coat green stain. The spore stain can also be used to visualize fungal endospores from molds and yeast, which are a diverse group of heterotrophic organisms comprising the higher fungi. Many are saprophytes and digest dead organic matter and waste, while others are parasitic and obtain their nutrients from the tissues of the other organisms. Most fungi, such as the molds, are multicellular, but yeasts are unicellular. Fluorescent staining: Chromofluors are chemicals that adsorb light at one wavelength to produce excited electrons, that emit light at another wavelength = fluorescence. To visualize these stains a fluorescent microscope is required, which will illuminate with ultraviolet light to produce the excitation wavelength required for the chromofluor emission. Filters are used to isolate emitted light at a particular wavelength and separate it from other wavelengths traveling to the ocular lens. A dark field condenser is used to create dark background. Chromofluors can

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be used to directly stain molecules within the cells, such as DNA or RNA, or they may be coupled to other probe molecules (usually antibodies) that bind specific components of the cell. DAY 1: GRAM AND SPORE STAIN. In this laboratory we will examine three bacterial species using differential staining procedures which will include the Gram and spore stains. A demonstration of fluorescent microscopy will also be presented. IMPORTANT: Notes on the use of the microscope Never dust a lens by blowing on it as harmful saliva will be deposited on lenses. Use only lens paper to clean after use - Never use facial tissues to clean lenses. Avoid touching lenses. Light fingerprints can seriously degrade image quality. Use proper immersion oil on immersion objectives as specified by manufacturer. Avoid getting immersion liquid on non-immersion objectives. Keep microscopes covered when not in use. Procedures: A. Preparation and Fixation of Bacteria. 1. With a wire loop, place a small drop of each of the three bacterial suspensions on a clean slide. (see Figure below) 2. For multiple samples use grease pen to prepare individual circles for each sample. 3. Spread the drop on the slide to form a thin film. 4. Allow film layer on slide to dry by holding high above (film side up) a Bunsen flame (Dont over heat!). 5. When film is dry, pass the slide three times through Bunsen flame. This is called heat fixing. The purpose of heat fixing is to kill the microorganism, coagulate the protoplasm of the cell, and cause it to adhere to the slide. B. Gram Stain Procedure 1. Cover the entire heat fixed smear with crystal violet dye and let stand for 30 seconds. 2. Tilt slide so as to drain off excess crystal violet to run off into sink and rinse very gently for 1-3 seconds with deionized water, drain. 3. Cover the smear with iodine solution (mordant) and let stand for 30 seconds. 4. Tilt slide to drain off excess iodine solution and rinse gently with deionized water. 5. Dry slide between blotting paper. 6. Flood slide with ethanol (decolorizing solution) for 10-20 seconds. 7. Rinse ethanol from slide with deionized water to stop the decolorizing process. 8. Cover the smear for 20 seconds with safranin counterstain. 9. Wash slide with running water, gently blot dry, and allow to air dry. 10. Examine the stained preparation using oil immersion objective. 11. Examine and make drawings the stained microorganisms for the following: Size and shape Arrangement (singly, pairs, chains, clusters, etc.) Pleomorphisms (the same organism having more than one form) Gram-positive vs. gram-negative coloration Important note: The Gram stain should not be taken as an absolute indicator of gram-positive or gram-negative because, in certain instances, the Gram stain will not give the characteristic or expected reaction. The Gram stain is based on how quickly cells lose the crystal violet-iodine

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complex during the decolorization step. It is possible for Gram-positives to appear Gramnegative reaction (or vise versa) under certain conditions. Factors that could possibly affect Gram stain reaction include the following: 1. Overheating the slide during the fixation step causing cells to burst. 2. Too many cells: The higher the number of cells the longer the decolorization takes or it may be incomplete altogether. This would result in the gram-negative cells appearing to be gram-positive. 3. The extent to which the smear is washed. Excess dye remaining from too little washing or over-decolorization if over washing occurs. 4. The age of the culture suspension from which the smear is made. C. Spore stain procedure:

1. Heat fix bacterial and fungal suspension as described above in Part I. 2. Cover smear with malachite green and place over boiling water for five minutes. (Keep smear saturated by adding additional malachite green if stain boils off.) 3. After 5 min, remove slide and cool. Gently wash with deionized water for 20 sec. 4. Counterstain with safranin dye for 30 seconds. 5. Gently wash with deionized water and blot dry. 6. Examine the stained endospore preparation using oil immersion objective. 7. Make drawings of the stained microorganism for the following: Size and shape Arrangement Polymorphism Presence of spores

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STUDY QUESTIONS: 1. Diagram the difference in the Gram negative and Gram positive cell walls and explain how these difference contribute to the results of the gram stain. 2. Why would you see differences in morphology of cells within a pure culture? 3. Based on your previous knowledge of microbiology, which strain would you identify as Escherichia coli, Bacillus subtilus, or Staphylococcus aureus? 4. What other test would you suggest for confirmed identification of species?

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LAB EXERCISE 4: ENUMERATION OF MICROORGANISMS In order to determine food product safety the total number of bacteria within a sample is frequently determined. This laboratory will examine advantages and disadvantages of two methods for enumerating microorganisms in foods: Direct Microscopic Count (DMC) and Standard Plate Counts (SPC). Direct microscopic count is also referred to as the Petroff-Hausser method and uses slides embedded with grids of etched squares that contain a specified volume of microorganisms to be counted. The etched area is covered with a glass slip at a fixed distance from the etched surface, and bacteria are counted using the high dry (40X) objective of the light microscope. This method is only accurate for food samples containing a large number of microorganisms (4 x 106 to 2 x 107 bacteria/ml). The major sources of error are inaccuracies in diluting samples and in filling the etched chambers. Also food particles mask the microorganisms and lead to an underestimation. The DMC has been used most extensively with milk. A measured volume of milk is spread over the surface of the area over the etched grid, dried, stained, and viewed under oil-immersion. To determine the number of bacteria per field, clusters, clumps, or chains are counted as one bacterial cell because each cluster would give rise to a single colony if plated on solid medium. Limitations of this method include the inability to determine low concentrations of cells, to count motile cells, and to distinguish viable and non-viable microorganisms (unless the method is combined with the use of vital fluorescent dyes described below for viability determination). Advantages are that results are obtained rapidly and inexpensively with relatively simple equipment. Standard plate counts. Bacteria may also be enumerated by either spread or pour plating to solid media for standard plate counts. Plating to agar media allows determination of viability, as numbers of bacteria are calculated by the number of colony forming units (CFUs). Samples are serially diluted and dilutions are applied to agar plates. For spread plates samples are spread over the agar surface with a glass rod, while the pour plate method uses molten agar mixed with sample and poured onto the agar surface. With the SPC procedure we assume that a single cell will give rise to a separate colony; however, this method may not always measure the actual total number of organisms. Cells in pairs or clusters can still produce a single colony and thus underestimate actual number of bacteria. Also, not all strains of microorganisms will always grow on all media under all conditions. Bacteria that are stressed or injured may become noncultureable and require special recovery media or specific environmental conditions to grow. It is important to remember that determining colony counts from a food product will depend on the growth characteristics of organism itself. Some species may spread or swarm over the plate and present difficulties in determining a single CFU. Swarming is the coordinated migration of multicellular colonies that results in the production of non-separate colonies. Therefore, the plate count technique is usually an estimation of the actual number of living bacteria in a sample. The counts obtained by these methods should not be reported as total viable cell counts but rather as CFU per unit of sample. Although there are inherent inaccuracies in this method, the plate count is still the most accurate and widely used method for enumerating bacteria. Rules for Counting Colonies on Plates and Recording Data: To calculate the Aerobic Plate Count, multiply the total number of colonies counted by the reciprocal of the dilution factor. The

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dilution factor is simply the amount of sample transferred over the total volume of the sample from which it was taken. 1. How many to count. To obtain the Aerobic Plate Count, count duplicate plates from dilutions that produce 25 to 300 colonies and average the two counts. Use a Quebec colony counter equipped with magnification and a guide plate ruled in cm2. 2.Consecutive dilutions. If plates from two consecutive serial dilutions yield 25 to 250 colonies, compute the count per milliliter for each dilution by multiplying the number of colonies per plate by the dilution used. Report the arithmetic average as the Aerobic Plate Count per milliliter, unless the higher computed count is more than twice the lower one. If this is the case report the lower computed count as the Aerobic Plate Count per milliliter or per gram, as applicable. 3. No plate with 25 to 250 colonies. When number of CFU per plate exceeds 250, for all dilutions, record the counts as too numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those portions of the plate that are representative of colony distribution. Mark calculated APC with EAPC to denote that it was estimated aerobic plate count from counts outside of 25-250 per plate range. 4. All plate with fewer than 25 colonies. If plates from all dilutions yield fewer than 25 colonies each, record the actual number of colonies on the lowest dilution and report the count as the Estimated Aerobic Plate Count per milliliter or per gram. 5. Plates with no colonies. If plates from all dilutions of any sample have no colonies and inhibitory substances have not been detected, report the count as less than (<) one times the corresponding lowest dilution. For example, if no colonies appear on the 1:100 dilution, report the count as less than 100 (<100) Estimated Aerobic Plate Count per milliliter or per gram. 6. Spreaders. If spreaders occur on the plate(s) selected, count colonies on the representative portions of the plate where colonies are well distributed in spreader free areas. If all the plates prepared from the original samples have excessive spreader growth or are known to be contaminated or are otherwise unsatisfactory, report as Spreaders (Spr) or Laboratory Accident (LA). Inhibitory substances in a sample may be responsible for the lack of colony formation. 7. Computing and recording counts. To compute the Aerobic Plate Count, multiply the total number of colonies or the average number per plate by the reciprocal of the dilution used. Record the dilutions used, and the number of colonies counted or estimated on each plate. When colonies on duplicate plates and/or consecutive dilutions are counted and the results are averaged prior to recording, round off counts to two significant figures (one decimal place) only at the time of conversion to the Aerobic Plate Count. Sample preparation is essential to enumeration of bacteria from solid foods. Liquid foods like milk can be examined directly, but solid food samples are generally homogenized in culture media or buffer prior to enumeration. The volume of food will depend upon the matrix and expected level of bacterial contamination. A weighed amount of solid food is blended mechanically with diluent such as phosphate buffered saline (PBS). Generally 1 to 25 grams of food are blended with equal volume of liquid in a blender or stomacher. Liquid foods are added directly to the diluent in measured amounts. Sterile water is not used as diluent because it can cause cell destruction due to osmotic lysis. Adequate sample mixing and changing pipettes to avoid carry over between dilutions is essential to prevent miscalculations.

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Day 1. STANDARD PLATE COUNTS: A. Serial Dilutions: Before samples are enumerated, they usually need to be diluted or there will be too many colonies on a plate to count. Decimal or serial 10 fold dilutions are employed because they are easier to manipulate mathematically (See Figure 1 below). Samples (1ml) are mixed or vortexed and transferred to dilutions tubes (9 ml) using a fresh sterile pipette with each transfer. Dilution protocol is shown below. Samples will be plated in duplicate to agar plates in order to determine CFU/ml. Figure 1. Serial dilutions

Materials: Sample culture (log or stationary?) Phosphate Buffered Saline Pipettes Test tubes Glass spreaders Alcohol Quebec colony counter L agar (LA) Procedure: 1. For sample, prepare 6 dilution tubes with 9 ml sterile PBS, using aseptic technique. Label a series of dilution tubes ranging from 10-1 to 10-6 for each sample, with corresponding plates that are labeled for 10-2 to 10-7. 2. Use a well-mixed sample so that the test portion represents the entire lot. 3. Using aseptic technique, pipette 1.0 ml of sample into 9-mL PBS dilution tube. Recap tube and mix the test tube thoroughly for 10 seconds. Using new pipette, apply 0.1-mL of this dilution to a previously marked Petri dish. Flame spreader and gently spread liquid around plate. Plate each dilution in duplicate. 4. Using new pipette, continue dilutions from first diluted sample and repeat as above until the end of the dilution series. 5. Allow plates to dry and then invert and incubate at 35C for 24 hours. 6. Calculate the number of bacteria from plate counts and convert the CFU/ml to LogCFU/ml. Average the numbers obtained from all groups and calculate the standard deviation. DAY 2: Direct Microscopic Count In this lab, DMC will be used to directly enumerate the number of bacteria in a samples derived either from liquid food products. Numbers obtained for all lab groups will be compiled and used to determine standard deviation. One of the most common counting chambers for DMC 19

is the hemocytometer, also used for counting blood cells. The counting grid is divided into 25 small squares, further divided into 16 smaller ones (Fig. 1). The depth of the hemocytometer chamber, 0.1 mm differs from Petroff-Hausser counter which is 0.02 mm, and thus calculations will also differ between methods. Figure 1. Hemocytometer grid
Large central square = 1mm2 with 25 small squares further subdivided into 16 squares

Materials: Gloves Biohazard waste container Hemacytometer slide and glass cover slip Microscope Disposable pipettes and Pipettors Methyl violet stain Bacterial culture Disinfectant Water bottle PBS dilution tubes (9 ml) Procedure 1. Add 1 drop of methyl violet stain to empty test tube 2. Add 1 ml of sample or 1:10 diluted sample to test tubes with dye. 3. Place 10l of stained cells onto the notch of the grid-etched slide. Cover drop with a cover glass and read the number of bacteria using the 40X objective. 4. The cells to be counted will appear green with a purple border. If there are more than 100 cells per 1mm large square, count the next dilution of sample. 5. Count the cells in the large 1mm center square containing 25 small squares (See Figure 1 below). If there is more than one cell per small square, count all the cells in five small squares (four corners and the center). Regard a clump of cells as one cell. Multiply number obtained by 5 to estimate the total for 1 mm2. 6. If there is less than one cell per square, count all 25 large squares. 7. Bacteria/ml = the number of bacteria in the 25 squares x 104 x dilution factor. 8. Count both chambers and use the mean of the two counts. 9. Rinse slides with disinfectant and then water to biohazard waste . STUDY QUESTIONS: 1. How do the two methods of enumeration compare in terms of sensitivity of detection? 2. What does the standard deviation indicate? 3. Do the numbers of cells obtained by DMC indicate they were all viable? 4. Can the food matrix influence the results?

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LAB EXERCISE 5: MOST PROBABLE NUMBER AND INDICATOR ORGANISMS Most probable number (MPN) is a procedure to estimate the population density of viable microorganisms in a test sample. It applies the theory of probability to positive growth responses in a standard dilution series or end point titration. Growth of bacteria is obtained in enrichment broth, which is generally a non-selective medium that encourages the growth of injured or stressed cells. A positive growth response is indicated by turbidity or gas production in fermentation tubes. The number of sample dilutions to be prepared is based on the expected population within the sample. Generally tenfold dilution is used with replicates of 3, 5 or 10 test tubes for each dilution in the MPN series. When a higher number of tubes are inoculated in the series, the confidence limits of the MPN are narrowed. Most reliable results occur when all tubes at the lower dilutions are positive and all tubes at the higher dilutions are negative. For large microbial populations, the MPN value is generally not as precise as population numbers derived from direct plating methods, and it should be emphasized that MPN values are only estimates. MPN values are, however, particularly useful when low concentrations of organisms (<100/g) are encountered in such materials as milk, food, water and soil or where the matrix may interfere with obtaining accurate colony counts. Coliforms. MPN is commonly used to estimate the numbers of coliform bacteria in food. These organisms are defined as gram-negative, non-sporeforming, facultative rods that ferment lactose with acid and gas formation within 48 hrs at 35C. These organisms are natural flora of the intestines of warm blooded animals, including humans. Collectively, coliforms are referred to as indicator organisms because they indicate the presence of animal or human fecal contamination. The historical definition of this group has been based on the methods used for their detection, primarily lactose fermentation. Although E. coli is nearly always found in fresh fecal pollution from warm-blooded animals, other coliform organisms may be found in the absence of E. coli. The genera Escherichia, Enterobacter, Klebsiella, and Citrobacter usually represent the majority of coliform isolates, with Enterobacter the most frequently isolated. It is important to note that not all coliforms originate from sewage, and E. coli may be more readily affected by conventional water treatment than other coliforms. Differentiation of coliform types is valuable in determining the source of increased coliform densities. Large numbers of coliforms of the same type in water source suggests that multiplication has occurred. Industrial waste containing high concentrations of bacterial nutrients are capable of promoting growth of coliforms in effluents and receiving waters. Elevated temperatures (45C) are needed to discriminate organisms of fecal origin from others in the coliform group. Thus, incubation at 45C is used to determine fecal coliform numbers, while total coliforms are determined by incubation at 35C. E. coli detection is considered to be a more accurate measure of fecal contamination and requires confirmatory assays or the use of media, such as EMB, which is selective and differential for the species. E. coli LST-MUG broth combines lactose medium (LST) with a substrate of methylumbelliferone glucose (MUG) to release fluorescent compound 4-methylumbelliferone in the presence of the enzyme glucuronidase, which is produced by the majority of E. coli (94%) but not by other coliforms.

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Day 1: Total and Fecal coliform MPN. In this exercise you will perform coliform MPNs from a water sample or from samples brought to class. Fecal coliform detection is a simple 24-48 h test using A-1 medium with a Durham tube. A-1 is a differential medium that uses the production of gas, from the fermentation of lactose, to indicate the presence of coliforms. Durham tubes are small tubes that are placed upside down inside larger tube to catch some of the gas that is produced during lactose fermentation. Samples of different amounts are inoculated directly into replicate tubes, alternatively samples can be diluted (usually 10 fold) and volumes of diluted samples can be used to inoculate tubes. In this case we will use 3 replicate tubes at each of 4 dilutions for a 3-tube MPN with 4 dilutions. Materials: A-1 MPN tubes Whirl-Pak bags 10-ml pipettes Pipettes and tips Portable UV light UV protective goggles EM agar plates Wooden applicator sticks Procedure: 1. Two racks with A-1 Medium with Durham tubes will be provided. 2. Set up and label a three-tube MPN rack with a row for each of four dilutions for the Total coliform MPN. The first row of three tubes should contain 10 ml of 2X or 1X A-1 media for liquid samples, depending on the total volume of sample. The remaining three rows of three tubes should contain 10mL of 1X A-1 media. (Solid samples will all use 10ml of 1X medium). 3. Shake sample bag (Whirl-Pak) or tube 25 times within 7 seconds in a one-foot arc to mix sample. Use a well-mixed sample so that the test portion represents the entire lot, dilute sample 10-1, 10-2, 10-3. 4. Inoculate each of the three tubes in the first row containing either 2X or 1X A-1 media with either 10 ml or 1 ml of undiluted sample, respectively. 5. Inoculate each of the three tubes in the next rows (containing 1X A-1 media) with 1.0 diluted water sample. 6. Incubate all tubes at 35 oC (0.5oC) for 48 hours for total coliform determination. 7. Repeat the above MPN method for the Fecal Coliform MPN with a second set of A-1 MPN tubes, and incubate tubes at 35oC (0.5oC) for 1 hour. 8. After 1-hour incubation, transfer these tubes to 44.5oC (0.2oC). 9. At 24 and 48 hour total incubation time, record gas production as positive. All tubes exhibiting a positive result from Fecal coliform MPN should be streaked for isolation onto EMB Medium for a confirmation of E.coli. Incubate plates at 35C for 24h and record results.

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DAYs 2 and 3. Computation of MPN Results: 1. Write down the number of positive tubes for all dilutions at 24 and 48 hours. To obtain 3 digit number for MPN table, begin with the first dilution that is completely negative or the last dilution with positives and the next two dilutions with positives. If all have positives, start with most diluted sample. (as an example: 3-2-0). 2. Most Probable Number (MPN) values are determined from established tables. Look up the corresponding three-digit number on the MPN table. This number should be reported as an MPN of Fecal Coliforms per g or ml of sample. LAB WRITE-UP: 1. How did the numbers compare for the different assays? 2. How did the numbers compare at 24 and 48h? 3. Which numbers would you expect to be higher: total, fecal, or E.coli MPN? 4. How can you adjust the sample size for MPN determinations? 5. What are some limitations of fecal coliform MPN?

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LAB EXERCISE 6: Escherichia coli Eschericia coli is a gram negative bacillus in the family Enterobacteriaceae, which can be classified as diarrheogenic or nondiarrheogenic according to the effects on the human host. The normal flora of the human intestine harbors nondiarrheogenic E. coli that are considered relatively harmless to the host. These generally harmless bacteria are frequently used as indicators of fecal contamination. Most strains will grow under conditions for fecal coliform analysis, but are differentiated by the following characteristics: 1. Methyl red positive 2. Voges-Proskauer negative 3. Does not use citrate as sole carbon source 4. Indole positive are E. coli type 1 and associated with mammalian intestines Methods of detection: Typical E. coli assay include the following: 1. IMViC tests: determine indole, methyl red, V-P, citrate characteristics but are time-consuming 2. MacConkeys agar: selective/differential agar used to detect coliforms such as E. coli in milk and water. It contains peptone, bile salts, NaCl, and lactose with an indicator dye to indicate fermentation. The inhibitory action of bile salts on the growth of gram-positive organisms allows for the isolation of gram-negative bacteria. Incorporation of the carbohydrate lactose, and the pH indicator neutral red permits differentiation of enteric bacteria on the basis of their ability to ferment lactose. Colonies that ferment lactose will appear pink or red, while nonlactose fermentors will be colorless. Typical appearances are as follows: E. coli red, non-mucoid; Klebsiella- pink, mucoid; Salmonella- colorless. 3. EMB (described in Lab 3) 4. Violet Red Bile Agar: modification of MacConkeys and includes crystal violet to inhibit gram positives and produce more red E. coli colonies. 5. MacConkeys Sorbitol Agar (MSA): uses sorbitol instead of lactose fermentation as an indicator. 6. MUG ASSAY: uses the substrate 4-methlumbelliferyl PD-glucuronidide (MUG), which will fluoresce upon enzymatic degradation. Most (93%) E. coli produce the enzyme B- glucuronidase, which is detected in this fluorogenic assay. 7. E.coli Petrifilm (3M Company): Contain ready-made media enriched with standard nutrients, a gelling agent, and indicator dyes. Includes Violet Red Bile (VRB) agar, which detects glucuronidase activity. Pathogens: Positive results for E. coli may indicate not only fecal contamination but also the presence of human pathogens. Six groups of E. coli are known as diarrheogenic including enteropathogenic (EPEC) strains that attach to the brush border of the intestinal epithelial cells and cause a specific type of cell damage called effacing lesions. Effacing lesions represent destruction of brush border microvilli adjacent to adhering bacteria. Enterotoxigenic (ETEC) strains produce two distinct enterotoxins, which are responsible for diarrhea and distinguished by their heat stability: heat stable enterotoxin (ST) and heat-labile enterotoxin (LT). The enteroinvasive (EIEC) strains cause diarrhea by penetrating and multiplying within the intestinal epithelial cells. The enteroaggregative (EaggEC) strains show unique localized regions with a

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stacked brick appearance. The diffuse adhering (DAEC) strains adhere over the entire surface of the epithelial cells and usually cause disease in immunocompromised or malnourished children. The enterohemorragic E. coli (EHEC) include E. coli 0157:H7. The first E. coli 0157:H7 outbreak occurred 1982 at fast-food restaurants in Oregon and Michigan. The first case documented was from a young girl who died from hemolytic uremic syndrome (HUS) as a result of eating an undercooked cheeseburger. During an investigation, the CDC team traced the bacteria back through the meat slaughtering and distribution system to the farm, pinpointing its reservoir in cattle. Entrance into the human food supply is through the contamination of meat and produce with fecal material. EHEC is considered a major health concern in the US and it is responsible for an estimated 10,000- 20,000 infections and 250 deaths per year. This dangerous serotype of ubiquitous and normally harmless genus has the ability to attach to human intestinal cells and produces potent toxins that produce bloody diarrhea. It may also enter the blood stream and the renal system, producing high mortality particularly in children. There is no cure for an infection with this toxin. Although bacteria are killed by antibiotics, toxin is already present by the time symptoms appear. Thus, antibiotics are not recommended, as they can set the stage for complications by destroying those harmless bacteria that compete the pathogen. The incubation period of hemorrhagic colitis usually lasts for 3 to 4 days with a range of approximately 2 to 8 days. It should be noted that EHEC is not detected in many of the standard tests for E. coli. Nearly all E. coli (93%) ferment sorbitol with the exception of EHEC, which exhibits clear colonies on MacConkey sorbitol agar. It also does not produce B- glucuronidase and therefore is negative for the MUG assay. EHEC is also unique from other E. coli strains in terms of growth characteristics: Although EHEC grows rapidly between 30-40C, it shows slow growth at 44 44.5C. Therefore, it is not be detected in standard screening procedures for fecal coliforms involving these temperatures ranges. However, EHEC resembles other pathogens, such as Salmonella, in that it can be acclimated to grow at very low pH (3.7- 4.0), such as that found in apple juice. Day 1. The purpose of this lab is to compare alternate methods for E. coli detection. Obtain sample and bacterial controls from your instructor. Alternatively, water sample or swabbed surface expected to be positive for E. coli may be used. Determine E. coli counts by 1) SPC by spread plating to petrrifilm vs. MacConkeys Sorbitol Agar and by 2) MPN using MUG broth and streaking to MacConkeys. Materials: Petrifilm, MSA, MUG-MPN tubes (10 ml-1X), pipettes, glass spreader, inoculation loops, burners, alcohol, water bath, and incubators. Prodedure: 1. Use a well-mixed sample so that the test portion represents the entire lot, dilute sample 10-1, 10-2, 10-3. 2. Distribute samples to MPN-MUG tubes as described in Lab 4 and incubate tubes at 35C (0.5C) for 1 hour and then transfer these tubes to 44.5C (0.2C) water bath. 3. Spread Plate samples to MacConkeys in duplicate as described in Lab 4. 4. Place the 3M Petrifilm (Note: Petrifilm should be stored below 8C) on a flat surface and dispense 1mL of your sample onto the center of the Petrifilm. 5. Distribute the sample on the Petrifilm in duplicate by applying downward pressure using a spreader to press the top film over the sample.

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6. Incubate the Petrifilm plates at 35C in stacks placed in a horizontal position with the clear side of the plate up. The stacks should not exceed 20 plates. Day 2 and 3. Record results at 24 and 48h 1. SVC on MSA: determine logCFU/ml as described in Lab 4. 2. Petrifilm: All of the colored dots indicate a colony and should be counted. The circular growth area is 20 cm. You may count the number of colonies in a square area and multiply by 20 to get the total number of colonies. 3. MPN-MUG: Examine the (MUG) test tubes for fluorescence by darkening the room and placing an ultraviolet light over the tube. (POSSIBLE EYE DAMAGE!!! WEAR SAFETY GLASSES!). For safety reasons please do not directly look into the ultraviolet light. Examine the turbidity of the (MUG) test tubes from the incubator at 44.5C. Streak all turbid samples after 24h incubation to MSA and incubate at 35C. Record results for SPC and MPN as logCFU/ml and post on board. Sample Group E. coli Numbers Plate Counts (CFU/ml) MPN (MPN/ml) Petrifilm SPC- MSA MUG 24h MUG 48h MSA

1 2 3 4 5 6 7 8 9 Reference: Harrigan, W. F. 1998. Laboratory Methods in Food Microbiology. Academic Press, London Lab Write-up: 1. Compare results from the different assays and time points. 2. Samples 1-5 and 6-10 are from identical sources respectively and represent duplicate samples. Calculate MPN using 3 vs. 15 tube replicates. How do these results differ statistically? 3. What are the advantages of using MPN-MUG assay? 4. How did the initial numbers of E. coli in sample alter the results? 5. Would any of the tests detect EHEC?

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LAB EXERCISE 7: FUNGI - MOLDS AND YEAST

Molds and Yeast are a diverse group of heterotrophic organisms comprising the higher fungi. Many are saprophytes, which digest dead organic matter and waste, while others are parasitic and obtain their nutrients from the tissues of the other organisms. Most fungi, such as the molds, are multicellular. Yeast are unicellular fungi closely related to the molds. They are ellipsoidal, spherical, or cylindrical cells, and are several times larger than the average bacterial cell. Because of their large size, numerous structures and inclusions may be readily seen using a light microscope. In the food industry, the presence of molds or yeast may be either beneficial or detrimental. Molds are used in the manufacture of cheeses such as Blue, Camembert, and Stilton as a ripening substrate. They are also employed to produce enzymes and acids as in the case of various Oriental foods such as soy sauce and miso. Yeast are used in the manufacture of a number of foods through fermentation, such as beer, wine, vinegar, and some cheeses, which are surface ripened. Yeast, like the molds, are grown for their enzymatic action in foods, but may also serve a source of single cell protein. A special strain of Saccharomyces cerevisiae, which produces large amounts of carbon dioxide, is added to bread dough to make it rise. Mold spoilage is frequently found in nuts and oilseeds and in refrigerated foods such as cheese and cured meats. Rhizopus nigricans is the most common bread mold, but several other species, which thrive in bread, are Penicillium, Aspergillus, and Monilla. The contamination of bread by Monilla, a pink bread mold, is extremely troublesome because it is difficult to eliminate from a bakery once it has become established. Bacillus species are the most likely to contaminate Rye bread, which hydrolyze proteins and starch and give the bread a stringy texture. Yeast can cause spoilage in certain foods such as sauerkraut, pickles, meats, and other foods. Fresh fruit juices, because of their high sugar and acid content, provide an excellent growth environment for molds and yeast. Penicillium expansum, which grows on apples, produces a toxin, patulin, that can contaminate cider. Other molds in foods are capable of producing other toxic compounds called mycotoxins, of which aflatoxins are an example. The body of a fungus is called a thallus. The thallus consists of a mass of intertwining branching threads called mycelium. A single strand of these thread-like structures is called a hypha. Much of the mycelium of each mold grows within or on the surface of the growth medium and functions to extract nutrients for its survival and growth. This is termed vegetative mycelium. Growing above this vegetative mycelium are specialized fruiting structures that produce asexual or sexual spores. Molds and yeast are grouped into four classes, based on their mode of sexual reproduction: 1. Phycomyces resembles algae but differ in lacking chlorophyll. This class includes both aquatic and terrestrial molds in which sexual spores, when produced, are borne exposed. The growth of molds in this class tend to spread over the entire surface of the culture plates, appearing not a single colony but as a fibrous coarse mass, whose borders are limited only by the walls of the petri plate. The vegetative mycelium of the Phycomyces is coenocytic, or nonseptate (without crosswalls) and consists of a continuous mass of multinucleate protoplasm within the tube-like cell walls of the mold. The fruiting, stalklike structures of the Phycomyces produce sporangiospores, an asexual spore enclosed in sporangia, or spore case. Less frequently, molds produce sexually by the fusion of two 27

hyphae, which give rise to gametes. From these gametes, sexual spores are produced. In certain Phycomyces genera, the sexual spores are formed singly and without a covering and are termed zygospores. 2. The class Ascomycetes are characterized by the formation of sexual spores called ascospores. These ascospores are produced in a sac like ascus with several spores being formed. Some genera of Ascomycetes do, however, produce asexual spores. These spores are termed condiospores and they are unenclosed. Molds of the class Ascomycetes tend to form discrete colonies on culture plates. 3. The class Basidiomycetes are the fleshy fungi and bear sexual spores, termed basidiospores, in highly developed structures called basidia. 4. The class Deuteromycetes (or Fungi Imperfecti) represent a taxonomic dumping ground for those molds and yeast in which no sexual reproduction has been observed. Within this class are pathogenic fungi such as the genus Trichophyton, which is the cause of athletes foot, and the species Candida albicans, the cause of a throat infection known as thrush. Procedure: Yeast morphology and growth 1. Prepare a suspension of bakers yeast as described on the back of package. Allow to grow for about 45 minutes at room temperature. 2. Make a methylene blue stain of the yeast culture and observe by using a microscope. 3. Make drawings of all observations. 4. Inoculate three tubes of yeast extract sucrose broth with bakers yeast and incubate at 50, 200 (room temp.) and 430C. Molds morphology Growth 1. Observe all mold cultures for characteristics such as color, texture, reverse color, 2. Make a slide mount of each mold culture. Take a small amount of mold growth and suspend it in water on a clean slide and cover with coverslip. Starting with low power magnification, use all three objectives, if necessary, to observe the following: Mycelium Spores if present Fruiting heads Special structures 3. Make drawings of all observations. 4. Select one of the mold cultures and inoculate some spores onto a Potato Dextrose agar plate and a Czapeks agar plate (general purpose media for the cultivation of both molds and yeast). Incubate plates at room temperature in a dark area. Do not invert mold plates during incubation. Check growth in 5 and 7 days. 5. Divide one Potato Dextrose agar (PDA) plate into three sections. Using one mold culture, the bakers yeast and a bacterial culture provided, inoculate each of the three sections on each plate. Incubate the plates and record results in 2 days and again at day 5.

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LAB EXERCISE 8: DETECTION OF SALMONELLA Salmonella are facultatively anaerobic, Gram-negative, rod-shaped bacteria. Salmonella are motile by peritrichous flagella, grow on citrate as a sole carbon source, and generally produce hydrogen sulfide (H2S), although H2S-negative serovars do exist. Well-established culture methods for the detection of Salmonella in various foods are available from the following: 1. FDA, Bacteriological Analytical Manual (BAM): http://vm.cfsan.fda.gov/~ebam/bamtoc.html 2. USDA, Microbiological Laboratory Handbook: http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp 3. American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods. 4th Edition. 4. Culture and rapid methods validated by the AOAC Currently, there are over 2,500 recognized serovars of Salmonella, however 10 serovars are responsible for the majority of illness in humans, known as salmonellosis. Salmonella is widely distributed throughout nature with the primary reservoir being the intestinal track of mammals, birds and reptiles. Salmonellosis is a communicable disease and is readily transmitted from animals to humans, but most often is acquired through the ingestion of contaminated food products. The actual number of cases may be between 400,000 and 4 million/yr. Clinical symptoms include stomach pain, diarrhea, headache, and fever accompanied by chills. After ingestion, bacteria reproduce in the small intestine, and onset of illness usually occurs 8 to 48 hours after ingestion. Symptoms can persist for 3 to 5 days. Salmonellosis can be fatal for the very young, infirm, and those with compromised immune systems. Salmonellosis is usually contracted from food products that have been abused during handling. Abuse can occur through exposure to warm temperatures and/or cross-contamination Media and test kits: The following media are often used for the enrichment/isolation of Salmonella from food. Primary enrichment media: a. Universal pre-enrichment broth (UPB): Used for the recovery of sublethally injured Salmonella and Listeria from food products. Sodium and potassium phosphate buffers provide sufficient buffering capacity to prevent the rapid decrease in pH, allowing for the repair of injured cells which may be sensitive to low pH. UPB also contains peptones, essential ions, dextrose as an energy source, and sodium pyruvate to aid in the recovery of stressed cells. Secondary (selective) enrichment media: a. Tetrathionate (TT) broth: Tetrathionate and excess thiosulfate suppress coliform microorganisms and other accompanying bacteria, whereas all tetrathionate-reducing bacteria (e.g. salmonellae and Proteus) can multiply more or less normally in this medium. Acidic tetrathionate decomposition products are formed, which are neutralized by calcium carbonate. TT broth contains bile salts, which largely inhibit all microorganisms that do not normally live in the intestine. The addition of brilliant green suppresses, above all, the Gram-positive microbial flora. An iodine solution is also added to provide an inhibitory effect. b. Rappaport-Vassiliadis (RV) medium: This medium is a modification of the Salmonella Enrichment Broth with lower concentrations of malachite green and magnesium chloride

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to improve the growth of Salmonella at 43 C. Peptone from soymeal is also used for the same reason. The pH of RV medium is 5.2 for increased selectivity. Isolation/plating media: a. Bismuth sulfite (BS) agar: Brilliant green and bismuth largely inhibit the accompanying bacterial flora. Colonies of H2S-positive salmonellae exhibit blackening due to the formation of iron sulfide. Reduction of bismuth ions to metallic bismuth produces a metallic lustre around the colonies. b. Xylose lysine desoxycholate (XLD) agar: Degradation of xylose, lactose and sucrose to acid causes phenol red to change its color to yellow. Production of hydrogen sulfide is indicated by thiosulfate and iron (III) salt, which react to form a precipitate of black iron sulfide in the colonies. Bacteria which decarboxylate lysine to cadaverine can be recognized by the appearance of a purple coloration around the colonies due to an increase in pH. These reactions can proceed simultaneously or successively, this may cause the pH indicator to exhibit various shades of color or it may change its color from yellow to red on prolonged incubation. The culture medium is weakly inhibitory. c. Hektoen enteric (HE) agar: When compared with other selective culture media (e.g. SS Agar, BPL Agar and Bismuth Sulfite Agar), HE agar has the advantage that it only slightly inhibits the growth of Salmonella and Shigella, thus giving high yields of these microorganisms, but at the same time ensures adequate inhibition of accompanying microorganisms. Lactose-positive colonies have a clearly different color from lactosenegative colonies due to the presence of the two indicators bromothymol blue and acidic fuchsin. This color difference is also observed for colonies, which can only slowly ferment lactose due to the presence of sucrose and salicin. These reactive compounds can be fermented more easily - false-positive pathogenic results are thus avoided. The combination of thiosulfate as a reactive compound with an iron salt as an indicator causes H2S-positive colonies to become black in color. The mixture of bile salts suppresses the growth of most of the accompanying microorganisms. Differential media: a. Triple sugar-iron (TSI): Designed to differentiate among the different groups of Enterobacteriaceae, which are capable of fermenting glucose with the production of acid. TSI slants contain lactose, sucrose, glucose and the acid base indicator phenol red. Degradation of sugar and accompanying acid production are detected by the pH indicator phenol red, which changes its color from red-orange to yellow, on alkalinization it turns deep red. Thiosulfate is reduced to hydrogen sulfide by several species of bacteria, the hydrogen sulfide reacts with an iron salt to give black iron sulfide. b. Lysine iron agar (LIA). Lysine is decarboxylated by LDC-positive microorganisms to give the amine cadaverine, which causes the pH indicator bromocresol purple to change its color to violet. As decarboxylation only occurs in an acidic medium (below pH 6.0), the culture medium must first be acidified by glucose fermentation. This medium can therefore only be used for the differentiation of glucose-fermenting microoganisms. LDC-negative, glucose-fermenting microoganisms cause the entire culture medium to turn yellow. On prolonged incubation alkalinization of the culture medium surface may occur, resulting in a color change to violet. H2S production causes a blackening of the culture medium due to the formation of iron sulfide.

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Enterotube II: The BBL Enterotube II is a prepared multimedia tube for the rapid identification of Enterobacteriaceae. Each compartment of the Enterotube II tests for a specific biochemical trait of the microorganism. After incubation, each compartment is observed for color change, which indicates a positive result. The results are recorded and a score for each result is tabulated. Using the provided score card, the scores are combined to form either a 5 or 6 digit code. The resulting code can be used to identify the unknown microorganism. In some cases, further biochemical testing is needed to differentiate among several possible identifications. Reveal for Salmonella: The Reveal for Salmonella test system is a presumptive qualitative test that detects the presence of Salmonella organisms in foods within 21 h total testing time. Foods are enriched with a proprietary resuscitation medium called Revive and then selectively enriched with either Selenite Cystine or Rappaport-Vassiliadis selective media. The enriched culture is used to inoculate the detection device, which initiates a lateral flow through a reagent zone containing anti-Salmonella antibodies conjugated to colloidal gold particles that capture antigens present in the culture. The antigen-antibody complex migrates farther and is captured by an additional anti-Salmonella antibody, causing the colloidal gold to precipitate and form a visual line, indicating a positive result. A procedural control line also will form regardless of the presence of Salmonella organisms to indicate the test is working properly. PROCEDURE: The following procedure is based on the U.S. Food and Drug Administrations Bacteriological Analytical Manual (BAM) Online, updated September 2005. A flow chart has been provided as an overview to the analytical procedure for the isolation of Salmonella. DAY 1 a) Each group will be given a samples that contain Salmonella positive control, a nonSalmonella negative control and two unknowns that have been incubated in Universal pre-enrichment broth (UPB) for 24 h at 35 2.0C. b) For each culture: Transfer 1.0 ml of the UPB enrichment to a 9.0 ml tube of tetrathionate (TT) broth. Transfer 0.1 ml of the UPB enrichment to a 9.9 ml tube of RappaportVassiliadis (RV) medium. c) Incubate RV medium 24 2 h at 42 0.2C (circulating, thermostatically controlled, water bath). Incubate TT broth 24 2 h at 35 2.0C. DAY 2 a) Vortex tubes and streak broths for isolation on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. In addition, streak a MacConkey plate (to be used for the Enterotube II). Incubate plates 24 2 h at 35C. b) Save the RV medium for use on DAY 4 at 4C. DAY 3 a) Examine BS, XLD, and HE plates for presence of colonies that resemble Salmonella.

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TYPICAL Salmonella COLONY MORPHOLOGY: 1. Hektoen enteric (HE) agar. Blue-green to blue colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies. 2. Xylose lysine desoxycholate (XLD) agar. Pink colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies. 3. Bismuth sulfite (BS) agar. Brown, gray, or black colonies; sometimes they have a metallic sheen. Surrounding medium is usually brown at first, but may turn black in time with increased incubation, producing the so-called halo effect. ATYPICAL Salmonella COLONY MORPHOLOGY: In the absence of typical or suspicious Salmonella colonies, search for atypical Salmonella colonies as follows: 1. HE and XLD agars. Atypically a few Salmonella cultures produce yellow colonies with or without black centers on HE and XLD agars. In the absence of typical Salmonella colonies on HE or XLD agars after 24 2 h incubation, then pick 2 or more atypical Salmonella colonies. 2. BS agar. Atypically some strains produce green colonies with little or no darkening of the surrounding medium. If typical or suspicious colonies are not present on BS agar after 24 2 h, then do not pick any colonies but reincubate an additional 24 2 h. If typical or suspicious colonies are not present after 48 2 h incubation, then pick 2 or more atypical colonies. b) Pick 2 or more colonies of Salmonella from each selective agar. Lightly touch the very center of the colony to be picked with sterile inoculating needle and inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 5-8C. Again without flaming, inoculate a tube of tryptic soy broth (TSB). c) Incubate TSI and LIA slants and TSB at 35C for 24 2 h. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H2S production. d) Select an isolated colony from the MAC plate and use to inoculate an Enterotube II as demonstrated by the instructor. Incubate at 35C for 24 2 h. e) Transfer the RV medium (saved from DAY 3) to a fresh RV tube by DAY 4 a) Examine/interpret the TSI and LIA slants as follows: a. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in TSI. b. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct yellow in butt of tube as acidic (negative) reaction. Do not

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eliminate cultures that produce discoloration in butt of tube solely on this basis. Most Salmonella cultures produce H2S in LIA. Some non- Salmonella cultures produce a brick-red reaction in LIA slants. b) Examine and score the Enterotube II. Use the codebook to determine the identity of your unknown microorganism. c) Describe your results, including any color changes seen in broth, agar plates and slants. What do these color changes tell you about the isolated microorganism(s)? d) What was the identity of your unknown? e) Were there any differences in the results obtained from enrichment in TT broth versus enrichment in RV broth? If so, describe those differences and why they might have occurred STUDY QUESTIONS: 1. What is the lowest initial Salmonella concentration detected by this FDA protocol? 2. The USDAs method for evaluating meat products for Salmonella uses a different medium for primary enrichment. What is the name of this broth and why does the use of it seem odd? (HINT: What are the biochemical traits of Salmonella?) Explain the rationale behind using this broth for the enrichment of Salmonella. 3. Are there any other bacteria in the Enterobacteriaceae that can produce typical colonies on the plating media used in this lab? If so, what are they and how could you determine if they are Salmonella or not using conventional culture techniques?

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LAB EXERCISE 9: Immunoassays Agglutination assay: Antibodies or immunoglobulins (Ig) are proteins produced by the immune system in response to infectious disease. They are generally specific for a particular organism and can bind with high affinity to a particular antigen on the cell surface. Common types of antigens include LPS (O antigen), capsular polysaccharide (K antigen), flagella (H antigen). These structures contain epitopes (the antibody-binding portion of the antigen) that are specific to a particular species or strain of bacteria and bind the unique Fab segment of the antibody. Some antigens also bind less specifically to the tail of Fc portion of the antibody and can detect all strains of a species. The antigen-antibody binding is commonly used for detection of foodborne pathgens. One of the simplest tests is the agglutionation assay, which is detected visulally due to the clumping of bacteria and antibody. An example of an agglutination assay is the one used for Staphylococcus aureus. These Gram positive, halophilic cocci can be highly pathogenic and produces a variety of toxins, which can be lethal to humans. It is responsible for 20-50% of all foodborne illness outbreaks in the United States. The bacterium is sensitive to heat and nearly all sanitizing agents. Therefore, detection of S. aureus in processed foods is an indication of poor sanitation, and post-processing contamination is usually the result of human contamination or contamination from unsanitary surfaces. Foods that are suspected to be the source of staph food poisoning are examined for the presence of the organism and/or its enterotoxins. Microbiological tests for S. arueus include the use of a differential/selective BairdParker medium. Colonies should appear as black, shiny, convex colonies with a narrow white margin, surrounded by a clear zone. The BBL Staphyloside Latex Test is an agglutination assay that can be used to detect bacterial surface structures that are unique to pathogenic S. aureus. One such component is coagulase enzyme, which the catylses the fomation of fibrin clots from the fibrinogen in plasma. It is produced as both a soluble and surface-associated form and is also referred to as clumping factor. Another surface factor is Protein A, which is able to bind the non-specific or Fc end of IgG antibody molecules and thus render them inactive. The Staphyloside Latex test consists of blue latex particles coated with human fibrinogen and IgG. Colonies of staphylococci which have clumping factor or Protein A present, will cross linking the latex and produce visible agglutination of latex particles. The agglutination will occur notably with S. aureus. If neither clumping factor nor Protein A is present, no agglutination will occur, and the result will be regarded as negative. The reaction is illustrated here.

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ELISA assay: Another very powerful immunoassay is the Enzyme-Linked ImmunoSorbent assay (ELISA). This method is also referred to as a sandwich ELISA, and the basic principles are outlined in this figure. 1.Bind Ab Basically in Step 1 a high affinity capture antibody 2.Add Ag (Ab) that is specific for the target organisms is absorbed 3.Wash Unbound Ag to the surface of a microtiter plate. If cells or antigen (Ag) are present that correspond to 4.Add labeled Ab this antibody, they will be captured by the antibody in 5.Wash unbound Ab Step 2. In Step 3 bound Ag will remain and unbound will 6. Appearance of green color as increasing concentrations of be removed during washing. enzyme convert substrate, In order to detect the bound indicating increased antigen, a labeled secondary concentrations of antigen antibody conjugated to an enzyme label and also specific for the antigen completes the sandwich in Step 4. This antibody will also bind the cells or antigen and all unbound labeled antibody is removed in the final washing in step5. The enzyme label can then be used for detection of a positive sample. In this assay the enzyme converts the substrate to a green color. Thus, presence of any potential pathogens will be noted by the appearance of a green color. The antibodies in this assay may detect all strains within a species or may be specific for a particular serotype. Antibody Lateral Flow devices: In these assays, reagents are loaded into one end of the device and the sample moves through the paper by lateral diffusion (wicking) until it reached the spot where antibody markers are bound on the paper. Anitbodies are gold conjugated Antibody Lateral Flow Devices and the complex of antibody and antigen continues to move and bind second Reagents are load in one end antibody which captures complex. The of the device captured complex then aggregates and Sample moves through the forms visual indication markers for paper where antibody markers are bound. positive sample.
Visual indicator marks presences.

Positive Result

Positive Control

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Day 1: Salmonella detection assays Microbiological detection of Salmonella spp may require days and may not always produce species-specific results. An alternative method is the enzyme-linked immunosorbent assay (ELISA) such as the TECRA Salmonella Visual Immunoassay. Although, kits like TECRA are rapid, other kits have been developed that require fewer steps and therefore give results more rapidly. An example of this is the Visual Immunoprecipitate Assay (VIP) Salmonella kit. While these kits are faster, they utilize different ELISA concepts. This kit uses a proprietary reagent system to form an antigen-antibody-chromogen complex. When the Salmonella antigen comes in contact with the antibody-chromogen complex, a precipitate forms and causes a color change, which is indicated by the colored line that appears in the detection window. Immunological protocols: Each group will be given two unknown cultures, and results for positive and negative controls will be provided by instructor. The purpose of this lab will be to identify the Salmonella spp. IMPORTANT: Change your pipette tip between reagent bottles. DO NOT use the same tip. Reveal Lateral Flow Device 1.Using the dropper, place 5 free-flowing drops of each unknown into the sample port on the Reveal for Salmonella device. 2.Read the results after 15 minutes at room temperature. Make sure that the device is level and is not disturbed during incubation. TECRA Salmonella Visual Immunoassay ELISA 1. Heat the broth cultures for 15min in a boiling water bath. After 15 min vortex the tubes before use. 2. The four Removawells that are already coated with the capture antibody. One for each sample, one for positive control and one for the negative control (label them accordingly on the top of the plastic cling wrap). 3. Using a new pipette tip for each sample add 200L of the controls and samples into individual wells. Cover the wells with plastic cling wrap film and incubate for 30 min at 35-37C. 4. Quickly invert the holder, emptying its contents into a contaminated waste container. Remove residual liquid by striking the wells firmly several times face down on a piece of absorbent paper towel. This is important for effective removal of sample residue. 5. Add 0.5 mL of wash solution in each well, taking care not to trap air bubbles in the bottom of the wells. Wash and completely empty the wells a total of three (3) times as outlined above. 6. Ensure the Removawells are empty before proceeding. 7. Add 200L of Conjugate [4] to each well. Cover the holder with plastic cling wrap and incubate for 30 min at 35-37C. 8. Empty the wells and wash them thoroughly a total of four (4) times as described in steps 6 and 7.

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9. Ensure the Removawells are empty before proceeding. 10. Add 200L of Substrate [6] to each well. Incubate at 20-25C (room temperature). 11. At 10min the positive control should be equivalent to either Panel 4 on the Color Card. Results can be read visually using the Color Card. Day 2 Agglutination assays Polyvalent Salmonella flagellar (H) test: 1. Add 2.5 ml formalinized physiological saline solution to 5 ml TSB enrichment. 2. Place 0.5 ml of appropriately diluted Salmonella polyvalent flagellar (H) antiserum in a 13 x 100 mm serological test tube. 3. Add 0.5 ml of the TSB enrichment. 4. Prepare saline control by mixing 0.5 ml formalinized physiological saline solution with 0.5 ml formalinized antigen. 5. Incubate the test mixture and the control mixture in a 48-50C water bath. Observe at 15 min intervals and read final results in 1 h.. Staphylococcus aureus agglutionation assay Materials: Baird-Parker medium, plates BBL Staphyloside Latex Test Inoculating loops Procedure: 1. You will be given a positive control (S. aureus), a negative control (E. coli) and two unknowns grown on L agar. 2. Streak these strains for isolation to the Baird-Parker plates and incubate overnight at 35C. Observe and record the colony appearance the next day. 3. BBL Staphyloside Latex Test: (Warning: The reagents contain material from human origin. All reagent materials have been tested for HIV or Hepatitis C, but extreme care must still be taken when handling all materials. The reagents also contain azide, which is very toxic by inhalation, in contact with skin, and if swallowed. Contact with acids liberates very toxic gas. After contact with skin, wash immediately with plenty of water.) 4. Assay positive and negative control strains and unknown strains using the latex test. (NOTE: Better results are obtained from non selective media.) 5. Mix the latex reagent by shaking; expel any latex from the dropper. 6. Dispense 1 drop each of Test Latex onto four circles on the reaction card, and add 1 drop of Control Latex onto another four circles. 7. Using a microbiological loop pick up and smear 5 suspect colonies from each of the strains onto a Test Latex circle and mix bacteria into the Test Latex reagent. 8. Spread to cover the circle. 9. Repeat step 8-9 with the Control Latex for each of the strains. 10. Pick up and gently rock the card for 20 seconds and observe and record agglutination reactions under normal lighting conditions. 11. Dispose of the reaction cards in the orange biohazard bucket.

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Interpretation of test results: Positive: A positive result is obtained if agglutination of the blue latex particles is seen in 20 seconds in the test circle, with no agglutination in the control circle. Negative: A negative result is obtained if no agglutination occurs and a smooth suspension remains at 20 seconds in the test circle. Lab Write-up QUESTIONS: 1. What are the cellular components that are detected by the S. aureus latex test? 2. What are the corresponding host components? 3. What is the difference between Fab and Fc regions of the antibody? 4. If both the test latex and negative control latex agglutinated, is that a false positive? False Negative? 5. Why the TECRA ELISA procedure termed a sandwich ELISA. 6. What are possible advantages of the TECRA ELISA procedure over the VIP kit? 7. What antigens might be the targets for antibodies is this kit? 8. Can immunoassays discriminate virulent vs. avirulent strains?

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LAB EXERCISE 10: DNA PROBE DETECTION OF VIBRIOS DNA probes are used for Colony Blot Hybridization to directly enumerate bacteria in food samples. In this procedure, colonies from standard plate counts are lifted from the L-agar plates to a sterile filter paper and then hybridized with a DNA oligonucleotide probe that is specific for a particular bacterial species. DNA probes are enzymatically labeled with an alkaline phosphatase enzyme. The alkaline phosphatase label to detect probe-positive colonies is visualized by color change of a substrate (NBT/BCIP solution) that reacts with this enzyme. Postive colonies produce brown/bluish colonies, when substrate is cleaved by the enzyme, while negative colonies appear yellow. This protocol is used for enumeration of bacterial colonies on spread plates to detect probe positive colonies in a background of other bacteria. It can be used in conjunction with growth on different media, although it will not work well on differential agar containing dyes. Vibrio species are gram-negative, halophiles that are common to estuarine environments. V. vulnificus remains one of the leading causes of seafood related deaths in humans with a mortality rate of approximately 50%. Disease can result from the consumption of raw or undercooked shellfish, such as oysters and clams, which harbor the bacteria and lead to fatal septicemia (cellular destruction due to accumulation of bacteria in the blood). Exposure of open cuts to the bacterium in seawater can also lead to wound infections. Fortunately, most people are not susceptible to this disease, and fatalities are primarily in individuals with underlying disorders that compromise the immune system, including HIV/AIDS, cancer, diabetes, liver disease or hemochromatosis. Vibrio species are quite variable biochemically and it is difficult to discriminate species based on traditional differential media, such as TCBS (see lab 2). Also growth on selective media generally requires pre-enrichment in broth used for end-point titration of MPN enumeration (see lab 3). Unfortunately, this process requires days and lacks species specificity. This probe is derived from the cytolysin gene (vvh), and has been shown to be very species specific. PROCEDURE: Positive and negative controls should be prepared with each batch of samples and processed simultaneously with unknown samples. Incubation should be done in a water bath or hybridization oven that is accurate within 1 C. DAY 1: 1. Scrub and shuck 4-6 oysters and weigh oyster meat in sterile Petri dish (need at least 50g). Place oysters in sterile blender jar. Add an equal weight or volume (1g: 1ml) of sterile phosphate buffered saline (PBS) to blender jar, using sterile grad cylinder. 2. Homogenize oyster sample for 2 min on hi-speed and add 1 ml of oyster homogenate to 9 ml of APW and continue to serially dilute using the 9 ml APW dilution tubes to the 10-3 dilution. 3. Plate 100 L from each dilution tube to duplicate plates each of LA and TCBS, yielding 2 LA and 2 TCBS plates per dilution (Remember: plating 100 L increases the dilution on the plate, i.e plating 100 L from a 10-6 dilution tube will result in 10-7 plate.) 4. Incubate plates (media side up) at 37C for approximately 24 hours. DAY 2: 1. Count colonies on L agar and TCBS plates and place plates in cabinet at room temperature.

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2. Record numbers and determine CFU/g for comparison to colony blot hybridization results. DAY 3: Preparation of Colony Lifts: 1. Label 85 mm Whatman #541 filters with pencil to record your group number and the dilution of the plate you are blotting. Handle filters aseptically. 2. Overlay colonies on spread plate with filter (pencil side up) so that there are no bubbles under filter by first lining up one edge of the filter with one edge of the plate. (DO NOT pick up filter and replace on agar.). Use a hockey stick to lightly press the filter directly against the agar surface and remove bubbles. 3. Allow filter to sit on plate for 15 min to ensure transfer of smaller colonies. 4. Place 100 mm Whatman #3 filter onto microwaveable tray and saturate with lysis solution (about 3ml) using squeeze bottle 5. Aseptically lift the #541 filter from the agar plate using tweezers and overlay the #541 filter onto the wet #3 filter with colony side up ensuring contact and no bubbles between filters. 6. Microwave filters on medium to high heat until the filters are dry but not brown (about 35 min.). 7. Place #541 filters into a clean container containing ammonium acetate neutralization buffer (about 20 ml per filter) colony side down and incubate at room temperature, with shaking (125 rpm) for 5 min. 8. Drain the neutralization buffer and rinse in 1x SSC buffer (20 ml per filter). Swirl briefly, drain and repeat. 9. Place filters, colony side up, onto absorbent paper and dry completely at room temperature over night. Dried filters may be stored at room temp. in a sealed plastic bag. Alternatively, filters are to be processed immediately and drying is not necessary. Proteinase K treatment: 1. The proteinase K working solution (40 g/mL) will be prepared for you by your TA. Solution is pre-warmed to 42C. 2. Transfer filters to a container with about 5 ml per filter of proteinase solution. 3. Incubate with shaking at 42 C for 30 min. 4. Empty the container and rinse with shaking in1x SSC buffer (20 ml/ filter) at room temp for 10 min. Repeat 3 times. 5. Filters can be dried and stored indefinitely.

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Hybridization Demonstration: 1. Include control filters with each bag. Place 5-10 filters into a bag (Whirl Pack 4.5 X 9) or tupperware container and add 100 ml of pre-warmed hybridization buffer (56 for VVAP). Exclude air from bag and seal or roll up. 2. Incubate with shaking (125 rpm) at temperature for 30 min. 3. In new bag add probe (5 picomoles/filter, usually 3 to 20 l of probe) to pre-warmed hybridization buffer (25-100 ml). 4. Transfer filters with forceps to fresh buffer with probe. Exclude air from bag and seal or roll up. Incubate for 1 h with shaking (125 rpm) at 56C. 5. Transfer filters to container with pre-warmed (56C) 1x SSC/1%SDS using 10 ml/filter with shaking (125 rpm) at appropriate temp for 10 min. 6. In the same container, rinse five times in 1x SSC buffer (20ml/filter) at room temperature for 5 min. each rinse. Filters can be combined at this point with appropriate increase in the volume of buffer. Probe Development Demonstration: 1. In a new petri-dish, develop each filter in 2 ml of NBT/BCIP solution. 2. Incubate at room temperature (or at 35C for faster results). Cover to omit light. Check development of the positive control every 1/2 hr. 3. To stop further development, place filters in new container and rinse 3 times at room temp with DI water for 10 min. each. NOTE: be sure to dispose of the NBT/BCIP correctly. It is a hazardous waste. 4. Place the filters on an absorbent paper in the dark and allow to dry. Positive colonies will have a bluish or brown color. Negative colonies should appear colorless or yellow. The filters should be stored in the dark to prevent color change STUDY QUESTIONS: 1. Create a table comparing your TCBS counts and your colony blot hybridization counts for each dilution. Do these counts differ? In what ways? Give an explanation of your results. 2. What accounts for the specificity of colony blot hybridization? 3. Is colony blot hybridization more specific than traditional plating? than the MPN method? Why or why not? 4. We saw in the ELISA lab that by increasing the speed of an assay we decreased the sensitivity. Is the same true when comparing traditional plating to colony blot hybridization? Why or why not? (hint: base your answer on your results)

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LAB EXERCISE 11: POLYMERASE CHAIN REACTION (PCR) LAB PCR is a method for rapid amplification of short pieces of DNA using DNA polymerase, and was first described by Kleppe, Ohtsuka, and Kleppe in 1971. Saiki and colleagues went on to develop a primer-directed enzymatic amplification of DNA using a thermo-stable polymerase. PCR is one of the most widely applied procedures in biology today. Applications include the following: Identification of species using species- or type-specific primers Molecular characterization: DNA fingerprinting DNA Sequencing: single primer is used to amplify one strand to be sequenced Cloning of targeted genes Gene expression studies using reverse transcriptase PCR (RT-PCR) to detect RNA transcripts of specific genes Quantification of bacteria or expressed genes PCR amplification is one of the most powerful diagnostic tools available for identification of food borne pathogens, providing extremely rapid and specific detection. PCR primers are currently available for every major pathogen, and some of the assays require less than an hour. PCR will often detect as little as one microorganism in a sample. Some sample media can be very complex with a large background of other organisms, such as fecal samples. Thus, this method is exceptionally useful for finding the needle in the haystack. This procedure is based on 1) the specificity of DNA sequences, called primers, which serve as species-specific markers, and 2) the thermal stability of the Taq polymerase enzyme. In PCR reactions, a gene target is selected that is specific for the organism or gene of interest. For example, PCR primers for Salmonella are based on the virulence gene invA. Thus, detection is not only specific for the species but may also determine if strains have virulence potential. Through a series of enzymatic reactions, a small amount of DNA is amplified exponentially to produce thousands of DNA fragments that can then be visualized on an agarose gel. Electrical charge is used to separate DNA fragments by size on the gel. There is a positive charge at one end of the gel box and a negative charge at the other end. DNA is negatively charged and will migrate toward the positive electrode. Smaller fragments of DNA move through the gel more quickly than larger fragments, which allows for separation by size. After migration, DNAspecific dyes are then used to visualize the PCR product. The reaction mixture is prepared in small volumes of 10 to 100L and contains the following components: a) Template or target DNA (10 to 1000 ng) from the microorganism of interest that you are tying to detect in your sample b) Primers or short pieces of DNA (14-45 bases) that correspond to your target DNA and match up or hybridize in different directions at 2 separate locations that are usually 500 to 3000 bp apart. c) Taq polymerase is a heat stable enzyme that functions optimally around 72C, but is stable at higher temperatures, which are needed for denaturation of DNA. Thus the enzyme is added only once but is repeatedly reused for the multiple cycles of the amplification reactions d) dNTPs are nucleotide bases (ATP,CTP,GTP,TTP) or building blocks of DNA. e) Buffers and salts at concentrations that are optimized for the reaction. Generally the MgCl2 concentration is adjusted for optimization. 42

The basic reaction is performed in a programmable incubator called thermocycler, and the procedure is outlined in the following steps and shown in Figure 1: a) Denaturation: The DNA is heated to 94-96C to denature or separate the double stranded template DNA. b) Annealing: The mixture is rapidly cooled to 30-60C, allowing the primers to match up and bind to specific sites on the target DNA. c) Elongation: The Taq polymerase extends the DNA strand begining where the primer binds to produce a new strand of DNA. 72C is the optimum temperature for enzymatic activity. d) Cycling: The steps above are repeated in the same sequence for 25-35 cycles to produce multiple new strand of DNA. e) Final elongation: The elongation temperature is held for about 10 min. to maximize reactions. Figure 1. Polymerase Chain Reaction

Potential Problems include the following: a) Interfering compounds: Heavy metals and other compounds can interfere with the enzymatic activity of Taq polymerase. Thus, sample preparation includes a step where DNA is extracted and isolated from the rest of the cellular material.

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b) Sample size: Some organisms may be present in very low numbers in foods. Although PCR can detect 1 organism in a sample, generally very small samples are analyzed. These problems can be addressed by testing multiple samples and enrichment of samples. c) Viability question: DNA can be extremely stable under certain conditions and PCR may detect DNA that is present from organisms that are not alive d) Quantification: The amount of product produced roughly reflects that amount of template. Therefore more heavily contaminated samples will produce more fragments and exhibit a stronger band. However, exact quantification requires more complex procedures or special equipment. PROTOCOL: Ground rules for molecular biology: 1. Always were gloves (DNases on hands destroy DNA, some of the reagents used are carcinogens). 2. Be aware of cross-contamination (change tips frequently and NEVER use the same tip for different reagents). 3. Always run controls (positive and negative). 4. Due to the extreme sensitivity of the PCR assay, false positive are possible if samples are cross contaminated. False negatives are possible due to experimental error usually arising from pipetting problems. Therefore, positive and negative controls are extremely important, and data is only acceptable when controls show the expected results. Colonies from the previous lab have been selected for DNA extraction and PCR analysis. The PCR primers are based on sequences from the invA gene and should produce a PCR product about 457 bp in length. You will perform PCR amplification with your Salmonella strain. Each group will perform PCR on the following samples: 1. Positive control: DNA from a known Salmonella strain that is positive for invA 2. Negative control for specificity: DNA from V. vulnificus 3. Negative control for contamination: PCR reaction with everything except the target DNA. Water will be added to compensate for the volume. DAY 1: Material needed: Salmonella culture Microcentrifuge tubes Sterile PBS DNA templates from target strain (which you will be extracting) PCR tubes PCR master mix reaction: water, DNA, Taq, dNTPs, buffer, and primers. PCR racks 10-20 l pipettors 200 l pipettors Filtered pipette tips Centrifuge Heat Block Thermocycler 44

DNA extraction: 1. Pipette 1 ml from each of the two (Vibrio spp. and Salmonella spp.) L Broth cultures into two microcentrifuge tubes. Label them accordingly. 2. Centrifuge your tubes at 10,000 rpm for 10 min. 3. Pour off the supernatant, taking care not to dislodge the pellet. 4. Add 400 l of sterile PBS to each microcentrifuge tube. 5. Vortex the tubes until the pellet is completely resuspended. 6. Boil the tubes at 100C for 5 min in the heat block. 7. Centrifuge tubes at 13,000 rpm for 3 min. 8. Transfer supernatants to sterile microcentrifuge tubes taking care not to disrupt the pellet. The supernatants contain your DNA, DO NOT DISCARD!! PCR amplification procedure: 1. Prepare a master mix in a separate tube on ice. Reaction mixture given below is for 1 sample. You will need to prepare a mixture for 4 samples (multiply volumes by 4). Write in the final volume before starting and check off each item as it is added. Volume Final Volume Primer forward 1.0 L Primer reverse 1.0 L dNTP 2.5 L 10x buffer 2.5 L Water 16.75 L Add last: Taq 0.25 L **Do not add Taq until you are ready to add the DNA to your PCR tube and you are ready to go into the thermocycler. Keep all your reagents on ice. Label PCR tubes to indicate sample and lab group. Add 24L of master mix to each tube. To your PCR tube, add 1L of your template (either DNA or water). Thermocycler should be started and preheated. Once temperature is up, place tubes in thermocycler, and start program. This gives you a hot start. DAY 2: Material needed: Gel boxes with combs and trays Power units UV source Camera/film 1% Agarose 1X TAE Loading dye Ethidium Bromide (EtBr) PCR rack 10-20 l pipettes Filtered tips Gel electrophoresis: 2. 3. 4. 5.

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1. Add 5L EtBr (CARCINOGEN!! WEAR GLOVES!) to a flask containing previously prepared 1% agarose gel that is kept warm in the water bath. 2. Set gel tray into gel box with rubber ends forming a seal with the side of the gel box. 3. Insert the comb with the 12 pronged-end facing downward. 4. Pour the gel into the gel tray. Avoid making bubbles in the gel. If bubbles do form use a sterile pipette tip to pop them immediately, before the gel sets. 5. When gel is completely solidified (approximately 15 min), remove comb and rotate gel tray so the wells are on the same side of the gel box as the negative electrode. 6. Carefully load 12L of the samples to any lane but the first on the gel. 7. Carefully load 5L of marker to the first lane. 8. Run gel at 100mV for h. 9. View gel on UV light (WEAR SAFETY GLASSES!). Molecular weight markers are run simultaneously to estimate the size of the fragments. Ethidium bromide is added to the gel to bind DNA and make it visible under UV light. Lab Questions 1. On what basis do you select primers used to identify target DNA, and how do they determine the specificity of PCR? 2. Define positive/negative controls and explain why they are important. 3. Why do you need a thermostable enzyme? 4. From the PCR products we obtained above, what other steps could be done to further characterize the DNA and the strains from which they were derived? 5. Define template, primers, and elongation. Helpful website: http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/

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LAB EXERCISE 13: MOLECULAR TYPING A variety of methods are available for the molecular typing of bacterial species. These methods generally provide DNA fragments of varying sizes that can be used like a barcode to discriminate either different species or strains of the same species. This genotypic characterization can be used to profile isolates from different geographic locations or from different sampling sources to establish geographic distribution among the strains or trace outbreak strains. The obtained profiles are usually analyzed using special imaging and data analysis software. One of the most widely accepted methods is Pulsed Field Gel Electrophoresis (PFGE). This method is the basis of PULSE NET, a CDC database for identification and tracking of bacterial pathogens. PFGE examines the entire bacterial chromosome and uses restriction enzymes cut up or digest chromosomal DNA into large fragments that can be visualized by agarose gel electrophoresis. The number of fragments is determined by the number of sites for restriction enzyme digestion in the chromosome. These large fragments will not enter an agarose gel using standard electrophoresis, but can be separated on a gel by alternating, pulsed electrical fields that help them move through the gel. The method requires precise calibration and technique in order to compare results from different sources. Many strains are untypeable by this method because they are not sensitive to the enzymes used for digestion under these conditions. Therefore, alternative strategies have been explored for more rapid and reliable typing systems. One of the alternatives to PFGE is repetitive extragenic palindromic DNA PCR (repPCR). These repetitive DNA element are distributed at various sites outside of specific genes throughout the chromosome and can serve as targets for PCR amplification. These extragenic sequences are panlidromic (same sequence in both directions, i.e., ATGCCCT followed by TCCCGTA) and therefore PCR primers derived from these sequences can be used to amplify adjacent DNA in both directions. Different types of repetitive elements, such as ERIC and BOX, have been identified in gram negative bacteria and are used for molecular typing at the genomic level. PCR products provide DNA fingerprints that discriminate interstrain variation based on amplicon size and intensity (Figure 1). Figure 1. Repetitive-DNA-sequence-based PCR. Primers hybridize with genomic repetitive elements and amplify intervening sequence to produce multiple PCR products.

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In this lab, we will compare the genetic profiles of V. vulnificus strains isolated from oysters and clinical case of septicemia. Rep-PCR genomic typing will be performed using DiversiLab Microbial Typing System (Bacterial Barcodes, Inc., Houston, TX). Extracted DNA will be amplified by PCR using the DiversiLab Salmonella kit (DL-SE01). PROTOCOL You will do PCR simultaneously on an unknown strain of V. vulnificus, a positive control, a negative control for a total of 3 PCR reactions/group. 1. Positive controls: DNA from 8 different V. vulnificus strains (one per group; 4 from oyster and 4 from environmental). 2. Negative control: Provided in the DiversiLab Kit. 3. Positive control : Provided in the DiversiLab Kit. Material needed: DNA templates Gel electrophoresis Equipment: PCR racks, forceps, 10-20 l pipettors (6), 100-200 l pipettors (6), Filtered tips (6 boxes each size), PCR tubes (6 containers with 10 in each), 1.5 ml tubes (6 containers with 10 in each), 6 gel boxes with combs, tray, 6 power units, UV source, Camera/film. DAY 1: PCR amplification procedure: 1. Prepare a master mix in a separate tube on ice. Reaction mixture given below is for 1 sample. You will need to prepare a mixture for 4 samples. In other words multiply volumes by 4 and add to one tube. Write in the final volume before starting and check off each item as it is added. Keep all your reagents on ice. Volume Final Volume Rep-PCR MM1 18.0 L GeneAmp 10x PCR Buf. 2.5 L Primer Mix P 2.0 L AmpliTaq DNA Polymerase 0.5 L 2. Label PCR tubes to indicate sample and group. Add DNA, positive, and negative control to each tube. 3. Add 23.0 L of master mix to each tube. 4. To each PCR tube, add 2L DNA, 2L of the positive, and 2L of the negative controls. 5. Vortex and return tubes in ice. 6. Program and preheat thermocycler. Once temperature is up, pause and place tubes in thermocycler. Start program. This gives you a hot start. DAY 2: Gel electrophoresis: The PCR reaction produces multiple DNA fragments for each strain and will be electrophoresed on an agarose gel. DNA is prepared in a loading buffer which contains tracking dye and glycerol to prevent the DNA from diffusing out of the well. The DNA is applied to the gel, which is placed in an electrical field, and DNA migrates to the positive electrode due to the net negative charge of DNA. The agarose acts as a molecular sieve with the shorter fragments of

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DNA migrating faster. Molecular weight markers are run simultaneously to estimate the size of the fragments. Ethidium bromide (Carcinogen!! Dispose properly and use gloves!) is added to the gel to bind DNA and make it visible under UV light. 1. Prepare 1x TAE buffer from 20x stock. 2. Prepare agarose gel (2% in TAE buffer or 2 g in 100 ml). Microwave to melt, cool to 50C, add 10 l EtBr (CARCINOGEN!! WEAR GLOVES!), and pour gel. 3. Add loading buffer to PCR product at a ratio of 1:6. Vortex and centrifuge. 4. Fill gel box with buffer, place gel in box. 5. Carefully load 12 l of sample or markers to each lane on gel. 6. Run gel at 100mV for h. 7. View gel on UV light (POSSIBLE EYE DAMAGE!!! WEAR SAFETY GLASSES!) Rep-PCR Demonstration in Building 866 The PCR products of the 8 V. vulnificus strains tested will be loaded on the Bacterial Barcode chip, to be analyzed by the Bioanalyzer (As shown in the flow chart below).

1. PCR amplicons are loaded on the chip

2. Chip is loaded in the Bioanalyzer

3. Band size and intensity is calculated as amplicons are scanned during microcapilary electrophoresis 4. Dendrogram are based on similarity of banding patterns and indicate the relatedness of the DNA targets among different strains.

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References 1. Beyer, W., F. Mukendi, M. Kimmig, and P. Bhm, 1998. Suitability of repetitive-DNAsequence-based PCR fingerprinting for characterizing epidemic isolates of Salmonella enterica serovar Saintpaul. J. Clin. Microbiol. 36:1549-1554. 2. Jensen, M. A., J. A. Webster, and N. Straus, 1993. Rapid Identification of Bacteria on the Basis of Polymerase Chain Reaction-Amplified Ribosomal DNA Spacer Polymorphism. Appl and Environ Microbiol. 59(4):945-942. 3. Versalovic, J., F. de Bruijn, and J. Lupski, 1998. Repetitive sequence-based PCR (repPCR) DNA fingerprinting of bacterial genomes. In: Bacterial Genomes: Physical Structure and Analysis. Chapman & Hall: New York. Lab Questions 1. How do the banding patterns on the gels relate to the origin of the strains?. 2. What are some sources of variability found in genetic typing assays? 3. Why where we able to use rep-PCR Salmonella typing kit for V. vulnificus typing? 4. Which assay would you expect to be more specific: regular PCR, rep-PCR or ELISA? Which assay would be more sensitive?

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