* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Environmental and Food Borne Pathogens Caused by Bacteria Lab
Survey
Document related concepts
Clostridium difficile infection wikipedia , lookup
Escherichia coli wikipedia , lookup
Staphylococcus aureus wikipedia , lookup
Unique properties of hyperthermophilic archaea wikipedia , lookup
Cyanobacteria wikipedia , lookup
Neisseria meningitidis wikipedia , lookup
Carbapenem-resistant enterobacteriaceae wikipedia , lookup
Phage therapy wikipedia , lookup
Small intestinal bacterial overgrowth wikipedia , lookup
Quorum sensing wikipedia , lookup
Anaerobic infection wikipedia , lookup
Bacteriophage wikipedia , lookup
Human microbiota wikipedia , lookup
Bacterial cell structure wikipedia , lookup
Transcript
Environmental and Food Borne Pathogens Caused by Bacteria Lab Part 1A: Culturing and Isolating Bacteria Introduction: Environmental and food borne pathogens are particularly Environmental and food borne illnesses are becoming a greater problem than ever. Every year there is an estimated 47.8 million food borne illnesses in the US with 128,000 being hospitalized and resulting in over 3,000 deaths. The bacteria we will be looking at are some of the bacteria responsible for these statistics. They are Escherichia coli, Enterobacter spp., Listeria innocua, Salmonella spp., and Staphylococcus aureus. See h t t p : / / a g g i e - h o r t i c u l t u r e . t a m u . e d u / e x t e n s i o n / p o i s o n . h t m l for more information on the pathogenic nature of these and a few other bacteria types. Many causes of food borne illnesses as well as the illness itself remain poorly understood. It is estimated that approximately 60% of the outbreaks are caused by unknown sources. This lab will investigate what bacteria may be involved and introduces the latest technology available for identifying the causes as well as the treatment. We will discover that due to the latest technology, treatment for previously deadly outbreaks can be made more readily available in a timely manner ultimately saving lives. We will obtain environmental and food borne bacteria from the sources of possible contamination including soil, surfaces, objects as well as food including meat, poultry, seafood products, fruits and juices, dairy products, infant formula, chocolate/bakery products, peanut butter, egg products and animal feed. Students can bring in various sample for us to test with this lab. Background: Bacteria is everywhere. In this lab, we want to concentrate on disease-causing bacteria. To do this, the bacteria that we are seeking has been limited to four types: Escherichia coli, Listeria innocua, Salmonella enterica and Staphylococcus aureus. E. coli and Salmonella are examples of enteric (found in the gut) pathogens that are gram negative. Listeria and Staph. .aureus are examples of gram positive bacteria. We will use a sterile swab to culture on enriched agar plates like various possible sources of bacteria. After incubating the plates at 37ºC for 24 hours, we will isolate the bacterium(a) using a sterile loop, onto another agar plate. We will incubate these isolated cultures overnight and prepare to identify the bacteria specifically using biochemical tests. Purpose: To culture, isolate and identify a bacteria culture, using the traditional methods similar to those done in the medical field. Procedure – First Day: 1.Obtain a sterile swab and an agar plate. Label your plate with your name, period and date on the agar side (bottom). Swab a possible contaminated sample assigned by the teacher. 2.Innoculate (GENTLY rub the swab on) an agar plate as directed (see diagram) and then using an inoculating loop to spread over the entire agar plate. 3.Incubate overnight at 37ºC. Procedure – Second Day: 1. Get agar plate from the incubator. Look for isolated colonies of bacteria. (Ones that are separated from each other). Ask the teacher for help as this part is very important, especially if you have more than one colony-type of bacteria. 2. With a new agar plate, use an inoculating loop to touch the bacterial colony and plate for isolation as follows: Refer to diagram on left: Section A: rub initial swab of sample or loop of bacteria here. Section B: Flame the loop and cool and go into A 2 or 3 times and continue to streak with loop. Section C and D: Repeat as in Section B for further isolation. On ¼ of the plate, gently rub your loop 3 or 4 times across the area Flame and cool in the agar on the side of the plate. Go into the first section a couple of times and streak in a narrow zigzag motion Repeat the previous step two more times with the rest of the sections of the plate. 3.Incubate overnight at 37ºC. Make a sketch of your plates below: Be sure to indicate if there is more than one type of bacteria by describing it. Include color, size and if wet/dry. Agar Plate: First Day Agar Plate: Second Day Lab Part 1B: Using Biochemical Testing to Identify Bacteria Introduction: To identify bacteria, there are some basic tests we can do to identify bacteria. The bacteria types that are the focus of this lab are divided into two basic types; gram negative and gram positive. Gram negative bacteria can be subdivided into bacteria found in the gut, called enteric and those not found in the gut, non-enteric. We will be focusing on two enteric bacteria that can be distinguished from each other by plating on MacConkey agar. Gram positive bacteria, such as Staphylococcus aureus and Streptococcus sp. can be can be distinguished from each other by a presence of catalase test. Background: See the H/O Part 1B: Understanding Biochemical Testing for Bacterial Identification Purpose: For each team’s isolated bacteria, a series of up to five biochemical tests will be done to definitively identify the bacteria you isolated. Procedure: The biochemical tests will require 2 days to complete: Day One: REMINDER: Label all plates and tubes CLEARLY with your initials, date and period! Students who have gram negative bacteria will perform Biochemical Test A Students who have gram positive bacteria, will perform Biochemical Test B Students who have both bacteria types, perform Biochemical Tests A and B A. MacConkey Agar: This agar will discriminate between gram negative bacteria (entric bacteria – found in the gut). If the culture is Escheresia coli from all other gram negative bacteria. Gram positive bacteria will not grow on this media. E. coli turns bright pink on this agar whereas all other bacteria will not. Use isolation technique to plate one colony of bacteria on your plate. If you have more than one gram negative bacteria, divide your plate in half to streak out the different colonies. Make sure that you use only one colony at a time. B. Presence of Catalase: Add a few drops of 3% hydrogen peroxide to each culture and look for foaming that indicates the release of oxygen as a result of hydrogen peroxide breakdown. Add a few drops of 3% hydrogen peroxide to each culture and look for foaming which indicates the release of oxygen as a result of hydrogen peroxide breakdown. This test can be used to differentiate between Staphylococcus aureus and Streptococcus lactis. Procedure: Day Two and Interpreting Results: A. MacConkey Agar: MacConkey agar is a selective and differential medium to isolate enteric bacteria from all other gram negative bacteria. The agar contains lactose plus a pH indicator that turns red (looks bright pink) for pH below 6.8. This means the bacteria are able to ferment lactose and the bacteria colonies will appear bright pink. If the bacteria do not ferment lactose, the colonies remain normally colored and exhibit no color change. This means they are unable to ferment lactose. B. Presence of Catalase Catalase is the name of an enzyme found in most bacteria which initiates the breakdown of hydrogen peroxide (H2O2) into water and free oxygen. If the bacterium produces the enzyme catalase, then the hydrogen peroxide added to the culture will be broken down into water and free oxygen. The oxygen will bubble through the water causing a surface froth to form. This is a catalase-positive bacterium. A catalase negative bacterium will not produce catalase to break down the hydrogen peroxide, and no frothing will occur. Results: see below for how to record your results For MacConkey agar: (+ = pink, - = colorless) For catalase: (+ = bubbles, no bubbles = -) Data Table: Test MacConkey Agar Catalase Result Discussion/Analysis Questions: 1. State the chemical nature and function of enzymes. 2. State the function of the enzyme catalase and describe a method of testing for catalase activity. 3. Identify your bacteria based on your biochemical tests as well as colony appearance. Conclusion Discussion: Include in your conclusion a discussion of the results of the biochemical test either with the MacConkey agar or the catalase test. Explain how the test helped you identify your specific bacteria. Lab Part 1: Catalase Test is adapted from and used with permission from Dr. Gary Kaiser; Lab 8:Identification of Bacteria Through Biochemical Testing. See website www.ccbcmd.edu