“What causes Yogurtness”? Microbes and Health: “What causes Yogurtness”?

Microbes and Health Instructors Stan Hitomi Coordinator – Math & Science San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Sherri Andrews, Ph.D. Curriculum and Training Specialist Bio-Rad Laboratories Essy Levy, M.Sc.

Why Teach Microbes and Health? Powerful teaching tool Laboratory extensions Real-world connections Link to careers and industry Standards based

Microbes and Health Kit – Core Content Alignment Scientific Inquiry Interpretation of experimental results Use of experimental controls Evaluation of hypothesis Microscopy Cell and Molecular Biology Bacterial metabolism Prokaryotic cell structure and cell division Effects of temperature and pH on bacterial growth Antibiotics Chemistry of Life Effects of pH on proteins Enzymes Protein structure and function Environmental and Health Science Epidemiology and disease Microbiology Evolution Adaptation to environment Bacterial defense mechanisms Genetics Variation in bacteria

Microbes and Health Kit Advantages Can be used in Biology, Microbiology, Health Sciences or Biotechnology Hands-on microbiology lab activity Application of Koch’s Postulates Sufficient materials for 8 student work stations (4 students per station) Easy preparation Can be used as on its own for any microbiology experiments or for independent study.

Workshop Time Line Introduction Preparation of microscope slides. Observe cultures and asses disease symptoms (pH, smell, texture) Isolate disease causing pathogens and grow in pure culture ( grow on LB sugar plates) Inoculate milk samples Assess disease symptoms (pH, smell, texture) from pre-inoculated milk samples and compare to the original bacteria Laboratory Extensions Timeline of lab Postulate 1 Assess disease symptoms: pH, smell, texture Demonstration of yogurt bacteria on slide with projector- include considerations for making the slides Postulate 2 Isolate disease causing pathogens Spread yogurt, milk and E.coli for single colonies on LB Sugar plates Julia Childs pregrown plates Demonstration of bacteria on slide with projector Postulate 3 Inoculate 5 tubes of milk with bacteria Julia Childs preinoculated milk Postulate 4 Demonstration of yogurt bacteria on slide with projector

What can you teach with the Microbes and Health Kit? Practice sterile microbial techniques commonly used in research Study the role of microbes in disease and health Learn how cells metabolize nutrients to form other products Utilize Koch’s Postulates to identify the causative agent for disease Your students will attempt to discover the causative agents that turn milk into yogurt

Bacteria in Yogurt lactic acid bacteria are found in yogurt Streptococcus thermophillus lactic acid bacteria are found in yogurt lactic acid lowers the pH in milk causing casein (milk protein) to denature and the milk to curdle Lactobacillus bulgaricus Lactobacillus acidophilus Lactobacillus casei Bifidobacterium Bifidum lactose pyruvic acid lactic acid

Robert Koch Robert Koch (pronounced “coke”) - German physician and bacteriologist - Lived 1843-1910 Developed a criteria for determining whether a given bacteria is the cause of a given disease: Known as Koch’s Postulates In 1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria for judging whether a given bacteria is the cause of a given disease. Koch's criteria brought some much-needed scientific clarity to what was then a very confused field. Koch's postulates are as follows: The bacteria must be present in every case of the disease. The bacteria must be isolated from the host with the disease and grown in pure culture. The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host. The bacteria must be recoverable from the experimentally infected host. However, Koch's postulates have their limitations and so may not always be the last word. They may not hold if: The particular bacteria (such as the one that causes leprosy) cannot be "grown in pure culture" in the laboratory. There is no animal model of infection with that particular bacteria. A harmless bacteria may cause disease if: It has acquired extra virulence factors making it pathogenic. It gains access to deep tissues via trauma, surgery, an IV line, etc. It infects an immunocompromised patient. Not all people infected by a bacteria may develop disease-subclinical infection is usually more common than clinically obvious infection. Despite such limitations, Koch's postulates are still a useful benchmark in judging whether there is a cause-and-effect relationship between a bacteria (or any other type of microorganism) and a clinical disease.

Koch’s Postulates The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms. 2. The microorganism must be isolated from a diseased organism and grown in pure culture. 3. The cultured microorganism should cause disease when introduced into a healthy organism. 4. The microorganism must be again isolated from the inoculated, diseased experimental host and identified as identical to the original specific causative agent.

Procedures Overview

Laboratory Quick Guide - There is a limited amount of loops so don’t use them for anything else unless the manual asks you to use them.

Postulate 1 The microorganism must be found in all organisms suffering from the disease, but not in healthy organisms. Compare yogurt and milk and define the symptoms of “yogurtness”: - microscopic observations - textures, consistency - smell - pH Milk simulates a “healthy” sample Yogurt simulates a “diseased” sample - When you take out a sample of the yogurt, you just take a small dap of sample from beneath the surface and you should be able to get a good sample. When preparing the slides for the microscope, use a small amount of yogurt (like a 1:10 dilution with the drop of water). The broken down protein will block the bacteria if there is too much yogurt on the slide. the pH of milk is 7.5 and the pH of yogurt is around 4.5 The organisms in the milk are smaller and darker when compared to the bacteria seen in yogurt. The numbers in the milk samples are usually lower than the amount of bacteria in yogurt. The bacteria you see in yogurt are round and they are usually chained together in formation of 2 or more individual bacteria. In some yogurt there are also rod shaped bacteria though not as abundant as the round ones. You will also see clumps of milk proteins (casein/curd) which is likely to take up most of the view of the slide if too much yogurt was used. This was done on a 400x microscope. You can probably see more detailed if you have access to a higher powered microscope

Postulate 2 The microorganism must be isolated from a diseased organism and grown in pure culture. 2. Observe the cultures using a microscope and compare the different types of colonies. 3. Inoculate 3 separate petri dishes: Heathy individual- milk Diseased individual- yogurt Control bacteria- E.coli (control) 4. Grow cultures overnight at 370C When streaking the yogurt on the LB sugar plates, very important to follow the streaking protocol to streak for single colonies. If not followed exactly, you may end up with colonies that are too small and crowded together which makes picking for individual colonies very tough. - After streaking milk, yogurt and HB101 on the sugar LB plates and incubate them at 37 for 24-48 hrs I only had colonies from the yogurt and HB101 plates. I had gotten 2 types of bacteria from some of my yogurt plates before, If you take a colony and dilute it in a drop of water, you will see round or rod shaped bacteria depending on the brand of yogurt. Usually the majority of the time I only get round shaped bacteria (individuals and in chains). The HB101 bacteria are rod shaped smaller in size when compared to the rod shaped bacteria from yogurt. If you have colonies from the milk plates, examine that too, but I have not seen any from my plates yet.

Postulate 3 The cultured microorganism should cause disease when introduced into a healthy organism. - After you inoculate the colonies into sterile milk and incubate for 24-48 hours, you examine as you did when you look at the original yogurt sample - After the incubation of inoculating sterile milk with yogurt bacteria and HB101 bacteria, open the tubes carefully when examining them, and beware of foul smell, especially in the HB 101 tubes and the tubes that do not appear to have become yogurt. The milk with amp should be fine. 5. Inoculate fresh milk with bacteria colonies from the petri dishes 6. Incubate overnight 370C 7. Assess symptoms of the subject (pH, smell, texture). Are these the same symptoms of “yogurtness”?

Postulate 4 The microorganism must be again isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent 8. Observe yogurt and milk under the microscope: Can the bacteria be matched to the original culture? Got Yogurt?

Bacteria, Bacteria, Bacteria The single most successful life form on earth Prokaryotic organisms Exist in soil, water, in and on animals, plants and humans Several distinct morphologies coccus – spherical, bacillus – rods, spiral forms Can orginize as single units, pairs, long strings, helical shapes, twisted spirochetes Divide by binary fission (some every 20 min!) Colonies originate from one bacterial cell (clonal growth) and can have different shapes Gram’s stain dye is taken up by bacteria with thick cell walls (Gram + or -) Bacteria, Bacteria, Bacteria Keep slide in ppt but do not plan to present http://www.neatorama.com

Good Bacteria, Bad Bacteria Bacteria as Pathogens Cholera – Vibrio cholerae Typhoid fever – Salomonella typhi Anthrax – Bacillus anthracis Tuberculosis – Mycobacterium tuberculosis Beneficial Bacteria Rhizobia – soil bateria important for nitrogen fixation Human bacterial flora – 500-100,00 species of bacteria live in the human body Lactobacillus species – convert milk to lactic acid Digestion of oil spills - Marine bacteria: Acinetobacter calcoaceticus RAG-1 Genetic engineering – use of E.coli in industry and reasearch Rhizobia (from the Greek words rhiza = root and bios = Life) are soil bacteria that fix nitrogen (diazotrophy) after becoming established inside root nodules of legumes (Fabaceae). The rhizobia cannot independently fix nitrogen, and require a plant host. Morphologically they are generally gram negative, motile, non-sporulating rods. Acinetobacter calcoaceticus RAG-1 is a marine bacteria which can utilize the hydrocarbons in oil as a source of carbon. When these bacteria are grown in a carbon minimal medium, they will breakdown hydrocarbons. RAG-1 releases an emulsan - a polysaccharide that will emulsify oil. Emulsan accumulates on RAG 1 cell surfaces as minicapsules and is released into the media as an active emulsifier as the cell growth approaches the stationary phase. The absence of a carbon nutrient source accelerates the release of emulsans. Salmonella typhimurium http://www.accessexcellence.org/AE/AEC/AEF/1994/brown_oil.html

Antibiotics and Drug Resistance Anti-bacterial antibiotics are one of the main theraputic tools to control and treat many bacterial infectious diseases. These may be: - Bactericidal – Kill bacteria Bacteriostatic – prevent bacteria from dividing Antibiotics have various modes of action - May inhibit important bacterial enzymes - May destroy cell wall components Antibiotic Resistance - Due to overuse/misuse of antibiotics - Some bacterial strains develop resistance as an outcome of natural selection pressures Antibiotics and Drug Resistance Genetic Transfer Genetic material can be transferred between bacteria by several means, most often by: Conjugation Transformation And, Transduction http://www.cdc.gov/drugresistance/community/#campaign

Laboratory Extensions Culture microbes from anywhere - Surfaces - Pets - Homes - School - Water Study the use of antibiotics Grow liquid culture to teach - Bacterial growth curves - Serial dilutions - Counting bacteria - Spectrophotometry