2“What causes Yogurtness”? Microbes and Health:“What causes Yogurtness”?
3Microbes and Health Instructors Stan Hitomi Coordinator – Math & ScienceSan Ramon Valley Unified School DistrictDanville, CAKirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High Schooland Delta College, Tracy, CASherri Andrews, Ph.D.Curriculum and Training SpecialistBio-Rad LaboratoriesEssy Levy, M.Sc.
4Why Teach Microbes and Health? Powerful teaching toolLaboratory extensionsReal-world connectionsLink to careers and industryStandards based
5Microbes and Health Kit – Core Content Alignment Scientific InquiryInterpretation of experimental resultsUse of experimental controlsEvaluation of hypothesisMicroscopyCell and Molecular BiologyBacterial metabolismProkaryotic cell structure and cell divisionEffects of temperature and pH on bacterial growthAntibioticsChemistry of LifeEffects of pH on proteinsEnzymesProtein structure and functionEnvironmental and Health ScienceEpidemiology and diseaseMicrobiologyEvolutionAdaptation to environmentBacterial defense mechanismsGeneticsVariation in bacteria
6Microbes and Health Kit Advantages Can be used in Biology, Microbiology, Health Sciences or BiotechnologyHands-on microbiology lab activityApplication of Koch’s PostulatesSufficient materials for 8 student work stations (4 students per station)Easy preparationCan be used as on its own for any microbiology experiments or for independent study.
7Workshop 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 samplesAssess disease symptoms (pH, smell, texture) from pre-inoculated milk samples and compare to the original bacteriaLaboratory ExtensionsTimeline of labPostulate 1Assess disease symptoms: pH, smell, textureDemonstration of yogurt bacteria on slide with projector- include considerations for making the slidesPostulate 2Isolate disease causing pathogensSpread yogurt, milk and E.coli for single colonies on LB Sugar platesJulia Childs pregrown platesDemonstration of bacteria on slide with projectorPostulate 3Inoculate 5 tubes of milk with bacteriaJulia Childs preinoculated milkPostulate 4Demonstration of yogurt bacteria on slide with projector
8What can you teach with the Microbes and Health Kit? Practice sterile microbial techniques commonly used in researchStudy the role of microbes in disease and healthLearn how cells metabolize nutrients to form other productsUtilize Koch’s Postulates to identify the causative agent for diseaseYour students will attempt to discover the causative agents that turn milk into yogurt
9Bacteria in Yogurt lactic acid bacteria are found in yogurt Streptococcus thermophilluslactic acid bacteria are found in yogurtlactic acid lowers the pH in milk causingcasein (milk protein) to denature and the milk to curdleLactobacillus bulgaricusLactobacillus acidophilusLactobacillus caseiBifidobacterium Bifidumlactose pyruvic acid lactic acid
10Robert Koch Robert Koch (pronounced “coke”) - German physician and bacteriologist- LivedDeveloped a criteria for determining whether a given bacteria is the cause of a given disease:Known as Koch’s PostulatesIn 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.
11Koch’s PostulatesThe 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.
13Laboratory 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.
14Postulate 1The 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- pHMilk simulates a “healthy” sampleYogurt 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 4.5The 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
15Postulate 2The 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- milkDiseased individual- yogurtControl bacteria- E.coli (control)4. Grow cultures overnight at 370CWhen 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 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.
16Postulate 3The cultured microorganism should cause disease when introduced into a healthy organism.- After you inoculate the colonies into sterile milk and incubate for 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 dishes6. Incubate overnight 370C7. Assess symptoms of the subject (pH, smell, texture). Are these the same symptoms of “yogurtness”?
17Postulate 4The microorganism must be again isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent8. Observe yogurt and milk under the microscope: Can the bacteria be matched to the original culture?Got Yogurt?
18Bacteria, Bacteria, Bacteria The single most successful life form on earthProkaryotic organismsExist in soil, water, in and on animals, plants and humansSeveral distinct morphologiescoccus – spherical, bacillus – rods, spiral formsCan orginize assingle units, pairs, long strings, helical shapes, twisted spirochetesDivide by binary fission (some every 20 min!)Colonies originate from one bacterial cell (clonal growth) and can have different shapesGram’s stain dye is taken up by bacteria with thick cell walls (Gram + or -)Bacteria, Bacteria,BacteriaKeep slide in ppt but do not plan to present
19Good Bacteria, Bad Bacteria Bacteria as Pathogens Cholera – Vibrio choleraeTyphoid fever – Salomonella typhiAnthrax – Bacillus anthracisTuberculosis – Mycobacterium tuberculosisBeneficial BacteriaRhizobia – soil bateria important for nitrogen fixationHuman bacterial flora – ,00 species ofbacteria live in the human bodyLactobacillus species – convert milk to lactic acidDigestion of oil spills -Marine bacteria: Acinetobacter calcoaceticus RAG-1Genetic engineering – use of E.coli in industry andreasearchRhizobia (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
20Antibiotics 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 bacteriaBacteriostatic – prevent bacteria from dividingAntibiotics have various modes of action- May inhibit important bacterial enzymes- May destroy cell wall componentsAntibiotic Resistance- Due to overuse/misuse of antibiotics- Some bacterial strains develop resistance as an outcome of natural selection pressuresAntibioticsand Drug ResistanceGenetic TransferGenetic material can be transferred between bacteria by several means, most often by:ConjugationTransformationAnd, Transduction
21Laboratory Extensions Culture microbes from anywhere- Surfaces- Pets- Homes- School- WaterStudy the use of antibioticsGrow liquid culture to teach- Bacterial growth curves- Serial dilutions- Counting bacteria- Spectrophotometry