Biology 229 Lecture 13 Ex 29 Oral Biofilms PDF

BIOLOGY 229 Subculture Environmental isolate onto fresh BHI slant LOOKING AHEAD…EXAM 2 ◼ October 18 ◼ Covers Exercises 17-23 and, 25-30 ◼ Chemical Control of Microbial Growth using...

BIOLOGY 229 Subculture Environmental isolate onto fresh BHI slant LOOKING AHEAD…EXAM 2 ◼ October 18 ◼ Covers Exercises 17-23 and, 25-30 ◼ Chemical Control of Microbial Growth using Disinfectant ◼ Differential media and staining profiles ◼ Biofilm formation ◼ Oral Microbiome ◼ Plant-microbe symbiosis ◼ Microbes in water, soil, foods ◼ 16S rRNA Days 1 & 2 ◼ Lab report Due Oct 23 TODAY’S TASKS Subculture and Analyze plates from WedEnvironmental Isolate BHI 🡪 BHI Quiz Oral Biofilms Microbiome of Plaque and Toothbrush Scientific Article II In-class Assignment – NOT COLLECTED QUIZ 15 MINUTES ORAL BIOFILM FORMATION SPREAD PLATE: REVIEW AND SAFETY ◼ Goal: cover the whole plate evenly with culture. ◼ Do: ◼ Make sure you are pipetting accurately ◼ Keep even pressure on the hockey stick ◼ Hold the hockey stick parallel with the table while flaming ◼ Do NOT: (unless you want to catch on fire) ◼ Hold the hockey stick vertical! ◼ Have the ethanol beaker ANYWHERE near the flame whatsoever ◼ Hold the flaming hockey stick anywhere near the ethanol ◼ Place a hot hockey stick back in the ethanol because it “didn’t flame well” ◼ If your ethanol catches fire, put the large beaker over the flame! And CALL ME OVER. It will burn out.  LEARNING OBJECTIVES ◼ Construct a timeline illustrating the development of the human oral microbiome from birth to adulthood, including outside factors that can influence the members of this cohort. ◼ Explain how quorum sensing influences the development of the oral microbiome. ◼ Explain how quorum sensing may influence horizontal gene transfer events within the oral microbiome environment. ◼ Explain the process by which cavities are formed and how this relates to biofilms. ◼ Compare and contrast dental caries and periodontitis. ◼ Provide examples why dental caries and periodontitis results from changes in the ecology of the oral biofilm rather than from acquisition of a new pathogen. ◼ Explain why the development of some human diseases is specifically linked to periodontal disease. ◼ Discuss factors that influence microbial populations on a toothbrush. ◼ Identify the components in each medium used in this experiment that make it selective and describe the function of each. Not all media used in this experiment are selective. ◼ Identify the components in each medium used in this experiment that make it differential and describe the function of each. Not all media used in this experiment are differential. ◼ Isolate members of one group member’s oral biofilm, calculate the CFU/ml of groups of organisms, and compare these concentrations to others in the class. FORM YOUR HYPOTHESIS ◼ Hypothesize with your group which microbial groups will be present on each of the agars we are testing today. ◼ Form a hypothesis about how the microbial groups will differ between toothbrush and plaque samples. ◼ Just for plaque: which microbial group do you expect to be the most/least abundant? Why? ◼ Just for toothbrushes: which microbial group do you expect to be the most/least abundant? Why? ORAL CAVITY AS A MICROBIAL HABITAT  DENTAL BIOFILMS ◼ “Plaque” – that stuff the dentist scrapes off your teeth ◼ Primarily bacterial film that forms around teeth, particularly beneath the gumline. ◼ Responsible for dental caries (AKA cavities), bad breath ◼ Attach to the tooth (via capsule) 🡪 Ferment sugars on teeth 🡪 Produce lactic acid 🡪 Acid degrades enamel  FACTORS CONTROLLING ORAL MICROBIOTA COMPOSITION ◼ Host innate immunity always in conflict with microbes ◼ Low biomass 🡪 negligible immune response (quickly resolved) ◼ Increased biomass 🡪 unresolved immune response ◼ plaque formation ◼ alterations to biofilm and stratum ◼ Prolonged exposure to immune response 🡪 tissue/enamel damage ◼ Allows greater infiltration of microbes ◼ Worsens immune response  “The oral microbiome includes up to 600 diverse species including Streptococci, Lactobacilli, Staphylococci, Corynebacteria, etc., residing in different microenvironments Oral Flora within the oral cavity” (Dewhirst et al., 2010). At Birth Natural 🡪 Mother’s Cesarean 🡪 Mother’s Vaginal Microbiome Skin Microbiome Tooth Emergence Tooth = Surface = Anaerobic Biofilms! Microenvironments Form Development of Adult Teeth Further increases in complexity Loss of Teeth Disappearance of Streptococci and anaerobes FACTORS INFLUENCING ORAL BIOFILM COMPOSITION Plaque Toothbrush ◼ Sanitary Habits ◼ Sanitary Habits ◼ Frequency of brushing ◼ Storage of toothbrush ◼ Type of toothpaste used? ◼ Age of toothbrush (aka replacement) ◼ Do you use mouthwash? ◼ Distance from toilet ◼ Diet and lifestyle ◼ Natural birth or C-section?  Bacteria detected in 3-month-old infant oral microbiomes who were delivered vaginally or via Caesarian section Holgerson et al. 2011 J Dental *** P < 0.005, ** P < 0.01 HOW CAN WE DISRUPT ORAL BIOFILMS? Chemical (Pre)Treatment Chemical Treatment Mechanical Action WHAT HABITS OR LIFESTYLE OF YOUR GROUP’S VOLUNTEER MAY INFLUENCE TODAY’S RESULTS? SHORT GROUP DISCUSSION WHAT WE ARE DOING TODAY ◼ Examination of Oral Biofilms- Exercise 29 ◼ Work in Groups of 4/5 ◼ 1-2 members do plaque, others do toothbrush ◼ Plating plaque and toothbrush on selective and differential media using spread plate method ◼ Make sure to label growth conditions and media clearly ◼ This will be your second lab report! Due Oct 23 ◼ Start writing your Intro and Methods now Culture Media MacConkey Columbia CNA Gram Negative +5% CO2 Lactose Gram Positive Fermentation Hemolysis Sabouraud Mannitol Salts Agar Dextrose Agar Halophiles Fungi Mannitol Do expect to see Fermentation yeast or mold?  TSA + 5% SHEEP BLOOD AKA: BLOOD AGAR Nonselective Allow the isolation of many types of bacteria Differential hemolysis patterns 5% sheep blood β- hemolytic α- hemolytic Clear zone indicating lysis 1 to 3mm greenish zone of incomplete hemolysis Ex. S. mutans Ex. Streptococcus pyogenes Includes group A Strep WILKINS-CHALGREN AGAR To cultivate anaerobes Non-selective Enriched medium for anaerobes Hemin and Vitamin K are essential for some anaerobes Anaerobic incubation allows for selection of anaerobes Why are we looking for anaerobes? himedialabs.com Which sample will have more?  PSEUDOSEL AGAR Used to culture Pseudomonas aeruginosa, an opportunistic pathogen Selective For Pseudomonas aeruginosa Cetrimide interferes with microbial membranes (quaternary ammonia) ◼ Encourages production of pyocyanin Blue/green secondary metabolite characteristic of P. aeruginosa MgCl2 and KSO4. MITIS SALIVARIUS AGAR ◼ Selective for Streptococcus mitis, S. salivarius and enterococci ◼ Components: ◼ Trypan blue, Crystal Violet, Tellurite ◼ Select against Gram Negative (Tellurite) and most Gram positive (CV) organisms ◼ Differential for several organisms: 1. Enterococci opportunistic pathogens (cavity, root canal infections, endocarditis, periodontitis) 2. Streptococcus salivarius opportunistic pathogen 3. Streptococcus mitis causes endocarditis  MITIS SALIVARIUS AGAR Selective for Streptococcus and Enterococcus crystal violet, 1% potassium tellurite, trypan blue Differential Absorption of Trypan Blue S. mitis- small blue colonies ◼ Does not hydrolyze sucrose S. salivarius- blue, mucoid “gum drop”, larger colonies ◼ Hydrolyzes sucrose = glucose and fructose (sticky) Enterococcus spp.- dark blue/black, shiny, slightly raised ◼ Reduce potassium tellurite  DIFFERENTIATION- WHY AGAIN? ◼ We are testing the metabolic potential of an organism ◼ Hemolysis ◼ Lactose Fermentation ◼ “pathogens” ◼ Lactose is a sugar unique to animals ◼ Red blood cells have lots of ◼ Identification of coliforms/ cariogenic nutrients for microbes to grow microbes on Why is identification useful? ◼ Mannitol fermentation ◼ Understanding of an environment ◼ Specifically common among Staphylococcal species ◼ Today: Risk factors for oral dysbiosis ◼ Useful for practical clinical identification FORM YOUR HYPOTHESIS ◼ Hypothesize with your group which microbial groups will be present on each of the agars we are testing today. ◼ Form a hypothesis about how the microbial groups will differ between toothbrush and plaque samples. ◼ Just for plaque: which microbial group do you expect to be the most/least abundant? Why? ◼ Just for toothbrushes: which microbial group do you expect to be the most/least abundant? Why? PLAQUE PLATING TODAY ◼ Plating oral microbes on many media ◼ Next week, you will enumerate CFUs on plates ◼ Review how to do this calculation. ◼ What does this number mean? How accurate is it? Direct or indirect method? ◼ Serial dilutions on some agar ◼ TSA + 5% sheep’s blood ◼ Wilkins-Chalgren ◼ Incubation ◼ Why 37°C ◼ Why 5% CO2  TODAY 1. Examination of Oral Biofilms- Exercise 29 1. Work in Groups of 4-5 1. 1-2 members do plaque, others do toothbrush. 2. Make sure to label growth conditions and media clearly 2. Use handout to help visualize dilutions 2. Keep your media and dilutions labeled! Be organized! Do not catch fire! Do not catch your neighbor on fire! 3. Subculture environmental isolate BHI -> BHI