Transcript Lab 8-9
Lab 8&9 Goals and Objectives: • Exercise 37: Morphological Study of Unknown Bacterium • Exercise 38: Cultural Characteristics • • • Discuss observations of the 12 possible organisms Discuss how to rule some out Start working on a dichotomous key of logic for solving all 12 unknowns Lab 8 Goals and Objectives: Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubes Make all Gram stain smears early so they can dry! Read cultural characteristic results on BHI broths FIRST, then make slides After heat fix, smears can be stored to stain later: make at least 5 smears of each today but only stain one pair (save some in case things don’t go well!) Collect all data and add to charts for your unknowns: Exercises 37 and 38 1. FTM tubes for O2 requirements (Ex. 38 pg. 267) 2. BHI broth for growth in liquid characteristics (Ex. 38 pg. 266-267) then Gram stain (Ex. 37 pg. 260) and get size, shape, and arrangement. 3. Gelatin stab culture for gelatin hydrolysis ability (Ex. 38 pg. 267) Ice for 10min before reading! Put on ice now so you don’t forget! 4. Three BHI plates each unknown grown at 25°C, 30°C, 37°C for optimal temp (Ex 37&38): compare colony size. Use best plate of colonies for colony characteristics (Ex. 38 pg. 267-268) 5. Motility test media stab (Ex. 37 pg. 262) Observe characteristics of all 12 potential unknowns in cultures supplied by PA: see these today while they are fresh, you can stain next lab if time is short Try to rule out some based on protease production or cultural characteristics! Save best streak for isolation plates (one each unknown) for use next class Data to collect for Exercise 37 & 38 Gram result, size, shape, arrangement Transfer to Data Chart Motility Amount of growth Color Opacity Transfer to Data Chart Form Surface (broth) Subsurface (broth) Sediment (broth) Growth (broth) Temperature Oxygen requirements (FTM) Gelatin Transfer to Data Chart Transfer to Data Chart Transfer to Data Chart Data chart requires you fill in info regarding the assay: Media used: Indicators: Biochemical aspect: Enzyme Substrate Product Make sure you understand what the assays are for!!! We are testing for particular biochemical reactions. The reactions performed by living cells involve enzymes. The enzymes act on a particular starting material called the substrate and enzymatically alter it (cause a reaction) that results in a particular ending material called the product. Nutrient Gelatin Stab Inoculation method: stab with needle Contains: beef extract, peptone, high gelatin concentration to gel (no agar) Must be set on ice 5-10 min before reading Discriminates organisms that can produce gelatinases to hydrolyze the gelatin into amino acids Results: liquid = gelatin hydrolysis, positive for gelatinase production solid = negative for gelatinase production Data chart requires you fill in info regarding the assay: Media used: Nutrient Gelatin Indicators: none Biochemical aspect: ability to produce gelatinases to hydrolyze gelatin into amino acids Enzyme = gelatinase Substrate = gelatin Product = amino acids 1 2 Bacillus subtilis 5 3 Proteus vulgaris 9 7 Pseudomonas aeruginosa 10 Staphylococcus aureus Escherichia coli Enterobacter cloacae 6 4 8 Salmonella typhimurium 11 Staphylococcus epidermidis Klebsiella pneumoniae Shigella flexnari 12 Enterococcus faecalis Streptococcus pyogenes For next lab: Fill in information you have collected thus far (Gelatinase, Gram stain result, size of cells, shape and arrangement of cells) in the data charts for each unknown. Read over formal lab report directions in packet. State a hypothesis “If the collective results of a profile of key biochemical assays are compared to the known results for limited subset of bacterial species, then a specific unknown culture from that subset of bacterial species can be correctly identified based on the results obtained from the biochemical assays.” The Dichotomous Key -a visible map of logic flow -divides a group of possible things into smaller and smaller groupings until it leads to the identification of just one -tracing the pathway backwards shows all the characteristics that collectively are unique to only that one -assesses only one characteristic per branch -lines all the answers up across the bottom of the key Example: explain with the least number of characteristics how to tell the difference between an old lady and her three pets: sort a human, a cat, a dog and a bird. Construct a Dichotomous key to ascertain the identity of each from this small group: Animals in the house External covering: feathers External covering: hair No tail Bird Human Tail Meow Woof Cat Dog 12 Possible Unknowns 1 2 Bacillus subtilis 5 3 Proteus vulgaris 9 7 Pseudomonas aeruginosa 10 Staphylococcus aureus Escherichia coli Enterobacter cloacae 6 4 8 Salmonella typhimurium 11 Staphylococcus epidermidis Klebsiella pneumoniae Shigella flexnari 12 Enterococcus faecalis Streptococcus pyogenes 12 Possible Unknowns Gram Positive Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus pyogenes Gram Negative Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Shigella flexnari 12 Possible Unknowns Gram Negative Gram Positive Gelatinase + Gelatinase - Gelatinase + Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Salmonella typhimurium Shigella flexnari Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus pyogenes Bacillus subtilis Gelatinase -