BIOL 2P98 2023 Fall Lab 4 PP draft.pdf

BIOL 2P98 Lab 4 Bacterial Growth and Identification Distinguishing Characteristics of Bacteria • Bacteria are commonly distinguished by various physiological and chemical traits: • • • • • • • Cell wall composition Cell shape, arrangement and size Motility Use of oxygen Growth on various substrat...

BIOL 2P98 Lab 4 Bacterial Growth and Identification Distinguishing Characteristics of Bacteria • Bacteria are commonly distinguished by various physiological and chemical traits: • • • • • • • Cell wall composition Cell shape, arrangement and size Motility Use of oxygen Growth on various substrates Production of specific enzymes Production of different end products Research the potential bacteria: your unknown is one Gram Stain 2 2-6 - Cell shape & arrangement Straight bacillus/rods:singly or pairs Colonial characteristics (pigment colour) Smooth convex grey moist or rough flat dry dull wrinkled Preferred Temperature Range (oC) Oxygen requirement (FTM) 21-37 Facultative anaerobe Catalase + *Glucose fermentation (24 hr KIA) A Lactose fermentation (24 hr KIA) - H2S (KIA or SIM) - Methyl Red Test + Voges-Proskauer - Citrate Test Indole Test Gelatin hydrolysis + - Motility (SIM) v Serratia marcescens D1 Cell length (mm) Pseudomonas fragi Risk Group Escherichia coli B inactive Escherichia coli DH5a inactive • Using Bergey’s Manual of Systematic Bacteriology (20052012), fill in the characteristics of each of the remaining bacteria in Table 4.1. The information for the inactive mutant E. coli DH5a is already completed for you. • These tests have been performed on an unknown bacteria already and you will need to determine which one of these bacteria on this table is the unknown. • Physiological and biochemical testing is a common way to identify an unknown. • Can you think of another way? Research the media and tests you are using to identify your unknown bacteria • Using A Photographic Atlas of the Microbiology Laboratory (Leboffe & Pierce, 2011) or other peer reviewed work, identify the components of the media and tests that indicate the mechanisms to aid in identification, into Table 4.2. • Understanding the mechanisms behind the biochemical tests is essential to identifying the unknown bacteria. Name of Test Purpose/Importance Name of Medium Differential /selective PhenylEthyl Alcohol Agar Differential /selective Columbia CNA Agar Differential /selective MacConkey Agar Differential /selective Eosin Methylene Blue Agar Differential /selective Endo Agar Aerotolerance Fluid Thioglycolate Medium Fermentation of glucose & lactose Kligler’s Iron Agar 24 hr H2S SIM & Kligler’s Iron Agar Motility SIM Indole Test SIM Methyl Red MRVP Voges Proskauer MRVP Citrate utilization Simmond’s Citrate Gelatin hydrolysis Nutrient Gelatin Indicator or Reagent + Reaction Result - Reaction Result Create a dichotomous Identification Key • A dichotomous key is one that only splits into two results at each choice (positive & negative). • You do not need to include all tests, just the ones that will lead you to one final organism. • All the bacteria in Table 4.1 are gram negative and bacillus shaped. You would not include that information on the key. • You will need to create an Identification Key for the 4 bacteria in Table 4.1. Test A Bacteria 3 Bacteria 4 Bacteria 5 + Bacteria 1 Bacteria 2 Test B Bacteria 1 Test C + Bacteria 2 + Bacteria 3 Bacteria 5 Test D + Bacteria 3 Bacteria 5 Bacteria 4 Gram Stain • Review the Gram Stain (Lab 2) • Always work over a stain tray • Record final colour after staining • Also record cell shape and arrangement, then calculate cell length (Lab 1) • In our Lab the liquid stain waste goes in the Liquid Waste jug in the Fume Hood Leboffe & Pierce, 2011, p. 45 Motility Test • Semisolid medium to allow movement. • Stab inoculate with inoculating needle. • Record if growth is spreading outward from the stab line in the SIM tube • Growth away from stab line indicates that the bacteria is motile. Inoculated SIM medium uninoculated Away from stab at stab line only Motility medium with TTC indicator Leboffe & Pierce, 2011, p. 83 Colony/Cultural Characteristics • Label one 100mm Tryptic Soy Agar plate with Group #’s, Lab day, Date, Unknown #, TSA • Quadrant streak the plate with your unknown • Record Colony Morphology / Cultural Characteristics (Lab 1) after incubation Aerotolerance - FTM • There is great diversity in how and if bacteria utilize oxygen • Fluid Thioglycolate Medium (FTM) tube kept in the water bath (CAUTION: Hot water makes steam) to drive off remaining oxygen. • Use tongs to remove the tube and place into your test tube rack • Pink colour indicates the presence of oxygen • When dry and cool, label FTM tube with Gp#, Lab day, Date, Unknown # and FTM • Stab inoculate with inoculating needle then incubate • Where growth occurs in the tube indicates the bacteria’s need for oxygen or lack thereof Leboffe & Pierce, 2011, p. 30 Aerotolerance - FTM • Aerotolerant anaerobe • Not inhibited or killed by oxygen (fermentation only, but have enzymes to convert harmful byproducts of oxygen) • Facultative anaerobe • Can grow in the presence or absence of oxygen (aerobic and anerobic respiration and fermentation) • Obligate anaerobe • Inhibited or killed by oxygen (anaerobic respiration and fermentation, lack enzymes to convert harmful by-products of oxygen) • Obligate aerobe • Requires the presence of oxygen for growth (aerobic respiration only) • Microaerophile • Requires a low concentration of oxygen for growth (aerobic respiration) • Madigan et. al., 2021, pp. 135-136 Leboffe & Pierce, 2011, p. 30 Selective and Differential Media • Label 100mm plates with Gp#, Lab day, Date, Unknown# and media name • Quadrant streak all 100mm plates • Record if there is growth and colony colour Endo Agar MacConkey Agar • NOT a Quadrant Streak! Leboffe & Pierce, 2011, p. 11-13, 15 EMB Agar PEA Agar Indole Test - Respiration Leboffe & Pierce, 2011, p. 74-75 Indole Test - Respiration • Pre-inoculated Sugar Indole Motility (SIM) tube. • Kovac’s reagent is in the BSC. Take the SIM tube in test tube rack over to the BSC to add 3 drops of the reagent. • Record final colour of Kovac’s reagent • Pink indicates the bacteria has the enzyme tryptophanase Sulfur Reduction • Look for Black precipitate (FeS) in SIM tube; black indicates the bacteria can reduce sulfur Leboffe & Pierce, 2011, p. 93-94 Carbon Sources & Fermentation - KIA • With inoculating needle, you would stab into the butt portion of the tube and then streak the slant portion on your way out of the tube. • Record the colour of the butt, slant and if there is black precipitate or cracking/bubbles Leboffe & Pierce, 2011, p. 75-76 Fermentation End Products Methyl Red-Vogues Proskauer Procedure • Pre-inoculated MRVP broth culture tube • Aseptically, remove 2ml from MRVP tube into empty sterile test tube, label as VP • Collect Methyl Red and VP reagents from TA • Add 5 drops of Methyl Red indicator into the 8ml of the original MRVP tube; record colour after swirling for a couple minutes • Add 250µl of VP reagent A to the 2 ml VP tube and gently swirl, then add 250µl of VP reagent B and swirl • Re-shake VP tube every 5 minutes up to 30 minutes and record final colour Methyl Red Test • Record the final colour in the MR tube • A red colour indicates that the bacteria is able to perform mixed-acid fermentation Leboffe & Pierce, 2011, p. 82 Voges-Proskauer Test • Record the final colour in the VP tube • A red colour indicates that the bacteria is able to produce acetoin from 2,3butanediol fermentation Leboffe & Pierce, 2011, p. 98 Carbon Sources - Citrate Test • Using inoculating loop, quadrant streak the 100mm plate or simple streak an agar slant. • Record any growth and change in colour of the media • If the bacteria can transport Citrate into the cell and convert it into pyruvate, then it will grow, and the media would turn blue. Leboffe & Pierce, 2011, p. 64-65 Gelatin Hydrolysis • Record the consistency (liquid or solid) of the nutrient gelatin media after inoculation and incubation • Indicates if the bacteria has gelatinase enzymes that will break down the gelatin, liquifying the medium Leboffe & Pierce, 2011, p. 73 Multi-test Systems RapID SS/u • This Multi-test system is made up of 10 wells with select biochemical tests to quickly identify an unknown bacteria after 2 hours of incubation. • A RapID SS/u test was performed for Unknown # and results will be provided in the viewing room and on Brightspace. References • Garrity, G. M., Brenner, D. J., Krieg, N. R., and Staley, J. T. (2005) Volume Two Proteobacteria Part B The Gammaproteobacteria of Bergey’s Manual of Systematic Bacteriology, 2nd edition. East Lansing, MI: Springer. • Leboffe, M. J. and Pierce, B. E. (2011) A Photographic Atlas for the Microbiology Laboratory, 4th edition. Morton Publishing Company, Colorado. • Madigan MT, Bender KS, Buckley DH, Sattley WM, and Stahl DA (2021) Brock Biology of Microorganisms, 16th edition. Pearson Education, Inc., New York.