_ + Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen

Transcription

1 Lab 11 Goals and Objectives: Catalase Test Exercise 39: Oxidation and Fermentation Tests (Catalase) Exercise 67: Staphylococci Identification (MSA & Coagulase) Exercise 68: Streptococci & Enterococci Identification (BAP Hemolysis & Bacitracin resistance) Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen Construct Dichotomous Key to solve for the 12 unknowns Identification of Unknown Bacteria: individual assignment Each person sign out one unknown culture Streak for isolation on BHIA _ + Figure Mannitol Salt Agar Inoculation method: surface streak with loop Contains: 7.5% NaCl, mannitol, Phenol red ph indicator: alkaline ph = red/pink, acidic ph = yellow Selective and differential medium: selects for growth of organisms salt tolerant to 7.5% (usually Staphylococci). Of those that grow, differentiates organisms that have the ability to ferment mannitol to acid. Results: Growth = salt tolerant to 7.5% Yellow = positive for mannitol fermentation to acid Pink = negative for mannitol fermentation to acid No growth = negative for salt tolerance to 7.5%, inconclusive for mannitol fermentation Growth = colonies Don t count a heavy streak only as growth! Rabbit Plasma: Coagulase Test Inoculation method: loop transfer Contains: rabbit plasma (fibrinogen) Discriminates organisms that can produce coagulase to catalyze the conversion of soluble fibrinogen into insoluble fibrin (clot) Results: Solid = positive for fibrin formation, positive for coagulase production Liquid = negative for coagulase production Figure _ Figure 67.2 Amy Warenda Czura, Ph.D. 1 SCCC BIO244 Lab 11 Notes

2 Blood Agar Inoculation method: surface streak and stab with loop Contains: BHIA, sheep blood Discriminates organisms that have the ability to hemolyse red blood cells completely through production of hemolysins (streptolysins or alpha-toxin) or partially through ability to degrade hemoglobin pigment into green products (biliverdin) Results: Complete clearing of RBCs = Beta-hemolysis, positive for production of hemolysins Partial clearing and greening of blood = Alpha-hemolysis, positive for degradation of hemoglobin pigment into biliverdin No clearing with or without rusting = Gamma-hemolysis, negative for hemolysis See Figure 68.2 Partial clearing and greening of blood = Alphahemolysis, positive for degradation of hemoglobin pigment into biliverdin Complete clearing of RBCs = Beta-hemolysis, positive for production of hemolysins No clearing with or without rusting = Gamma-hemolysis, negative for hemolysis Figure 68.2 Gamma Alpha Beta Bacitracin Susceptibility or Resistance Resistant = organism growth not Susceptible = organism killed affected by bacitracin: organism bacitracin: zone of no growth grows around and under disk around disk Staphylococcus aureus 4 Escherichia coli Enterobacter cloacae Proteus vulgaris Amy Warenda Czura, Ph.D. 3 8 Salmonella typhimurium 11 Staphylococcus epidermidis Klebsiella pneumoniae Shigella flexneri 12 Enterococcus faecalis Streptococcus pyogenes SCCC BIO244 Lab 11 Notes

3 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 Tail Meow Woof Bird Human Cat Dog Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Salmonella typhimurium Shigella flexneri Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes Amy Warenda Czura, Ph.D. 3 SCCC BIO244 Lab 11 Notes

4 Go to Exercise 69 Amy Warenda Czura, Ph.D. 4 SCCC BIO244 Lab 11 Notes

5 Figure 69.1 Lactose + Lactose - Figure 69.1 EXAMPLE!!!! Lactose + Lactose - H 2 S+ H 2 S- Pseudomonas aeruginosa Escherichia coli Klebsiella pneumoniae Enterobacter cloacae Proteus vulgaris Salmonella typhimurium Shigella flexneri Amy Warenda Czura, Ph.D. 5 SCCC BIO244 Lab 11 Notes

6 ? Staphylococcus Streptococcus and Enterococcus EXAMPLE!!!! Staph assays: Choose one -Mannitol fermentation -Coagulase Strep/Entero assays: Choose one -Hemolysis type -Bacitracin sensitivity Coagulase + Coagulase - Bacitracin R Bacitracin S Bacillus subtilis Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes Amy Warenda Czura, Ph.D. 6 SCCC BIO244 Lab 11 Notes

7 All Unknown Identification Gelatinase - Gelatinase + Mannitol + Coagulase + Mannitol - Coagulase - " Hemolytic Bacitracin R! Hemolytic Bacitracin S Indole + Citrate - Lactose + Lactose - Indole - Citrate + H 2 S + Motile H 2 S - Nonmotile Day 1: Day 2: Each person sign out one unknown Make streak for isolation on BHIA Put your name on original slant and return Gram stain Catalase test Inoculate MacConkey Agar Inoculate Gelatin Deep Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes Escherichia coli Urea + MR + VP - Klebsiella pneumoniae Urea - MR - VP + Enterobacter cloacae Urea + Urea - Proteus vulgaris Salmonella typhimurium Shigella flexneri Day 3: Based on Gram, Gelatinase, Catalase, Lactose, set up assays to solve: A. If Staph: MSA and Coagulase B. If Strep/Entero: BAP and Bacitracin C. If Gram Neg: SIM, Citrate, MR, Urea Lab 11 Goals and Objectives: Exercise 39: Oxidation and Fermentation Tests (Catalase) Exercise 67: Staphylococci Identification (MSA & Coagulase) Exercise 68: Streptococci & Enterococci Identification (BAP Hemolysis & Bacitracin resistance) Construct Dichotomous Key to solve for the 12 unknowns Identification of Unknown Bacteria: individual assignment Each person sign out one unknown culture Streak for isolation on BHIA Return slant for storage Amy Warenda Czura, Ph.D. 7 SCCC BIO244 Lab 11 Notes