Exercise 39: Oxidation and Fermentation Tests (Catalase) 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

Catalase Test Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen _ + Figure 39.11

Don’t count a heavy streak only as growth! 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! Figure 67.2

_ + 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 67.4 _ +

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 Gamma Alpha Beta

Partial clearing and greening of blood = Alpha-hemolysis, 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

Bacitracin Susceptibility or Resistance Susceptible = organism killed bacitracin: zone of no growth around disk Resistant = organism growth not affected by bacitracin: organism grows around and under disk

1 2 3 4 5 6 7 8 9 10 11 12 Bacillus subtilis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae 5 6 7 8 Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Shigella flexneri 9 10 11 12 Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes

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

External covering: feathers External covering: hair 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

12 Possible Unknowns

1 2 3 4 5 6 7 8 9 10 11 12 Bacillus subtilis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae 5 6 7 8 Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Shigella flexneri 9 10 11 12 Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes

12 Possible Unknowns Gram Positive Gram Negative

12 Possible Unknowns Gram Positive Gram Negative

12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Pseudomonas aeruginosa Bacillus subtilis

Go to Exercise 69 12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Gelatinase - Gelatinase + Gelatinase - Go to Exercise 69 Pseudomonas aeruginosa Bacillus subtilis

Figure 69.1

12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Lactose + Lactose - Pseudomonas aeruginosa Bacillus subtilis

Figure 69.1

EXAMPLE!!!! Gram Negative Gelatinase + Gelatinase - Lactose + H2S+ H2S- Pseudomonas aeruginosa Escherichia coli Klebsiella pneumoniae Enterobacter cloacae Proteus vulgaris Salmonella typhimurium Shigella flexneri

? 12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Bacillus subtilis

Streptococcus and Enterococcus 12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Gelatinase - Catalase + Catalase - Staphylococcus Streptococcus and Enterococcus Bacillus subtilis

-Mannitol fermentation -Coagulase Strep/Entero assays: Choose one Gram Positive Gelatinase + Gelatinase - Catalase + Catalase - Staph assays: Choose one -Mannitol fermentation -Coagulase Strep/Entero assays: Choose one -Hemolysis type -Bacitracin sensitivity Bacillus subtilis

EXAMPLE!!!! Gram Positive Gelatinase + Gelatinase - Catalase + Coagulase + Coagulase - Bacitracin R Bacitracin S Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes

All 12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Gelatinase - Gelatinase - Gelatinase + Catalase + Catalase - Lactose + Lactose - Mannitol + OR Coagulase + Mannitol - OR Coagulase -  Hemolytic OR Bacitracin R  Hemolytic OR Bacitracin S Indole + OR Citrate - Indole - OR Citrate + H2S + OR Motile H2S - OR Nonmotile Urea + OR MR + VP - Urea - OR MR - VP + Urea + Urea - Staphylococcus epidermidis Pseudomonas aeruginosa Staphylococcus aureus Enterococcus faecalis Streptococcus pyogenes Klebsiella pneumoniae Salmonella typhimurium Enterobacter cloacae Bacillus subtilis Escherichia coli Proteus vulgaris Shigella flexneri

Unknown Identification Day 1: Each person sign out one unknown Make streak for isolation on BHIA Put your name on original slant and return Day 2: Gram stain Catalase test Inoculate MacConkey Agar Inoculate Gelatin Deep 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

Exercise 39: Oxidation and Fermentation Tests (Catalase) 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