Exercise 41: Multiple Test Media: Read and record results Lab 13 Goals and Objectives: Exercise 41: Multiple Test Media: Read and record results Exercise 71: Gram Negative Intestinal Pathogens Read and record results, observe PA provided controls Exercise 69: Staphylococci Identification - Read lab Perform according to supplemental directions (packet pg. 61) Exercise 70: Streptococci & Enterococci Identification – Read lab Perform according to supplemental directions (packet pg. 62) Each pair will need: 6 blood agar plates 5 Mannitol Salt Agar plates 5 salt tolerance tubes 5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes Control broth cultures: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes Exercise 22: Mold Cultures Set up one set of mold cultures (2 cultures total) per pair for use next week (needs 7 days to grow) as per Fig 22.1, but inoculate sides of block after placing on slide instead of top and bottom while holding One culture of each from spore suspensions: Rhizopus stolonifer Penicillium notatum *Save old plates in case you need to buy back or repeat any observations next class

Klebsiella pneumoniae - + - + page 289 cloacae Klebsiella pneumoniae - + - +

SIM Medium Inoculation method: stab deep with needle Contains: casein (source of tryptophan and cysteine), ferrous salts (reacts with H2S to produce black ferrous sulfide), 0.7% agar (semisolid) Additional reagents added: Kovac’s reagent, reacts with indole to produce a red product Discriminates three characteristics: S = “sulfide”, discriminates organisms that can produce cysteine desulfurase to hydrolyze the amino acid cysteine into pyruvic acid, ammonia and hydrogen sulfide I = “indole”, discriminates organisms that can produce tryptophanase to hydrolyze the amino acid tryptophan into indole, ammonia and pyruvic acid M = “motility” discriminates motility (presence of flagella), ability to “swim” through media

SIM Medium Results: S Black = formation of ferrous sulfide, hydrolysis of cysteine into hydrogen sulfide, positive for cysteine desulfurase production Colorless = negative for cysteine desulfurase production I Red with Kovac’s = cleavage of tryptophan into indole, positive for tryptophanase production Colorless = no indole present, negative for tryptophanase production M Organism growing only in line of inoculation = non-motile Organism appears as haze beyond line of inoculation = motile

IMViC: tests to differentiate lactose +, gram -, enterics I = Indole (tryptophan degradation) M = Methyl Red (mixed acid fermentation of glucose) V = Voges Proskauer (butanediol fermentation of glucose) C = Citrate (use of citrate as carbon source) cloacae Klebsiella pneumoniae - + - +

MacConkey Agar Inoculation method: surface streak with loop Contains: bile salts, crystal violet and sodium desoxycholate to inhibit Gram positive growth, lactose, Neutral Red pH indicator: neutral pH = red, acidic pH = bright hot pink Selective and differential medium: selects for growth of Gram negative organisms by inhibiting growth of Gram positives. Of those that grow, differentiates ability to ferment lactose Results: Growth = Gram negative Bright pink = positive for lactose fermentation Pale pink/colorless = negative for lactose fermentation No growth = Gram positive, inconclusive for lactose fermentation lactose + lactose - No growth = Gram positive *Dead organisms cannot be scored for lactose fermentation!* Growth = Gram negative

Russell’s Double Sugar Agar Inoculation method: streak and stab slant with needle Contains: glucose (0.1%), lactose (1%), peptone, Phenol red pH indicator: alkaline pH = red, acidic pH = yellow Discriminates organisms that can ferment only glucose to acid from those that can ferment both glucose and lactose or lactose alone to acid. Organisms that ferment only glucose will show alkaline reversion of the slant when the glucose is exhausted. Results: Yellow slant / yellow butt = positive for fermentation of either lactose alone or both glucose and lactose to acid Red slant / yellow butt = positive for fermentation of glucose only to acid Red slant / red butt = negative for fermentation of either glucose or lactose

Russell’s Double Sugar Agar Results: Yellow slant / yellow butt = positive for fermentation of either lactose alone or both glucose and lactose to acid Red slant / yellow butt = positive for fermentation of glucose only to acid Red slant / red butt = negative for fermentation of either glucose or lactose Alkaline/no change lactose - glucose - Alkaline/acid glucose only Acid/acid lactose + glucose unknown

Fig 71.1 H2S+ (Confirm answer with IMViC)

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

Fig 71.1 H2S+ (Confirm answer with IMViC)

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

Exercise 22

Exercise 41: Multiple Test Media: Read and record results Lab 13 Goals and Objectives: Exercise 41: Multiple Test Media: Read and record results Exercise 71: Gram Negative Intestinal Pathogens Read and record results, observe PA provided controls Exercise 69: Staphylococci Identification - Read lab Perform according to supplemental directions (packet pg. 61) Exercise 70: Streptococci & Enterococci Identification – Read lab Perform according to supplemental directions (packet pg. 62) Each pair will need: 6 blood agar plates 5 Mannitol Salt Agar plates 5 salt tolerance tubes 5 coagulase (rabbit serum) tubes 5 Bile Esculin tubes Control broth cultures: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes Exercise 22: Mold Cultures Set up one set of mold cultures (2 cultures total) per pair for use next week (needs 7 days to grow) as per Fig 22.1, but inoculate sides of block after placing on slide instead of top and bottom while holding One culture of each from spore suspensions: Rhizopus stolonifer Penicillium notatum *Save old plates in case you need to buy back or repeat any observations next class