Answer
Separate and identify the two bacteria by:
Doing a Gram stain (S. aureus = Gram-positive cocci; Klebsiella = Gram-negative rods).
Plating the culture on selective/differential media (e.g., Mannitol Salt Agar for Staph aureus and MacConkey agar for Klebsiella).
Confirm with simple biochemical tests (e.g., catalase/coagulase for S. aureus, lactose fermentation and IMViC-type tests for Klebsiella) and colony appearance (mucoid colonies for Klebsiella).
Work Step by Step
Smear + Gram stain
Make two smears from the mixed culture, air dry, heat fix, and Gram stain.
Under the microscope: Staphylococcus aureus appears as purple (Gram-positive) clusters of round cells (cocci). Klebsiella appears as pink (Gram-negative) short rods.
This immediately shows you there are two different types.
Plate on selective/differential media
Mannitol Salt Agar (MSA): high salt concentration inhibits many Gram-negatives and many other organisms; S. aureus tolerates salt and often ferments mannitol turning the medium yellow. Klebsiella will not grow well on MSA.
MacConkey agar: selects for Gram-negative bacteria and differentiates lactose fermenters. Klebsiella is a lactose fermenter → pink colonies on MacConkey. S. aureus (Gram+ ) won’t grow or will be inhibited.
Streak the mixed sample onto both plates. After incubation you will get separated colonies of each bacterium on the appropriate plate.
Colony morphology
Klebsiella often forms mucoid, glistening colonies because of its capsule.
S. aureus forms golden-yellow or white, round colonies on nutrient agar and ferments mannitol on MSA.
Simple biochemical tests to confirm
Catalase test: Staphylococcus (catalase +) bubbles with H₂O₂; Klebsiella (also catalase + sometimes) but Gram result differentiates.
Coagulase test: S. aureus is usually coagulase positive (distinguishes it from other Staph species).
Oxidase (negative for both here) and IMViC/fermentation tests help confirm Klebsiella identity (indole, methyl red, Voges-Proskauer, citrate).
