Here is the eight steps identification of gram negative bacteria in microbiology.
- Start: Unknown Bacteria
- Perform a Gram Stain to confirm the bacteria is Gram-negative (pink cells).
- Step 1: Cell Morphology and Arrangement
- Observe under a microscope:
- Bacilli (Rod-shaped): Continue to Step 2.
- Cocci (Spherical): Test for oxidase activity to differentiate Neisseria and Moraxella (oxidase-positive) from other cocci.
- Observe under a microscope:
- Step 2: Oxidase Test
- Oxidase-positive: Likely Pseudomonadaceae or Vibrionaceae.
- Perform extra tests like motility and glucose fermentation.
- Oxidase-negative: Enterobacteriaceae family; proceed to Step 3.
- Oxidase-positive: Likely Pseudomonadaceae or Vibrionaceae.
- Step 3: Lactose Fermentation (MacConkey Agar or TSI Test)
- Lactose fermenters (pink on MacConkey agar): Possible genera:
- Escherichia coli: Confirm with indole and methyl red (positive).
- Klebsiella: Non-motile, urease-positive.
- Enterobacter: Motile, ornithine decarboxylase-positive.
- Non-lactose fermenters (no color change on MacConkey):
- Proceed to Step 4.
- Lactose fermenters (pink on MacConkey agar): Possible genera:
- Step 4: H2S Production (TSI Slant or SIM Test)
- H2S-positive: Likely Proteus, Salmonella, or Edwardsiella:
- Proteus: Swarming motility, urease-positive.
- Salmonella: Lysine decarboxylase-positive, citrate-positive.
- Edwardsiella: Indole-positive, found in aquatic environments.
- H2S-negative: Proceed to Step 5.
- H2S-positive: Likely Proteus, Salmonella, or Edwardsiella:
- Step 5: Urease Test
- Urease-positive: Likely Proteus or Klebsiella.
- Proteus: Swarming motility, phenylalanine deaminase-positive.
- Urease-negative: Proceed to citrate utilization test.
- Urease-positive: Likely Proteus or Klebsiella.
- Step 6: Citrate Utilization Test
- Citrate-positive: Citrobacter, Salmonella.
- Citrate-negative: Likely Shigella (non-motile, lactose-negative).
- Step 7: Motility Test
- Motile: Differentiates motile genera (e.g., E. coli, Proteus) from non-motile (e.g., Shigella, Klebsiella).
Final Identification
Combine the results from all tests, including:
- Gram stain
- Cell morphology
- Oxidase test
- Lactose fermentation
- H2S production
- Urease activity
- Citrate utilization
- Motility test