Identification of Gram Negative Bacteria Lab

Identification of Gram Negative Bacteria

All gram-negative bacteria usually appear as gram-negative small rods with microscope
5 biochemical tests are used to identify genus
1. Carbohydrate Fermentation Test
2. SIM Test (Hydrogen-Sulfide, Indole, Motility)
3. Citrate Test
4. Phenolalanine Deaminase Test
5. Urease Test
Gram negative bacteria are usually cultivated on MacConkey agar, which contains bile salts and crystal violet to inhibit gram-positive bacteria. Agar also contains lactose and a neutral red dye to indicate pH changes. Lactose-positive colonies are red to pink in color, lactose-negative are not.
Carbohydrate Fermentation Test
- usually in broth with phenol red pH indicator
- Phenol Red lactose broth or Phenol red glucose broth is used
- Durham tube inverted in base of test tube traps gas if organism produces gas when fermenting carbohydrate
- Organism must have beta-galactosidase to break down lactose into glucose and galactose
- When organism ferments simple sugars, it produces acidic end products, which lower pH and change broth to yellow color.
- Test result possibilities:
  1. Negative - red broth - no fermentation
  2. A/G Positive - produces acid and gas, forming bubble in Durham tube and changing broth to yellow.
  3. Acid Positive - changes broth to yellow, but no bubble produced.
SIM Test -
- SIM agar inoculated by stabbing culture on loop straight into agar.
- SIM medium is used to determine if organism can metabolize certain amino acids.
- 3 tests in one:
  1. Hydrogen Sulfide (H₂S)
    - Cystamins and Metathiamine contain sulfur, which allow formation of hydrogen sulfide if organism can metabolize them.
    - Lead nitrate in agar traps hydrogen sulfide, forming a black precipitant when lead sulfide is formed. Agar turns black in hydrogen sulfide positive test.
    - Negative result = no change in color of medium.
  2. Indole Test
    - Aromatic amino acid Tryptophan (an ring shaped amino acid with an amine group and indole) is present in medium. If Bacteria can break this molecule amino group and indole, test is positive.
    - After bacteria has had time to grow, Kovac's reagent will react with indole to form a bright red ring at the surface of the tube.
    - If test is negative, no change will occur when Kovac's reagent is added.
  3. Motility test
    - If organism is motility positive, it will spread throughout the medium from the stab
    - If negative, bacteria will only grow where it was stabbed into the medium.
Citrate Test
- Surface of citrate agar (green) slant streaked with bacteria
- Agar contains citric acid, which is a tricarboxylic acid (has 3 carboxyl groups) and Bram Cresol Agar.
- Bacteria with citrate permease can uptake citric acid, causing alkaline end products that change pH indicator to blue.
- If test is positive, slant changes to blue
- If negative, slant remains green.
Phenylalanine (PA) Deaminase Test
- Bacteria are smeared on phenylalanine agar slant.
- Phenylalanine is an aromatic amino acid (has ring structure associated)
- If bacteria possess phenylalanine deaminase, it can remove amino group, leaving phenylpyruvic acid.
- To complete test (after bacterial growth), 5% solution of ferric chloride is added to slant.
- If test is positive, slant will turn olive green on surface
- If negative, no change
- Members of the genus Proteus are generally positive for this test.
Urease Test
- Urea broth (orange), which contains phenol red indicator in low concentration is medium for test.
- Urea, which is usually toxic, is the end product of amino acid metabolism. Some organisms contain urease, which allows them to break down urea to form CO₂ and ammonia. Ammonia reacts with water to form ammonium hydroxide.
- Broth becomes red-purple color if test is positive due to production of ammonium hydroxide.
- If negative, broth remains orange.

Bacteria Identification Flow Chart

Lactose Fermentation
- positive = coliforms
  - Citrate test
    - positive
      - H₂S test
        
        positive = Citobacter
        
        negative
        
        motility test
        
        positive = Enterobacter
        
        negative = Klebsiella
    - negative (also Indole Positive, motility positive, H₂S negative) = Escherichia coli.
- negative = noncoliforms
  - Urease test
    - positive
      - Phenylalinine deaminase test
        
        positive = Proteus
    - negative
      - Phenylalinine deaminase test
        
        negative
        
        Motility test
        
        negative = Shigella (also produces Acid with glucose)
        
        positive = Salmonella (also produces Acid and Gas with glucose)
- Glucose negative
  - Oxidase positive = Pseudomonis

	Lactose	Glucose	Citrate	H₂S	Indole	Motility	Urease	PA deaminase	Oxidase
Escherichia coli	pos	neg	neg	neg	pos	pos
Citobacter	pos		pos	pos
Enterobacter	pos		pos	neg		pos
Klebsiella	pos	A/G	pos	neg		neg
Proteus	neg	pos					pos	pos
Salmonella	neg	A				pos	neg	neg
Shigella	neg	A/G				neg	neg	neg
Pseudomonas	neg	neg							pos

Antibiotic Lab

Kirby Bauer test is used to evaluate antibiotic resistance by placing antibiotic discs on agar where bacteria are growing
Limitation to Bauer test is that it is only a qualitative test; it cannot indicate how much antibiotic is needed to control microorganism.
Candida albicans is sensitive only to nystatin, which is used to control yeast, not bacteria.
Pseudomonas is resistant to Penicillin, tatracycline, erythromyacin; sensitive to cyproflexin and genomyacin.
E. coli is resistant to penicillin, but sensitive to ampacillin