- Postitive - Purple Stain - Produce exotoxin (specific - nephro, neuro, etc)
- Coccus - round/sphere shape
- Staphylococcus aureus
- Staphylococcus epidermis
- Streptococcus pneumoniae
- Streptococcus salvarious
- Streptococcus mitis
- Streptococcus pyogenes
- Micrococcus Luteus
- Bacillus - rod shape
- Bacillus cereus
- Bacillus megaterium
- Cornynebacterium pseudodiphthericum
- Negative - Pink Stain - produce exotoxin and endotoxin (from lipopolysaccharide)
- Coccus - round/sphere shape
- Neisseria meningidis
- Branhamella/moraxella catarrhalis
- Neisseria lactamica
- Bacillus - rod shape
- Eschericha coli
- Proteus vulgaris
- Pseudomonas aeruginosa
- Serratia marcesens
- Enterobacter Cloacae
Hemolytic Activity
- Coccus
- Beta
- Staphylococcus Aureus
- Streptococcus pyogenes
- Alpha
- Streptococcus Pneumoniae
- Streptococcus mitis
- Neisseria lactamica
- Neisseria meningitidis
- Non
- Stapholococcus epidermidis
- Streptococcus salvarius
- Branhamella/Moraxella catarrhalis
- Micrococcus luteus
- Bacillus*
- Beta
- Bacillus cereus
- Non
- Bacillus megaterium
- Corvnebacterium pseudodiphthericum
*Hemolytic activity of the gram negative bacillus will not be used as a characteristic by which they are identified. Instead, an ENTEROTUBE II will be used (See Exercise 44). Note: There is no blue plastic strip on your enterotube. This strip hid air vents to eight of the 12 chambers. You will need to create these vents. (Ask instructor).
Catalase test (aka Peroxidase) reaction of the Cocci
2H2O ---Catalase--> 2H2O + 02
- Catalase Positive
- All staphylococcus Species
- Micrococcus luteus
- All Neisseria speciies
- Branhamella/Moraxella catarrhalis
- Catalase negative
- All streptococcusspecies
Procedure for catalase (peroxidase) test: Place a small dab of the organism on the inside of the petri dish cover of your culture with the inoculating loop. DO NOT USE WATER. Place a drop of the hydrogen peroxide (H2O2) on the dab of organism and observe for the generation of oxygen as indicated by the equation above, which should occur within a few seconds and should be vigorous. Organisms positive for and negative for catalase should be used as controls. See your instructors for these.
Reactions of Gram positive Cocci on mannitol salt Agar (MSA)
Reactions of Gram positive Cocci on mannitol salt Agar (MSA)
- Growth
- Staphylococcus
- Utilization of mannitol, acid production - Staphylococcus aureus
- Non-utilization of mannitol, no acid production - All other staphylococcus
- No Growth
- Streptococcus
- Micrococcus
Micrococcaceae (family) differentiation
- Random clusters
- Staphylococcus auerus
- Staphyloccus Epidermidis
- Tetrad shape
- Micrococcus luteus
Resistance/ sensitivity of the micrococcaceae to bacitracin and erythromycin (see procedure for sensitivity/resistance to ethyl hydrocuprein, below, but use TSA for this)
- Resistant
- Staphylococcus aureus
- Staphylococcus epidermidis
- Sensitive
- Micrococcus luteus
Identification of gram negative cocci will be done by the Neisseria Tube test (NET). Directions and interpretation of the test are as follows:
- Net Tube. Reactive ingredients per tube in crystallized form.
- Hydrolysis of this by beta-galactosidase yields blue color
- Hydrolysis of this by gamma-glutamylamino-peptidase yeilds yellow color
- Hydrolysis of this by prolylaminopeptidase yields free beta-napthylamine which reacts with the PRO reagent to form a red color
- Phosphate buffered saline
- PRO reagent
Procedure:
- Add 3 drops of PBS into the NET tube and shake
- Use a sterile plastic loop to harvest 2 or 3 colonies and mix in PBS
- Incubate at 35 degrees Celsius for 40 minutes
- Read colors reaction
- Yellow
- N. Meningitidis
- Blue
- N. Lactamica
- Colorless - Add 1 drop of PRO reagent and mix
- Pink to Red
- N. gonorrheae
- Slightly Yellow
- Moraxella
- Colorless
- Branhamella catarrhalis
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