Module 1: Niesseria and Moraxella Flashcards
What 4 genera are in the Nisseriaceae family?
Acinitobacter
Kingella
Neisseria
Moraxella
What are the 2 main Neisseria pathogens
N. meningitidis and N. gonorrhoeae
What basic media is used to isolate Neisseria?
Chocolate agar (enriched)
How is chocolate agar prepared?
Same as blood then slowly heat to 75-85 degrees for 10-15 minutes to lyse the blood and release nutrients
Cool to 50 degrees and add supplements (supplement B and Iso-Vitale X)
What typically used plate does N. gonorrhoeae not grow well on?
Blood agar
What selective media is used to isolate N. gonorrhoeae? Why?
Modified Thayer Martin (MTM)
New York City (NYC)
Because genital, rectal, and oropharyngeal swabs typically have many normal flora which will overgrow the N. gonorrhoeae
How is MTM media made?
GC agar base and hemoglobin rehydrated and autoclaved separately
Cooled to 50 degrees, IsoVitaleX and antibiotics added
What antibiotics are present in MTM? What do each of them do?
Vancomycin - inhibit gram positive (concentration is important, too much slows growth)
Colistin - inhibit gram negative and “non-pathogenic” Neisseria
Nystatin - inhibits yeast
Trimethoprim lactate - prevents Proteus swarming
How is NYC media made?
Lysed horse RBC, horse plasma, yeast dialysate, and glucose added to agar
Antimicrobials added
What antimicrobials are in NYC media? What do each of them do?
Vancomycin - inhibit gram positive
Colistin - inhibit gram negative
Amphotericin B - inhibit yeast
Trimethoprim lactate - inhibit Proteus swarming
What two types of transport media can be used for N. gonorrhoeae swabs if they cannot be immediately planted?
Non-nutrient holding media (Amie’s charcoal) and nutrient growing media
What is a recommended non-nutrient holding media for N. gonorrhoeae? How does it work?
Amie’s charcoal transport media
Maintains viability without growth in a buffered semi-solid media
Charcoal absorbs any fatty acids (vagina, cotton swabs) that may kill N. gonorrhoeae
What swabs are recommended for transporting N. gonorrhoeae on?
Synthetic ones such as calcium alginate
Cotton swabs should be soaked in charcoal or placed in Amie’s charcoal media
What is a growing transport media?
A selective plate is planted and placed in container with CO2 generating system, usually bicarbonate and citric acid, incubated 18-24 hours before transport and sent
What is the genus test for Neisseria and moraxella?
Oxidase positive
How does the oxidse test work?
Detects cytochrome oxidase enzyme
Uses aromatic amine (dimethyl or tetramethyl-para-phenylenediamine dihydrochloride) that is colorless in a reduced state and purple in an oxidized state
What are the precautions about the oxidase reagent?
1% solution (Kovac’s oxidase) is unstable and is oxidized by oxygen and turns dark purple
How is the oxidase reagent stabilized? What is a downside of doing this?
Stabilized with ascorbic acid
- 2% will be stable for a week
- 02% will be stable for a day
More ascorbic acid = less sensitive
What are some oxidase test methods that can be used?
Filter paper
Swab
Drop on colonies
What are the filter paper and swab oxidase methods?
Oxidase on filter paper, colonies rubbed on
Swab put in oxidase, touched to colony
Positive = purple
What is the drop on colonies oxidase method?
Drop oxidase reagent on isolated colonies
Colonies turning purple = positive
What are some precautions with the oxidase test?
Iron/nicrome loops cannot be used, causes false positives
Neisseria loses reactivity with age
Inhibitory/selective media may cause false negatives
Carbohydrate utilization media may cause acid production and cause false negatives
MAC may cause false positive (already pink/purple)
What is the gram stain for Neisseria species?
Gram negative diplococci
“Jellybean” shaped
May be seen intracellularly in PMNs
What unique gram stain feature may N. meningitidis have that N. gonorrhoeae will not?
Capsules
Seen as a “halo” around cells
