Gram Negative identification Flashcards
What is the name for bacteria that require CO2 to grow?
Capnophilic
What do Microaerophilic bacteria require?
grows in a low concentration of oxygen, but not in its absence or in ambient air
What are 5 features common to all Enterobacteriaceae?
- Gram negative rods
- Facultative anaerobes
- Ferment Glucose
- Grow rapidly
- Reduce Nitrate to nitrite
What are 2 other features that are common to most Enterobacteriaceae?
- Oxidase Negative (except Plesiomonas)
2. Many are motile (except Klebsiella, Shigella)
Explain how MacConkey agar is both selective and differential
Selective: contains bile salts and crystal violet that inhibit the growth of gram positive organisms
Differential: contains lactose as a CHO source. Only gram negatives capable of fermenting lactose will grow. Effectively cuts the Enterobacteriaceae in half
- media will turn pink if a lactose fermenter is growing
What is the oxidase test checking for?
presence of cytochrome oxidase involved in the reduction of oxygen at the end of the electron transport chain
Are the Enterobacteriaceae oxidase positive or negative?
All oxidase negative (except 1)
What 3 species of gram negative bacteria are oxidase + ?
Vibrio
Pseudomonas
Aeromonas
What is the nitrate test used for?
Detect the presence of nitrate reductase (ability to turn nitrate into nitrite)
What is a positive test for the Nitrate test? Why do you sometimes get false negatives? How can you test for that?
Presence of red colour after addition of reagents = positive
If the bacteria further reduces the nitrite to ammonia then no red will be observed
Adding zinc will confirm the test
- change to red = negative nitrate test
- no change = positive nitrate test
What are the reagents in a TSI slant test?
1% glucose, 10% lactose & 10 % sucrose agar
Sodium thiosulfate for H2S production
FeSO4 for H2S detection
Phenol red indicator
Explain the significance of the red/yellow colouring in a TSI slant
Fermentation of glucose will lower pH in tube and turn agar yellow (from red)
Once glucose is depleted, organism must switch to lactose/sucrose or break down amino acids
If the organism can break down the other sugars, the pH remains low and the tube stays yellow
If it cannot break down the other sugars, it will resort to amino acids, increasing the pH and the tube will turn red again
- only on the slant as this is an aerobic reaction
What produces the black colouring sometimes seen in TSI slants?
Sodium thiosulfate is used by some bacteria to produce H2S which reacts with the iron salts to produce black precipitation
What is the significance of a TSI slant test where the tube remains red?
Means that the organism is an Afermenter
Where are Vibrio and Aeromonas found typically? what do they cause?
Primarily found in water sources
May cause gastrointestinal disease
What features do Aeromonas and Vibrio share with the Enterobacteriaceae?
Gram-negative
Facultative anaerobes
Fermentative (glucose)
What is a key difference between Aeromonas + Vibrio and the Enterobacteriaceae?
Aeromonas and Vibrio are oxidase positive
What 3 tests can be used to confirm the presence of Vibrio? What should the results be?
- Growth in 6% NaCl (see growth)
- Oxidase test (positive)
- Susceptibility to O/129 (susceptible)
What is O/129?
A vibriostatic agent that most other bacteria are resistant to
What selective agar is used to culture Vibrio?
TCBS = Thiosulfate Citrate Bile Salts Sucrose Agar
How is TCBS selective? (4 features)
Contains Oxgall which inhibits the growth of gram positives
Sodium thiosulfate / ferric chloride can detect H2S
Sucrose CHO source
Vibrio = yellow on agar
What is the morphology of Haemophilus influenzae? atmospheric requirements? Oxidase test?
Gram negative bacilli, (cocco-bacilli)
Oxidase positive
Facultative anaerobic, (CO2 enhance growth)
What is Haemophilus influenzae primarily associated with?
Respiratory tract infections
What media will Haemophilus influenzae grow on, which will it not? Why?
Won’t grow on MacConkey or SBA
Will grow on chocolate agar because the RBCs have been lysed and the heme and NAD released which Haemophilus influenzae requires
What are the two factors that Haemophilus influenzae needs to grow?
X: protoporphyrin IX (basically heme)
V: NAD (nicotinamide adenine dinucleotide)
What is the Porphyrin test?
determines an isolates X factor requirement.
What is the method for conducting the porphyrin test?
Heavy suspension of organism in aminolevulinic acid, incubated 4 hr and then illuminated with UV light —> examined for red fluorescence
What is a positive/negative porphyrin test?
positive: Fluorescence indicates enzymatic conversion of aminolevulinic acid to porphyrins and therefore X-factor independence
negative: no fluorescence means no conversion of ALA to porphyrins = haemophilus influenzae
Where are afermenters normally found
In the water and soil
As part of the normal flora on the mucous membranes of man and animals
What groups the Afermenters together?
No family designation (i.e. comprise many families)
Includes many genera whose names are continually changing
**By definition they do not ferment glucose
What is the typical morphology of the Aftermenters? What kind of atmosphere do they require?
Gram neg bacilli or cocco-bacilli
Most are obligate aerobes
The means of isolating Aftermenters is similar to which other type of bacteria?
Enterobacteriaceae
How long do afermenters typically take to grow? At what temperature?
24-48 hours
usually around 35º
What are 4 diagnostic clues that an organism is an afermenter?
Lack of glucose fermentation (TSI= K/K)
Often oxidase +
May fail to grow on a MacConkey plate
May be unusually resistant to antibiotics
Name 4 aftermenters
- Stenotrophomonas maltophilia
- Acinetobacter
- Elizabethkingia meningosepticum
- Burkholderia
Describe the clinical features of Stenotrophomonas maltophilia. Where is it normally found? what is it susceptible to?
Found transiently in the flora of hospital patients. Highly prevalent in the ICU
- 2nd most commonly isolated aftermenter
Susceptible to TMP/SMX
Where are Acinetobacter usually found? What are they susceptible to?
typically found in the soil and colonizing the skin
Susceptible to Colisten
Elizabethkingia meningosepticum is associated with what diseases? It is also one of three organisms that is susceptible to…
Occasionally associated with meningitis and septicemia (very high mortality rate)
One of 3 gram negative bacteria susceptible to Vancomycin IN VITRO
What 3 species of Burkholderia are true pathogens?
B. cepacia, B. mallei and B. pseudomallei
What is the most clinically relevant species of pseudomonas?
aeruginosa
What is the morphology of Pseudomonas aeruginosa?
Gram negative rod
What 2 tests is P. aeruginosa positive for?
Oxidase and nitrate tests
What is are 2 common things to observe when growing P. aeruginosa ?
Often has a metallic sheen
Greenish discoloration of media (may be other colours)
What is different about the growth temperature of P. aeruginosa?
can grow at up to 42 degrees
