Practical 5: Investigation of Gram negative Aerobic bacilli/coccobacilli (NOT FINISHED) Flashcards
List the four genus of gram negative bacilli
Haemophilus
Psuedomonas
Enterobacterales
- Escherichia
- Enterobacter
- Salmonella
- Proteus
List the four enterobacterales
Escherichia
Enterobacter
Salmonella
Proteus
What are the 2 genus of lactose fermenting enterobacterales
Escheruchua
Enterobacter
What are the 2 genus of non lactose fermenting enterobacterales
Salmonella
Proteus
How can you tell a bacteria is a gram negative bacilli from the plate?
GNBs don’t grow well on blood agar
They may not grow at all on MacConkey
List the five gram negative aerobic bacilli
Haemophilus
Bordetella
Brucella
Pseudomonas
Enterobacterales
How do you differentiate pseudomonas from enterobacterales
Pseudomonas is oxidase positive
List the five clinically significant species of Haemophilus
H. influenzae
H. parainfluenzae
H. ducreyi
H. aegyptius
H. aphrophilus
How can you identify capsular H. influenzae from the case study
Isolated from nasopharynx
Bloodstream infections
Pneumoniae
Acute bacterial meningitis
How do you identify non capsulated H. influenzae (2)
Ear infections
Sinusitis in children
How do you identify H. ducreyi
Causes chancroid STD
What test do you carry out to confirm ID of Haemophilus species
X and V factors
What is the principle behind the X and V factor test for Haemophilus
(3)
Haemophilus species are all dependant on one or both of the growth factors, X and V
X = haemin
V = nicotinamide adenine dinucleotide (NAD)
Explain why Haemophilus species can grow on chocolate agar
(3)
Horse blood agar contains X factor normally
Horse blood agar contains a NADase enzyme which inactivates factor V(NAD)
However when the blood is heated to 80 degrees (chocolate agar) the NADase activity is destroyed and thus makes the V factor available
What growth factors does H. influenzae need?
X factor and V factor
What growth factors does H. parainfluenzae need?
V factor only
What growth factors does H.ducreyi need?
X factor only
What growth factors does H. aegyptius need?
X factor and V factor
What growth factors does H. aphrophilus need?
Variable growth with X only
What is the main clinically significant species of Bordetella
B. pertussis
What infection does Bordetella pertussis cause?
Whooping cough
How would you identify Bordetella pertussis?
(3)
WONT GROW ON BLOOD AGAR
Its not dependent on X or V growth factors -> it will grow all over the plate
It needs a highly nutritious medium (without peptone) for primary isolation e.g. chocolate agar or Boredt-Gengou medium (won’t grow on normal blood agar)
What are the clinically significant strains of brucella
(2)
Brucella abortus
Brucella melitensis
What infection does Brucella abortus cause
Causes brucellosis in cattle
What strain of brucella causes Brucellosis in humans
Brucella melitensis
How does a human get brucellosis via brucella melitensis?
Its associated with the consumption of unpasteurised milk and soft cheeses made from the milk of infected animals
How do you carry out an X/V test for Haemophilus
(6)
Ask for X, V, X/V discs, a bijou jar and a Diagnostic Sensitivity Agar (DST) plate
Make a light suspension of the test organism by touching one or more colonies and emulsifying in a reduced volume (avoid picking up chocolate agar)
Lawn the bacterial suspension evenly
Position the three discs on the inoculum
Do the same for your controls
Incubate in 5% CO2 at 35-37 degrees
What controls could you use for the X/V factor test?
Use a H.influenzae control -> needs X and V
How should you incubate the V/X test
Incubate in 5% CO2 at 35-37 degrees
List the four lactose fermenting enterobacterales
Escherichia
Citrobacter
Klebsiella
Enterobacter
Why are lactose fermenters so called?
They produce acid or acid and gas rapidly from lactose
What are lactose fermenters also called?
coliform bacilli
What infections does E.Coli cause
UTIs
Infantile gastroenteritis
Bloodstream infection
What infections can Klebsiella pneumoniae cause
Bacterial pneumonia
HCA UTI and wound infections particularly in immunocompromised
Where is citrobacter found and what infection does it cause
Found in soil
Incriminated in UTI and wound sepsis
Where is enterobacter found and what infection does it cause
Found in soil and water
Associated with systemic infection
List the non lactose fermenters
(5)
Salmonella
Yersinia
Shigella
Proteus
Serratia
What infections does salmonella cause
Foodborne infection and enteric infection
What infection does shigella cause
Bacillary dysentery
What species of proteus are clinically significant
Proteus mirabilis
Proteus vulgaris
How can you identify proteus on an agar plate
Swarming colonies
What infections do proteus cause
UTIs and in suppurating wounds
What are the clinically significant strains of pseudomonas
P. aeruginosa
P. fluorescence
How can you identify P. aeruginosa from the case study
Opportunistic UTI, respiratory tract infections, soft tissue infections, bloodstream infections particularly in cystic fibrosis and severe burns or immunosuppressed
Resistant to antibiotics and disinfectants
What two tests do you need to carry out for Pseudomonas
Growth at different temperatures -> 4 and 42 degrees
King’s A Media
How do you carry out a growth at different temperatures test for pseudomonas?
(5)
Ask for 2 blood/nutrient agar plates
Split plate into 3, test and controls
Zig-zag inoculum of test and controls (+ P. aeruginosa, - P. fluorescence)
Incubate at 42 degrees
Repeat this but reverse controls and incubate at 4 degrees
At what temperature does P. aeruginosa grow at
42 degrees
At what temperature does P. fluorescence grow at
4 degrees
How do you carry out a King’s A Medium test
(5)
Ask for King’s A Medium, P. fluorescence and P. aeruginosa
Separate plate into 3 for test and controls
+ control = P. aeruginosa, - control = P. fluorescence
Streak inoculate the test onto the agar
Incubate at 37 degrees
What is a positive result for King’s A Medium
P. aeruginosa grows at 37 degrees with green fluorescence
What is a negative result for King’s A Medium
P. fluorescence will grow but won’t be fluorescent green
E.Coli will not grow at all
What is the principle behind King’s A Medium
(3)
Contains magnesium chloride and potassium sulphate which enhances pigment profuction
Selective supplement = antimicrobial cocktail comprising centrimide (antiseptic) and nalidixic acid (antibiotic)
P. aeruginosa is resistant to both these selective agents
What is the principle behind Brilliance Agar used to grow enterobacterales
Contains specific chromogenic substrates (Red Gal) which are cleaved by the B-galactosidase enzymes prouced by E. Coli and coliforms, resulting in pink colonies
Medium also contains phenylalanine and tryptophan, as an indication of tryptophan deaminase activity, indicating the presence of Proteus species, morganella and providencia species which appear brown
What testing is carried out for Enterobacterales
Biochemical testing
List the 8 biochemical tests
Citrate utilisation
Indole production
Methyl Red
Voges Proskauer Test
Hydrolysis of Urea
Peptone water sugars
Amino acid decarboxylase
Amino acid deaminase
List the ten components of the biochemical test
Inoculate a sterile water
- Peptone (water) indole
- Buffered glucose Methyl Red
- Buffered glucose Voges Proskauer Test
- Peptone sugar dulcitol
- Lysine decarb base control
- Lysine decarb test
- Phenylalanine agar
- Urea agar
- MacConkey purity plate
- Citrate slope
What must you make sure to do when inoculating your biochemical tests
Label each cap and loosen them
Use a single well isolated colony in about 5mls water
Don’t flame between each test
Inoculate by sliding loop along side of meniscus and shaking
Zig zag over agar slopes and stab
Leave cap loose on citrate -> stab citrate
Add mineral oil to two decarboxylase reactions
NEED TO DO THEORY OF BIOCHEMICAL TESTS FOUND IN PRACTICAL 7
NEED TO DO THEORY OF BIOCHEMICAL TESTS FOUND IN PRACTICAL 7
