Practical 3: Investigation of Gram-positive bacilli Flashcards
List the possible gram positive genus of bacilli
Bacillus species
Corynebacteriae
Listeria
Lactobacilli
List the clinically significant strains of Bacillus species
(4)
Bacillus anthracis
Bacillus cereus
Bacillus subtilis
Bacillus thuringenesis
What would indicate B. anthrax in a case study?
(3)
Food poisoning from eating contaminated fried rice
Contact with animals
Anthrax -> skin (blister), lung, intestine, injection
What would indicate B. cereus in a case study?
Food poisoning -> not from fired rice
How could you identify B. anthrax from a gram stain?
(3)
Medusa head -> large tangled chains of bacilli
Large, grey-white colonies
No haemolysis
What species of Bacillus have B-haemolysis
Bacillus cereus
Bacillus thuringenesis
What species of Bacillus have alpha-haemolysis
B.anthrax
B. subtilis
What are the two tests used to speciate bacillus species
PEMPA agar
Motility
What is the principle behind PEMBA agar
(5)
B. cereus selective agar
Agar contains a peptone level of 0.1% and the addition of sodium pyruvate improves egg yolk precipitation and enhances sporulation
Bromothymol blue is added as a pH indicator to detect mannitol utilisation
The medium is made selective by addition of Polymyxin B
B. cereus = peacock blue colony with no clearance
What does B. cereus look like on PEMBA?
Peacock blue colonies with no clearance
What does B. thuringenesis look like on PEMBA
The same as B.cereus
Peacock blue colonies with no clearance
What does B. anthracis look like on PEMBA
Blue colonies with a small narrow zone of clearance
What does B. subtilis look like on PEMBA
Yellow colonies with no zone of clearance
How do you carry out a PEMBA test?
Ask for PEMBA, a B. cereus control and a B. subtilis control
Streak inoculate
Incubate at 37 degrees Celsius
What is the principle behind the motility test
(3)
Through a hanging drop preparation
Essential to distinguish between motility and Brownian motion
Bacteria move in a definite direction but Brownian = continuous purposeless undirected agitation
How do you carry out the motility test
(6)
Apply a small amount of vaseline to the four corners of a clean coverslip
Add one drop of broth culture of test organism is added to centre of the slip
The cavity slide is inverted and centred over the drop of culture and lowered onto the coverslip
The slide is quickly turned right side up so that the hanging drop is suspended in the well
Place the slide on the microscope under low intensity
Locate the edge of the drop
Which species of bacillus are motile
B. cereus
B. thuringenesis
B. subtilis
What species of bacillus is non motile
B. anthracis
What tests are used for the speciation of Corynebacteria
Trehalose fermentation
Urease production
Tinsdale’s Medium
What is the principle behind the trehalose test
(5)
Peptone water ‘sugars’ comprise of a simple peptone medium plus 0.5% ‘sugar’ and Phenol Red indicator
Peptones yield alkaline products on breakdown, so a change in colour of the indicator only occurs when acid produced from fermentation of the ‘sugar’ exceeds alkali from the peptones
If gas is produced is collected in an inverted tube (Durham tube)
Peptone water sugars can be used to test for fermentation of a wide range of sugars including glucose, lactose, maltose, mannitol, sucrose, dulcitol and trehalose
Trehalose is pink, if fermented it turns yellow
What is the principle behind urease test?
(4)
Certain microbes e.g. Proteus mirabilis or Helicobacter pylori produce the hydrolytic enzyme urease
Urease decomposes urea to ammonia and carbon dioxide
Urea is a neutral substance and its decomposition is accompanied by the production of alkli
Phenol red indicator in the medium changes from a yellow-orange shade at pH 6.8 to pink at alkaline pH8.1
How do you carry out the Trehalose and Urease biochemical tests
(4)
Ask for a trehalose peptone water sugar bijou and a urease agar bijou
Label a blood agar plate to be used as a purity plate
Inoculate the trehalose peptone water sugar and sequentially inoculate the urease agar and then the purity plate without flaming
Incubate each at 37 degrees Celsius
What is the best way of inoculating the trehalose agar?
(2)
Tilt the liquid media to the right and gently rub the inoculated loop on the left side of the tube just below the meniscus
Turn the tube upright and gently shake to inoculate the broth
What is the best way of inoculating the urea slope?
Zig-zag across the surface of the agar and stab the slope with the loop
What is a positive trehalose
Yellow
What is a negative trehalose
Pink
What is a positive urea
pink
What is a negative urea
Yellow
What is the principle behind the Tinsdale Medium
(5)
Tinsdale medium contains serum, tellurite, cystine and blood
Tinsdale differentiates C. diphtheriae and the diphtheroids found in the upper respiratory tract
This differentiation was based on the ability of C. diphtheriae to produce black colonies, surrounded by a brown/black halo
The dark halo is due to the production of H2S from cystine, interacting with the tellurite salt
Diphtheroids do not produce this halo
How do you carry out a Tinsdale test
Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep
How do you carry out a Tinsdale test
Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep
Incubate at 37
How would you know if you have a corynebacteria and not a listeria species
Corynebacteria grow on Tinsdale but listeria wont
