Practical 3: Investigation of Gram-positive bacilli

Question 1

List the possible gram positive genus of bacilli

Answer 1

Bacillus species

Corynebacteriae

Listeria

Lactobacilli

Question 2

List the clinically significant strains of Bacillus species
(4)

Answer 2

Bacillus anthracis

Bacillus cereus

Bacillus subtilis

Bacillus thuringenesis

Question 3

What would indicate B. anthrax in a case study?
(3)

Answer 3

Food poisoning from eating contaminated fried rice

Contact with animals

Anthrax -> skin (blister), lung, intestine, injection

Question 4

What would indicate B. cereus in a case study?

Answer 4

Food poisoning -> not from fired rice

Question 5

How could you identify B. anthrax from a gram stain?
(3)

Answer 5

Medusa head -> large tangled chains of bacilli

Large, grey-white colonies

No haemolysis

Question 6

What species of Bacillus have B-haemolysis

Answer 6

Bacillus cereus

Bacillus thuringenesis

Question 7

What species of Bacillus have alpha-haemolysis

Answer 7

B.anthrax

B. subtilis

Question 8

What are the two tests used to speciate bacillus species

Answer 8

PEMPA agar

Motility

Question 9

What is the principle behind PEMBA agar
(5)

Answer 9

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

Question 10

What does B. cereus look like on PEMBA?

Answer 10

Peacock blue colonies with no clearance

Question 11

What does B. thuringenesis look like on PEMBA

Answer 11

The same as B.cereus

Peacock blue colonies with no clearance

Question 12

What does B. anthracis look like on PEMBA

Answer 12

Blue colonies with a small narrow zone of clearance

Question 13

What does B. subtilis look like on PEMBA

Answer 13

Yellow colonies with no zone of clearance

Question 14

How do you carry out a PEMBA test?

Answer 14

Ask for PEMBA, a B. cereus control and a B. subtilis control

Streak inoculate

Incubate at 37 degrees Celsius

Question 15

What is the principle behind the motility test
(3)

Answer 15

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

Question 16

How do you carry out the motility test
(6)

Answer 16

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

Question 17

Which species of bacillus are motile

Answer 17

B. cereus

B. thuringenesis

B. subtilis

Question 18

What species of bacillus is non motile

Answer 18

B. anthracis

Question 19

What tests are used for the speciation of Corynebacteria

Answer 19

Trehalose fermentation

Urease production

Tinsdale’s Medium

Question 20

What is the principle behind the trehalose test
(5)

Answer 20

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

Question 21

What is the principle behind urease test?
(4)

Answer 21

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

Question 22

How do you carry out the Trehalose and Urease biochemical tests
(4)

Answer 22

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

Question 23

What is the best way of inoculating the trehalose agar?
(2)

Answer 23

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

Question 24

What is the best way of inoculating the urea slope?

Answer 24

Zig-zag across the surface of the agar and stab the slope with the loop

Question 25

What is a positive trehalose

Answer 25

Yellow

Question 26

What is a negative trehalose

Answer 26

Pink

Question 27

What is a positive urea

Answer 27

pink

Question 28

What is a negative urea

Answer 28

Yellow

Question 29

What is the principle behind the Tinsdale Medium
(5)

Answer 29

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

Question 30

How do you carry out a Tinsdale test

Answer 30

Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep

Question 31

How do you carry out a Tinsdale test

Answer 31

Ask for a Tinsdale agar, C. diphtheriae and a diphtheroid such as haemophilus, neisseria, staphylococcus and strep

Incubate at 37

Question 32

How would you know if you have a corynebacteria and not a listeria species

Answer 32

Corynebacteria grow on Tinsdale but listeria wont