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2.2 Microbiology > Practical 3: Investigation of Gram-positive bacilli > Flashcards

Practical 3: Investigation of Gram-positive bacilli Flashcards

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Biology 101 flashcards
1
Q

List the possible gram positive genus of bacilli

A

Bacillus species

Corynebacteriae

Listeria

Lactobacilli

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2
Q

List the clinically significant strains of Bacillus species
(4)

A

Bacillus anthracis

Bacillus cereus

Bacillus subtilis

Bacillus thuringenesis

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3
Q

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

A

Food poisoning from eating contaminated fried rice

Contact with animals

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

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4
Q

What would indicate B. cereus in a case study?

A

Food poisoning -> not from fired rice

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5
Q

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

A

Medusa head -> large tangled chains of bacilli

Large, grey-white colonies

No haemolysis

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6
Q

What species of Bacillus have B-haemolysis

A

Bacillus cereus

Bacillus thuringenesis

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7
Q

What species of Bacillus have alpha-haemolysis

A

B.anthrax

B. subtilis

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8
Q

What are the two tests used to speciate bacillus species

A

PEMPA agar

Motility

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9
Q

What is the principle behind PEMBA agar
(5)

A

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

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10
Q

What does B. cereus look like on PEMBA?

A

Peacock blue colonies with no clearance

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11
Q

What does B. thuringenesis look like on PEMBA

A

The same as B.cereus

Peacock blue colonies with no clearance

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12
Q

What does B. anthracis look like on PEMBA

A

Blue colonies with a small narrow zone of clearance

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13
Q

What does B. subtilis look like on PEMBA

A

Yellow colonies with no zone of clearance

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14
Q

How do you carry out a PEMBA test?

A

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

Streak inoculate

Incubate at 37 degrees Celsius

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15
Q

What is the principle behind the motility test
(3)

A

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

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16
Q

How do you carry out the motility test
(6)

A

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

17
Q

Which species of bacillus are motile

A

B. cereus

B. thuringenesis

B. subtilis

18
Q

What species of bacillus is non motile

A

B. anthracis

19
Q

What tests are used for the speciation of Corynebacteria

A

Trehalose fermentation

Urease production

Tinsdale’s Medium

20
Q

What is the principle behind the trehalose test
(5)

A

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

21
Q

What is the principle behind urease test?
(4)

A

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

22
Q

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

A

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

23
Q

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

A

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

24
Q

What is the best way of inoculating the urea slope?

A

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

25
Q

What is a positive trehalose

A

Yellow

26
Q

What is a negative trehalose

A

Pink

27
Q

What is a positive urea

A

pink

28
Q

What is a negative urea

A

Yellow

29
Q

What is the principle behind the Tinsdale Medium
(5)

A

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

30
Q

How do you carry out a Tinsdale test

A

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

31
Q

How do you carry out a Tinsdale test

A

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

Incubate at 37

32
Q

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

A

Corynebacteria grow on Tinsdale but listeria wont

2.2 Microbiology (60 decks)

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