A laboratory pours its own sheep blood agar. A batch made by a new staff member sets as opaque, chocolate-brown plates instead of bright red ones. The agar base comes from the same lot as the last good batch, the pouring volume is correct and the sterility check is clean. QC plates incubated overnight at 35 °C in 5% CO2 give these results: Streptococcus pyogenes, growth but no clear zone of beta-hemolysis; Streptococcus pneumoniae, growth with no visible green zone; Haemophilus influenzae, good growth across the whole plate, with no staphylococcal streak present. The most likely error in preparing the batch is:

Blood added to agar that was still too hot

Agar base autoclaved for much too long

Plates stored too long before use

Blood used after its expiry date

QC plates read after overnight incubation in the CO2 incubator: Haemophilus influenzae on chocolate agar, scant growth; Streptococcus pneumoniae on sheep blood agar, scant growth; Escherichia coli on MacConkey agar, normal growth. All media are in date and the thermometer reads 35 °C. The most likely cause is:

The CO2 supply to the incubator has failed

The QC strains have lost viability

The lot of chocolate agar is faulty

The incubator temperature is too low

In thioglycolate broth, an isolate grows only as a narrow band a few millimetres below the surface, with no growth at the surface or at the bottom of the tube. In which atmosphere should its plates be incubated?

About 5% O2 with 10% CO2 (microaerobic)

No O2, with H2 and CO2 (anaerobic)

Culture media and incubation

15 DOH Abu Dhabi (HAAD) lab licensing practice questions on Culture media and incubation, part of Microbiology. The first 15 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A laboratory pours its own sheep blood agar. A batch made by a new staff member sets as opaque, chocolate-brown plates instead of bright red ones. The agar base comes from the same lot as the last good batch, the pouring volume is correct and the sterility check is clean. QC plates incubated overnight at 35 °C in 5% CO2 give these results: Streptococcus pyogenes, growth but no clear zone of beta-hemolysis; Streptococcus pneumoniae, growth with no visible green zone; Haemophilus influenzae, good growth across the whole plate, with no staphylococcal streak present. The most likely error in preparing the batch is:
1. AAgar base autoclaved for much too long
2. BBlood added to agar that was still too hot
3. CPlates stored too long before use
4. DBlood used after its expiry date
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Answer: B · Blood added to agar that was still too hot
Blood is added to the base only after it has cooled to about 45 to 50 °C. Added to hotter agar, the red cells lyse and are heated, which turns the medium brown, just as when chocolate agar is made on purpose. Heating also releases NAD and destroys the NADases of sheep blood, so Haemophilus influenzae, which needs X and V factors, grows without a staphylococcal streak, and hemolysis can no longer be read. Over-autoclaving can darken the base but frees no V factor, and old blood or old plates do not support H. influenzae either. The batch should be discarded and the cooling step checked with a thermometer.
- A: Over-autoclaving can darken the base but does not free V factor for Haemophilus.
- C: Long storage dries plates out; it does not turn them brown or free V factor.
- D: Old blood may hemolyze, but sheep blood NADases would still stop H. influenzae growing.
Question 2 · Medium
A gram-negative rod was subcultured to the same agar and incubated for 48 hours in the conditions shown. The organism is best described as:
Figure: MedExams
1. ACapnophilic
2. BFacultatively anaerobic
3. CObligately anaerobic
4. DMicroaerophilic
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Answer: D · Microaerophilic
The isolate grows only in the reduced-oxygen atmosphere of about 5% oxygen and 10% carbon dioxide, at both 35 and 42 °C, and fails in air, in a CO2 incubator and in an anaerobic jar. That defines a microaerophile, which needs some oxygen but is harmed by the 21% in air, or the nearly equal level in a CO2 incubator. Campylobacter jejuni behaves this way, and its growth at 42 °C helps select it from stool flora. A capnophile would grow in the CO2 incubator, and an anaerobe would grow in the anaerobic jar.
- A: A capnophile would grow in air with added CO2.
- B: A facultative anaerobe grows both in air and anaerobically.
- C: An obligate anaerobe would grow in the anaerobic atmosphere.
Question 3 · Hard
Dehydrated MacConkey agar is prepared at 50 g/L. A technologist needs 30 plates of 20 mL each and makes 10% extra to allow for losses. How much powder should be weighed?
1. A3.3 g
2. B27 g
3. C30 g
4. D33 g
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Answer: D · 33 g
Volume needed = 30 plates x 20 mL = 600 mL; adding 10% gives 600 + 60 = 660 mL, or 0.66 L. Powder = 0.66 L x 50 g/L = 33 g. Leaving out the extra gives 30 g, subtracting 10% gives 27 g, and a decimal slip when converting mL to L gives 3.3 g. The powder is dissolved in purified water by boiling, autoclaved and cooled to about 50 °C before pouring.
- A: 3.3 g comes from a decimal error when converting mL to L.
- B: 27 g subtracts the 10% instead of adding it.
- C: 30 g leaves out the 10% extra.
Question 4 · Hard
QC plates read after overnight incubation in the CO2 incubator: Haemophilus influenzae on chocolate agar, scant growth; Streptococcus pneumoniae on sheep blood agar, scant growth; Escherichia coli on MacConkey agar, normal growth. All media are in date and the thermometer reads 35 °C. The most likely cause is:
1. AThe QC strains have lost viability
2. BThe lot of chocolate agar is faulty
3. CThe incubator temperature is too low
4. DThe CO2 supply to the incubator has failed
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Answer: D · The CO2 supply to the incubator has failed
H. influenzae and S. pneumoniae both grow best in 5% to 10% CO2, while E. coli grows well in air. Poor growth of two capnophiles on two different media, with normal growth of E. coli and a correct temperature, points to the atmosphere rather than to one medium. The CO2 level is checked with an independent gas analyzer and the gas supply inspected. Patient plates incubated during the failure are re-incubated in a correct atmosphere and their results reviewed.
- A: Two different strains failing together in the same way points to a shared condition.
- B: A chocolate agar fault would not explain poor pneumococcal growth on blood agar.
- C: The thermometer reads 35 °C, and a low temperature would also slow E. coli.
Question 5 · Hard
In thioglycolate broth, an isolate grows only as a narrow band a few millimetres below the surface, with no growth at the surface or at the bottom of the tube. In which atmosphere should its plates be incubated?
1. ARoom air with 5% to 10% CO2
2. BAbout 5% O2 with 10% CO2 (microaerobic)
3. CAmbient room air alone
4. DNo O2, with H2 and CO2 (anaerobic)
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Answer: B · About 5% O2 with 10% CO2 (microaerobic)
In thioglycolate broth, oxygen is highest at the surface and falls with depth. Growth limited to a band just below the surface shows an organism that needs some oxygen but is harmed by the level in air: a microaerophile. Such organisms, for example Campylobacter and Helicobacter, are incubated in about 5% oxygen, 10% CO2 and 85% nitrogen, using a gas-generating pack or a tri-gas incubator. Obligate aerobes grow at the surface, obligate anaerobes at the bottom and facultative organisms throughout.
- A: A CO2 incubator still holds near-atmospheric oxygen, too much for a microaerophile.
- C: Room air has about 21% oxygen, which inhibits a microaerophile.
- D: An anaerobic atmosphere removes the oxygen a microaerophile needs.
Question 6 · Easy
Phenylethyl alcohol (PEA) agar is used mainly to:
1. ASupply X and V factors for Haemophilus
2. BInhibit gram-negative bacilli so gram-positive cocci grow
3. CDifferentiate lactose fermenters from non-fermenters
4. DFavor fungi through its acid pH
Answer and explanation in LearnFree account
Question 7 · Medium
In an anaerobic jar that uses a hydrogen-generating gas pack, the palladium catalyst:
1. AReleases carbon dioxide for capnophiles
2. BAbsorbs oxygen into the palladium metal
3. CChanges color once anaerobic conditions form
4. DCombines hydrogen with oxygen to form water
Answer and explanation in LearnFree account
Question 8 · Easy
In thioglycolate broth, sodium thioglycolate serves as:
1. AA pH indicator that shows fermentation
2. BA selective agent against gram-positives
3. CA source of X factor (hemin)
4. DA reducing agent that removes oxygen
Answer and explanation in LearnFree account
Question 9 · Medium
When a new lot of MacConkey agar is checked, which organism shows that its selective agents are working?
1. AEnterococcus faecalis
2. BProteus mirabilis
3. CPseudomonas aeruginosa
4. DEscherichia coli
Answer and explanation in LearnFree account
Question 10 · Medium
A candle extinction jar is suitable for incubating:
1. AObligate anaerobes such as Bacteroides
2. BDermatophytes from skin and nail samples
3. CMicroaerophiles such as Helicobacter pylori
4. DCapnophiles such as Neisseria and Haemophilus
Answer and explanation in LearnFree account
Question 11 · Easy
Which medium is both selective and differential?
1. AMacConkey agar
2. BSheep blood agar
3. CThioglycolate broth
4. DChocolate agar
Answer and explanation in LearnFree account
Question 12 · Medium
Sabouraud dextrose agar favors fungi over most bacteria mainly because of its:
1. AIncubation temperature of 42 °C
2. BBile salts and crystal violet
3. CAcid pH and high glucose content
4. DHigh salt content (7.5% sodium chloride)
Answer and explanation in LearnFree account
Question 13 · Medium
Chromogenic agars used for urine culture distinguish organisms mainly by:
1. AHydrogen sulfide blackening of colonies
2. BEnzyme substrates that give colored colonies
3. CThe type of hemolysis around colonies
4. DLactose fermentation shown by a neutral red indicator
Answer and explanation in LearnFree account
Question 14 · Easy
Which term describes a liquid medium, such as selenite broth, that favors one group of stool pathogens while holding back the normal flora for some hours?
1. ADifferential medium
2. BEnrichment broth
3. CEnriched medium
4. DTransport medium
Answer and explanation in LearnFree account
Question 15 · Easy
Which agar is selective and differential for Salmonella and Shigella in stool?
1. ATCBS agar
2. BLöwenstein-Jensen medium
3. CChocolate agar
4. DXLD or Hektoen enteric agar
Answer and explanation in LearnFree account

Culture media and incubation

A gram-negative rod was subcultured to the same agar and incubated for 48 hours in the conditions shown. The organism is best described as:

Dehydrated MacConkey agar is prepared at 50 g/L. A technologist needs 30 plates of 20 mL each and makes 10% extra to allow for losses. How much powder should be weighed?

In thioglycolate broth, an isolate grows only as a narrow band a few millimetres below the surface, with no growth at the surface or at the bottom of the tube. In which atmosphere should its plates be incubated?

Phenylethyl alcohol (PEA) agar is used mainly to:

In an anaerobic jar that uses a hydrogen-generating gas pack, the palladium catalyst:

In thioglycolate broth, sodium thioglycolate serves as:

When a new lot of MacConkey agar is checked, which organism shows that its selective agents are working?

A candle extinction jar is suitable for incubating:

Which medium is both selective and differential?

Sabouraud dextrose agar favors fungi over most bacteria mainly because of its:

Chromogenic agars used for urine culture distinguish organisms mainly by:

Which term describes a liquid medium, such as selenite broth, that favors one group of stool pathogens while holding back the normal flora for some hours?

Which agar is selective and differential for Salmonella and Shigella in stool?