A 63-year-old man has had a stiff, painful shoulder for a year, beginning 6 months after a shoulder arthroplasty. He has no fever, and his CRP and ESR are normal. At revision surgery, five periprosthetic tissue samples are sent for culture. The Gram stain shows rare neutrophils and no organisms. The laboratory's routine tissue protocol is sheep blood, chocolate and MacConkey agars in 5% CO2 for 4 days, plus anaerobic blood agar and thioglycolate broth for 5 days. All cultures are negative at day 5, but the surgeon still suspects a low-grade infection. Which change to the setup is most likely to recover the expected organism?

Hold anaerobic plates and broth for 14 days

Add fungal media at 30 °C for 4 weeks

Add mycobacterial media for 8 weeks

Hold MacConkey agar in CO2 for 14 days

A wound swab was streaked for isolation on sheep blood agar; the plate is shown after 24 hours, with the quadrants numbered in streaking order. Before identification tests, the technologist should:

Pick isolated colonies of each type from quadrants 3 and 4

Pick growth from quadrant 1, where it is heaviest

Discard the plate as a contaminated culture

Mix colonies of both types into one suspension

After 18 hours, the methylene blue indicator in an anaerobic jar is still blue, and the Bacteroides fragilis control plate shows no growth. The most likely cause is:

Oxygen was not removed from the jar

The jar was incubated at 35 °C

B. fragilis needs 5 days to grow

Too little CO2 was added to the jar

Specimen processing: preparation, media, inoculation and incubation

16 ASCP MLS practice questions on Specimen processing: preparation, media, inoculation and incubation, part of Microbiology. The first 16 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 63-year-old man has had a stiff, painful shoulder for a year, beginning 6 months after a shoulder arthroplasty. He has no fever, and his CRP and ESR are normal. At revision surgery, five periprosthetic tissue samples are sent for culture. The Gram stain shows rare neutrophils and no organisms. The laboratory's routine tissue protocol is sheep blood, chocolate and MacConkey agars in 5% CO2 for 4 days, plus anaerobic blood agar and thioglycolate broth for 5 days. All cultures are negative at day 5, but the surgeon still suspects a low-grade infection. Which change to the setup is most likely to recover the expected organism?
1. AAdd fungal media at 30 °C for 4 weeks
2. BAdd mycobacterial media for 8 weeks
3. CHold anaerobic plates and broth for 14 days
4. DHold MacConkey agar in CO2 for 14 days
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Answer: C · Hold anaerobic plates and broth for 14 days
Late, low-grade infection of a shoulder prosthesis with normal inflammatory markers is typical of Cutibacterium acnes, a slow-growing anaerobic gram-positive rod that lives in the skin of the shoulder and axilla. Its colonies often appear only after 5 to 7 days or later, so cultures held for the usual 5 days miss many infections. Holding anaerobic plates and broth for about 14 days, with several tissue samples, raises recovery, and the same organism in two or more samples supports true infection. Fungi and mycobacteria are rare causes of prosthetic joint infection and are cultured only when specifically suspected.
- A: Fungal prosthetic joint infections are rare and do not fit this classic C. acnes picture.
- B: Mycobacteria are rare in prosthetic joints and are cultured only when suspected.
- D: MacConkey agar inhibits gram-positive bacteria such as C. acnes, however long it is held.
Question 2 · Easy
A wound swab was streaked for isolation on sheep blood agar; the plate is shown after 24 hours, with the quadrants numbered in streaking order. Before identification tests, the technologist should:
Figure: MedExams
1. APick growth from quadrant 1, where it is heaviest
2. BDiscard the plate as a contaminated culture
3. CMix colonies of both types into one suspension
4. DPick isolated colonies of each type from quadrants 3 and 4
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Answer: D · Pick isolated colonies of each type from quadrants 3 and 4
Quadrant streaking thins the inoculum, so the later quadrants hold isolated colonies. This plate has heavy, confluent growth in quadrants 1 and 2 and two colony types, large white and small yellow, standing apart in quadrants 3 and 4. Each test should start from a single isolated colony of one type, so that every result belongs to one organism. Growth from the heavy quadrants is likely to be mixed and gives unreliable identifications and susceptibility results.
- A: Quadrant 1 is confluent and mixed; colonies there are not isolated.
- B: Two colony types are common in wounds; each is worked up, not discarded.
- C: Mixing two organisms gives a mixed suspension and meaningless results.
Question 3 · Medium
At what temperature, and for how long, are most fungal cultures incubated before being reported as negative?
1. A30 °C for about 4 weeks
2. B35 °C for 48 hours
3. C42 °C for 7 days
4. D25 °C for 24 hours
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Answer: A · 30 °C for about 4 weeks
Most pathogenic fungi grow best at 25-30 °C, and many, such as dimorphic fungi and dermatophytes, grow slowly. Cultures are therefore held for about 4 weeks (30 days) and read regularly before being called negative. Yeasts such as Candida often appear within 2-3 days, but a short hold would miss slow molds. Plates are sealed with gas-permeable tape to prevent drying and accidental opening.
- B: 48 hours is far too short for slow-growing molds.
- C: 42 °C is not used for routine fungal culture.
- D: 24 hours misses nearly all molds.
Question 4 · Hard
After 18 hours, the methylene blue indicator in an anaerobic jar is still blue, and the Bacteroides fragilis control plate shows no growth. The most likely cause is:
1. AThe jar was incubated at 35 °C
2. BOxygen was not removed from the jar
3. CB. fragilis needs 5 days to grow
4. DToo little CO2 was added to the jar
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Answer: B · Oxygen was not removed from the jar
Methylene blue is blue when oxidized and turns colorless once oxygen is removed; resazurin turns from pink to colorless in the same way. A blue indicator after incubation means the jar never became anaerobic, usually because of a spent or damp palladium catalyst, an exhausted gas-generating sachet or a leaking lid. B. fragilis grows within 24-48 hours, so its failure confirms the problem. The system is checked, and the specimens are set up again if possible.
- A: 35 °C is the correct temperature for anaerobic culture.
- C: B. fragilis grows within 24 to 48 hours.
- D: Low CO2 would not keep the oxygen indicator blue.
Question 5 · Easy
Chocolate agar plates for routine bacterial cultures are incubated at 35 °C in:
1. AAmbient air only
2. BAn anaerobic atmosphere
3. C5-10% CO2
4. DA microaerobic mix (5% O2)
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Answer: C · 5-10% CO2
Chocolate agar is used for fastidious organisms such as Haemophilus and Neisseria, which are capnophilic and grow best with extra carbon dioxide. A CO2 incubator set to 5-10% CO2, or a candle jar giving about 3%, provides it. Campylobacter needs a microaerobic mix instead, and anaerobes need an oxygen-free atmosphere. Plates are usually examined at 24 and 48 hours.
- A: Haemophilus and Neisseria grow poorly without added CO2.
- B: Anaerobic conditions are for anaerobic plates, not routine chocolate agar.
- D: A microaerobic mix is used for Campylobacter and Helicobacter.
Question 6 · Easy
Why are agar plates incubated with the lid facing down (agar side up)?
1. ATo let more CO2 reach the colonies
2. BTo keep anaerobes from growing
3. CTo stop condensation dripping onto the agar
4. DTo make hemolysis easier to see
Answer and explanation in LearnFree account
Question 7 · Hard
Stool in Cary-Blair from a patient with bloody diarrhea is plated on Campylobacter-selective agar and incubated at 42 °C in a 5% CO2 incubator. There is no growth at 72 hours, but a multiplex GI PCR detected Campylobacter. The most likely error is that:
1. ACampylobacter needs 7 days of incubation
2. BThe plate should have been held at 25 °C
3. CA microaerobic atmosphere was not used
4. DCary-Blair medium killed the organism
Answer and explanation in LearnFree account
Question 8 · Easy
What makes MacConkey agar selective for gram-negative bacilli?
1. AVancomycin, colistin and nystatin
2. BColistin and nalidixic acid
3. CPhenylethyl alcohol
4. DCrystal violet and bile salts
Answer and explanation in LearnFree account
Question 9 · Medium
A patient in diabetic ketoacidosis has a black, necrotic lesion in the nose, and a biopsy is sent for fungal culture. How should the tissue be prepared?
1. ASwabbed, and the swab plated
2. BMinced into small pieces with a scalpel
3. CDecontaminated with NALC-NaOH first
4. DGround thoroughly in a tissue grinder
Answer and explanation in LearnFree account
Question 10 · Easy
For tissue and normally sterile fluids, a thioglycolate broth is often inoculated along with the plates because it:
1. ASelects for gram-negative rods only
2. BGives an accurate colony count
3. CRecovers small numbers of aerobes and anaerobes
4. DInhibits normal skin flora
Answer and explanation in LearnFree account
Question 11 · Medium
A routine sputum culture is plated on sheep blood, chocolate and MacConkey agars. Which pathogen is the chocolate agar mainly there to recover?
1. AKlebsiella pneumoniae
2. BHaemophilus influenzae
3. CStreptococcus pneumoniae
4. DPseudomonas aeruginosa
Answer and explanation in LearnFree account
Question 12 · Medium
When one specimen is inoculated onto several plates, the plates should be inoculated in order from:
1. ADifferential to nonselective media
2. BMost selective to least selective
3. CLeast selective to most selective
4. DBroth first, then the agar plates
Answer and explanation in LearnFree account
Question 13 · Medium
In an anaerobic culture, Bacteroides bile esculin (BBE) agar is used to:
1. AIsolate pigmented Prevotella species
2. BSelect and identify the Bacteroides fragilis group
3. CSelect gram-positive anaerobes such as clostridia
4. DSupport all anaerobes as a nonselective medium
Answer and explanation in LearnFree account
Question 14 · Hard
A suprapubic aspirate is plated with a 0.01 mL calibrated loop, and 35 colonies of E. coli grow. What colony count should be reported?
1. A3,500 CFU/mL
2. B350 CFU/mL
3. C35,000 CFU/mL
4. D35 CFU/mL
Answer and explanation in LearnFree account
Question 15 · Easy
Which medium selects for gram-positive cocci in a wound specimen that also contains gram-negative rods?
1. AMacConkey agar
2. BEosin methylene blue (EMB) agar
3. CColistin-nalidixic acid (CNA) agar
4. DXylose lysine deoxycholate (XLD) agar
Answer and explanation in LearnFree account
Question 16 · Medium
Routine blood agar is made with sheep blood. In throat cultures, which advantage does this give?
1. AIt inhibits Haemophilus haemolyticus, a strep mimic
2. BIt supplies V factor for Haemophilus influenzae
3. CIt suppresses alpha-hemolytic viridans streptococci
4. DIt makes Streptococcus pyogenes grow faster
Answer and explanation in LearnFree account

Specimen processing: preparation, media, inoculation and incubation

A wound swab was streaked for isolation on sheep blood agar; the plate is shown after 24 hours, with the quadrants numbered in streaking order. Before identification tests, the technologist should:

At what temperature, and for how long, are most fungal cultures incubated before being reported as negative?

After 18 hours, the methylene blue indicator in an anaerobic jar is still blue, and the Bacteroides fragilis control plate shows no growth. The most likely cause is:

Chocolate agar plates for routine bacterial cultures are incubated at 35 °C in:

Why are agar plates incubated with the lid facing down (agar side up)?

Stool in Cary-Blair from a patient with bloody diarrhea is plated on Campylobacter-selective agar and incubated at 42 °C in a 5% CO2 incubator. There is no growth at 72 hours, but a multiplex GI PCR detected Campylobacter. The most likely error is that:

What makes MacConkey agar selective for gram-negative bacilli?

A patient in diabetic ketoacidosis has a black, necrotic lesion in the nose, and a biopsy is sent for fungal culture. How should the tissue be prepared?

For tissue and normally sterile fluids, a thioglycolate broth is often inoculated along with the plates because it:

A routine sputum culture is plated on sheep blood, chocolate and MacConkey agars. Which pathogen is the chocolate agar mainly there to recover?

When one specimen is inoculated onto several plates, the plates should be inoculated in order from:

In an anaerobic culture, Bacteroides bile esculin (BBE) agar is used to:

A suprapubic aspirate is plated with a 0.01 mL calibrated loop, and 35 colonies of E. coli grow. What colony count should be reported?

Which medium selects for gram-positive cocci in a wound specimen that also contains gram-negative rods?

Routine blood agar is made with sheep blood. In throat cultures, which advantage does this give?