Classification and the diagnostic laboratory
Introduced| English | Meaning |
|---|---|
| culture/ˈkʌltʃə/ | culture |
| Gram stain | Thick peptidoglycan stays purple (positive). |
| peptidoglycan/ˈpeptɪdɒɡlɪkən/ | A material made of sugar chains cross-linked by short peptides that gives most bacterial cell walls their strength. |
| polymerase chain reaction/ˌpɒlɪməˈreɪz tʃeɪn rɪˈækʃn/ | polymerase chain reaction |
A bottle flags at 2 a.m.
- A blood culture bottle signals growth in the night. By morning the laboratory reports "Gram-positive cocci in clusters"; the next day, "Staphylococcus aureus, sensitive to flucloxacillin".
- Each line of that report is one fork in a decision tree, and reading it is a clinical skill.
The Gram stain
- The Gram stain 革兰染色 runs four steps: crystal violet, iodine, a decolouriser (alcohol or acetone), then a pink counterstain.
- Gram-positive bacteria stay purple; Gram-negative bacteria lose the purple and turn pink.
Put the steps of the Gram stain in order.
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The decolouriser is the deciding step; the counterstain shows the cells that lost the purple.
Why the colours differ
- Gram-positive cells have a thick wall of peptidoglycan 肽聚糖 that traps the crystal violet–iodine complex.
- Gram-negative cells have a thin peptidoglycan layer inside an outer membrane; the decolouriser washes the complex out. The same wall structure later predicts which drugs work.
Why do Gram-negative bacteria stain pink?
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The decolouriser removes the complex from thin-walled cells, which then take up the counterstain.
Walking the tree
- After the colour comes shape (cocci or bacilli) and arrangement: clusters suggest staphylococci, chains streptococci.
- Then quick tests: catalase separates staphylococci (positive) from streptococci (negative), and coagulase separates S. aureus (positive) from skin staphylococci.
Match each finding to what it tells you.
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Each test is one fork in the tree.
Culture is the reference
- Culture 培养 grows the organism: most bacteria show in 24–48 hours, while slow growers such as tuberculosis need weeks.
- Only a living culture can be tested against antibiotics, so culture stays the reference standard.
PCR is the fast lane
- The polymerase chain reaction 聚合酶链反应 (PCR) copies a target DNA sequence, doubling it each cycle, and can name an organism in 1–2 hours.
- It shows that the DNA is there, not that the organism is alive or which drugs will work.
A positive PCR shows that the organism is alive and tells you which antibiotics will work.
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PCR detects DNA, which dead organisms also leave behind; drug testing needs a living culture.
PCR doubles the target each cycle. Starting from one copy, about how many billion copies are there after 30 cycles?
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2³⁰ = 1 073 741 824, about 1.07 billion.
Worked example: reading the report
- (Fictional) Blood culture at 48 hours: Gram-positive cocci in clusters, catalase positive, coagulase positive.
- Clusters → staphylococci; catalase positive → not streptococci; coagulase positive → S. aureus, not skin flora. Three lines, three forks.
Gram-positive cocci in clusters, catalase positive, coagulase positive. What is the organism?
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Clusters and catalase give staphylococci; coagulase picks out S. aureus.
Worked example: real or contaminant?
- (Fictional) Two blood-culture sets are taken from a well-looking patient. One bottle in one set grows coagulase-negative staphylococci after 3 days; the other set stays negative.
- These skin bacteria in only one of two sets most likely came from the skin during sampling. Taking two sets is what makes this judgement possible.
One of two blood-culture sets grows coagulase-negative staphylococci after 3 days in a well patient. What is most likely?
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These are common skin bacteria; growth in only one of two sets points to contamination.
Three common slips
- Reading a Gram stain as a species. It gives a colour-and-shape class, not a name.
- "PCR replaces culture." Live organisms and drug testing still need culture.
- Treating every positive bottle as infection. Skin contaminants are common.
Recap
- Gram stain: thick peptidoglycan stays purple (positive); thin wall plus outer membrane turns pink (negative).
- Then shape, arrangement, catalase and coagulase.
- Culture (24–48 hours) is the reference; PCR names DNA in 1–2 hours but cannot test drugs.