About Microbiology
Microbiology questions in a medical or research recruitment paper cover the whole breadth of the subject - the bacterial cell, staining, growth and nutrition, sterilisation, media, enumeration, identification, antibiotics, immunology, viruses, fungi and parasites - and most of them carry a number that has to be worked out rather than recalled. This topic covers that whole breadth on one page, with the counting that goes with it.
What you need to understand
- Bacteria are prokaryotes: no nucleus, a single circular chromosome, ribosomes of the 70S kind, and a peptidoglycan cell wall. Fungi, protozoa and helminths are eukaryotes with a true nucleus, which is why antibacterial drugs can be selective.
- The Gram stain divides bacteria by the thickness of the peptidoglycan layer. Gram positive cells retain the crystal violet and look purple; Gram negative cells have a thin layer under an outer membrane, lose the stain and take up the counterstain.
- Bacteria grow by binary fission, so the population doubles at a fixed interval - the generation time. In a closed culture the growth curve runs through lag, log, stationary and death phases, and only the log phase is exponential.
- The population after n generations is the starting population times two to the power of n. It is the logarithm of the fold increase that counts the generations, not the fold increase itself.
- A viable count works back from the plate: the colonies grew from the small volume that was plated, and that volume had already been diluted, so the count per millilitre is the colonies multiplied by the dilution and divided by the volume plated.
- A count of 30 to 300 colonies on a plate is the range that can be counted accurately. Below it the sampling error is large and above it the colonies merge.
- Sterilisation is measured in log reductions: one log is a tenfold kill, two logs a hundredfold. A sterility assurance level of 10 to the minus six means six logs below the starting population.
- Moist heat kills by coagulating protein, which is why the autoclave at 121 degrees Celsius for 15 minutes works; dry heat needs far longer and higher temperatures because it kills by oxidation instead.
- A minimum inhibitory concentration is read from a dilution series as the lowest concentration in the first clear well. A two fold series halves at every step, so the first well is no steps along and each subsequent well is one more halving.
- Antibiotic resistance arrives by mutation or by transfer: transformation takes up free DNA, conjugation passes a plasmid through a pilus, and transduction carries genes in a bacteriophage.
- Viruses are obligate intracellular parasites with either DNA or RNA and a protein coat, and they are classified by their nucleic acid, their symmetry and whether they have an envelope. They cannot be grown on ordinary media.
- Fungi are eukaryotes with ergosterol in the membrane instead of cholesterol, which is why antifungal drugs target ergosterol and antibacterial drugs miss them entirely.
- An antigen is anything the immune system recognises; an antibody is the protein made against it. IgM appears first in an infection, IgG later and for longer, which is what makes paired sera useful.
- Disinfection reduces the number of organisms on a surface but does not sterilise it. Antisepsis is the same thing applied to living tissue, and asepsis is working so that nothing gets in at all.
- The magnification of a micrograph is the image size divided by the specimen size, and the two have to be in the same unit before they are divided - a millimetre is a thousand micrometres.
How to work through these questions
- Read the question for the shape of the arithmetic before reaching for the science. Words like diluted, doubles, reduced to and percentage each fix one relation.
- For a viable count, write the dilution as a fraction and the plated volume separately, then decide which way each one acts. The answer is always larger than the colony count when less than a millilitre was plated.
- For a growth question, find the number of generations first - either from the time divided by the generation time, or from the logarithm to base two of the fold increase.
- For a log reduction, ask for the power of ten that separates the two counts. It is a subtraction of logs, not a division of the numbers.
- For a dilution series, count the steps to the well in question rather than the wells themselves, because the first well is no steps along.
- Check the answer against the science: a viability above a hundred per cent, a magnification below one, or a count lower than the colonies on the plate all signal a slip.
Mistakes that cost marks
- Dividing by the volume plated instead of multiplying, which reports a count lower than the colonies that were actually counted.
- Forgetting that the dilution has been applied twice when a sample is diluted and then diluted again, which loses the whole factor.
- Multiplying the starting population by the number of generations instead of by two to that power, which turns an exponential into a straight line.
- Quoting the fold increase as the number of generations - a thousandfold increase is ten generations, not a thousand.
- Counting the first well of a dilution series as one step along, which halves the minimum inhibitory concentration.
- Dividing the specimen size by the image size, or forgetting that a millimetre is a thousand micrometres.
- Confusing sterilisation with disinfection: an autoclave sterilises, an alcohol wipe disinfects, and only the first destroys spores reliably.
- Treating the average of a total count and a plate count as if it were a viability, rather than taking the viable count as a fraction of the total.
Worked example
A culture is diluted 1 in 100 and then that dilution is diluted 1 in 100 again. Plating 0.1 mL of the final dilution gives 80 colonies. What is the viable count of the original culture? A culture of 1000 cells doubles every 30 minutes: how many cells are there after 3 hours?
- For the count, the dilution as a whole is 1 in 100 x 100, which is 1 in 10 000. The 80 colonies therefore represent 80 / 10 000 organisms in each millilitre of the dilution.
- Only 0.1 mL was plated, so that figure has to be scaled up tenfold to give the count per millilitre: 80 x 10 000 / 0.1 = 8 000 000 colony forming units per millilitre.
- Check the direction: plating less than a millilitre must give a count HIGHER than the colonies suggest, and 8 million is well above 80. That check catches the commonest slip in the whole topic.
- For the growth, 3 hours is 180 minutes, and at 30 minutes a generation that is 180 / 30 = 6 generations.
- The population is the starting count times two to the power of the generations: 1000 x 2 to the sixth, which is 1000 x 64 = 64 000 cells. The point is that it is a power law - each generation doubles what is already there, so the rise is not a fixed amount per generation. Adding 1000 cells per generation would give 7000, which is out by nearly a factor of ten.
Answer: 8 000 000 colony forming units per millilitre; 64 000 cells after six generations
Practice questions with answers
A few Microbiology questions with the full solution shown, so you can see
how the method is applied before you attempt the timed set.
Question 1
A sample is diluted 1 in 100, and the dilution is then diluted 1 in 100. Plating 1 mL of the final dilution gives 45 colonies. What is the viable count of the original sample, in colony forming units per millilitre?
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A
225000
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B
450000
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C
900000
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D
45
Answer: Option B — with explanation
A viable count is worked back from the plate, not read off it. The colonies that grew came from the small volume that was plated, and that volume had already been diluted twice.
The dilution as a whole is 1 in 100 x 100, which is 1 in 10000, so the 45 colonies represent 45 / 10000 organisms in every millilitre of the dilution. Dividing by the volume plated scales that up to one millilitre.
That is 45 x 10000 / 1 = 450000 colony forming units per millilitre. Dividing by the volume instead of multiplying is the commonest slip: plating less than a millilitre means the count is HIGHER than the colonies suggest, never lower.
Common mistakes
- 45 is not the answer: 45
- 225000 is not the answer: 225000
- 900000 is not the answer: 900000
Question 2
A culture of 250 cells doubles every 60 minutes. What is the population after 6 hours?
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A
32000
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B
8000
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C
16000
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D
1500
Answer: Option C — with explanation
Every generation doubles the population, so the number after n generations is the starting number times two to the power of n. It is a power law, not a straight line - the population does not rise by a fixed amount each hour.
In 6 hours there are 360 minutes, and at 60 minutes a generation that is 360 / 60 = 6 generations.
The population is therefore 250 x 2 to the 6, which is 250 x 64 = 16000 cells. Multiplying the starting number by the number of generations, rather than by two to that power, is what the other answers are built on.
Common mistakes
- 32000 is not the answer: 32000
- 8000 is not the answer: 8000
- 1500 is not the answer: 1500
Question 3
A culture rises from 1000 cells to 16000 cells. How many generations have passed?
Answer: Option D — with explanation
Each generation doubles the population, so the number of generations is the power of two by which the population has grown: it is the logarithm to base two of the fold increase, not the fold increase itself.
The population has gone from 1000 to 16000, which is 16 times as many cells, and 2 to the 4 is 16, so 4 generations have passed.
Quoting the fold increase as the number of generations is the mistake the other answers are built on. Ten generations is a thousandfold increase, and a thousandfold increase is not a thousand generations.
Common mistakes
- 3 is not the answer: 3
- 16 is not the answer: 16
- 5 is not the answer: 5
Question 4
A specimen 2 micrometres across appears 40 millimetres across in a photograph. What is the magnification?
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A
20000
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B
20
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C
40000
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D
1/20000
Answer: Option A — with explanation
Magnification is how many times larger the image is than the object, so it is the image size divided by the specimen size - and the two have to be in the same unit before they are divided.
A millimetre is a thousand micrometres, so the image of 40 mm is 40000 micrometres across, and the magnification is 40000 / 2 = 20000 times.
Leaving the conversion out, or dividing the specimen by the image, both give answers of the wrong size or the wrong kind - a magnification is always a number larger than one, because an image that was smaller than the object would be a reduction.
Common mistakes
- 20 is not the answer: 20
- 40000 is not the answer: 40000
- 1/20000 is not the answer: 1/20000
Question 5
A population of 100000000000 organisms is reduced by a sterilisation process to 10 organisms. What is the log reduction achieved?
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A
11
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B
12
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C
10000000000
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D
10
Answer: Option D — with explanation
A log reduction is the logarithm to base ten of the ratio of the population before to the population after. One log reduction is a tenfold kill, two is a hundredfold, and so on - the logarithm counts the noughts, it is not the number left.
The population has gone from 10 to the 11 to 10 to the 1, a fall of 10 powers of ten, so the reduction is 10 log.
A sterility assurance level of 10 to the minus six means six log reductions below the starting population, which is why the answer is a small number and not a large one.
Common mistakes
- 10000000000 is not the answer: 10000000000
- 12 is not the answer: 12
- 11 is not the answer: 11
Frequently asked questions
Why is the plate count only ever an estimate?
Because a colony can come from one cell or from a clump of several, so the count is of colony forming units rather than of cells. The dilution and the pipetting carry their own errors too, which is why counts are usually quoted to one or two significant figures.
Why must the plate count lie between 30 and 300?
Below about thirty colonies the chance variation is large - a count of ten could easily be five or twenty. Above about three hundred the colonies crowd each other and merge, so several are counted as one. Both ends spoil the statistics.
What is the difference between a log reduction and a percentage kill?
A log reduction counts powers of ten, so six logs is a millionfold kill. A percentage is easy to misread at the last step: a 99.9 per cent kill leaves a thousand organisms in a million, which is only three logs. Logs are used because they do not saturate as the kill gets better.
Why does a Gram negative cell stain differently?
Because its peptidoglycan layer is thin and covered by an outer membrane. The decolouriser washes the crystal violet out through the outer membrane, so the cell takes up the safranin counterstain and looks pink or red instead of purple.
How do I remember which way a dilution works?
Think of the plate: it saw only a small part of the sample, so the true count must be larger. Every dilution step and every fraction of a millilitre makes the true count larger still. If your answer is smaller than the colony count, something is upside down.
Why can an antibiotic work against bacteria and not against the patient?
Because it targets something the bacterium has and the human cell does not - the peptidoglycan wall, the 70S ribosome, or a pathway of folate synthesis. That difference is called selective toxicity, and it is the whole basis of antibiotic therapy.
