Microbiology

A bacterial cell has a cell wall made of murein (peptidoglycan), which is not found in human cells.

Its genetic material is a single circular molecule of DNA lying free in the cytoplasm (the nucleoid), rather than linear DNA enclosed in a nucleus.

It also contains 70S ribosomes, which are smaller than the 80S ribosomes of a human cell. (Other acceptable answers: plasmids, a capsule, a flagellum or pili.)

A virus is described as non-living because it has no cytoplasm, no organelles, no ribosomes and no metabolism of its own. It cannot respire, grow or carry out any chemical reactions independently.

It is also unable to reproduce by itself. A virus can only replicate by entering a living host cell and using the host's ribosomes, enzymes and raw materials to make copies of its components.

Because it must be inside a living host cell to replicate, a virus is described as an obligate (intracellular) parasite.

The plate is taped so that the lid does not come off accidentally. This protects the investigator from the microbes growing inside and stops unwanted airborne microbes entering and contaminating the culture.

However, the lid is taped but not sealed all the way round, so some air, and therefore oxygen, can still reach the culture. If it were completely sealed, anaerobic conditions could develop and favour the growth of harmful anaerobic pathogens.

The plate is incubated at no more than 25 °C, which is below human body temperature (around 37 °C). Microbes that are pathogenic to humans grow best at body temperature, so keeping the temperature lower means they are unlikely to grow, greatly reducing the risk of culturing a dangerous human pathogen.

In the lag phase, the number of bacteria increases very little. The bacteria are adjusting to their new environment, synthesising the enzymes, proteins and DNA they need before they can begin to divide rapidly, so reproduction is limited by this preparation time.

In the exponential, or log, phase the number increases exponentially, doubling at a constant rate. Nutrients and space are plentiful and few toxic waste products have built up, so the rate of reproduction is at its maximum and far exceeds the rate of death. On a logarithmic scale this phase appears as a straight line.

In the stationary phase the population size stays roughly constant. Nutrients are now becoming limited, space is restricted and toxic waste products have accumulated, so the rate of reproduction has fallen until it equals the rate of death.

In the death, or decline, phase the number falls. The nutrients have been exhausted and toxic wastes have built up to harmful levels, so the rate of death now exceeds the rate of reproduction and the population declines.

A total count is a count of all the cells present in a sample, both living and dead. It can be made directly, for example using a counting chamber under a microscope or by measuring the turbidity (cloudiness) of a broth. It does not distinguish living cells from dead ones.

A viable count, by contrast, counts only the living cells that are able to reproduce. The sample is diluted and spread on agar, where each living cell divides to form one visible colony after incubation. The result is therefore expressed in colony-forming units (cfu) rather than as a direct cell count.

A viable count may slightly underestimate the true number of living cells because if two or more living cells lie very close together on the agar, they grow into a single colony rather than separate ones. Each colony is therefore counted as one, even though it may have arisen from more than one cell, which is exactly why the result is reported as colony-forming units.

(a) Three ×10 dilution steps give an overall dilution factor of 10 × 10 × 10 = 10³ (a 1 in 1000 dilution).

(b) The number of viable bacteria per cm³ is calculated using:

cfu per cm³ = (number of colonies ÷ volume plated) × dilution factor.

Only 0.1 cm³ was plated, so first divide by the volume plated: 56 ÷ 0.1 = 560. Then multiply by the dilution factor: 560 × 10³ = 560 000.

So the original milk contained 5.6 × 10⁵ cfu cm⁻³.

(c) A plate from the undiluted milk would contain far too many bacteria. The colonies would overlap and merge into a confluent lawn, so individual colonies could not be distinguished and counted. Diluting first brings the number into the countable range of about 30–300 colonies per plate, giving a reliable count.

HIV consists of a protein capsid surrounding its genetic material, which is RNA rather than DNA. Around the capsid is a lipid envelope, derived from the host cell membrane, studded with surface glycoproteins that allow the virus to attach to and enter host cells. Like all viruses it has no organelles or ribosomes of its own.

Because its genetic material is RNA, HIV cannot directly insert its genes into the host's DNA. It carries the enzyme reverse transcriptase, which catalyses the synthesis of a complementary DNA strand using the viral RNA as a template — the reverse of normal transcription, hence the name retrovirus.

This newly made viral DNA can then be inserted into the host cell's genome, so that the host cell's machinery transcribes and translates the viral genes to make new virus particles. Reverse transcriptase is therefore essential for HIV to convert its RNA into DNA and so replicate inside the host.

(a) The number of divisions is the total time divided by the mean division time. Converting 2.5 hours to minutes gives 150 minutes, so:

n = 150 ÷ 25 = 6 divisions.

The number after n divisions is N = N₀ × 2ⁿ. With N₀ = 4.0 × 10² and n = 6:

N = 4.0 × 10² × 2⁶ = 4.0 × 10² × 64 = 25 600.

So there are 2.56 × 10⁴ bacteria.

(b) The bacteria could not continue to grow exponentially because they are in a closed system with finite resources. As the population rose, the nutrients in the medium became limited and toxic waste products accumulated, while space also became restricted. These factors slowed the rate of reproduction until it no longer exceeded the rate of death, so the population entered the stationary phase rather than continuing to double.

Both a bacterium and a virus contain genetic material in the form of nucleic acid, and both have an outer protein-containing layer — a murein cell wall in the bacterium and a protein capsid in the virus. Both are also far smaller than a eukaryotic cell.

However, the differences are more numerous and more important. A bacterium is a living cell: it has cytoplasm enclosed by a cell surface membrane, a circular DNA chromosome, 70S ribosomes, and often plasmids, a capsule, a flagellum and pili. Because it has ribosomes and enzymes it can carry out its own metabolism, respire and reproduce independently by binary fission.

A virus, in contrast, is not a cell. It consists only of a protein capsid surrounding a core of nucleic acid, which may be either DNA or RNA, sometimes with a lipid envelope. It has no cytoplasm, no membrane-bound or other organelles and no ribosomes, so it has no metabolism of its own. It cannot reproduce independently and must enter a living host cell to replicate, making it an obligate parasite.

In summary, although both contain nucleic acid and an outer protein layer, only the bacterium is a living, metabolising cell, whereas the virus is a non-living, acellular particle that depends entirely on a host cell.

To obtain a viable count, a measured sample of the culture is taken and diluted in a series of equal ×10 steps, for example by transferring 1 cm³ into 9 cm³ of sterile water and mixing, then repeating. This serial dilution lowers the cell concentration so that, after a known volume is spread on agar and incubated, a countable number of separate colonies (about 30–300) forms, each grown from one living cell. Multiplying the colony count by the dilution factor and dividing by the volume plated gives the viable count in cfu cm⁻³, and repeating plates at each time point and taking a mean improves reliability.

Aseptic technique is essential throughout: equipment and media are sterilised, the inoculating loop or pipette is flamed or used sterile, and transfers are made near a Bunsen burner with the dish barely open. This prevents contaminating microbes from entering and being counted as part of the culture, which would make the results invalid.

The method has limitations. First, a viable count can underestimate the true number of living cells, because cells that are clumped together grow into a single colony and are counted only once — this is why the result is reported as colony-forming units. Second, it is slow: the plates must be incubated for many hours before colonies appear, so the result is not available immediately, making it less suitable for following rapid changes than a direct method such as turbidity.

On balance, a viable count is valuable because it measures only living, reproducing cells, but the clumping error and the time delay mean the technique must be carried out carefully and the figures interpreted as colony-forming units rather than absolute cell numbers.