Topic 6 - Microbiology A2, Immunity and Forensics

Microbial culture requires sterile media, equipment and technique so that the organism being studied is not outcompeted or confused with contaminants. Aseptic handling protects the sample, the investigator and the environment.

Sterilise media and apparatus, disinfect the work area, minimise exposure of plates or broths, and label controls. Compare growth with a negative control to detect contamination.

If a plate exposed briefly during inoculation grows colonies outside the intended streak, those colonies may be contaminants rather than evidence about the tested organism.

For viable count, make serial dilutions aseptically, spread a known volume, incubate consistently and use a countable plate. $\text{CFU per cm}^3=\text{colonies}/(\text{dilution}\times\text{volume plated in cm}^3)$. Include dilution and units in the answer.

During the exponential phase, $k=\dfrac{\log_{10}N_t-\log_{10}N_0}{0.301t}$, where $N_0$ and $N_t$ are living-cell numbers and $t$ is elapsed time. The constant $k$ is generations per unit time because $\log_{10}2\approx0.301$. Generation time is $1/k$ in the matching time unit.

If $N_0=900$, $N_t=14000$ after 2 h, then $k=(4.146-2.954)/(0.301\times2)=1.98$ generations h$^{-1}$ to three significant figures.

1. Disinfect the bench and aseptically add the same inoculum to sterile, labelled nutrient broth flasks; include an uninoculated blank.
2. Keep medium volume, nutrient composition, pH, aeration and actual incubation temperature controlled.
3. At fixed intervals, mix consistently and withdraw samples aseptically.
4. Zero a colorimeter with the blank, measure absorbance at a suitable wavelength and dilute readings above the calibrated range.
5. Use biological replicates, plot mean absorbance or calibrated cell count against time and calculate rate over the exponential phase.

Use only the organism, temperature and containment authorised by the local risk assessment; keep cultures closed except for aseptic sampling, wear specified protection, disinfect spills and sterilise cultures and contaminated materials before disposal. Minimise culture volume and exposure consistent with reliable results.

In a lytic infection, the viral genome directs synthesis and assembly of new particles, followed by host-cell lysis and release. In latency, viral genetic material persists with little or no particle production—often integrated into host DNA—and can later reactivate into productive infection.

Tuberculosis begins when inhaled $Mycobacterium tuberculosis$ reaches alveoli and is engulfed by macrophages. The bacteria can survive inside these cells, including by resisting normal phagosome digestion. Immune cells form tubercles that may contain a latent infection; reactivation damages lung tissue and can spread through blood to other organs.

Active pulmonary TB can cause persistent cough, blood in sputum, fever, night sweats and weight loss. Gas-exchange damage and disseminated infection can become fatal, especially when immunity is weak.

HIV envelope protein binds CD4 and co-receptors on T helper cells. Viral RNA enters; reverse transcriptase makes DNA, integrase inserts it into host DNA, and the provirus may remain latent or direct new virus production. Budding and immune destruction progressively reduce functional T helper cells.

Loss of T helper signalling weakens activation of B cells, killer T cells and macrophages. Advanced immunodeficiency allows opportunistic infections and cancers; these consequences, rather than one immediate effect of viral entry, can cause death.

Pathogens can enter through broken skin, food and drink, the respiratory tract or mucosal surfaces. Skin, clotting, gut flora, stomach acid and lysozyme reduce entry or survival before a specific immune response is needed.

Each barrier uses a different mechanism: intact skin blocks access, a clot seals damage, resident microbes compete, acid harms swallowed pathogens and lysozyme damages bacterial walls.

A cut bypasses the skin barrier, but clotting limits further entry; a swallowed pathogen must also survive stomach acid and compete with gut flora before reaching intestinal tissue.

Non-specific responses act against many pathogens and begin rapidly. Histamine-driven inflammation changes blood flow and capillary permeability; interferons limit viral replication; phagocytes engulf and digest foreign material.

During phagocytosis, a phagocyte recognises non-self antigens, encloses the pathogen in a vacuole, lysosomes fuse with it and digestive enzymes break it down. Antigen presentation can then initiate a specific response.

A splinter can trigger local swelling as histamine recruits fluid and phagocytes. A virus-infected cell can release interferons that warn nearby cells and activate immune defences.

1. A macrophage engulfs a pathogen, digests it and displays pathogen antigen, acting as an antigen-presenting cell.
2. A complementary T helper cell is activated and releases cytokines that help activate a matching B cell.
3. The selected B cell divides clonally and differentiates into B effector (plasma) cells and memory cells.
4. Plasma cells secrete large quantities of the specific antibody.

Antibody binding can neutralise toxins or attachment sites, agglutinate pathogens and opsonise their surfaces. Macrophages then bind antibody-tagged material more readily and remove it by phagocytosis.

An antigen-presenting macrophage can activate T helper cells. Helper cytokines support both the humoral B-cell response and cell-mediated T-cell response, so the cell types cooperate without performing interchangeable jobs.

Active immunity follows exposure to an antigen and creates memory cells. It can be natural after infection or artificial after vaccination. Passive immunity transfers antibodies from another source and does not create memory.

Active responses are slower initially but usually longer-lasting; passive protection is immediate but temporary. Vaccination presents antigen without needing the disease itself to establish memory.

Antibodies crossing the placenta are natural passive immunity, while a vaccine is artificial active immunity. An antitoxin injection gives passive antibodies and may need repeating.

An evolutionary arms race occurs when pathogen variants that evade immunity reproduce more successfully while host defences favour recognition and removal. The changing pathogen and host populations create reciprocal selection pressures.

HIV can vary its antigens, kill helper T cells and reduce antigen presentation; Mycobacterium tuberculosis can interfere with lysosome fusion and persistence inside phagocytes.

Memory cells against one viral strain may not recognise a mutated antigen, so the host faces a new primary-like response while the new strain spreads.

Selective toxicity arises because drugs target bacterial features such as murein-wall synthesis, 70S ribosomes or bacterial DNA-processing enzymes. Viruses lack these bacterial targets, and host toxicity must still be assessed.

To compare antibiotics, spread a known bacterial culture on sterile agar, place equal-sized antibiotic discs and measure the clear zones after the same incubation. A larger zone suggests greater inhibition under those conditions.

Use aseptic technique, a distilled-water negative control, equal disc size and concentration where possible, replicate plates and consistent incubation. Invert plates to reduce condensation and use safe temperature/containment.

If tetracycline produces a 14 mm zone and methicillin 8 mm on the same strain, tetracycline inhibited growth more in that assay; the result does not automatically establish clinical choice.

Hospital-acquired infection risk is reduced by interrupting transmission: identify the pathogen and reservoir, clean or sterilise equipment, isolate cases, use hand hygiene and protect vulnerable patients.

Different controls act on different links. A disinfectant reduces environmental load, aseptic technique prevents transfer during procedures, and antibiotic stewardship reduces selection for resistant strains.

A catheter-associated infection needs both sterile insertion and ongoing site care; treating only the patient after symptoms appear leaves the route of transmission unchanged.

Bacteria and fungi secrete enzymes that digest large molecules in dead tissue into smaller molecules. They absorb and respire these products, releasing carbon dioxide and sometimes methane.

Decomposition returns carbon to the atmosphere and leaves nutrients available for reuse. Oxygen availability changes the pathway and the gases produced.

In an aerobic compost heap, decomposers release carbon dioxide while breaking down plant material; an anaerobic saturated site can produce methane instead.

PCR copies a selected DNA sequence in vitro. Primers define the region, heat separates strands, primers anneal at a lower temperature and thermostable DNA polymerase extends complementary strands.

Each cycle can double the target, so a tiny starting sample becomes enough for profiling or analysis. The polymerase must survive the high denaturation temperature.

A 20-cycle run can produce roughly a million copies from a single target molecule under ideal doubling; a missing primer or wrong annealing condition prevents selective amplification.

DNA is negatively charged, so an electric field moves it toward the positive electrode. In an agarose gel, smaller fragments pass through pores more easily and travel farther than larger fragments.

Load samples with a known DNA standard, keep the polarity correct, run the gel consistently and compare band positions. A stain or fluorescent tag makes fragments visible.

A sample band aligning with the same-size standard indicates similar fragment length. A matching pattern across several markers is stronger evidence than one shared band.

For identification, agreement at several independent variable loci makes a common source more likely. The evidential strength depends on the combined profile frequency, sample quality, contamination controls and chain of custody; a profile match is not by itself proof of an event or guilt.

For parentage or relatedness, each offspring allele should be explainable by its parents. Across plants or animals, more shared profile markers generally support a closer genetic relationship, provided homologous loci and suitable reference populations are compared. Profiles can also distinguish cultivars, individuals or breeding lines.

Measure core and ambient temperature, record rigor distribution and decomposition stage, collect insects from several body locations and preserve some while rearing others for identification. Use species-specific temperature-development data, then compare the resulting intervals with scene and succession evidence.