Culturing and controlling microorganisms

What is a culture medium and which conditions (nutrients, temperature, pH, oxygen) are controlled when culturing bacteria in the laboratory? State the difference between a solid and a liquid medium.

Explain what aseptic technique is in the microbiology laboratory and discuss the purpose of two specific practices: flaming the inoculating loop until it is red hot and working close to the burner.

Viruses do not grow on agar plates. Discuss why (what do they lack in order to multiply on their own?) and indicate which systems laboratories use to culture them (cell cultures, embryonated eggs).

Design an experiment to compare the efficacy of two disinfectants against a skin bacterium: agar plates, controls, measurement of inhibition zones, and criteria for concluding which is more effective, stating the variables you would keep constant.

The graph represents the growth curve of a closed bacterial culture, with its four phases labelled with numbers (1 to 4). Name each phase and explain what happens to nutrients and waste in each.

A bacterium divides every $20$ minutes. Starting from $1\,000$ cells in the exponential phase, calculate how many there will be after two hours (how many generations fit in?). Then discuss why exponential growth cannot be sustained in a closed culture (which factors slow it down?).

Differentiate between sterilisation, disinfection, and antisepsis, and give an example of a situation (operating theatre, kitchen worktop, superficial wound) in which each is applied.

Name four methods of microbial control in the food industry (pasteurisation, UHT sterilisation, refrigeration and freezing, salting or curing) and explain the principle each one is based on (does it kill, inhibit or hinder growth?).

Explain what pasteurisation consists of (temperature and time, for example $72\ \text{°C}$ for $15$ seconds), why it does not sterilise the food (what survives?) and which historical diseases it helped control in milk (tuberculosis, brucellosis).

Compare the effectiveness of bleach, $70\ \%$ alcohol and soap against bacteria and against enveloped viruses. Explain why $70\ \%$ alcohol disinfects better than nearly pure alcohol (the role of water in protein denaturation and in penetration) and what soap exactly does to a lipid viral envelope.

Following a school banquet, 48 diners develop acute gastroenteritis caused by Salmonella enterica. During the inspection, samples are taken from the food consumed (homemade mayonnaise, roast chicken, and cake) and from the kitchen staff. The laboratory carries out plate isolation and analyzes the genetic profile using pulsed-field gel electrophoresis (PFGE).

1. a) The culture tests positive for S. enterica in both the mayonnaise and raw chicken scraps found on the countertop. However, the DNA banding pattern of the sick individuals matches the one isolated in the mayonnaise 100 % and differs from the one isolated in the raw chicken. Interpret this result and deduce the origin of the food poisoning, justifying why molecular identification is indispensable compared to simple species-level taxonomic culture.
2. b) The mayonnaise was prepared with eggs at 7:30 h and remained at room temperature (24.0 °C) until consumed at 14:30 h (7 hours in total). If the initial contamination in the batch was 10 bacteria/g and the doubling time at that temperature is 30 minutes, calculate the theoretical final concentration per gram and justify how keeping the dish refrigerated at 4.0 °C from the moment it was prepared would have modified the risk.
3. c) Design a hazard analysis and critical control points (HACCP) system for the school's kitchen that simultaneously prevents initial biological contamination, cross-contamination, and microbial proliferation.

In a home canning workshop, two groups prepare fermented cabbage (sauerkraut), whose preservation relies on the epiphytic lactic acid microbiota (Leuconostoc and Lactobacillus):

• Group 1: Chops the cabbage, adds 2.5 % table salt by weight, presses the mixture until the liquid completely covers the vegetable, and seals the jar preventing air entry, maintaining it at 20.0 °C. After three weeks, the preparation has a pH of 3.6 and a fresh acidic smell.

• Group 2: Chops the cabbage, adds no salt to "make it more heart-healthy", leaves the jar open to the air, and adds hot water at 45.0 °C. After 4 days, the pH remains at 6,2, there is mold on the surface, and it gives off a foul odor.

1. a) Justify the dual selective role of the 2.5 % salt and the anoxic immersion in Group 1's procedure against competing bacteria of telluric origin and enterobacteria.
2. b) Explain the metabolic change that causes the drop in pH to 3.6 and reason why this decrease inhibits the development of Clostridium botulinum endospores, a strict anaerobic bacillus that tolerates anoxia but is sensitive to acidity.
3. c) Predict what biochemical and ecological failures Group 2's protocol caused and why the combination of a temperature of 45.0 °C and the presence of oxygen prevented spontaneous lactic acid fermentation.

In a hospital sterilization unit, they must process:

1) Boxes of stainless steel surgical instruments (forceps, retractors).

2) Laparoscopes and endoscopic optics with fiberglass and thermosensitive plastics (maximum tolerated temperature: 55.0 °C).

3) Disposable plastic syringes and catheters bulk-packaged in sealed cardboard boxes.

1. a) Assign and justify the optimal method for each material from: pressurized steam autoclave, ethylene oxide gas chamber, and ionizing gamma radiation, detailing why the discarded methods in each case would be less suitable (they would ruin the material, fail to penetrate adequately, or be less practical or safe).
2. b) During an autoclave cycle (134.0 °C for 5 minutes), the thermometer and pressure gauge recorded the prescribed values, and the external indicator tape changed color correctly. However, upon incubating the biological indicator (vial with 10^6 Geobacillus stearothermophilus endospores placed in the center of the load) at 56.0 °C for 24 hours, the broth turns from purple to yellow and exhibits turbidity.

Interpret what occurred, decide with reasoning whether the use of the instruments can be authorized, and justify why biological monitoring takes precedence over physical and chemical controls.

Distinguish between a bactericidal and a bacteriostatic antibiotic, give an example of each type (penicillin versus tetracyclines) and explain what happens to the bacterial population when each one is discontinued.

What is an antibiogram and how is it performed (antibiotic discs on the inoculated plate)? Explain what it tells the doctor (sensitive, intermediate, resistant) and why an antibiotic should not be prescribed without this information when it is possible to obtain it.

Explain, using natural selection, how a bacterial population resistant to an antibiotic arises when treatment is stopped early, and why the antibiotic does not 'create' resistance.

A patient diagnosed with the flu insists on being prescribed amoxicillin "in case the infection moves down to the chest", while a farmer continuously administers low doses of tetracycline in the drinking water of healthy pigs to "accelerate their fattening and prevent diarrhea".

1. a) Refute the patient's demand based on biochemical and cytological foundations: justify why beta-lactam antibiotics are completely harmless to viruses and predict what immediate selective impact that amoxicillin will have on the patient's own commensal microbiota.
2. b) Interpret the described farming practice from the perspective of natural selection: explain why continuous subtherapeutic concentrations represent the optimal scenario for selecting resistant bacteria compared to full lethal doses.
3. c) Explain the horizontal gene transfer mechanisms (conjugation, transformation, and transduction) through which a resistance gene generated in a commensal livestock bacterium can end up in a human pathogenic strain in a hospital setting.

What is horizontal gene transfer and how does it differ from 'vertical' inheritance from parents to offspring? Name its three mechanisms in bacteria.

Describe the three mechanisms of bacterial horizontal gene transfer — conjugation, transformation, and transduction — indicating which vehicle transports the DNA in each case and which one is involved in the spread of resistance.

Many antibiotics are natural products of soil bacteria (streptomycetes). Explain what evolutionary advantage manufacturing an antibiotic gives its producer (chemical warfare for nutrients) and analyse why the discovery of new antibiotics has slowed down in recent decades.

An industrial bioreactor produces yoghurt, beer or insulin on a large scale. Explain which parameters are controlled continuously (temperature, pH, dissolved oxygen, stirring, foam) and discuss what happens to the product if two of them, of your choice, go out of control.

In a hospital ICU, an outbreak of carbapenem-resistant Klebsiella pneumoniae is detected, affecting four patients admitted with venous catheters and assisted mechanical ventilation.

1. a) Analyze the epidemiological chain of the outbreak: identify the probable source, the most plausible transmission mechanism (vehicle and passive vector), and justify what physiological factors make these patients especially vulnerable.
2. b) Design a contingency plan with control measures ranked in order of priority (isolation and physical barriers, active screening, and fomite disinfection), biologically justifying why hand hygiene between patients is more critical for halting transmission than environmental fumigation.
3. c) Critically evaluate the following proposal: "To eradicate the outbreak at its root, we will administer prophylactic colistin to all patients admitted to the ICU, whether or not they have symptoms." Predict the selective and evolutionary consequences of this measure in the medium term.

Colistin is a polypeptide antibiotic that destabilizes the outer membrane of Gram-negative bacteria and was considered a medical "last resort" for years. In 2015, the mcr-1 gene, capable of conferring colistin resistance, was detected in pig farms located on a conjugative plasmid.

1. a) Justify why the discovery of the mcr-1 gene on a plasmid generated incomparably greater global health alarm than if the resistance were due to a point mutation in the bacterial chromosome.
2. b) For decades, colistin was administered at continuous low doses in pigs as a growth promoter. Explain from the theory of natural selection how this livestock practice created the ideal conditions for the selection and fixation of the mcr-1 gene.
3. c) Drawing on the "One Health" approach, argue why legislation banned the preventive use of colistin in livestock farming and refute the stance of a farmer who claims that "bacteria from livestock intestines have no way of reaching patients in a human ICU".

A patient with cystic fibrosis suffers from a chronic lung infection caused by a pan-resistant strain of Pseudomonas aeruginosa (resistant to all available antibiotics). The medical team considers the compassionate use of phage therapy.

1. a) In the laboratory, two types of bacteriophages are available: phage ΦA (strictly lytic) and phage ΦB (lysogenic or temperate phage). Justify which of the two must be selected for the treatment and what severe clinical risk administering the lysogenic phage would pose.
2. b) After administering an inhaled dose of lytic phages, the bacterial load in sputum drops in 72 hours from 8.0 · 10^7 to 3.0 · 10^2 CFU/mL, while the phage titer increases from 1.0 · 10^5 to 4.5 · 10^9 PFU/mL. Compare these kinetics with the pharmacokinetics of a traditional chemical antibiotic and deduce what advantage the bacteriophage presents regarding dosage at the site of infection.
3. c) On day 14, the patient's bacterial load rebounds to 6.0 · 10^6 CFU/mL. Analysis shows that the surviving bacteria have lost the OprM membrane receptor that the phage used for attachment. Explain why a therapeutic design based on a "cocktail" of multiple phages targeting independent cell receptors minimizes treatment failure compared to the use of a single viral strain.

Unit challenge. Is your kitchen an ideal culture medium for pathogens or a hostile environment worthy of an extremophilic archaeon? Design a food biosecurity protocol for your home.

1. Data collection: Use a thermometer to measure the interior temperature of your refrigerator and countertop, or check the current temperature of your municipality on the Met Éireann website. Record both values (for example, $4.1\text{ }^\circ\text{C}$ and $21.8\text{ }^\circ\text{C}$).

2. Microbial analysis: Prepare a table comparing the optimal growth range of common mesophilic eubacteria with the adaptations of extremophilic archaea (psychrophiles or thermophiles), deducing which microorganisms would thrive in each measured area.

3. Control strategies: Propose three bacterial growth control measures for everyday foods (such as heat above $72.5\text{ }^\circ\text{C}$, osmotic salting, or refrigeration), explaining how they alter the viability of the bacterial wall or membrane.

4. Final product: Write a brief one-page report containing your measurements, the eubacteria-archaea comparative table, and your reasoned preservation protocol.

Assessment criteria:

• Rigorous recording of real thermal data using the decimal point.

• Correct biological differentiation between eubacterial and archaeal physiology.

• Scientific justification of the culture and microbial control methods applied.