In the Gram stain, why do Gram-negative bacteria lose the crystal violet–iodine complex during decolourisation?

Alcohol dissolves their lipid-rich outer membrane, and the thin peptidoglycan layer cannot hold the complex

They have no peptidoglycan at all, so crystal violet never binds to their cell walls in the first place

Iodine cannot cross their outer membrane, so no insoluble complex forms inside these cells at any stage

Safranin displaces the crystal violet from their walls because it binds lipopolysaccharide more tightly

A mould isolated from spoiled bread has broad aseptate hyphae, root-like rhizoids and runner-like stolons, with unbranched sporangiophores arising at the nodes above the rhizoids, each ending in a black sporangium. Which genus is it?

Rhizopus, which bears sporangia on sporangiophores arising from stolons above tufts of rhizoids

Aspergillus, which bears conidia in chains on a swollen vesicle at the tip of each conidiophore

Penicillium, which bears chains of conidia on a branched, brush-like penicillus of septate hyphae

Mucor, which bears sporangia on its sporangiophores but has neither rhizoids nor stolons

Rhizopus, which bears sporangia on sporangiophores arising from stolons above tufts of rhizoids

Which of the following statements about bacterial endospores and fungal spores are correct?

Calcium dipicolinate and a dehydrated core contribute to the heat resistance of endospores

Small acid-soluble spore proteins bind and protect the DNA of endospores

Calcium dipicolinate and a dehydrated core contribute to the heat resistance of endospores

Mould conidia are generally more heat-resistant than bacterial endospores

Small acid-soluble spore proteins bind and protect the DNA of endospores

Endospores are formed by Staphylococcus and Salmonella when nutrients are exhausted

UHT milk, commercially sterile and aseptically packed, turns bitter and forms a gel after four months at room temperature. The raw milk had been held cold for several days before processing. What is the most likely cause?

Heat-stable proteases secreted by psychrotrophic Pseudomonas in the cold raw milk survived UHT and acted in storage

Spores of Bacillus cereus that survived UHT germinated in the pack and produced a rennet-like protease

Heat-stable proteases secreted by psychrotrophic Pseudomonas in the cold raw milk survived UHT and acted in storage

Pseudomonas cells survived the UHT treatment and continued to grow slowly in the sealed pack at room temperature

Lactic acid bacteria that entered through a faulty seal soured the milk, and the acid coagulated the casein

The characteristic fishy odour of spoiling chilled marine fish arises mainly from

trimethylamine, formed when bacteria such as Shewanella putrefaciens reduce trimethylamine oxide

histamine, formed when bacteria such as Morganella decarboxylate the free histidine of fish muscle

hexanal and other aldehydes formed by the autoxidation of fish polyunsaturated lipids

ammonia released when fish muscle enzymes break down urea stored in the tissue

Which of the following statements about the spoilage of foods are correct?

Lysozyme of egg albumen attacks peptidoglycan, so Gram-positive bacteria rarely spoil whole eggs

Rope in bread is caused by Bacillus spores that survive baking and later make the crumb sticky and stringy

Lysozyme of egg albumen attacks peptidoglycan, so Gram-positive bacteria rarely spoil whole eggs

Field fungi such as Fusarium grow in stored grain at lower water activity than storage fungi such as Aspergillus

Rope in bread is caused by Bacillus spores that survive baking and later make the crumb sticky and stringy

Spoilage of vacuum-packed chilled meat is dominated by strictly aerobic Pseudomonas

Several guests vomited 2 to 4 hours after eating a cream-filled pastry that had been prepared by hand, left at room temperature for hours and then warmed thoroughly before serving. Which organism and mechanism best explain the outbreak?

Staphylococcus aureus, whose heat-stable enterotoxin formed in the pastry before reheating

Salmonella, whose cells invaded the intestinal wall after surviving the reheating step

Clostridium perfringens, whose spores germinated in the gut and released enterotoxin on sporulation

Clostridium botulinum, whose neurotoxin formed in the cream and blocked acetylcholine release

Staphylococcus aureus, whose heat-stable enterotoxin formed in the pastry before reheating

Why is pH 4.6 the dividing line between acid and low-acid canned foods?

Below pH 4.6 Clostridium botulinum spores cannot germinate and grow to produce toxin

Below pH 4.6 the spores of Clostridium botulinum are killed by the acid alone during storage

Below pH 4.6 any botulinum toxin already formed is broken down by acid hydrolysis

Below pH 4.6 Clostridium botulinum spores cannot germinate and grow to produce toxin

Below pH 4.6 heat-resistant flat-sour bacteria cannot survive, so no heat process is needed

Which of the following statements about food-borne pathogens and their toxins are correct?

The emetic toxin of Bacillus cereus, cereulide, is heat-stable, so reheating fried rice does not make it safe

Escherichia coli O157:H7 produces Shiga toxins and can cause haemolytic uraemic syndrome

The emetic toxin of Bacillus cereus, cereulide, is heat-stable, so reheating fried rice does not make it safe

Shigella needs millions of cells to cause dysentery, so food handlers rarely spread it

Escherichia coli O157:H7 produces Shiga toxins and can cause haemolytic uraemic syndrome

Aflatoxin M1 in milk is produced by Aspergillus growing in the milk itself

In a yoghurt culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, acid develops faster than with either organism alone. Which description of their interaction is correct?

The lactobacillus releases peptides and amino acids that stimulate the streptococcus, which supplies formic acid and CO₂ that stimulate the lactobacillus

The streptococcus releases peptides and amino acids that stimulate the lactobacillus, which supplies formic acid and CO₂ that stimulate the streptococcus

The lactobacillus produces nisin that suppresses competitors, while the streptococcus produces lactase that frees glucose for both organisms

Each organism ferments a different half of lactose, the streptococcus using glucose and the lactobacillus using only the galactose

Which organism is chiefly responsible for leavening idli batter during its overnight natural fermentation?

Leuconostoc mesenteroides, a heterofermentative lactic acid bacterium that produces CO₂

Acetobacter aceti, which oxidises ethanol in the batter to acetic acid and gas

Leuconostoc mesenteroides, a heterofermentative lactic acid bacterium that produces CO₂

Propionibacterium freudenreichii, which converts the batter's lactate into propionate and CO₂

Aspergillus oryzae, which grows on the rice and black gram as a solid-state koji

Which of the following statements about probiotics, prebiotics and synbiotics are correct?

Inulin and fructo-oligosaccharides resist human digestive enzymes and are fermented by colonic bifidobacteria

A synbiotic combines a probiotic with a prebiotic substrate

Saccharomyces boulardii is a yeast used as a probiotic

Inulin and fructo-oligosaccharides resist human digestive enzymes and are fermented by colonic bifidobacteria

Paneer is a probiotic food because its curd is formed by lactic acid bacteria

A synbiotic combines a probiotic with a prebiotic substrate

Saccharomyces boulardii is a yeast used as a probiotic

Food Microbiology: Microorganisms, Growth and Death Kinetics, Spoilage, Pathogens, Fermented and Probiotic Foods

This chapter answers Section XL5.2, Food Microbiology, of XL5 Food Technology, one of the five optional sections of the Life Sciences (XL) paper (the Engineering Sciences (XE) paper sets the same four headings as its section XE6 Food Technology, one of nine, so this chapter serves a candidate of either paper): “Characteristics of Microorganisms: Morphology of bacteria, yeast, mold and actinomycetes, spores and vegetative cells, gram-staining, Resistant bacteria. Microbial Growth: D-value, Z-value, F-value, Growth and death kinetics, serial dilution technique. Food Spoilage: Spoilage microorganisms in different food products including milk, fish, meat, egg, cereals and their products. Toxins from Microbes: Pathogens and non-pathogens including Staphylococcus, Salmonella, Shigella, Escherichia, Bacillus, Clostridium, and Aspergillus genera, Food borne diseases. Fermented Foods and Beverages: Curd, yoghurt, cheese, pickles, soya-sauce, sauerkraut, idli, dosa, vinegar, alcoholic beverages, and sausage. Probiotic foods: probiotic microorganisms, their benefits, prebiotics, synbiotics.” The numericals are exponential growth, D-, z- and F-values, log reductions under the 12D rule, and plate counts back-calculated through a dilution series.

1. Bacteria, yeasts, moulds and actinomycetes

Group	Cell form	Reproduction and spores	Role in foods
Bacteria	Prokaryotic, about 0.5–5 µm; cocci (clusters in staphylococci, chains in streptococci), rods (bacilli), curved and spiral forms; peptidoglycan wall	Binary fission; some genera form endospores	Fastest spoilers of moist, near-neutral foods; most food-borne pathogens
Yeasts	Unicellular eukaryotic fungi, oval, about 5–10 µm	Budding (Saccharomyces) or fission (Schizosaccharomyces); sexual ascospores	Ferment sugars to ethanol and CO₂; spoil sugary and acid foods (osmophilic Zygosaccharomyces)
Moulds	Filamentous fungi: hyphae forming a mycelium; septate in Aspergillus and Penicillium, aseptate (coenocytic) in Mucor and Rhizopus	Asexual conidia on conidiophores (a swollen vesicle in Aspergillus, a brush-like penicillus in Penicillium) or sporangiospores in sporangia (Mucor; Rhizopus, which also has rhizoids and stolons)	Tolerate lower water activity and pH than most bacteria; spoil bread, fruit and grain; some make mycotoxins
Actinomycetes	Gram-positive bacteria with branched filaments about 1 µm wide; high G+C (Streptomyces)	Chains of conidia at hyphal tips	Earthy, musty taints (geosmin) in water and fish; antibiotic producers

Two consequences follow for foods. Bacteria multiply fastest and so dominate moist, near-neutral foods, while yeasts and moulds win where acid, sugar, salt or dryness hold bacteria back. A mould is identified by its spore-bearing structures, seen in a mount from the colony edge, and a yeast by its size and budding.

2. Gram staining, spores and resistant bacteria

Gram staining runs crystal violet (primary stain), Gram's iodine (mordant, forming a crystal violet–iodine complex), alcohol or acetone (decolouriser) and safranin (counterstain). Gram-positive cells have a thick peptidoglycan wall with teichoic acids that alcohol dehydrates and tightens, trapping the complex, so they stay purple. Gram-negative cells have a thin peptidoglycan layer under a lipopolysaccharide outer membrane; alcohol dissolves the membrane, the complex washes out and the cells take the pink counterstain. Old Gram-positive cultures may stain variably. Bacillus, Clostridium, Staphylococcus, Listeria and the lactic acid bacteria are Gram-positive; Salmonella, Shigella, Escherichia, Pseudomonas, Campylobacter and Vibrio are Gram-negative.

Endospores are dormant cells formed inside the vegetative cell by Bacillus (aerobic or facultative) and Clostridium (anaerobic) when nutrients run out. A dehydrated core rich in calcium dipicolinate, small acid-soluble proteins coating the DNA, a peptidoglycan cortex and protein coats make them resistant to heat, radiation, drying and disinfectants. They germinate back into vegetative cells, often after a sublethal heat shock, when triggered by nutrients such as L-alanine. Vegetative cells are the growing, toxin-making form and are killed by pasteurisation. Fungal spores are reproductive and far less heat-resistant, with exceptions such as the ascospores of Byssochlamys that spoil heat-processed fruit.

Heat-resistant spore-formers: Geobacillus stearothermophilus (flat-sour spoilage of low-acid cans: acid without gas), Clostridium botulinum, and thermophilic anaerobes that swell cans with H₂ and CO₂.
Thermoduric organisms survive pasteurisation without growing at its temperature: Microbacterium, Micrococcus, enterococci and spores.
Psychrotrophs grow at refrigeration temperatures: Pseudomonas, Listeria monocytogenes, Yersinia enterocolitica and non-proteolytic C. botulinum.
Other resistances: osmophilic yeasts in syrups, halophiles that turn salted fish pink, acid-tolerant Alicyclobacillus in fruit juices, radiation-resistant Deinococcus, and antimicrobial-resistant strains that move through the food chain from animal production.

3. Growth and death kinetics: D-, z- and F-values and serial dilution

In a closed batch a population passes through lag (adaptation, no increase), exponential growth at a constant generation time, stationary phase (nutrients exhausted, wastes accumulating) and death. During exponential growth N = N₀ × 2ⁿ, where the number of generations n = (log N − log N₀) ÷ log 2 = 3.32 (log N − log N₀); the generation time g = t ÷ n and the specific growth rate μ = ln 2 ÷ g. Worked example: 10² cfu g⁻¹ with g = 25 min and no lag reach 10² × 2¹² = 4.1 × 10⁵ cfu g⁻¹ after 5 h. Lower temperature, water activity or pH lengthen the lag and the generation time, which is how preservation buys shelf life.

Thermal death is first order: at constant temperature log N falls linearly with time, so log(N₀ ÷ N) = t ÷ D. The D-value is the time at a stated temperature for a 90 % (one-log) reduction. The z-value is the temperature change that alters D ten-fold: log(D₁ ÷ D₂) = (T₂ − T₁) ÷ z. The F-value is a process's lethality expressed as minutes at a reference temperature: F = D × (log N₀ − log N), and t minutes at temperature T are worth t × 10^((T − Tref)/z) minutes at Tref, the lethal rate. For low-acid foods, F₀ uses Tref = 121.1 °C and z = 10 °C.

The 12D concept: low-acid canned foods (pH above 4.6, water activity above 0.85) must receive enough heat to reduce Clostridium botulinum spores by 12 log cycles. With D₁₂₁.₁ = 0.21 min, 12D = 2.52 min, the minimum botulinum cook. Worked example: F₀ = 3.0 min on spores with D = 0.25 min gives 3.0 ÷ 0.25 = 12 log reductions, so 100 spores per can leave 10² × 10⁻¹² = 10⁻¹⁰, one survivor in 10¹⁰ cans. Milk pasteurisation targets Coxiella burnetii, the most heat-resistant non-spore-forming pathogen of milk: 63 °C for 30 min (batch) or 72 °C for 15 s (HTST).

Serial dilution and plate count: 10 g of food in 90 mL of diluent is the 10⁻¹ dilution, and each 1 mL into 9 mL adds a further ten-fold step. A known volume is pour- or spread-plated, plates in the countable range (about 25 to 250 or 30 to 300 colonies, by the standard used) are counted, and cfu g⁻¹ = mean colonies ÷ (volume plated in mL × dilution). Worked example: 0.1 mL of the 10⁻⁵ dilution gives 45 and 51 colonies; 48 ÷ (0.1 × 10⁻⁵) = 4.8 × 10⁷ cfu g⁻¹. Each colony is assumed to arise from one cell or clump, hence colony-forming units.

4. Spoilage of milk, meat, fish, eggs and cereals

Which organism wins depends on intrinsic factors (pH, water activity, nutrients, natural antimicrobials) and extrinsic ones (temperature, atmosphere). Moist, near-neutral, protein-rich foods go to Gram-negative psychrotrophs in the cold; acid foods to yeasts, moulds and lactic acid bacteria; dry foods to moulds.

Food	Main spoilage organisms	Signs and chemistry
Raw milk, warm	Lactic acid bacteria (Lactococcus lactis, lactobacilli, enterococci)	Souring and curdling as lactose becomes lactic acid
Raw milk, refrigerated	Psychrotrophic Pseudomonas	Heat-stable proteases and lipases survive pasteurisation and UHT, later causing bitterness, gelation and rancidity
Pasteurised milk	Spore-formers, notably Bacillus cereus; post-process contaminants	Sweet curdling by a rennet-like protease; “bitty cream” from lecithinase
Fresh meat, chilled in air	Pseudomonas, Brochothrix thermosphacta	Off-odours once glucose runs out and amino acids are attacked; slime at about 10⁷–10⁸ cfu cm⁻²
Vacuum-packed meat	Lactic acid bacteria; Shewanella in high-pH meat	Slow, mild souring; H₂S and greening where Shewanella grows
Marine fish	Shewanella putrefaciens, Pseudomonas, Photobacterium phosphoreum	High post-mortem pH and much non-protein N; trimethylamine oxide reduced to fishy trimethylamine; H₂S; total volatile basic nitrogen rises; histidine decarboxylated to histamine in tuna and mackerel
Eggs	Gram-negative rods: Pseudomonas (green rot), Proteus (black rot); moulds	Shell, membranes, lysozyme, iron-binding ovotransferrin, avidin and alkaline albumen (pH about 9) hold off Gram-positives; spoilers enter through cracks and wet shells
Grain and flour	Field fungi (Fusarium, Alternaria) need high water activity; storage fungi (Aspergillus, Penicillium) grow at lower water activity	Mustiness, heating, mycotoxins
Bread	Rhizopus stolonifer, Penicillium, Aspergillus; Bacillus subtilis and B. licheniformis	Mould growth; “rope”, a soft, sticky, stringy crumb with a fruity odour from spores that survive baking, prevented by calcium propionate and acidification

5. Microbial toxins and food-borne disease

Food-borne disease is an infection (cells multiply in the gut: Salmonella, Shigella, Campylobacter, Listeria), an intoxication (toxin preformed in the food: Staphylococcus aureus, emetic Bacillus cereus, Clostridium botulinum, mycotoxins) or a toxico-infection (ingested cells make toxin in the gut: Clostridium perfringens, diarrhoeal B. cereus). Intoxications have short incubation and can strike even after the cells are killed.

Organism	Features	Toxin or mechanism	Foods and illness
Staphylococcus aureus	Gram-positive cocci in clusters; facultative; salt-tolerant; coagulase-positive	Heat-stable enterotoxins preformed in food; boiling does not destroy them	Handled cooked foods, cream, custard, khoa sweets; vomiting in 1–6 h
Salmonella	Gram-negative motile rods (Enterobacteriaceae)	Invasive infection	Poultry, eggs, raw milk: gastroenteritis with fever in 6–72 h; S. Typhi causes typhoid via food or water contaminated with human faeces
Shigella	Gram-negative non-motile rods close to E. coli; humans the only reservoir	Invades the colon; S. dysenteriae type 1 makes Shiga toxin	Salads handled by carriers; bacillary dysentery from as few as 10–100 cells
Escherichia coli	Gram-negative; mostly harmless gut commensals, used as faecal indicators	Pathotypes: ETEC (enterotoxins LT and ST), EPEC, EIEC, and EHEC such as O157:H7 (Shiga toxins)	Undercooked minced beef, raw milk, sprouts; O157:H7 causes bloody diarrhoea and haemolytic uraemic syndrome
Bacillus cereus	Gram-positive aerobic spore-forming rods	Emetic cereulide (heat-stable, preformed); diarrhoeal enterotoxins (heat-labile, made in the gut)	Emetic: cooked rice held warm, 0.5–6 h; diarrhoeal: meat, vegetables, sauces, 8–16 h
Clostridium botulinum	Gram-positive obligately anaerobic spore-forming rods; no growth below pH 4.6	Heat-labile neurotoxin that blocks acetylcholine release: flaccid paralysis	Under-processed low-acid canned or vacuum-packed foods; honey in infant botulism
Clostridium perfringens	Gram-positive anaerobic spore-forming rods; generation time about 10 min near 45 °C	Enterotoxin released when cells sporulate in the gut	Large batches of meat and gravy cooled slowly; diarrhoea and cramps in 8–24 h
Aspergillus	Septate moulds: A. flavus, A. parasiticus, A. ochraceus	Aflatoxins B1, B2, G1, G2 (blue and green fluorescence in UV), B1 a potent liver carcinogen excreted as M1 in milk; ochratoxin A from A. ochraceus	Groundnuts, maize, spices and cottonseed stored moist; the toxins are heat-stable

ℹ️ Non-pathogens of the named genera

The same genera hold useful non-pathogens: Aspergillus oryzae and A. sojae make koji for soy sauce and A. niger makes citric acid and enzymes; Bacillus subtilis ferments natto and B. coagulans is a probiotic; Staphylococcus carnosus and S. xylosus reduce nitrate in fermented sausage; most E. coli strains are harmless commensals.

6. Fermented foods and beverages

Product	Key microorganisms	Main changes
Curd (dahi)	Mesophilic lactic acid bacteria: Lactococcus lactis, Leuconostoc, lactobacilli	Lactose → lactic acid; casein coagulates near pH 4.6; diacetyl aroma
Yoghurt	Streptococcus thermophilus with Lactobacillus delbrueckii subsp. bulgaricus at about 42–45 °C	Fast acidification by protocooperation; acetaldehyde flavour
Cheese	Starter lactic acid bacteria and rennet; ripening flora such as Propionibacterium freudenreichii (Swiss eyes), Penicillium roqueforti (blue), P. camemberti (surface mould)	Acidification, coagulation, whey drainage; ripening by proteolysis and lipolysis
Pickles, sauerkraut	Vegetables in brine, or shredded cabbage with about 2–3 % salt; succession of Leuconostoc mesenteroides (heterofermentative, CO₂) → Levilactobacillus brevis → Lactiplantibacillus plantarum (homofermentative, acid-tolerant), both formerly Lactobacillus	Salt draws out juice and selects lactic acid bacteria; the acid preserves
Idli and dosa	Batter of soaked rice and black gram; natural flora: Leuconostoc mesenteroides (leavening CO₂), Enterococcus faecalis, pediococci, yeasts	Overnight acidification and leavening; black-gram mucilage holds the gas
Soy sauce	Koji of Aspergillus oryzae or A. sojae on soybean and roasted wheat; brine (moromi) stage with Tetragenococcus halophilus and the yeast Zygosaccharomyces rouxii	Proteins → amino acids (glutamate), starch → sugars; months of ageing
Vinegar	Saccharomyces cerevisiae, then acetic acid bacteria (Acetobacter, Gluconobacter, Komagataeibacter)	C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂, then aerobically C₂H₅OH + O₂ → CH₃COOH + H₂O
Beer, wine, spirits	S. cerevisiae (ale, top-fermenting) or S. pastorianus (lager, bottom-fermenting); Oenococcus oeni for malolactic fermentation of wine	Malting makes amylases; hops add bitter iso-α-acids; malic → lactic acid softens wine; distillation concentrates ethanol
Fermented sausage	Pediococcus and Latilactobacillus sakei (acid); Staphylococcus carnosus or S. xylosus (nitrate reductase, flavour)	Falling pH sets the meat-protein gel and suppresses pathogens; drying lowers water activity

In yoghurt the two cultures help each other (protocooperation): the proteolytic lactobacillus releases peptides and amino acids that stimulate S. thermophilus, which in turn removes oxygen and supplies formic acid and CO₂ that stimulate the lactobacillus, so together they acidify faster than either alone. Paneer and chhena, by contrast, are made by acid–heat coagulation, not fermentation.

7. Probiotics, prebiotics and synbiotics

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Common ones are lactobacilli (Lactobacillus acidophilus, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum), bifidobacteria (Bifidobacterium longum, B. animalis subsp. lactis), the yeast Saccharomyces boulardii and spore-forming Bacillus coagulans. A strain must be safe, survive gastric acid and bile, persist in or adhere to the gut, antagonise pathogens and stay viable through shelf life; benefits are strain-specific. Supported benefits include easier lactose digestion (the β-galactosidase of yoghurt cultures) and shorter infectious and antibiotic-associated diarrhoea, through competitive exclusion, organic acids, bacteriocins, a stronger gut barrier and immune modulation.

Prebiotics are substrates selectively used by host microorganisms that confer a health benefit: inulin and fructo-oligosaccharides (β-2,1 fructosyl links that human enzymes cannot split), galacto-oligosaccharides (made from lactose by the transgalactosylating action of β-galactosidase), lactulose and human-milk oligosaccharides. They reach the colon intact and are fermented, chiefly by bifidobacteria, to short-chain fatty acids (acetate, propionate, butyrate) that lower colonic pH and feed colonocytes. Synbiotics combine a probiotic with a prebiotic substrate, either complementary or chosen so that the substrate feeds the co-administered strain, as in Bifidobacterium with fructo-oligosaccharides.

Key takeaways

Gram-positive walls (thick peptidoglycan) keep the crystal violet–iodine complex; Gram-negative walls (thin layer plus outer membrane) lose it. Endospores of Bacillus and Clostridium owe their resistance to a dry, dipicolinate-rich core and DNA-protecting proteins.
Growth: N = N₀ × 2ⁿ with n = t ÷ g. Death: log(N₀ ÷ N) = t ÷ D; z is the rise that cuts D ten-fold; F = D × log reductions, with the lethal rate 10^((T − Tref)/z). Low-acid foods get at least a 12D botulinum cook (F₀ about 2.5 min).
Plate count: cfu g⁻¹ = mean colonies ÷ (volume plated × dilution), counting only plates in the countable range.
Spoilage follows the food: lactic souring and Pseudomonas enzymes in milk, Pseudomonas slime on chilled meat, trimethylamine in fish, Gram-negative rots in eggs, moulds and Bacillus rope in cereals and bread.
Preformed heat-stable toxins (S. aureus enterotoxin, B. cereus cereulide, aflatoxins) survive reheating; botulinum toxin is heat-labile but C. botulinum spores are not, and the organism cannot grow below pH 4.6.
Fermented foods rely on lactic acid bacteria, yeasts, acetic acid bacteria and moulds in characteristic successions; probiotics are live strains with proven benefit, prebiotics are selectively fermented substrates, and synbiotics combine the two.

Practice questions (18)

Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.

In the Gram stain, why do Gram-negative bacteria lose the crystal violet–iodine complex during decolourisation?
1. Alcohol dissolves their lipid-rich outer membrane, and the thin peptidoglycan layer cannot hold the complex
2. They have no peptidoglycan at all, so crystal violet never binds to their cell walls in the first place
3. Iodine cannot cross their outer membrane, so no insoluble complex forms inside these cells at any stage
4. Safranin displaces the crystal violet from their walls because it binds lipopolysaccharide more tightly
Show answer
Answer: A — Alcohol dissolves their lipid-rich outer membrane, and the thin peptidoglycan layer cannot hold the complex
Gram-negative walls have a thin peptidoglycan layer beneath an outer membrane; alcohol extracts the membrane lipids and the complex washes out of the thin, porous layer, so the cells take up pink safranin. They do contain peptidoglycan, the complex does form in them, and the counterstain only colours cells that are already decolourised.
A mould isolated from spoiled bread has broad aseptate hyphae, root-like rhizoids and runner-like stolons, with unbranched sporangiophores arising at the nodes above the rhizoids, each ending in a black sporangium. Which genus is it?
1. Aspergillus, which bears conidia in chains on a swollen vesicle at the tip of each conidiophore
2. Penicillium, which bears chains of conidia on a branched, brush-like penicillus of septate hyphae
3. Mucor, which bears sporangia on its sporangiophores but has neither rhizoids nor stolons
4. Rhizopus, which bears sporangia on sporangiophores arising from stolons above tufts of rhizoids
Show answer
Answer: D — Rhizopus, which bears sporangia on sporangiophores arising from stolons above tufts of rhizoids
Aseptate hyphae with sporangia place the mould among the Mucorales; rhizoids and stolons, with sporangiophores at the nodes, identify Rhizopus (R. stolonifer is the common bread mould). Mucor is the close alternative but lacks rhizoids and stolons. Aspergillus and Penicillium have septate hyphae and bear conidia, not sporangia.
Which of the following statements about bacterial endospores and fungal spores are correct?
1. Calcium dipicolinate and a dehydrated core contribute to the heat resistance of endospores
2. Mould conidia are generally more heat-resistant than bacterial endospores
3. Small acid-soluble spore proteins bind and protect the DNA of endospores
4. Endospores are formed by Staphylococcus and Salmonella when nutrients are exhausted
Show answer
Answer: A — Calcium dipicolinate and a dehydrated core contribute to the heat resistance of endospores; C — Small acid-soluble spore proteins bind and protect the DNA of endospores
The low water content of the core, stabilised by calcium dipicolinate, and the small acid-soluble proteins that coat the DNA underlie endospore resistance to heat and radiation. Fungal conidia are killed by mild heat, far below what endospores need, apart from a few heat-resistant ascospores. Among food bacteria endospores are formed by Bacillus, Clostridium and their relatives, not by Staphylococcus or Salmonella.
A cooked rice dish contains 500 cfu g⁻¹ of a bacterium whose generation time at the holding temperature is 20 min. Assuming no lag and exponential growth, what is the count after 3 h?
1. 4.5 × 10³ cfu g⁻¹
2. 2.56 × 10⁵ cfu g⁻¹
3. 5.0 × 10⁵ cfu g⁻¹
4. 1.28 × 10⁵ cfu g⁻¹
Show answer
Answer: B — 2.56 × 10⁵ cfu g⁻¹
3 h = 180 min = 9 generations, so N = 500 × 2⁹ = 500 × 512 = 2.56 × 10⁵ cfu g⁻¹. Multiplying 500 by 9 treats growth as linear (4.5 × 10³), and stopping one generation short gives 1.28 × 10⁵. This is why cooked rice must not be held warm.
Spores of a mesophilic Clostridium heated at 115 °C fall from 3.0 × 10⁶ to 3.0 × 10² per mL in 9.2 min, following first-order kinetics. The D-value at 115 °C, in minutes to one decimal place, is ____.
Numerical answer — type the value.
Show answer
Answer: 2.3
Log reduction = log(3.0 × 10⁶ ÷ 3.0 × 10²) = log 10⁴ = 4. D = t ÷ (log N₀ − log N) = 9.2 ÷ 4 = 2.3 min, the time for each 90 % reduction. Dividing by the ratio (10⁴) instead of its logarithm is the usual error.
The D-value of a spore suspension is 18.0 min at 110 °C and 2.0 min at 118 °C. The z-value, in °C to one decimal place, is ____.
Numerical answer — type the value.
Show answer
Answer: 8.4
z = (T₂ − T₁) ÷ log(D₁ ÷ D₂) = (118 − 110) ÷ log(18.0 ÷ 2.0) = 8 ÷ log 9 = 8 ÷ 0.954 = 8.38, i.e. 8.4 °C, the temperature rise that cuts D ten-fold. Using the natural logarithm gives 3.6 °C, a common slip.
A can is held at 115.0 °C for 20 min; ignore the heating and cooling lags. Taking Tref = 121.1 °C and z = 10 °C, the F₀ value delivered, in minutes to two decimal places, is ____.
Numerical answer — type the value.
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Answer: 4.91
Lethal rate L = 10^((T − Tref)/z) = 10^((115.0 − 121.1)/10) = 10^(−0.61) = 0.2455. F₀ = 20 × 0.2455 = 4.91 min: 20 min at 115 °C are worth about 4.9 min at 121.1 °C, more than the 2.52 min botulinum cook. Putting the temperature difference the wrong way round gives 81.5 min.
A low-acid canned food contains 100 spores of Clostridium botulinum per can before processing. The spores have D₁₂₁.₁ = 0.20 min, and the process delivers F₀ = 3.0 min. If the probability that a processed can holds a surviving spore is 10⁻ˣ, what is x?
Numerical answer — type the value.
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Answer: 13
Log reductions = F₀ ÷ D = 3.0 ÷ 0.20 = 15. Survivors per can = 10² × 10⁻¹⁵ = 10⁻¹³, so one can in 10¹³ is expected to hold a spore and x = 13. Forgetting the initial load of 10² gives 15. A bare 12D process would be F₀ = 12 × 0.20 = 2.4 min, so this process exceeds it.
Ten grams of minced meat are blended with 90 mL of diluent, and further ten-fold dilutions are made by transferring 1 mL into 9 mL. Duplicate spread plates receiving 0.1 mL of the 10⁻⁴ dilution give 148 and 164 colonies. The count in the meat, expressed in units of 10⁶ cfu per g, is ____.
Numerical answer — type the value.
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Answer: 15.6
Mean = (148 + 164) ÷ 2 = 156 colonies from 0.1 mL of the 10⁻⁴ dilution. cfu g⁻¹ = 156 ÷ (0.1 × 10⁻⁴) = 1.56 × 10⁷ = 15.6 × 10⁶. The 10 g in 90 mL step is already the first 10⁻¹; forgetting that only 0.1 mL was plated gives 1.56 × 10⁶.
UHT milk, commercially sterile and aseptically packed, turns bitter and forms a gel after four months at room temperature. The raw milk had been held cold for several days before processing. What is the most likely cause?
1. Spores of Bacillus cereus that survived UHT germinated in the pack and produced a rennet-like protease
2. Heat-stable proteases secreted by psychrotrophic Pseudomonas in the cold raw milk survived UHT and acted in storage
3. Pseudomonas cells survived the UHT treatment and continued to grow slowly in the sealed pack at room temperature
4. Lactic acid bacteria that entered through a faulty seal soured the milk, and the acid coagulated the casein
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Answer: B — Heat-stable proteases secreted by psychrotrophic Pseudomonas in the cold raw milk survived UHT and acted in storage
Psychrotrophic Pseudomonas grow in raw milk during cold storage and secrete proteases (and lipases) that withstand UHT although the cells die; the enzymes slowly hydrolyse casein in the pack, causing bitterness and age gelation. The milk is commercially sterile, so surviving or growing cells are ruled out (B. cereus spores do not survive a correct UHT process), and souring through a faulty seal would give an acid curd quickly, not a slow bitter gel.
The characteristic fishy odour of spoiling chilled marine fish arises mainly from
1. trimethylamine, formed when bacteria such as Shewanella putrefaciens reduce trimethylamine oxide
2. histamine, formed when bacteria such as Morganella decarboxylate the free histidine of fish muscle
3. hexanal and other aldehydes formed by the autoxidation of fish polyunsaturated lipids
4. ammonia released when fish muscle enzymes break down urea stored in the tissue
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Answer: A — trimethylamine, formed when bacteria such as Shewanella putrefaciens reduce trimethylamine oxide
Marine fish hold trimethylamine oxide as an osmolyte; spoilage bacteria such as Shewanella putrefaciens use it as an electron acceptor and reduce it to trimethylamine, the fishy smell, which is why TMA and total volatile basic nitrogen serve as freshness indices. Histamine causes scombroid poisoning but has no fishy odour, so fish can be toxic while smelling acceptable. Lipid oxidation gives rancid notes, and large urea stores belong to sharks and rays, not typical marine fish.
Which of the following statements about the spoilage of foods are correct?
1. Lysozyme of egg albumen attacks peptidoglycan, so Gram-positive bacteria rarely spoil whole eggs
2. Field fungi such as Fusarium grow in stored grain at lower water activity than storage fungi such as Aspergillus
3. Rope in bread is caused by Bacillus spores that survive baking and later make the crumb sticky and stringy
4. Spoilage of vacuum-packed chilled meat is dominated by strictly aerobic Pseudomonas
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Answer: A — Lysozyme of egg albumen attacks peptidoglycan, so Gram-positive bacteria rarely spoil whole eggs; C — Rope in bread is caused by Bacillus spores that survive baking and later make the crumb sticky and stringy
Lysozyme, together with ovotransferrin, avidin and the high pH of albumen, suppresses Gram-positives, so eggs are spoiled mainly by Gram-negative rods entering through the shell. Rope is caused by B. subtilis and B. licheniformis, whose spores survive baking. Field fungi need high water activity and invade grain before harvest, whereas storage fungi such as Aspergillus and Penicillium grow at lower water activity. Vacuum packing excludes O₂, so lactic acid bacteria, not aerobic Pseudomonas, dominate.
Several guests vomited 2 to 4 hours after eating a cream-filled pastry that had been prepared by hand, left at room temperature for hours and then warmed thoroughly before serving. Which organism and mechanism best explain the outbreak?
1. Salmonella, whose cells invaded the intestinal wall after surviving the reheating step
2. Clostridium perfringens, whose spores germinated in the gut and released enterotoxin on sporulation
3. Clostridium botulinum, whose neurotoxin formed in the cream and blocked acetylcholine release
4. Staphylococcus aureus, whose heat-stable enterotoxin formed in the pastry before reheating
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Answer: D — Staphylococcus aureus, whose heat-stable enterotoxin formed in the pastry before reheating
A short incubation with vomiting points to a preformed toxin. S. aureus from a handler's hands grows in cream held warm and makes enterotoxins that survive reheating, so killing the cells does not help. Salmonella infection takes 6 to 72 h and brings fever and diarrhoea; C. perfringens causes diarrhoea after 8 to 24 h; botulism causes descending paralysis after 12 to 36 h, and its toxin is destroyed by thorough heating.
Why is pH 4.6 the dividing line between acid and low-acid canned foods?
1. Below pH 4.6 the spores of Clostridium botulinum are killed by the acid alone during storage
2. Below pH 4.6 any botulinum toxin already formed is broken down by acid hydrolysis
3. Below pH 4.6 Clostridium botulinum spores cannot germinate and grow to produce toxin
4. Below pH 4.6 heat-resistant flat-sour bacteria cannot survive, so no heat process is needed
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Answer: C — Below pH 4.6 Clostridium botulinum spores cannot germinate and grow to produce toxin
C. botulinum does not grow or make toxin below pH 4.6, so acid foods need only a process that kills vegetative cells, yeasts, moulds and acid-tolerant spoilers, whereas low-acid foods need a full botulinum cook. Acid does not kill the spores or destroy preformed toxin, and acid foods still need a heat process.
Which of the following statements about food-borne pathogens and their toxins are correct?
1. The emetic toxin of Bacillus cereus, cereulide, is heat-stable, so reheating fried rice does not make it safe
2. Shigella needs millions of cells to cause dysentery, so food handlers rarely spread it
3. Escherichia coli O157:H7 produces Shiga toxins and can cause haemolytic uraemic syndrome
4. Aflatoxin M1 in milk is produced by Aspergillus growing in the milk itself
Show answer
Answer: A — The emetic toxin of Bacillus cereus, cereulide, is heat-stable, so reheating fried rice does not make it safe; C — Escherichia coli O157:H7 produces Shiga toxins and can cause haemolytic uraemic syndrome
Cereulide forms in cooked rice held warm and survives reheating; O157:H7 is a Shiga-toxin-producing E. coli whose toxins damage the kidney's blood vessels. Shigella has a very low infective dose (about 10 to 100 cells) and spreads readily from carriers. Aflatoxin M1 is the hydroxylated metabolite of aflatoxin B1 that cows excrete in milk after eating contaminated feed.
In a yoghurt culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, acid develops faster than with either organism alone. Which description of their interaction is correct?
1. The lactobacillus releases peptides and amino acids that stimulate the streptococcus, which supplies formic acid and CO₂ that stimulate the lactobacillus
2. The streptococcus releases peptides and amino acids that stimulate the lactobacillus, which supplies formic acid and CO₂ that stimulate the streptococcus
3. The lactobacillus produces nisin that suppresses competitors, while the streptococcus produces lactase that frees glucose for both organisms
4. Each organism ferments a different half of lactose, the streptococcus using glucose and the lactobacillus using only the galactose
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Answer: A — The lactobacillus releases peptides and amino acids that stimulate the streptococcus, which supplies formic acid and CO₂ that stimulate the lactobacillus
This protocooperation runs both ways: the proteolytic L. bulgaricus supplies peptides and amino acids to the weakly proteolytic S. thermophilus, which in turn produces formic acid and CO₂ (and removes O₂) that stimulate the lactobacillus. The reversed roles are the tempting distractor. Nisin is made by Lactococcus lactis, not by either yoghurt organism, and both yoghurt organisms ferment the glucose of lactose while excreting much of the galactose.
Which organism is chiefly responsible for leavening idli batter during its overnight natural fermentation?
1. Acetobacter aceti, which oxidises ethanol in the batter to acetic acid and gas
2. Leuconostoc mesenteroides, a heterofermentative lactic acid bacterium that produces CO₂
3. Propionibacterium freudenreichii, which converts the batter's lactate into propionate and CO₂
4. Aspergillus oryzae, which grows on the rice and black gram as a solid-state koji
Show answer
Answer: B — Leuconostoc mesenteroides, a heterofermentative lactic acid bacterium that produces CO₂
Leuconostoc mesenteroides from the raw ingredients ferments sugars heterofermentatively to lactic acid, ethanol and CO₂; the gas, trapped by black-gram mucilage, raises the batter, while acid develops with Enterococcus faecalis and pediococci. Acetobacter needs ethanol and plenty of air, Propionibacterium makes the eyes of Swiss cheese, and Aspergillus oryzae is the koji mould of soy sauce.
Which of the following statements about probiotics, prebiotics and synbiotics are correct?
1. Inulin and fructo-oligosaccharides resist human digestive enzymes and are fermented by colonic bifidobacteria
2. Paneer is a probiotic food because its curd is formed by lactic acid bacteria
3. A synbiotic combines a probiotic with a prebiotic substrate
4. Saccharomyces boulardii is a yeast used as a probiotic
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Answer: A — Inulin and fructo-oligosaccharides resist human digestive enzymes and are fermented by colonic bifidobacteria; C — A synbiotic combines a probiotic with a prebiotic substrate; D — Saccharomyces boulardii is a yeast used as a probiotic
The β-2,1 links of inulin and FOS are not split by human enzymes, so these fructans reach the colon and selectively feed bifidobacteria; a synbiotic pairs such a substrate with live probiotic organisms; and S. boulardii is a well-studied probiotic yeast. Paneer is made by acid–heat coagulation of hot milk with no fermentation, and being heated it carries no live culture.