Food Science & Technology · Foundations · also Fundamentals of Microbiology, Introductory Microbiology, Microbiology I
The microbial world before it meets food: cell structure, staining, growth, nutrition, genetics and control of bacteria, yeasts, moulds and viruses. Food microbiology (phase 2) builds directly on it.
In the exams: GATE XE-G and XL-U Section 2 (characteristics of microorganisms, growth and death kinetics, serial dilution)
General microbiology is the study of microorganisms as organisms: their structure, how they are seen and stained, how they are classified, what they need to grow, how fast they grow and how growth is measured, how they are killed, and how their genes change and move. It deals with bacteria, archaea, yeasts, moulds, protozoa and viruses before any of them meets a food. Food microbiology, food safety and fermentation technology all build directly on it.
The subject matters because every practical decision in a food laboratory or plant starts from these basics. A plate count is only meaningful if the analyst understands serial dilution and the countable range. A thermal process is designed from death kinetics. A cleaning programme depends on knowing why spores and biofilms survive what kills vegetative cells. A dairy that loses a fermentation to bacteriophage, or a pathogen that acquires an antibiotic-resistance plasmid, is a lesson in microbial genetics.
General microbiology is examined in food technology, food science, microbiology and biotechnology degrees everywhere, and in the food microbiology sections of national postgraduate entrance examinations, food analyst and food technologist licensure examinations and food safety officer recruitment. Examiners repeatedly ask about the history of the science, Gram and other staining reactions, differences between prokaryotic and eukaryotic cells, endospores, culture media, the phases of growth, generation time and growth-rate arithmetic, serial dilution and plate counts, D- and z-values, sterilisation and disinfection agents, and the three routes of gene transfer.
This manual follows the curriculum units in order and keeps the arithmetic explicit, because numerical-answer questions on generation time, colony counts and death kinetics are a fixture of these papers.
Microorganisms were first seen by Antonie van Leeuwenhoek, a cloth merchant in Delft, who in the 1670s used single-lens microscopes of his own making to describe "animalcules" in rainwater, plaque and infusions. Robert Hooke had earlier described moulds and coined the word "cell". For two centuries the main question was where microbes came from. Francesco Redi showed in 1668 that maggots did not arise from meat kept away from flies, and Lazzaro Spallanzani showed that boiled, sealed broths stayed clear, but supporters of spontaneous generation argued that heating destroyed a "vital force" in the air. Louis Pasteur settled the matter around 1861 with swan-neck flasks: boiled broth open to air through a curved neck stayed sterile because dust and microbes settled in the bend, and turned cloudy as soon as the neck was broken. John Tyndall showed that some infusions needed repeated heating, explained by Ferdinand Cohn's discovery of heat-resistant bacterial endospores; intermittent steaming on successive days is still called tyndallisation. Pasteur also showed that fermentation and spoilage are caused by living microbes, that different microbes make different products (lactic acid, ethanol, butyric acid), and that wine could be protected by mild heating, the origin of pasteurisation. Nicolas Appert had already preserved food in sealed, heated containers around 1810, without knowing why it worked. Joseph Lister applied the germ theory to surgery with phenol antisepsis in 1867. Robert Koch proved that a specific bacterium causes a specific disease (anthrax, 1876; tuberculosis, 1882) and set out Koch's postulates: the organism is found in every case of the disease, is isolated in pure culture, causes the disease when inoculated into a healthy host, and is re-isolated from that host. Koch's laboratory introduced solid media with agar, suggested by Fanny Hesse, and Julius Petri's dish (1887), which made pure cultures routine. Christian Gram published his staining method in 1884.
Sergei Winogradsky and Martinus Beijerinck founded environmental microbiology with enrichment cultures, chemolithotrophy and nitrogen fixation; Beijerinck and Dmitri Ivanovsky showed that tobacco mosaic disease was caused by an agent that passed through bacteria-proof filters, the first virus. Bacteriophages were described by Frederick Twort in 1915 and Félix d'Herelle in 1917. Alexander Fleming observed penicillin in 1928, and Howard Florey and Ernst Chain developed it as a drug around 1940. Frederick Griffith's transformation experiment (1928) and the demonstration by Oswald Avery, Colin MacLeod and Maclyn McCarty (1944) that the transforming principle is DNA opened molecular biology, followed by the discovery of conjugation and transduction. Carl Woese's comparison of 16S ribosomal RNA sequences in 1977 showed that life falls into three domains: Bacteria, Archaea and Eukarya. Microbiology today spans medical, veterinary, agricultural, environmental, industrial and food microbiology, and the tools range from the light microscope to whole-genome sequencing. Microbes cycle carbon, nitrogen and sulphur, fix nitrogen in legume nodules, make antibiotics, enzymes, vitamins and organic acids, and ferment bread, cheese, yoghurt, soy sauce, vinegar and beer. A minority cause disease. For a food technologist the scope is threefold: microbes that spoil food, microbes that cause food-borne illness, and microbes used to make food and ingredients. General microbiology supplies the common language for all three.
The compound light microscope has an objective lens and an eyepiece (ocular); total magnification is their product, so a 10× eyepiece with a 100× objective gives 1000×. Magnification is useless without resolution, the smallest distance at which two points are still seen as separate. Resolution depends on the wavelength of light (λ) and the numerical aperture (NA) of the objective; by the Abbe relation d = λ ÷ (2 NA), and a commonly used alternative form is d = 0.61 λ ÷ NA. With visible light and the best oil-immersion objectives (NA about 1.25 to 1.4), the limit is about 0.2 µm. Immersion oil has a refractive index close to that of glass (about 1.5), so light leaving the slide is not bent away from the objective, raising the effective NA. Shorter wavelengths, such as blue light or ultraviolet, improve resolution. In bright-field microscopy the specimen is seen dark against a bright background; unstained bacteria have little contrast, so they are usually fixed and stained. Dark-field microscopy uses a condenser that blocks direct light, so only light scattered by the specimen enters the objective and cells appear bright on a black background; it shows thin spirochaetes and motility in living cells. Phase-contrast microscopy, invented by Frits Zernike, converts small differences in refractive index and thickness within an unstained cell into differences in brightness. It lets living, unstained cells be examined, and it shows endospores as bright, refractile bodies. Differential interference contrast gives a similar, three-dimensional-looking image.
Fluorescence microscopy uses ultraviolet or blue light to excite fluorochromes, which emit light of a longer wavelength. Cells may fluoresce naturally, be stained with dyes such as acridine orange or DAPI that bind nucleic acids, or be tagged with fluorescent antibodies (immunofluorescence) or probes (fluorescence in situ hybridisation). Live/dead stains distinguish cells with intact and damaged membranes. In food laboratories the direct epifluorescent filter technique (DEFT) filters a sample, stains the cells on the membrane and counts them under an epifluorescence microscope within an hour, a rapid method once widely used for raw milk. Confocal laser scanning microscopy builds sharp optical sections and three-dimensional images, and is used to study biofilms on food-contact surfaces. Electron microscopes use a beam of electrons, whose wavelength is far shorter than that of light, focused by electromagnets in a vacuum. The transmission electron microscope (TEM) passes electrons through ultrathin sections or negatively stained particles and resolves internal structure such as membranes, ribosomes and virus capsids, with resolution far below 1 nm under good conditions. The scanning electron microscope (SEM) scans a beam over a metal-coated surface and records emitted electrons, giving three-dimensional images of surfaces, useful for spores, hyphae and biofilms on stainless steel. Specimens for conventional electron microscopy are dead, fixed and dehydrated, which can create artefacts. Cryo-electron microscopy examines rapidly frozen, hydrated samples and has transformed the study of virus and protein structure. Atomic force microscopy probes surfaces with a fine tip and can image living cells.
Prokaryotes, the Bacteria and Archaea, have no membrane-bound nucleus; their DNA, usually one circular chromosome, lies in a nucleoid region of the cytoplasm, often with smaller plasmids. They have 70S ribosomes, made of 30S and 50S subunits, no mitochondria or endoplasmic reticulum, and divide by binary fission. Most are 0.5 to 5 µm long. The cytoplasmic membrane is a phospholipid bilayer with proteins that carries out respiration, transport and energy generation. Outside it, almost all bacteria have a cell wall containing peptidoglycan (murein): chains of alternating N-acetylglucosamine and N-acetylmuramic acid linked by β-1,4 bonds, cross-linked by short peptides. Lysozyme, found in egg white and tears, breaks these β-1,4 bonds; penicillin blocks the transpeptidation that forms the cross-links. Gram-positive bacteria have a thick peptidoglycan wall, about 20 to 80 nm, with teichoic acids. Gram-negative bacteria have a thin peptidoglycan layer in the periplasm, covered by an outer membrane whose outer leaflet contains lipopolysaccharide (LPS); the lipid A part of LPS is endotoxin, which causes fever and shock and is not destroyed by ordinary heat. The outer membrane also makes Gram-negative bacteria more resistant to some detergents, dyes and antibiotics. Mycobacteria have walls rich in mycolic acids, the basis of acid-fast staining. Archaea lack peptidoglycan and have ether-linked membrane lipids. Outside the wall, many bacteria have a capsule or slime layer of polysaccharide, which aids attachment and protects against drying and phagocytosis and is part of biofilm matrix. Flagella give motility and are arranged as one polar flagellum (monotrichous), a tuft at one end (lophotrichous), flagella at both ends (amphitrichous) or all over the cell (peritrichous). Fimbriae and pili attach cells to surfaces; the sex pilus joins cells in conjugation. Inclusions store carbon and energy (glycogen, poly-β-hydroxybutyrate) or phosphate (volutin).
Eukaryotic microorganisms, the fungi, algae and protozoa, have a nucleus with a nuclear membrane, several linear chromosomes, 80S cytoplasmic ribosomes (40S and 60S subunits), mitochondria (which have their own 70S ribosomes, a sign of bacterial ancestry), endoplasmic reticulum and Golgi apparatus. Fungal membranes contain ergosterol rather than cholesterol, and fungal cell walls are built of chitin and glucans; yeast walls are mainly glucan and mannoprotein. Eukaryotic cells divide by mitosis and many reproduce sexually by meiosis. Endospores are dormant, highly resistant cells formed inside a vegetative cell by Bacillus, Clostridium and related genera such as Geobacillus, when nutrients run short. One vegetative cell forms one spore, so sporulation is survival, not multiplication. The spore has a dehydrated core containing DNA protected by small acid-soluble proteins and a large amount of calcium dipicolinate, surrounded by an inner membrane, a cortex of modified peptidoglycan, and protein coats. Core dehydration is the main reason for heat resistance; dipicolinic acid and the protective proteins add resistance to heat, ultraviolet radiation, drying and chemicals. Spores can survive boiling, which is why low-acid canned foods are processed at 121 °C to destroy spores of Clostridium botulinum. When conditions improve, nutrients such as L-alanine or a heat shock trigger germination; the spore loses its resistance, takes up water and grows out as a vegetative cell. Vegetative cells, the growing form of any bacterium, are killed by pasteurisation, while spores of Bacillus cereus and Clostridium perfringens survive cooking and germinate if food is cooled slowly. Spore position (central, subterminal or terminal) and whether the spore swells the cell help identification.
Staining starts with a smear: a thin film of culture spread on a slide, air-dried and heat-fixed by passing through a flame, which kills cells and fixes them to the glass. Most stains are basic (cationic) dyes such as methylene blue, crystal violet, safranin and basic fuchsin, which bind to negatively charged nucleic acids and wall components. A simple stain uses one dye and shows size, shape and arrangement. In negative staining, an acidic dye such as nigrosin or India ink colours the background and leaves cells and capsules clear; cells are not heat-fixed, so shape is not distorted. The Gram stain is a differential stain that divides bacteria into two groups according to wall structure. The steps are: crystal violet (primary stain) for about a minute; Gram's iodine (mordant), which forms a crystal violet–iodine complex inside the cell; decolourisation with alcohol or acetone–alcohol; and safranin (counterstain). In Gram-positive cells the thick peptidoglycan dehydrates and shrinks during decolourisation and traps the dye complex, so cells stay purple. In Gram-negative cells the solvent dissolves the outer membrane and the thin peptidoglycan cannot hold the complex, so cells lose the violet colour and take up the red-pink safranin. Decolourisation is the critical step: too long makes Gram-positive cells look negative, too short leaves Gram-negative cells purple. Old cultures, damaged cells and some genera stain Gram-variable, so young cultures, of 18 to 24 hours, are used. A quick check without staining is the potassium hydroxide string test: Gram-negative cells lyse in 3 per cent KOH and their DNA makes the suspension stringy.
Acid-fast staining identifies bacteria with mycolic-acid-rich walls, chiefly Mycobacterium (the agents of tuberculosis and leprosy) and some Nocardia. In the Ziehl–Neelsen method, carbol fuchsin is driven into the cells by steaming, the smear is decolourised with acid-alcohol, and methylene blue is used as counterstain. Acid-fast cells hold the red dye; others lose it and appear blue. The Kinyoun method uses a stronger carbol fuchsin without heat. Mycobacterium bovis in raw milk is one reason milk pasteurisation began. Endospores resist ordinary stains because of their coats. In the Schaeffer–Fulton method, malachite green is steamed into the spores, the smear is rinsed with water, which removes the dye from vegetative cells but not from spores, and safranin is applied as counterstain. Spores appear green and vegetative cells red. Spores can also be seen unstained under phase contrast as bright bodies, and in a Gram stain they appear as unstained holes in the cells. Other special stains include capsule stains (a negative stain combined with a cell stain, so a clear halo is seen around a coloured cell), flagella stains that build up the thin flagella with a mordant until visible, and stains for storage granules such as metachromatic (volutin) granules. Fungi are commonly stained with lactophenol cotton blue, which kills, preserves and stains hyphae and spores for identification by their structure. Fluorescent stains extend these methods, and live/dead staining is used to study disinfectant action.
Bacteria are described first by shape and arrangement. Cocci are spherical; bacilli are rods; short, oval rods are coccobacilli; vibrios are curved rods; spirilla are rigid spirals and spirochaetes flexible spirals. Cocci dividing in one plane form pairs (diplococci) or chains (streptococci, also lactococci and enterococci); in two planes they form tetrads; in three planes, cubes (sarcinae); and in random planes, grape-like clusters (staphylococci, micrococci). Shape, Gram reaction, spore formation, motility, oxygen requirement, catalase and oxidase reactions and sugar fermentations are the classical keys to identification. Classification places organisms in a hierarchy from domain to species, and names are binomial: genus and species, written in italics, as in Escherichia coli. The reference for bacterial taxonomy is Bergey's Manual. Modern classification is phylogenetic, based on 16S rRNA gene sequences and whole-genome comparisons; two strains with average nucleotide identity above about 95 to 96 per cent are usually considered one species. Below the species level, strains, serotypes and genotypes matter in food safety, because different strains of one species differ in virulence or spoilage potential. Actinomycetes are Gram-positive bacteria, rich in G+C, that grow as branching filaments and, in Streptomyces, form chains of spores on aerial hyphae, superficially like moulds. They are common in soil, produce most of the antibiotics used in medicine, and make geosmin, the compound responsible for earthy and musty taints in drinking water, fish and some foods.
Fungi are eukaryotic, heterotrophic organisms with chitin-containing walls. Yeasts are unicellular fungi, usually oval, 3 to 10 µm, that reproduce mostly by budding (Saccharomyces, Candida, Zygosaccharomyces) or, in a few genera, by binary fission (Schizosaccharomyces). Many form sexual ascospores in an ascus. Saccharomyces cerevisiae ferments sugar to ethanol and carbon dioxide in bread, beer and wine; Zygosaccharomyces rouxii and Z. bailii tolerate high sugar or preservatives and spoil syrups, juice concentrates and dressings. Moulds are filamentous fungi. Their threads, hyphae, form a mass called a mycelium. Hyphae are septate (divided by cross-walls) in the Ascomycota and Basidiomycota, and coenocytic (largely without cross-walls) in the Mucoromycota, the group formerly called zygomycetes, such as Mucor and Rhizopus. Moulds are identified mainly by how they make asexual spores. Rhizopus and Mucor bear sporangiospores inside a sac (sporangium) on a sporangiophore; Rhizopus also has root-like rhizoids and runners (stolons). Aspergillus bears conidia in chains on phialides covering a swollen vesicle at the tip of the conidiophore. Penicillium bears chains of conidia on a branched, brush-like structure called a penicillus. Fusarium forms canoe- or sickle-shaped multicelled macroconidia. Sexual spores are zygospores in the Mucoromycota, ascospores in the Ascomycota and basidiospores, as in mushrooms, in the Basidiomycota. Many moulds grow at lower water activity than bacteria, which is why they spoil bread, cereals, nuts and jams; some produce mycotoxins, and others are used to make tempeh (Rhizopus), soy sauce and miso (Aspergillus oryzae) and mould-ripened cheese (Penicillium roqueforti and P. camemberti).
Viruses are not cells. A virus particle (virion) consists of a nucleic acid genome, either DNA or RNA, single- or double-stranded, inside a protein coat called a capsid, which is built of repeating subunits (capsomeres). Some viruses also have a lipid envelope taken from the host cell membrane, carrying viral glycoproteins. Capsids are helical, icosahedral or complex. Most viruses are 20 to 300 nm across, smaller than the resolution limit of the light microscope, and pass through bacteria-retaining filters. They have no metabolism of their own and multiply only inside living host cells, so they cannot grow in food; but a food can carry them from an infected handler or contaminated water to a consumer. The general replication cycle has attachment to specific receptors, penetration or injection of the genome, uncoating, synthesis of viral nucleic acid and proteins using host machinery, assembly, and release by lysis or budding. The Baltimore system groups viruses by genome type and the way they make messenger RNA. Enveloped viruses are relatively easy to inactivate with detergents, alcohols and drying; non-enveloped viruses such as norovirus and hepatitis A virus, the main food-borne viruses, resist many sanitisers, low pH and drying, and survive freezing.
Bacteriophages (phages) are viruses that infect bacteria. In the lytic cycle, as in phage T4 of E. coli, the phage attaches, injects its DNA, takes over the cell, makes new phages and lyses the cell to release them. A one-step growth experiment shows the latent period (from infection to the first release of phage), within which is the eclipse period (when no infectious phage can be recovered from inside the cell), followed by a rise in phage numbers; the average number of phages released per infected cell is the burst size. Phages are counted as plaque-forming units (PFU): a dilution of phage is mixed with host bacteria in soft agar over a plate, and each infective particle makes a clear zone (plaque) in the bacterial lawn. Temperate phages, such as lambda, can instead follow the lysogenic cycle: their DNA integrates into the host chromosome as a prophage and is copied with it until a stress, such as ultraviolet light, induces the lytic cycle. Lysogeny matters because prophages can carry genes for toxins, for example the Shiga toxin genes of enterohaemorrhagic E. coli, and can move genes between bacteria by transduction. Phages are the main cause of slow or failed fermentations in cheese and yoghurt making, because starter bacteria such as Lactococcus lactis and Streptococcus thermophilus grow in large numbers in an open vat. Control measures are rotation of starter strains with different phage sensitivities, phage-resistant strains, phage-inhibitory media for bulk starter, direct-to-vat starters, separation of whey handling from the starter room, and strict sanitation. Phages are also used for good: lytic phages specific for Listeria, Salmonella or E. coli O157 are applied to some foods and surfaces as biocontrol agents in several jurisdictions, and phage-based detection methods are used in laboratories.
Microbial cells need water, a source of carbon and energy, nitrogen, phosphorus, sulphur, potassium, magnesium, calcium and iron, trace elements such as zinc, copper, manganese, cobalt and molybdenum, and, for some organisms, growth factors they cannot make, such as vitamins, amino acids, purines and pyrimidines. Lactic acid bacteria are nutritionally fastidious and need many growth factors, which is one reason they grow well in milk, meat and vegetables but poorly on simple media. Organisms are grouped by their sources of energy and carbon. Phototrophs use light and chemotrophs use chemical energy; autotrophs fix carbon dioxide and heterotrophs use organic carbon. Chemoorganoheterotrophs, which use organic compounds for both energy and carbon, include all food spoilage organisms and pathogens. Chemolithoautotrophs such as nitrifying bacteria oxidise inorganic compounds for energy. Organisms also differ in their response to oxygen. Obligate aerobes need oxygen for respiration (Pseudomonas, most moulds). Facultative anaerobes respire with oxygen and ferment without it (E. coli, Staphylococcus, Saccharomyces). Microaerophiles need low oxygen (Campylobacter). Aerotolerant anaerobes, such as most lactic acid bacteria, ferment whether or not oxygen is present. Obligate anaerobes, such as Clostridium, are harmed by oxygen because they lack enzymes such as catalase and superoxide dismutase that destroy toxic oxygen species. By temperature, psychrophiles grow best at about 15 °C or below; psychrotrophs grow at refrigeration temperatures but have optima of 20 to 30 °C (Listeria, Pseudomonas); mesophiles grow best at about 30 to 40 °C; thermophiles at about 55 to 65 °C; hyperthermophiles above 80 °C.
A culture medium supplies nutrients in a suitable physical and chemical environment. Defined (synthetic) media have known chemical composition. Complex media contain extracts and digests, such as peptone, beef extract, yeast extract and tryptone, whose exact composition is unknown; nutrient broth and plate count agar (tryptone, yeast extract and glucose) are examples. Media are liquid (broth), solid, or semi-solid (for motility). Agar, a polysaccharide from red seaweeds, is used at about 1.5 per cent to solidify media; it melts near 85 to 100 °C and sets near 40 °C, so it can be held molten at 45 to 50 °C for pour plates without killing cells, and very few microbes degrade it. Selective media contain agents that suppress unwanted organisms, such as bile salts and crystal violet against Gram-positive bacteria, high salt in mannitol salt agar for staphylococci, or antibiotics. Differential media show a visible difference between organisms, often by a pH indicator: on MacConkey agar, which is both selective and differential, lactose-fermenting colonies are pink-red because acid turns neutral red, while non-fermenters are colourless. On eosin methylene blue agar, E. coli gives a green metallic sheen; on blood agar, haemolysis zones are seen. Enrichment media favour a target organism present in low numbers among many competitors; for Salmonella, a non-selective pre-enrichment in buffered peptone water is followed by selective enrichment in Rappaport–Vassiliadis or tetrathionate broth. Media for fungi are often acidified or contain antibiotics, as in potato dextrose agar acidified with tartaric acid or dichloran rose bengal chloramphenicol agar. Media are sterilised by autoclaving at 121 °C, usually for 15 minutes for small volumes; heat-sensitive components such as some antibiotics, sugars and blood are sterilised by membrane filtration or added aseptically after the medium has cooled. Each batch is checked with positive and negative control strains.
When bacteria are inoculated into fresh medium in a closed vessel (batch culture), the population follows a typical curve. In the lag phase numbers barely change while cells adjust to the new environment, repair damage and make enzymes; lag is long when cells are injured, cold, starved or moved to a very different medium, and short when actively growing cells move to the same medium. In the exponential (logarithmic) phase cells divide at a constant rate and a plot of log number against time is a straight line; cells here are most uniform and most sensitive to antimicrobials. In the stationary phase growth stops because a nutrient runs out or waste products such as acids accumulate; numbers are steady as division and death balance, and spore-formers sporulate. In the death phase numbers fall, usually exponentially. During exponential growth N = N0 × 2^n, where n is the number of generations. Taking logarithms, n = (log N − log N0) ÷ log 2 = 3.3 × (log N − log N0). The generation (doubling) time is g = t ÷ n. For example, an increase from 10³ to 10⁷ cells/mL in 4 hours is 4 log cycles, which is 13.3 generations, so g = 240 ÷ 13.3 = 18 minutes. The specific growth rate μ (per hour) relates to doubling time by μ = ln 2 ÷ g = 0.693 ÷ g. Generation times range from about 10 to 20 minutes for fast pathogens such as Clostridium perfringens or Vibrio parahaemolyticus at their optimum to many hours at refrigeration temperatures, which is why chilling extends shelf life. Exam problems often ask for the number after a given time (N = N0 × 2^(t/g)) or for g from two counts.
The relation between specific growth rate and the concentration of a limiting substrate S is described by the Monod equation: μ = μmax × S ÷ (Ks + S), where μmax is the maximum specific growth rate and Ks is the substrate concentration at which μ is half of μmax. At S much greater than Ks, growth runs near μmax; at low S, growth rate is roughly proportional to S. The yield coefficient Y(X/S) is the mass of cells formed per mass of substrate used. In a chemostat, fresh medium flows in at rate F and culture flows out at the same rate, keeping the volume V constant. The dilution rate is D = F ÷ V. At steady state the specific growth rate equals the dilution rate (μ = D), because cells must grow as fast as they are washed out; the steady-state substrate concentration is set by D through the Monod equation. If D is raised above μmax, cells cannot keep up and are washed out. Chemostats allow growth at a controlled, constant rate and are used in research and some industrial fermentations; the turbidostat instead holds cell density constant by adjusting flow. Two other patterns appear in exams. Diauxic growth, described by Jacques Monod, is the two-step growth curve seen when a culture is given two sugars, such as glucose and lactose: the cells use glucose first, pause (a second lag) while lactose-using enzymes are induced, then grow on lactose, an effect of catabolite repression. Synchronous cultures, in which all cells divide at once, are made by selection or by starvation and are used to study the cell cycle. In food microbiology the same growth kinetics, measured at different temperatures, pH values and water activities, form the basis of predictive microbiology models.
Food and cultures contain too many organisms to count directly, so samples are diluted in tenfold steps. A typical first step is 10 g of food blended or stomached with 90 mL of diluent such as peptone saline or buffered peptone water, giving a 10⁻¹ dilution; then 1 mL is transferred to 9 mL of diluent to give 10⁻², and so on. Each transfer uses a fresh sterile pipette. Peptone in the diluent protects cells from osmotic shock during the short time before plating. In the pour plate method, 1 mL of a dilution is placed in an empty Petri dish and mixed with molten agar cooled to about 45 °C; colonies grow within and on the agar. In the spread plate method, usually 0.1 mL is spread over the surface of a pre-poured plate; colonies are all on the surface, which helps with heat-sensitive organisms and colony recognition. After incubation, plates in the countable range are counted. Many standards use 30 to 300 colonies per plate; some recent standards use lower limits for small plates or for confirmation. Each colony is assumed to arise from one cell or one clump, so results are expressed as colony-forming units (CFU). Count per g or mL = colonies ÷ (volume plated × dilution). So 156 colonies from 0.1 mL of the 10⁻⁵ dilution means 156 ÷ (0.1 × 10⁻⁵) = 1.6 × 10⁸ CFU/mL. When two successive dilutions give countable plates, ISO 7218 uses a weighted mean: N = ΣC ÷ [V × (n1 + 0.1 n2) × d], where ΣC is the sum of colonies on all counted plates, n1 and n2 are the numbers of plates at the first and second dilutions, V is the volume plated and d is the first (less diluted) dilution. Plate counts measure only cells able to grow on that medium under those conditions; injured, viable-but-non-culturable or fastidious organisms are missed.
The most probable number (MPN) method estimates numbers statistically from the pattern of positive and negative tubes in a dilution series, usually three or five tubes at each of three successive dilutions. It is used when numbers are low or when the food contains particles that interfere with plating, and is classic for coliforms and E. coli in water and food: tubes of lactose broth with an inverted Durham tube are read for gas (presumptive test), positives are subcultured to brilliant green lactose bile broth (confirmed test), and the pattern of positive tubes is read from an MPN table, which gives the estimate and its confidence limits. MPN has wide confidence intervals. Membrane filtration passes a large volume of water or clear liquid through a membrane with pores about 0.45 µm wide, which retains bacteria; the membrane is placed on a selective medium and colonies are counted. It is the usual method for low counts in drinking water. Direct microscopic counts use a counting chamber (haemocytometer or Petroff–Hausser chamber) of known depth and grid area, or a stained smear of known area, as in the Breed smear for milk. They are fast but count dead cells as well as live ones and need high numbers. Indirect methods measure something proportional to cell mass or activity. Turbidity, measured as optical density with a spectrophotometer, often at 600 nm, rises with cell mass; it is fast and non-destructive but needs about 10⁷ cells/mL or more to detect, and must be calibrated against plate counts. Dry weight, protein or nitrogen content, ATP bioluminescence, impedance or conductance changes, flow cytometry and metabolic activity (acid or gas production, dye reduction such as the methylene blue and resazurin tests for milk) are other options.
Sterilisation destroys or removes all viable microorganisms, including spores. Disinfection destroys most pathogens on inanimate objects but not necessarily spores. Sanitisation reduces microbes on food-contact surfaces to levels judged safe by public-health standards. Antisepsis is disinfection of living tissue. Agents ending in -cidal kill; those ending in -static only stop growth, which resumes when the agent is removed. Moist heat kills by denaturing proteins and is far more effective than dry heat at the same temperature. The autoclave uses saturated steam at 121 °C, reached at about 103 kPa (15 psi) above atmospheric pressure, typically for 15 minutes for small loads, longer for large volumes; air must be removed because an air–steam mixture is cooler than pure steam at the same pressure. Boiling at 100 °C kills vegetative cells but not all spores. Pasteurisation kills vegetative pathogens with a mild heat treatment. Dry heat in a hot-air oven, usually 160 to 170 °C for about two hours, kills by oxidation and is used for glassware and materials that steam cannot penetrate, such as oils and powders. Incineration and flaming sterilise loops. Filtration through membranes with 0.2 or 0.22 µm pores removes bacteria from heat-sensitive liquids and air (HEPA filters), but not most viruses. Ultraviolet light near 260 nm is absorbed by DNA and forms pyrimidine (thymine) dimers; it penetrates poorly and is used for surfaces, air and clear water. Ionising radiation (gamma rays, electron beams and X-rays) produces free radicals and breaks DNA, penetrates packages and is used to sterilise medical devices and, at regulated doses, to treat some foods. Low temperature, drying and high osmotic pressure are static rather than cidal.
Chemical agents differ in spectrum and in how organic matter affects them. Alcohols (ethanol or isopropanol at 60 to 90 per cent, commonly 70 per cent) denature proteins and dissolve membranes; some water is needed, so absolute alcohol works less well, and alcohols do not kill spores. Halogens include chlorine compounds (hypochlorite, chlorine dioxide), which oxidise cell components and are cheap and broad-spectrum but inactivated by organic matter, and iodine and iodophors. Phenolics damage membranes; the phenol coefficient compares a disinfectant with phenol as the dilution of the test agent that kills a test organism in a set time divided by the corresponding dilution of phenol. Quaternary ammonium compounds are cationic detergents active against Gram-positive bacteria and less so against Gram-negatives, spores and non-enveloped viruses. Aldehydes (glutaraldehyde, formaldehyde) alkylate proteins and nucleic acids and can sterilise with long contact. Peroxygens (hydrogen peroxide, peracetic acid) are strong oxidisers; ethylene oxide gas sterilises heat-sensitive items. Microbes differ in resistance: in broad order from most to least resistant are prions, bacterial spores, mycobacteria, non-enveloped viruses, fungi and vegetative bacteria, and enveloped viruses. Death by heat and by many chemicals follows first-order kinetics: the log number of survivors falls linearly with time. The decimal reduction time, D, is the time at a stated temperature to kill 90 per cent of the population (one log reduction). If counts fall from 10⁶ to 10² in 12 minutes, that is four log reductions and D = 3 minutes. The number of survivors after time t is log N = log N0 − t/D. The z-value is the temperature change that changes D tenfold; if D at 121 °C is 0.2 min and at 111 °C is 2 min, z = 10 °C. The thermal death time F is the time to achieve a stated reduction: F = D (log N0 − log N). The 12D concept for low-acid canned foods requires a process that would reduce C. botulinum spores by 12 log cycles; with D121 = 0.21 min, F = 12 × 0.21 = 2.52 min at 121 °C. Death curves sometimes show shoulders or tails, from clumping, mixed populations or protective food components.
A mutation is a heritable change in the base sequence of DNA. Spontaneous mutations arise from replication errors and occur at low rates, typically about one in a million to one in a hundred million per gene per generation; induced mutations are caused by mutagens. Point mutations change one base pair: a transition swaps a purine for a purine or a pyrimidine for a pyrimidine; a transversion swaps a purine for a pyrimidine. They may be silent, missense (a different amino acid) or nonsense (a stop codon). Insertions or deletions of one or two bases shift the reading frame (frameshift mutations), usually destroying the protein. Mutagens include ultraviolet light, which makes thymine dimers; base analogues such as 5-bromouracil; chemicals that modify bases, such as nitrous acid and alkylating agents like ethyl methanesulphonate; and intercalating agents such as acridine dyes, which cause frameshifts. The Luria–Delbrück fluctuation test (1943) and the Lederbergs' replica plating showed that mutations such as phage resistance arise spontaneously before selection, not in response to it. Replica plating transfers colonies from a master plate to several media with a velvet pad, letting auxotrophic mutants, which cannot make a needed nutrient, be found. The Ames test uses histidine-requiring Salmonella mutants and counts revertants that regain the ability to grow without histidine to screen chemicals for mutagenicity, often after activation with liver enzymes. Plasmids are small, usually circular, self-replicating DNA molecules separate from the chromosome. They are not essential under ordinary conditions but carry useful genes: resistance (R) plasmids carry antibiotic and heavy-metal resistance; fertility (F) plasmids carry the genes for conjugation; other plasmids carry toxin, bacteriocin or metabolic genes. In Lactococcus lactis, lactose fermentation and the cell-envelope proteinase needed for fast growth in milk are plasmid-encoded, so strains that lose these plasmids become slow acid producers. Plasmids can be lost (cured) by growth at high temperature or with certain chemicals.
Bacteria exchange genes by three routes. Transformation is uptake of free DNA from the environment by competent cells. Griffith showed in 1928 that a heat-killed virulent, capsulated pneumococcus could convert a living non-capsulated strain to the virulent form, and Avery's group showed the agent was DNA. Some species are naturally competent; others are made competent in the laboratory by calcium chloride treatment and heat shock or by electroporation, the basis of introducing recombinant plasmids into E. coli. Transduction is transfer of bacterial DNA by a phage. In generalised transduction, a phage mistakenly packages any fragment of host DNA during lytic growth and injects it into a new host. In specialised transduction, a temperate phage such as lambda excises imprecisely from the chromosome and carries only the genes next to its integration site. Conjugation is transfer of DNA through direct cell contact, using a sex pilus. An F+ cell, which carries the F plasmid, transfers a copy of it to an F− cell, which becomes F+. In Hfr (high-frequency recombination) strains the F plasmid is integrated into the chromosome, so chromosomal genes follow it into the recipient in a fixed order, which was used to map the E. coli chromosome by interrupted mating; the recipient usually stays F− because the whole chromosome is rarely transferred. An F′ plasmid carries some chromosomal genes. Conjugation, together with transposons (jumping genes) and integrons, spreads antibiotic resistance between species, including from animal and food bacteria to human pathogens, which is why antimicrobial resistance in the food chain is monitored. CRISPR–Cas systems are adaptive immune systems of bacteria and archaea: short pieces of phage DNA are stored as spacers in the CRISPR locus and used to guide Cas nucleases to destroy matching invading DNA. The system was first shown experimentally in Streptococcus thermophilus, a yoghurt and cheese starter, in 2007, and is used to select phage-resistant starter strains; Cas9 was later adapted as a tool for genome editing.
Aseptic technique is the set of practices that keeps cultures, media and samples free of contamination and keeps the worker and the environment free of the organisms being handled. Its core habits are simple: work surfaces are disinfected before and after work; inoculating loops and needles are sterilised by flaming until red hot and cooled before use, or disposable sterile loops are used; tubes and bottle necks are flamed briefly after opening and before closing where a flame is used; caps are held in the hand, not put down on the bench; Petri dish lids are lifted only as much as needed; sterile pipettes and tips are used once; and nothing sterile is touched with fingers. Pipetting by mouth is never done. Work is organised so that clean materials do not pass over used ones. Streak plating isolates pure colonies: a loopful of culture is streaked over one sector of a plate, the loop is sterilised, and successive sectors are streaked from the end of the previous one, thinning the cells until single cells give separate colonies. A pure culture is kept by subculturing from a single colony. Cultures are preserved short-term on slants in a refrigerator, and long-term frozen at −70 °C or below with a cryoprotectant such as glycerol, or freeze-dried. Reference cultures for quality control come from recognised culture collections. In food laboratories aseptic technique also covers sampling: sterile tools and containers, labelling, keeping samples chilled and testing them quickly, and avoiding cross-contamination between samples. Media sterility checks, blank plates of diluent and air-exposure plates detect contamination introduced by the laboratory itself.
Biosafety is the containment of biological agents to protect laboratory workers, others and the environment. WHO's Laboratory Biosafety Manual and national rules classify microorganisms into risk groups by the hazard they pose: risk group 1, unlikely to cause disease; risk group 2, can cause disease but is unlikely to spread and effective treatment exists, which includes most food-borne bacterial pathogens such as Salmonella, Listeria monocytogenes and Staphylococcus aureus; risk group 3, serious disease with available treatment, such as Mycobacterium tuberculosis and some E. coli strains under certain national schemes; risk group 4, serious disease, readily transmitted, usually without treatment, such as Ebola virus. Laboratories are built and run at matching biosafety levels 1 to 4, with stricter facilities, equipment and practices at each step; the current WHO manual emphasises local risk assessment over fixed categories. Classification of particular organisms varies between national schemes. Biological safety cabinets (BSCs) are the main containment equipment. A Class I cabinet draws room air in past the worker and exhausts it through a HEPA filter; it protects the worker and environment but not the sample. A Class II cabinet adds a downward flow of HEPA-filtered air over the work, protecting worker, sample and environment, and is the workhorse of microbiology laboratories. A Class III cabinet is a sealed glove box for the highest-risk work. A horizontal laminar-flow clean bench blows filtered air towards the worker and protects only the product, so it must not be used for pathogens. Other measures include personal protective equipment (laboratory coat, gloves, eye protection), hand washing, no eating or drinking, decontamination of all cultures and contaminated waste by autoclaving before disposal, procedures for spills (cover with absorbent, flood with disinfectant, wait, clean up), training, vaccination where relevant, and records. Accredited food laboratories document these practices as part of their quality system.
A student uses this subject in every microbiology practical and in the most predictable exam arithmetic. They prepare tenfold dilutions, pour and spread plates and convert colony counts to CFU per gram, calculate generation time from two counts and D-values from survivor curves, and read a Gram stain under oil immersion. They learn which media select and which differentiate, why agar is held at 45 °C for pour plates, how an autoclave cycle works and why loops are flamed. Knowing the structure of the Gram-negative envelope and of the endospore explains later topics: why Salmonella survives some sanitisers, why canned foods are processed at 121 °C and why pasteurised milk can still spoil.
Five years into a food microbiology laboratory or a quality role, a technologist runs and checks plate counts, MPN tests and enrichment procedures, chooses dilutions that land in the countable range, applies the weighted-mean rule when two dilutions count, and decides when a result is an estimate. They validate media with control strains, maintain cultures, verify autoclave cycles with biological and chemical indicators, and work at the correct biosafety level with Class II cabinets. In a dairy they track starter performance and recognise the signs of phage attack.
A senior microbiologist with twenty-five years of experience designs the laboratory: its biosafety level, cabinets, waste flow and quality system, and its methods and their validation. They advise on thermal process design from D- and z-values, on sanitiser choice from resistance hierarchies, and on investigations of unusual results, such as injured cells missed by plate counts or a strain that has gained resistance. They manage starter strain rotation and phage control programmes, and train analysts across several sites.
A researcher with a doctorate and long experience works on the edges of the classical picture: viable-but-non-culturable cells, heterogeneity within populations, non-linear survival curves, spore germination and dormancy, CRISPR-based phage defence and its use in strain design, horizontal transfer of resistance genes in food environments, and genome-based taxonomy that reorganises the species food scientists rely on. They review methods for standards bodies and supervise doctoral projects on microbial physiology.
This manual does not give MPN tables, detailed biochemical identification schemes, metabolic pathways (glycolysis, respiration, fermentation routes) in depth, immunology, or the classification of protozoa and algae. Risk-group assignments for named organisms differ between national schemes and should be checked locally. Resolution figures, countable ranges and autoclave times are standard teaching values; laboratories must follow the method standard they are accredited to.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions.
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