Dairy Science & Technology · Foundations · also Fundamentals of Microbiology, Introductory Microbiology
Microbial cells, growth, metabolism, genetics and laboratory technique: the base for every dairy microbiology subject.
In the exams: ICAR AICE subject 36 Units 1–2; GATE XL-U Section 2 and XL-S; ICAR AIEEA-PG group 16
Microbiology is the study of organisms too small to be seen clearly with the naked eye: bacteria, archaea, yeasts and moulds, microscopic algae and protozoa, and the viruses that infect all of them. For a dairy scientist it is a working science. Milk leaves a healthy udder nearly sterile but picks up micro-organisms from the teat skin, the milking equipment, the air and the people handling it, and it is an excellent growth medium. Some of those organisms spoil it, some cause disease, and some, the lactic acid bacteria, propionibacteria, yeasts and moulds of starter cultures, turn it into yoghurt, cheese, kefir and cultured butter. Every later dairy microbiology subject, from raw milk quality to starter cultures, pathogens and rapid methods, assumes the general principles taught here.
This manual covers those principles. It begins with the history and scope of microbiology and the microscopes used to see micro-organisms. It then describes the structure of the bacterial cell, the classification of bacteria, yeasts, moulds and viruses, and the bacteriophages that are the main biological threat to dairy fermentations. It explains how micro-organisms are fed and cultured, how they grow and how growth is measured, how temperature, pH, water activity and oxygen control growth, how they obtain energy by respiration and fermentation, and how their genes mutate and move between cells. It ends with the methods that kill or remove micro-organisms, sterilisation and disinfection, and with the aseptic and pure-culture techniques on which every laboratory result depends.
Postgraduate entrance and doctoral fellowship examinations in dairy microbiology and food technology ask this material directly: the Gram stain and cell wall structure, the phases of the growth curve and generation-time calculations, temperature groups of bacteria, types of fermentation, mechanisms of gene transfer, the lytic and lysogenic cycles of phages, and autoclave and disinfectant conditions.
Antonie van Leeuwenhoek, a cloth merchant of Delft, used simple single-lens microscopes of his own making to describe bacteria and protozoa, which he called animalcules, in letters to the Royal Society of London from 1676. For two centuries afterwards the main question was where such organisms came from. The doctrine of spontaneous generation held that they arose from non-living matter. Louis Pasteur settled the question in the early 1860s with swan-necked flasks: boiled broth stayed sterile as long as the curved neck trapped dust, but became cloudy within days once the neck was broken.
Pasteur also showed that fermentations are caused by living organisms: yeasts make alcohol and other organisms make lactic acid, and different organisms give different products. His work on spoilage of wine and beer led to the mild heat treatment that bears his name, pasteurisation, later applied to milk to prevent disease. Robert Koch, working in Germany, proved that a specific bacterium causes a specific disease, anthrax in 1876 and tuberculosis in 1882, and stated the criteria now called Koch's postulates: the organism is present in every case, is isolated in pure culture, causes the disease when inoculated into a healthy host, and is recovered again from that host.
Koch's laboratory gave microbiology its basic tools. Solid media allowed single cells to grow into separate colonies, each a pure culture. Gelatin was tried first but melts at body temperature and is digested by many bacteria; agar, a seaweed polysaccharide suggested by Fanny Hesse, replaced it because it stays solid up to about 85 °C and few organisms can break it down. Julius Petri introduced the shallow covered dish in 1887. Hans Christian Gram devised his differential stain in 1884.
Dairy microbiology grew up alongside these discoveries. Joseph Lister obtained what was probably the first pure culture of a bacterium, the lactic acid organism now called Lactococcus lactis, from soured milk in 1878 by serial dilution. Around 1890 dairy scientists in northern Europe, among them Storch in Denmark and Weigmann in Germany, introduced pure starter cultures for cream ripening, replacing the uncontrolled souring of earlier butter making. Orla-Jensen's monograph of 1919 laid the basis for classifying the lactic acid bacteria by shape, temperature of growth and fermentation products, a scheme that shaped the field for decades.
The twentieth century added antibiotics (Fleming, 1928), the electron microscope, DNA as the genetic material and, from the 1970s, Carl Woese's ribosomal RNA comparisons, which revealed three domains of life.
Modern microbiology is divided by organism (bacteriology, mycology, virology) and by application: medical, veterinary, environmental, industrial and food microbiology.
Dairy microbiology is a branch of food microbiology with three concerns. The first is quality: the bacteria that grow in raw milk during cooling and storage, especially psychrotrophs, and the spoilage of every dairy product. The second is safety: pathogens such as Listeria monocytogenes, Salmonella, pathogenic Escherichia coli, Staphylococcus aureus, Bacillus cereus, Campylobacter and Brucella, and toxins they or moulds produce. The third is fermentation: the selection, propagation and protection of starter and probiotic cultures.
A compound light microscope has two lens systems: the objective, close to the specimen, and the eyepiece or ocular. Total magnification is the product of the two; a 100× oil-immersion objective with a 10× eyepiece gives 1,000×. Magnification is useless without resolution, the ability to show two close points as separate. The limit of resolution is set by the wavelength of light and the numerical aperture (NA) of the objective. A common form, from Abbe, is d = λ/(2NA); with green light of about 550 nm and an oil objective of NA 1.25 to 1.4, the limit is about 0.2 µm. Most bacteria are 0.5 to 5 µm long, so they can be seen, but their internal detail cannot.
Immersion oil has nearly the same refractive index as glass. Placed between the slide and the objective, it stops light from bending away as it leaves the slide, raising the numerical aperture and the resolution. Without oil, the 100× objective gives a dim, blurred image. In bright-field microscopy the specimen is seen dark against a bright background, and most bacteria, being nearly transparent, must be stained.
Phase-contrast microscopy, invented by Frits Zernike, converts small differences in refractive index within an unstained specimen into differences in brightness. Living cells, their motility, bacterial endospores (which appear bright and refractile) and yeast budding can be seen without staining or killing.
Fluorescence microscopy illuminates the specimen with short-wavelength light, usually ultraviolet or blue, and views the longer-wavelength light emitted by fluorescent dyes. In epifluorescence microscopes the light comes through the objective, and filters let only the emitted light reach the eye. Dyes such as acridine orange and DAPI stain nucleic acids; antibodies tagged with fluorescent labels identify particular organisms; and dyes that enter only damaged membranes distinguish live from dead cells. The direct epifluorescent filter technique (DEFT), in which milk is filtered through a membrane and the trapped bacteria stained with acridine orange and counted, gives a raw-milk count in under an hour. Confocal laser scanning microscopy builds sharp optical sections and is used to study biofilms and cheese structure.
Electron microscopes use a beam of electrons, whose wavelength is far shorter than that of light, focused by magnetic lenses in a vacuum. The transmission electron microscope (TEM) passes electrons through very thin sections stained with heavy metals and resolves detail well below one nanometre in physical terms; in biological specimens the practical resolution is a few nanometres. It shows cell walls, membranes, ribosomes, phages and the structure of casein micelles. The scanning electron microscope (SEM) scans a focused beam over a metal-coated surface and builds an image of surface topography with great depth of field; it shows biofilms, the surface of powder particles and the network of cheese curd.
Stains remain the everyday tool of the light microscope. A simple stain uses one basic dye, such as methylene blue, to show shape and arrangement. The Gram stain is differential: cells are stained with crystal violet, treated with iodine as a mordant, decolourised with alcohol or acetone, and counterstained with safranin. Gram-positive cells keep the violet complex in their thick peptidoglycan; Gram-negative cells lose it and appear red. The Ziehl–Neelsen acid-fast stain detects mycobacteria, whose waxy walls retain carbol fuchsin against acid alcohol. Endospores are stained with malachite green by the Schaeffer–Fulton method, and capsules are shown by negative staining with nigrosin. In the Breed smear, 0.01 mL of milk is spread over 1 cm² of a slide, stained with methylene blue, and the cells or clumps per field are multiplied by a microscope factor to give a direct microscopic count.
Bacteria and archaea are prokaryotes: their DNA lies in a nucleoid without a nuclear membrane, they have no mitochondria or other membrane-bound organelles, and their ribosomes are 70S. Yeasts and moulds are eukaryotes, with a true nucleus, organelles and 80S cytoplasmic ribosomes. Bacterial shapes are spheres (cocci), rods (bacilli), curved rods (vibrios) and spirals; cocci may form pairs, chains (streptococci and lactococci), clusters (staphylococci) or tetrads.
The cell wall of almost all bacteria is built on peptidoglycan, a mesh of glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid joined by β(1→4) bonds, cross-linked by short peptides. It gives the cell its shape and resists the osmotic pressure of the cytoplasm. Gram-positive bacteria have a thick, many-layered peptidoglycan wall threaded with teichoic and lipoteichoic acids; lactic acid bacteria, staphylococci, bacilli and clostridia are Gram-positive. Gram-negative bacteria have a thin peptidoglycan layer lying in the periplasm between the cytoplasmic membrane and an outer membrane. The outer membrane contains lipopolysaccharide, whose lipid A part is the endotoxin that causes fever, and porins that let small molecules through. It makes Gram-negative bacteria such as pseudomonads, coliforms and Salmonella more resistant to some detergents, dyes and antibiotics. Lysozyme cleaves the glycan chain and lyses many Gram-positive cells; penicillins block the cross-linking transpeptidases.
The cytoplasmic membrane is a phospholipid bilayer with embedded proteins. It is the cell's permeability barrier, the site of active transport, and in respiring bacteria the site of the electron transport chain and ATP synthesis. Bacterial membranes generally lack sterols. Inside, the cytoplasm holds the nucleoid, usually one circular chromosome, often one or more plasmids, many ribosomes, and storage granules such as polyphosphate or poly-β-hydroxybutyrate.
Many bacteria secrete a layer of polysaccharide or protein outside the wall, the glycocalyx. A firmly attached, organised layer is a capsule; a loose one is a slime layer. Capsules protect pathogens from phagocytosis. Extracellular polysaccharides (exopolysaccharides) of some lactic acid bacteria give ropy milk when they come from contaminants, and the desirable smooth, viscous texture of stirred yoghurt and Nordic fermented milks when they come from selected strains. Polysaccharides also form the matrix of biofilms on milk-contact surfaces.
Flagella are long helical filaments of the protein flagellin, turned by a rotary motor in the membrane powered by the proton-motive force. Their arrangement is used in identification: monotrichous (one at one pole), lophotrichous (a tuft at one pole), amphitrichous (at both poles) and peritrichous (all over the surface). Pseudomonads are polar-flagellated and Enterobacteriaceae such as E. coli are peritrichous; lactic acid bacteria are generally non-motile. Fimbriae, short hair-like pili, attach cells to surfaces and host tissues, and sex pili join cells during conjugation.
Some Gram-positive rods, principally the genera Bacillus, Clostridium and their relatives such as Geobacillus and Paenibacillus, form endospores when nutrients run out. One endospore forms inside each cell and is released when the mother cell lyses. The spore is dormant and extremely resistant to heat, drying, radiation and chemicals. Its structure explains this: a core with very little water, containing DNA protected by small acid-soluble proteins and a large amount of calcium dipicolinate; a cortex of modified peptidoglycan; and protein coats. Spores of some species survive boiling for hours, and the most resistant need temperatures of 121 °C or higher for minutes to be destroyed.
When conditions improve, spores germinate in response to specific nutrients, such as certain amino acids or sugars, lose their resistance and grow out as vegetative cells. Germination can be triggered deliberately by a mild heat shock, a principle used in spore counting: a sample is heated, typically at 80 °C for 10 minutes, to kill vegetative cells and activate spores before plating.
Endospores matter throughout dairying. Bacillus cereus spores survive pasteurisation, then germinate and grow in chilled milk, causing sweet curdling and bitty cream and, in large numbers, food poisoning. Thermophilic spore-formers such as Geobacillus stearothermophilus grow in evaporators and limit milk-powder quality. Clostridium tyrobutyricum spores from silage survive pasteurisation and cause late blowing of hard and semi-hard cheeses. Clostridium botulinum is the reason low-acid canned foods receive a sterilising process.
Taxonomy has three parts: classification (arranging organisms into groups), nomenclature (naming them) and identification. Organisms are named by the binomial system of Linnaeus: a capitalised genus and a lower-case species epithet, written in italics in print, such as Lactococcus lactis. Below species, bacteria are divided into subspecies, biovars and strains; a strain is a population descended from a single isolate, and starter cultures are sold and studied as strains. The ranks above genus are family, order, class, phylum and domain.
Older classifications of bacteria relied on phenotype: shape, Gram reaction, motility, spore formation, oxygen relations, temperature range and biochemical tests. Since Woese's work, classification has been based increasingly on genes, first the 16S ribosomal RNA gene and now whole-genome comparisons such as average nucleotide identity; two genomes sharing more than about 95 to 96% average nucleotide identity are generally regarded as the same species. The reference for bacterial classification is Bergey's Manual of Systematics of Archaea and Bacteria, and valid names are published under the International Code of Nomenclature of Prokaryotes.
Names change as knowledge grows, and dairy students must recognise both old and new names. In 2020 the large genus Lactobacillus was divided into 25 genera on genomic evidence. Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus and L. acidophilus remain in Lactobacillus, but the former L. casei, L. paracasei and L. rhamnosus are now Lacticaseibacillus, L. plantarum is Lactiplantibacillus plantarum, and L. fermentum is Limosilactobacillus fermentum. Several phyla have also been renamed, Firmicutes becoming Bacillota.
Most dairy bacteria fall into a few groups. The low-G + C Gram-positive bacteria (phylum Bacillota) include the lactic acid bacteria of the order Lactobacillales: Lactococcus, Streptococcus thermophilus, the lactobacilli and their new genera, Leuconostoc, Pediococcus, Enterococcus and Carnobacterium. They are Gram-positive, catalase-negative, non-sporing cocci or rods that produce lactic acid as the main end product of carbohydrate fermentation. The same phylum includes the spore-formers Bacillus and Clostridium, the staphylococci and Listeria.
The high-G + C Gram-positive bacteria (phylum Actinomycetota) include Bifidobacterium, used as a probiotic; Propionibacterium, which forms the eyes and flavour of Swiss-type cheeses; the coryneform bacteria Brevibacterium and Corynebacterium on the rind of smear-ripened cheeses; Microbacterium, a thermoduric contaminant; and Mycobacterium, which includes the agents of tuberculosis in cattle and humans.
The Gram-negative bacteria (phylum Pseudomonadota) include the psychrotrophic pseudomonads, which dominate spoilage of chilled raw and pasteurised milk; Acinetobacter and related genera; and the family Enterobacteriaceae and its relatives, with the coliforms (Escherichia, Klebsiella, Enterobacter, Citrobacter), Salmonella, Cronobacter, Serratia and Hafnia. Coliforms ferment lactose with acid and gas and are used as indicators of hygiene and post-pasteurisation contamination.
Fungi are eukaryotes with cell walls of chitin and glucans and membranes containing ergosterol. Yeasts are unicellular fungi that reproduce mostly by budding or, in a few genera, by fission. Some ferment sugars to ethanol and carbon dioxide. Dairy yeasts include Kluyveromyces marxianus and K. lactis, which ferment lactose and occur in kefir and cheese; Debaryomyces hansenii, salt-tolerant and common on cheese surfaces; Saccharomyces species; and Candida, Yarrowia and Rhodotorula, which spoil yoghurt, fruit preparations, butter and cream. Many yeasts tolerate low pH, high sugar and low temperatures.
Moulds are filamentous fungi. Their threads, hyphae, form a mat called a mycelium, and they reproduce by spores formed on specialised structures: the brush-like conidiophores of Penicillium, the swollen-headed conidiophores of Aspergillus, and the sporangia of Mucor and Rhizopus. Penicillium roqueforti makes the blue veins of blue cheeses and P. camemberti the white rind of Camembert and Brie; Geotrichum candidum, a yeast-like mould, contributes to the rinds of many soft cheeses. Other moulds spoil cheese, butter and yoghurt, and some, such as Aspergillus flavus, produce mycotoxins in feed that can lead to aflatoxin M1 in milk.
Viruses are not cells. Each virus particle, a virion, has a genome of either DNA or RNA, single- or double-stranded, inside a protein coat, the capsid; some also have a lipid envelope. They have no metabolism of their own and replicate only inside living host cells. Viruses that infect bacteria are bacteriophages.
Bacteriophages, or phages, are viruses that infect bacteria. Most dairy phages are tailed phages with double-stranded DNA: an icosahedral head holding the genome, attached to a tail that recognises the host. Phages are highly specific, usually infecting only certain strains of one species, because they must bind to particular receptors on the cell surface, such as wall polysaccharides or membrane proteins.
A virulent phage follows only the lytic cycle. It adsorbs to the receptor, injects its DNA, takes over the cell's machinery to replicate its genome and make phage proteins, assembles new particles, and lyses the cell with enzymes such as endolysin and holin, releasing the progeny. The time from infection to lysis is the latent period, and the number of phages released per cell is the burst size. For lactococcal phages the latent period is often under an hour and the burst size can be one hundred or more, so a few phage particles in a vat can multiply to destroy the starter population within a few hours.
A temperate phage can either lyse the cell or enter the lysogenic cycle, in which its genome integrates into the bacterial chromosome as a prophage and is copied with it. The lysogenic cell is immune to infection by the same phage. Stress, such as ultraviolet light or some chemicals, can induce the prophage to excise and enter the lytic cycle. Many starter strains carry prophages, which can be released and become a source of new phages in a plant.
Phages are counted by the plaque assay. A dilution of the sample is mixed with a sensitive host in soft agar and poured over a nutrient agar base. The host grows into a lawn, and each infective phage produces a clear zone, a plaque, where cells have been lysed. The count is expressed as plaque-forming units (PFU) per millilitre. Spot tests, in which drops of whey are placed on a lawn, show quickly whether a whey sample contains phages active against a given starter strain. Phages can also be detected by the failure of acid production in a test of the culture's activity in milk, and identified by PCR targeting genes of known phage groups.
Phages of Lactococcus lactis, the most studied, fall into several groups; three of them, the groups formerly called 936, c2 and P335, cause most problems in cheese plants worldwide. Streptococcus thermophilus phages are the main problem in yoghurt and in cheeses made with thermophilic starters. Most dairy phages need calcium ions to adsorb or complete infection, which explains the use of phage-inhibitory media: phosphate or citrate in the bulk-starter medium binds calcium and protects the culture during propagation. Phages are also tools: in transduction, in typing strains for outbreak work, and, where regulations permit, in biocontrol of pathogens such as Listeria.
A phage attack in a fermentation shows as slow or failed acid development: a yoghurt that does not set, or a cheese vat that stays sweet, with poor curd, high moisture and a risk of pathogen growth. Phages enter with raw milk, persist in whey, on equipment, in the air near whey separators and on workers' clothing, and multiply in each vat in which the starter is sensitive. Because whey is so rich in phages, whey handling and the reuse of whey products in the same plant are frequent sources.
Control combines hygiene, culture management and plant design. Hygiene means effective cleaning and sanitising of vats, lines and the starter room, often with chlorine or peracetic acid, which inactivate phages; separating the starter room from cheese-making and whey areas; filtered, positive-pressure air; and keeping whey in closed systems. Culture management means using phage-resistant or unrelated strains, rotating cultures so that no single strain is used day after day, and testing whey routinely to detect rising phage titres. Direct-vat-set concentrated cultures avoid propagation of bulk starter in the plant, removing one step at which phages multiply.
Bacteria have evolved several natural defences against phages: blocking adsorption by changing or masking receptors, preventing DNA injection, restriction–modification systems that cut foreign DNA, abortive-infection systems in which the infected cell dies without releasing phages, and CRISPR–Cas systems, an adaptive immunity that stores fragments of phage DNA and uses them to destroy matching DNA. The first CRISPR experiments that demonstrated this function were made with S. thermophilus, a dairy organism. These mechanisms, often carried on plasmids in lactococci, are used to build phage-resistant starter strains.
Every cell needs a source of energy, a source of carbon, and the elements from which its components are made: hydrogen, oxygen, nitrogen, phosphorus and sulfur in large amounts; potassium, magnesium, calcium and iron in smaller amounts; and trace elements such as manganese, zinc, cobalt and copper. Some also need growth factors, organic compounds they cannot make: amino acids, purines and pyrimidines, and vitamins.
Micro-organisms are grouped by how they obtain energy and carbon. Phototrophs use light and chemotrophs use chemical energy. Autotrophs use carbon dioxide as their carbon source and heterotrophs use organic compounds. Almost all organisms of concern in dairy science are chemoorganoheterotrophs, using organic compounds for both energy and carbon.
Organisms differ greatly in how much they can make for themselves. E. coli grows on glucose and mineral salts alone. Lactic acid bacteria are fastidious: having adapted to rich environments such as milk, plants and the gut, they have lost many biosynthetic pathways and need several amino acids, vitamins of the B group and nucleic acid bases. In milk they obtain amino acids by breaking down casein with a cell-envelope proteinase and peptidases; strains that lose the proteinase grow slowly in milk, a cause of slow starter activity.
A culture medium supplies the nutrients needed to grow micro-organisms in the laboratory. A defined or synthetic medium has an exactly known chemical composition and is used in physiological studies. A complex medium contains ingredients of variable composition, such as peptones (partial digests of protein), meat or yeast extract and milk solids, and supports a wide range of organisms. Media are liquid (broths), solid (with about 1.5% agar) or semi-solid (with about 0.3 to 0.5% agar, used to show motility). Agar melts near 85 °C and, once melted, stays liquid until it cools to about 40 °C, so it can be mixed with a sample in a pour plate without killing the organisms.
Media are designed for particular purposes. A selective medium contains substances that suppress unwanted organisms and allow the target to grow: bile salts and crystal violet suppress Gram-positive bacteria in media for coliforms; antibiotics such as chloramphenicol suppress bacteria in media for yeasts and moulds; and lithium chloride and tellurite help select Staphylococcus aureus. A differential medium contains an indicator that makes different organisms look different: in violet red bile agar, lactose-fermenting coliforms form purple-red colonies because acid changes the neutral red indicator. Many media are both. An enrichment medium favours a target present in small numbers among many competitors, as in the pre-enrichment and selective enrichment steps of Salmonella and Listeria detection.
Standard dairy methods name specific media. Plate count agar, also called standard methods agar, contains tryptone, yeast extract and glucose, with milk powder added in some versions; it is used for the aerobic mesophilic count of milk and dairy products, incubated at 30 °C for 72 hours under the international standard method, with other time-temperature combinations specified in some national methods. Violet red bile agar, or violet red bile lactose agar, is used for coliforms. Baird-Parker agar, containing egg yolk and tellurite, is used for coagulase-positive staphylococci, which form black colonies with clear zones. Yeast extract glucose chloramphenicol agar or acidified potato dextrose agar is used for yeasts and moulds.
Lactic acid bacteria need special media. MRS medium, developed by de Man, Rogosa and Sharpe, supports lactobacilli and related genera; M17 medium, buffered with β-glycerophosphate, supports lactococci and Streptococcus thermophilus, which acidify ordinary media so fast that they inhibit themselves. Selective variants and incubation conditions are used to count the components of yoghurt and probiotic products separately. Media must be prepared to the stated pH and sterilised without overheating, which browns them and destroys nutrients, and each batch is checked with reference strains.
Bacteria reproduce by binary fission: a cell enlarges, copies its chromosome and divides into two. When a few cells are placed in a fresh batch of medium, their numbers follow a characteristic growth curve with four phases. In the lag phase there is little or no increase in number while cells adjust to the new environment, synthesising enzymes and repairing damage; its length depends on the organism, its previous history and the conditions, and it is longer for cells that are injured, cold-shocked or moved to a very different medium. In the exponential or logarithmic phase cells divide at a constant rate, the population doubling at every generation; a plot of the logarithm of numbers against time is a straight line. In the stationary phase, nutrients are exhausted or waste products such as acid accumulate, and growth balances death so the count stays nearly constant. In the death or decline phase the number of viable cells falls, often exponentially.
The growth curve has practical meaning. Keeping milk cold lengthens the lag phase and slows exponential growth, so a good cold chain keeps counts low for days. A starter culture added to milk is chosen and prepared so that its lag phase is short and acid is produced quickly. When an organism is offered two sugars, it may use the preferred one first, pause and then use the second, giving two exponential phases; this diauxic growth was described by Monod using glucose and lactose with E. coli.
During exponential growth the number of cells after n generations is N = N₀ × 2ⁿ. Taking base-10 logarithms, log N = log N₀ + n log 2, so the number of generations is n = (log N − log N₀)/0.301, or approximately 3.3(log N − log N₀). The generation time, or doubling time, is g = t/n, where t is the time of exponential growth.
A worked example: raw milk contains 1 × 10⁴ cfu/mL and, after 6 hours at 30 °C in exponential growth, 1.6 × 10⁵ cfu/mL. Then log N − log N₀ = 5.204 − 4.000 = 1.204, n = 1.204/0.301 = 4 generations, and g = 6 h/4 = 1.5 h, or 90 minutes. The specific growth rate μ, used in kinetic models, is ln 2/g, here 0.46 per hour.
Generation times vary widely, from about 20 minutes for E. coli in rich medium at 37 °C to several hours for pseudomonads at 4 °C. A small change in temperature makes a large difference over a long storage time, which is why the temperature of milk in a farm tank is so important.
Growth can be measured as cell numbers, cell mass or activity. Direct microscopic counts in a counting chamber of known volume, or on a Breed smear, count all cells, living and dead, and are rapid; flow cytometry counts fluorescently stained cells in a stream and, with suitable dyes, distinguishes live from dead.
Viable counts count only cells able to multiply and form colonies. In the pour-plate method a measured volume of a dilution, usually 1 mL, is mixed with molten agar at about 45 °C in a Petri dish; in the spread-plate method 0.1 mL is spread over the surface of set agar. After incubation the colonies are counted, and the result is reported as colony-forming units (cfu) per mL or per g, because a colony may arise from a clump or chain rather than a single cell. Plates with between about 30 and 300 colonies, or the 10 to 300 range in some current international methods, give reliable counts. The most probable number method inoculates replicate tubes of several dilutions and estimates the count statistically from the pattern of positive tubes; it is used for low numbers, as for coliforms in some products.
Indirect methods include turbidity, measured as optical density with a spectrophotometer, which is proportional to cell mass over a limited range; dry weight; ATP bioluminescence; and impedance or conductance, which detect the change in electrical properties of a medium as organisms metabolise. These faster methods must be calibrated against plate counts.
Each organism has three cardinal temperatures: a minimum below which it does not grow, an optimum at which it grows fastest, and a maximum above which it does not grow. The optimum usually lies nearer the maximum than the minimum. Organisms are grouped by these temperatures. Psychrophiles have an optimum around 15 °C or below and a maximum around 20 °C; they live in polar seas and are of little importance in dairying. Psychrotrophs grow at refrigeration temperatures, 7 °C or below, although their optimum is 20 to 30 °C; this is the group that matters in chilled milk, including Pseudomonas, Acinetobacter, some Bacillus species and the pathogens Listeria monocytogenes and Yersinia enterocolitica. Mesophiles grow best at 30 to 40 °C and include most pathogens, the coliforms and the mesophilic starter bacteria (Lactococcus, Leuconostoc). Thermophiles grow best at 45 to 60 °C or above; S. thermophilus and L. delbrueckii subsp. bulgaricus, the yoghurt bacteria, are often called thermophilic starters because they grow at 40 to 45 °C, and Geobacillus stearothermophilus is a true thermophile.
Thermoduric is a different idea: a thermoduric organism survives pasteurisation without necessarily growing at high temperature. Thermoduric bacteria in milk include spore-formers, micrococci, some enterococci and Microbacterium. Their numbers in raw milk reflect poorly cleaned equipment, where they build up on surfaces.
Most bacteria grow best near neutral pH, 6.5 to 7.5, and few grow below pH 4. Lactic acid bacteria are acid-tolerant and produce enough acid to inhibit most competitors, which is the basis of preservation by fermentation. Yeasts and moulds tolerate lower pH, many growing at pH 3 or below, and are therefore the typical spoilage organisms of yoghurt, fruit preparations and acid cheeses. Organic acids are more inhibitory than strong mineral acids at the same pH, because the undissociated acid crosses the membrane and releases protons inside the cell.
Water activity, aw, is the ratio of the vapour pressure of water in a food to that of pure water at the same temperature, and it measures the water available for microbial growth. Pure water has aw = 1.0, fresh milk about 0.99, cheeses about 0.90 to 0.99 depending on salt and moisture, sweetened condensed milk about 0.83 to 0.85, and milk powder about 0.2 to 0.3. Most bacteria need an aw above about 0.91; Staphylococcus aureus grows down to about 0.86 aerobically; most yeasts need about 0.88 and most moulds about 0.80, while xerophilic moulds and osmophilic yeasts grow at 0.60 to 0.65. No organism grows below about 0.6, so dried milk is microbiologically stable while it stays dry, although organisms already present may survive for long periods.
Organisms differ in their relation to oxygen. Obligate aerobes, such as Pseudomonas and moulds, need oxygen for respiration. Microaerophiles, such as Campylobacter, need oxygen but at lower concentration than in air. Facultative anaerobes, such as E. coli, staphylococci and yeasts, use oxygen when it is present but can ferment or respire anaerobically without it. Aerotolerant anaerobes, including most lactic acid bacteria, do not use oxygen for energy but are not harmed by it. Obligate anaerobes, such as Clostridium, are killed or inhibited by oxygen. The difference lies partly in enzymes that destroy toxic oxygen species: superoxide dismutase and catalase, present in aerobes and absent or limited in anaerobes. Lactic acid bacteria lack catalase, which is the basis of the catalase test used in their identification.
The oxidation–reduction (redox) potential, Eh, measures the tendency of a medium to accept or donate electrons. Fresh milk has a positive Eh of about +200 to +300 mV, but starter bacteria rapidly lower it, and the interior of a cheese is strongly reducing, which favours anaerobes and the development of certain sulfur flavours.
In real foods these factors act together. Leistner's hurdle concept describes the combined use of several moderate barriers, such as mild heat, reduced aw, low pH, chilling and preservatives, so that no organism can overcome all of them, even though no single hurdle would suffice. Cheese is a classic example: low pH, salt, low redox potential, competing starter bacteria and their bacteriocins, and cold storage together inhibit pathogens.
Metabolism is the sum of the chemical reactions of a cell: catabolism, which breaks down nutrients to release energy, and anabolism, which uses that energy to build cell material. Chemoorganotrophs release energy by oxidising organic compounds and capture it as ATP and as reduced coenzymes such as NADH.
In aerobic respiration the electrons removed from the substrate pass through an electron transport chain in the cytoplasmic membrane to oxygen, the terminal electron acceptor, and the proton gradient generated drives ATP synthesis by oxidative phosphorylation. Glucose is oxidised completely to carbon dioxide and water, giving a high yield of ATP per molecule. Pseudomonads, bacilli, micrococci and moulds respire aerobically. Many bacteria can also respire anaerobically, using an inorganic acceptor other than oxygen: nitrate (reduced to nitrite or nitrogen gas), sulfate (reduced to hydrogen sulfide) or ferric iron. Anaerobic respiration yields less energy than aerobic respiration.
Two simple tests probe respiratory enzymes. The catalase test detects the enzyme that breaks down hydrogen peroxide to water and oxygen, seen as bubbling when a colony is mixed with hydrogen peroxide; staphylococci and micrococci are positive, and streptococci, lactococci and other lactic acid bacteria are negative. The oxidase test detects cytochrome c oxidase; pseudomonads are oxidase-positive and Enterobacteriaceae oxidase-negative.
In fermentation, the electrons removed from the substrate are passed to an organic molecule made from the substrate itself, such as pyruvate, so that NAD⁺ is regenerated without an external electron acceptor. ATP is made only by substrate-level phosphorylation, so the yield is low, usually one or two ATP per glucose, and large amounts of end products accumulate. The end products identify the type of fermentation.
In homolactic fermentation, carried out by lactococci, S. thermophilus and homofermentative lactobacilli, almost all the glucose is converted to lactic acid by glycolysis and lactate dehydrogenase. In heterolactic fermentation, carried out by Leuconostoc and heterofermentative lactobacilli through the phosphoketolase pathway, each glucose gives lactic acid, carbon dioxide, and ethanol or acetic acid. Alcoholic fermentation by yeasts gives ethanol and carbon dioxide, as in kefir and koumiss. Propionic fermentation by propionibacteria converts lactate to propionic acid, acetic acid and carbon dioxide; the carbon dioxide forms the eyes of Emmental-type cheese and propionic acid gives part of its nutty, sweet flavour. Butyric fermentation by Clostridium tyrobutyricum converts lactate to butyric acid, carbon dioxide and hydrogen; in cheese it causes the cracks and rancid flavour of late blowing. Mixed-acid fermentation by E. coli and other Enterobacteriaceae gives several acids, carbon dioxide and hydrogen; in cheese made from contaminated milk it causes early blowing, a spongy curd within the first days. Butanediol fermentation by Enterobacter and Klebsiella produces 2,3-butanediol and acetoin.
Fermentation patterns are used to identify bacteria. Carbohydrate fermentation tests show acid, with a colour change in a pH indicator, and gas, collected in a small inverted Durham tube. The IMViC series distinguishes coliforms: indole production from tryptophan, the methyl red test for strong mixed-acid fermentation, the Voges–Proskauer test for acetoin from butanediol fermentation, and the use of citrate as sole carbon source. Typical E. coli is positive, positive, negative, negative, and Enterobacter aerogenes the reverse.
Micro-organisms in milk also attack its proteins and fat, and these activities decide the defects they cause. Proteolytic bacteria, especially psychrotrophs, produce extracellular proteases that hydrolyse casein, giving bitter flavours and, in UHT milk, gelation. Lipolytic organisms produce lipases that release free fatty acids, giving rancid and soapy flavours. Some organisms coagulate milk by acid (lactic acid bacteria, coliforms) and some by enzyme, the sweet curdling caused by Bacillus cereus. Others produce exopolysaccharides (ropiness), pigments, or reduce the redox indicator dyes used in old platform tests, such as methylene blue and resazurin, whose reduction time is shorter the more active the bacteria in the milk. Many of these reactions are detected in litmus milk, a simple test medium that shows acid, alkali, reduction, coagulation and peptonisation.
The bacterial genome usually consists of one circular chromosome of a few million base pairs; lactic acid bacteria have relatively small genomes of about 2 to 3 million base pairs, reflecting their reliance on nutrients from their environment. Many bacteria also carry plasmids, small circular DNA molecules that replicate independently of the chromosome. Plasmids are not essential for life in ordinary conditions but often carry useful traits: resistance to antibiotics or heavy metals, toxin production, or new metabolic abilities. In Lactococcus lactis, plasmids frequently carry the genes for lactose transport and metabolism, the cell-envelope proteinase needed for fast growth in milk, citrate uptake, phage-resistance mechanisms and some bacteriocins. Loss of such a plasmid, which happens spontaneously, gives slow or non-acidifying variants and is one cause of unreliable starter performance.
A mutation is a heritable change in the DNA sequence. Point mutations change a single base; they may be silent, change one amino acid (missense) or create a stop codon (nonsense). Insertions or deletions of bases can shift the reading frame. Mutations arise spontaneously from replication errors, or are induced by mutagens: ultraviolet light forms thymine dimers, ionising radiation breaks DNA, and chemicals such as nitrous acid and alkylating agents alter bases. Cells repair much of this damage. Mutants selected without genetic engineering, such as strains that make less post-acidification or more exopolysaccharide, are widely used as dairy cultures.
Bacteria exchange genes by three main mechanisms, collectively called horizontal gene transfer. In transformation, a cell takes up free DNA from its surroundings and incorporates it. Griffith demonstrated it in 1928 with pneumococci, and Avery, MacLeod and McCarty showed in 1944 that the transforming principle is DNA. Only cells in a special state, called competence, take up DNA naturally; others can be made to take it up in the laboratory by treatment with calcium chloride or by electroporation, brief electric pulses that open pores in the membrane. Electroporation is the usual way to introduce DNA into lactic acid bacteria.
In conjugation, DNA passes directly from a donor to a recipient cell in contact. Lederberg and Tatum showed it in E. coli in 1946. In Gram-negative bacteria the F plasmid encodes a sex pilus that draws the cells together; when the F plasmid has integrated into the chromosome, as in Hfr strains, chromosomal genes can be transferred too. Many Gram-positive bacteria, including lactococci, conjugate by other mechanisms. Conjugation is the main route by which antibiotic resistance spreads among bacteria, and it has been used to transfer lactose and phage-resistance plasmids into dairy strains without recombinant DNA technology, producing improved starters acceptable under food-grade rules.
In transduction, a bacteriophage carries bacterial DNA from one cell to another. In generalised transduction, any fragment of host DNA is packaged by mistake into a phage head during the lytic cycle. In specialised transduction, a temperate phage that excises imprecisely carries the genes next to its integration site.
Transposable elements are DNA segments that can move from one location to another in a genome. Insertion sequences carry only the genes needed for their own movement; transposons carry other genes as well, often antibiotic resistance. Their insertion can disrupt genes, and they help assemble resistance genes on plasmids. In L. lactis the genes for the bacteriocin nisin and for sucrose fermentation lie on a large conjugative transposon, which can be transferred between strains by conjugation.
Genetic engineering, or recombinant DNA technology, cuts DNA with restriction enzymes, joins fragments with DNA ligase, and introduces them into a host on a vector such as a plasmid. Food-grade vectors for lactic acid bacteria use selection markers other than antibiotic resistance, such as the ability to use lactose, and carry only DNA from food-grade organisms. Precision fermentation, the production of specific proteins such as β-lactoglobulin or chymosin by engineered micro-organisms, depends on these methods; most cheese worldwide is now coagulated with chymosin produced by fermentation in this way. Because rules on genetically modified live cultures differ between countries, classical mutation, selection and conjugation remain the main ways of improving starter strains.
Sterilisation is the destruction or removal of all living organisms, including bacterial endospores. In practice sterility is expressed as a probability of a surviving organism, the sterility assurance level. Disinfection is the destruction of pathogenic and most other vegetative organisms on inanimate surfaces, without necessarily killing spores. Antisepsis is disinfection of living tissue. Sanitisation reduces the number of organisms on food-contact surfaces to levels judged safe; dairy sanitisers are often required to give a stated log reduction, commonly five logs, of test organisms in a set contact time. Agents that kill are called -cidal (bactericidal, sporicidal, fungicidal, virucidal) and those that only stop growth are -static.
Microbial death under a constant lethal treatment usually follows first-order kinetics: a constant proportion of the survivors dies in each unit of time. The decimal reduction time, D, is the time for a tenfold reduction at a stated condition. The z value is the temperature change that alters D tenfold. Effectiveness depends on the number and type of organisms (spores are the most resistant, then mycobacteria, then other vegetative bacteria), the concentration or intensity of the agent, the contact time, the temperature, the pH and the presence of organic matter such as milk residues, which protect organisms and consume chemical sanitisers. Cells in biofilms are far more resistant than the same cells suspended in liquid, which is why surfaces must be cleaned before they are sanitised.
Moist heat kills by denaturing proteins and is more effective than dry heat at the same temperature. The autoclave uses saturated steam under pressure; the standard cycle is 121 °C, reached at about 103 kPa (15 lb per square inch) above atmospheric pressure, for 15 minutes for small loads, with longer times for large volumes. Air must be removed completely, because a mixture of air and steam is cooler than pure steam at the same pressure. Media, glassware and contaminated waste are sterilised this way. Boiling at 100 °C kills vegetative cells but not all spores.
Dry heat in a hot-air oven kills by oxidation and needs higher temperatures and longer times, commonly 160 °C for 2 hours or 170 °C for 1 hour; it is used for dry glassware and metal instruments. Flaming and incineration are used for inoculating loops and waste. Pasteurisation is a mild heat treatment that kills pathogens and many spoilage organisms but is not sterilisation.
Filtration removes organisms from heat-sensitive liquids and gases: membrane filters with a pore size of 0.2 or 0.22 µm retain bacteria, and high-efficiency particulate air (HEPA) filters clean the air of laminar-flow cabinets and clean rooms. Radiation is also used. Ultraviolet light near 260 nm damages DNA but penetrates poorly, so it is used for surfaces, air and clear water. Ionising radiation, gamma rays or electron beams, penetrates deeply and sterilises disposable laboratory plastics and some packaging.
Chemical agents differ in their spectrum, speed and drawbacks. Alcohols, ethanol or isopropanol at 60 to 80%, with 70% widely used, denature proteins and dissolve membranes; they are fast but do not kill spores, and absolute alcohol is less effective because some water is needed. Chlorine compounds, such as sodium hypochlorite, act through hypochlorous acid, which is more active at slightly acid than at alkaline pH; they are cheap, broad-spectrum and widely used for dairy surfaces, but are inactivated by organic matter and corrode steel at high concentrations. Iodophors are used as sanitisers and teat dips. Quaternary ammonium compounds are cationic detergents that disrupt membranes; they are stable and non-corrosive but weaker against Gram-negative bacteria and spores and are inactivated by anionic detergents.
Peroxygen compounds are important in modern plants. Peracetic acid is effective at low temperature, kills spores at higher concentrations and breaks down to acetic acid, water and oxygen. Hydrogen peroxide, used hot at concentrations of about 30 to 35%, sterilises packaging material in aseptic filling machines before the residue is removed. Aldehydes, glutaraldehyde and formaldehyde, are sporicidal but toxic.
Disinfection must be verified. Biological indicators, spores of known resistance, check sterilisers: Geobacillus stearothermophilus for steam and hydrogen peroxide, and Bacillus atrophaeus for dry heat and ethylene oxide. Chemical indicators change colour with exposure, and surface swabs, contact plates and ATP tests check plant hygiene.
Aseptic technique is the set of practices that prevent contamination of cultures, media and samples by organisms from the environment, and prevent the escape of organisms from cultures to the worker and the environment. Its elements are simple and must become habit: working on a disinfected bench, near a flame or in a biological safety cabinet; flaming inoculating loops to red heat and letting them cool before use; flaming the necks of tubes and bottles when opened; holding lids and caps so that their inner surfaces face downwards and are not set on the bench; opening plates only as much as needed; using sterile pipettes and tips once only; and never pipetting by mouth.
Laboratories are designed and operated according to the risk of the organisms handled. Micro-organisms are placed in risk groups 1 to 4 by their ability to cause disease and spread, and laboratories are built to matching biosafety levels. Most dairy quality-control work, with starter cultures and indicator organisms, is at biosafety level 1 or 2. Work with pathogens such as Listeria monocytogenes, Salmonella or Brucella requires at least level 2 practices, and Brucella and Mycobacterium bovis cultures generally require level 3 containment. Class II biological safety cabinets, with filtered downward airflow, protect the worker, the sample and the environment; laminar-flow clean benches protect only the sample and must not be used for pathogens. Pathogen laboratories are kept separate from production areas.
A pure culture contains only one kind of organism, ideally the progeny of a single cell. Pure cultures are needed to identify an organism, to study its properties and to use it reliably as a starter or a reference strain. They are obtained by separating cells on solid media so that each grows into a distinct colony.
In the streak-plate method a loopful of the mixed sample is spread over one sector of an agar plate, and the loop is flamed and drawn from that sector across a fresh sector, two or three times, so that the cells are progressively thinned out; in the last streaks single cells are deposited far enough apart to form isolated colonies. In the pour-plate method serial dilutions of the sample are mixed with molten agar, so that colonies grow both in and on the agar. In the spread-plate method a small volume of a dilution is spread over the surface. Serial dilution, usually in tenfold steps in sterile diluent such as peptone saline or quarter-strength Ringer's solution, brings the number of cells into a range that gives separate colonies.
A typical colony is picked, restreaked and checked for purity by uniform colony appearance and by microscopy of a Gram-stained smear. Enrichment culture, devised by Winogradsky and Beijerinck, favours an organism present in small numbers by providing conditions that suit it better than its competitors, and is the first step in isolating pathogens such as Salmonella and Listeria from foods.
Colony counts are converted to counts per mL or g of the original sample by allowing for the dilution and the volume plated. Under the international standard method for counting, ISO 7218, when two successive dilutions give countable plates, the count is N = ΣC/[V(n₁ + 0.1n₂)d], where ΣC is the sum of colonies on all plates counted, V the volume plated in mL, n₁ and n₂ the numbers of plates at the first and second dilutions, and d the first dilution counted. For example, if duplicate plates of 1 mL at the 10⁻² dilution have 168 and 215 colonies and those at 10⁻³ have 14 and 25 colonies, N = (168 + 215 + 14 + 25)/[1 × (2 + 0.2) × 10⁻²] = 422/0.022 = 19,182, reported as 1.9 × 10⁴ cfu/mL. Some national methods use simpler averages; the method used must be stated.
Cultures must be maintained without contamination, loss of viability or genetic change. Short-term methods are repeated subculture and storage at refrigeration temperature, but repeated transfer selects variants and risks contamination. Long-term methods are freezing at −70 to −80 °C in a cryoprotectant such as 15 to 20% glycerol, or in liquid nitrogen, and freeze-drying (lyophilisation), in which cells are frozen and the ice removed by sublimation under vacuum; freeze-dried cultures in sealed ampoules remain viable for many years. Starter cultures are supplied as frozen or freeze-dried concentrates in the same way. Authenticated reference strains come from national and international culture collections.
Master the laboratory basics before the theory: a clean Gram stain, a streak plate with isolated colonies, a serial dilution and pour plate whose counts agree between duplicates, and correct use of the autoclave. Then learn the patterns examiners ask for: wall structure of Gram-positive and Gram-negative cells, the growth curve and generation-time formula, temperature groups with dairy examples, fermentation types and the cheese defects they cause, and the three routes of gene transfer. Keep a notebook of the media and incubation conditions of every method you use.
A dairy microbiologist five years into the job applies these principles daily: choosing media and incubation conditions for raw milk, product and environmental testing; interpreting why a count is high (poor cooling, post-pasteurisation contamination, thermodurics from equipment); investigating slow starter activity (phage, antibiotics, plasmid loss); validating sanitisers and autoclave cycles with biological indicators; and running a laboratory to biosafety and accreditation requirements. Typical decisions are whether a result reflects the product or the laboratory, and whether a deviation needs a hold or recall.
A quality director or laboratory head with 25 years' experience sets the microbiological strategy of a business: which organisms to monitor, at what points, with which methods, and how results feed decisions on raw milk payment, product release and shelf life. Phage-control policy, culture supply and rotation, biosafety design of laboratories, and the choice of sanitisers for cleaning-in-place are senior decisions. Understanding the general microbiology behind each is what allows them to be defended to auditors, customers and regulators, and changed when the evidence changes.
A professor or research leader builds on general microbiology to study the physiology, genetics and ecology of dairy organisms: phage–host interactions and resistance systems, stress responses of starter and probiotic strains, the behaviour of pathogens in cheese, and the taxonomy of new isolates from traditional products. Teaching the fundamentals with dairy examples, revising curricula as taxonomy changes, and setting examination papers that test understanding rather than memory are among the most lasting contributions of a senior scientist, alongside curating strain collections for future work.
Archaea, algae and protozoa are only mentioned. Viral replication strategies other than those of bacteriophages, immunology, and the detailed biochemistry of microbial pathways are not covered. National variants of standard methods (incubation times, media and calculation rules) differ and are named only where an international standard exists. Detailed coverage of milk-borne pathogens, starter cultures, rapid methods and molecular microbiology is left to the dairy microbiology subjects.
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.
These notes are free to read here, with no account.
What the Laisha app adds:
Free for ten days, then a subscription. We would rather write that here than have you find it later.