Spontaneous generation and the proof of biogenesis

The decisive difference was not the boiling; it was what the flask allowed in. Spallanzani melted the necks of his flasks shut, and that single act handed his critics a confounder they used for sixty years: if no air could enter, perhaps the vegetative force or vital principle that supposedly generated life had also been excluded, so the absence of microbes proved nothing about generation. Needham’s corks, and the loose plugs used before Pasteur’s cotton-wool plugs, by contrast, let air in but also let dust in, so his cloudy infusions could not distinguish between a spontaneous origin and aerial contamination. Pasteur removed both objections with one piece of glass.

His flask was open, so ordinary untreated, unfiltered atmospheric air moved freely over the broth, but the neck was drawn into a long S-shaped curve, so particles carried by that air were trapped in the lower bend by gravity and inertia while the gas itself passed round the corner. The broth stayed limpid indefinitely. The apparatus then carried its own positive control: when the flask was gently tilted so that the sterile broth ran into the bend and touched the trapped dust, the liquid became turbid within about twenty-four to forty-eight hours, and subculture showed the same organisms that had been lodging in the glass. When the neck was snapped off, or when a flask was opened in a dusty room, spoilage followed the same way.

Pasteur reinforced the argument with sealed flasks opened at different altitudes, including above the Mer de Glace in the Alps, where thinner, cleaner air produced far fewer contaminated cultures than the dusty air of a city, and with the observation that a cotton-wool plug that filtered air protected a culture while an open tube did not. Each variant reproduced the same relationship: airborne particulate matter, not air itself, is the vehicle. That is why the curved-neck flask ended the controversy where simple sealing could not, and why the same logic now underpins the autoclave, the sterile pack and the laminar-flow bench.

Pasteurisation is a targeted heat treatment, not a method of sterilisation, and the distinction deserves stating precisely because the two words are casually confused. The process raises the temperature of a liquid to a defined level for a defined time and then cools it rapidly: for milk, the holder method holds 63 °C for thirty minutes, while the high-temperature short-time method holds 72 °C for fifteen seconds, and both are followed by immediate chilling to below 4 °C. Pasteur’s own wine experiment used roughly 50 to 60 °C for a few minutes, which is where the technique began. What such heating reliably destroys is the vegetative form of most pathogens that milk can carry — Coxiella burnetii, the organism of Q fever and the classical biological marker of the process, together with Brucella species, Mycobacterium bovis, Salmonella, Listeria monocytogenes, Campylobacter and Shiga-toxin-producing Escherichia coli.

It therefore prevents brucellosis, bovine tuberculosis, several enteric fevers and a great deal of food-borne gastroenteritis. What it leaves behind is equally important: heat-resistant bacterial endospores such as those of Bacillus cereus and Clostridium species, thermoduric and thermophilic organisms, and harmless spoilage bacteria. Because living organisms survive, pasteurised milk is not sterile and still spoils; it merely keeps longer under refrigeration and must never be left at room temperature. Verification is biochemical rather than visual: the alkaline phosphatase test confirms that the required temperature was reached, because that enzyme is inactivated at about the same point as the target pathogens.

Ultra-heat treatment, 135 to 150 °C for one to four seconds, achieves commercial sterility in a hermetically sealed package and needs no refrigeration until the pack is opened. For nursing practice the consequences are concrete: patients who are pregnant, infants, older adults and people who are immunosuppressed should never receive raw milk or raw-milk cheeses; powdered infant formula is not sterile and must be reconstituted with water above 70 °C; expressed breast milk for a milk bank is Holder-pasteurised at 62.5 °C for thirty minutes; and boiling destroys immunoglobulin A and other protective proteins, which is a loss rather than a gain.

The controversy survived for two hundred years partly because nobody could see a bacterial endospore, and a spore is the reason a boiled flask could still spoil. When Bacillus and Clostridium species meet starvation or heat, they build a dehydrated core wrapped in a tough cortex and coat, and that structure resists boiling water for many minutes; ordinary vegetative cells die at 100 °C within seconds to minutes, but the spore does not. Tyndall’s chamber showed that filtered, dust-free air left a sterilised broth unchanged, and his intermittent method exploited the difference deliberately: the medium is steamed at 100 °C on three successive days, with cooling intervals at room temperature between them.

The first heating kills vegetative cells; during the interval surviving spores germinate into heat-sensitive vegetative forms; the next heating kills those, and the third disposes of the late germinators. This fractional sterilisation works only if the medium can support germination and if the intervals are respected, which is why it has largely been replaced by steam under pressure at 121 °C for fifteen to twenty minutes, a single exposure that destroys spores directly. The design lesson is larger than the history. An experiment is only as strong as its controls: the untreated vessel shows that the material could have supported growth, the treated vessel shows what the treatment did, and every remaining difference between them must be a variable the investigator intended.

Redi needed an open jar beside his covered one; Spallanzani’s sealed neck created a new difference his opponents could attack; Pasteur’s tilt supplied the positive control that closed the argument. Nurses apply the same discipline whenever they ask whether a dressing, a solution or a piece of equipment is genuinely sterile, and whenever they recognise that boiling a bowl on the ward disinfects it but cannot make it sterile. The germ theory that grew out of these flasks is the direct ancestor of aseptic technique, of the autoclave, and of the hand hygiene that interrupts transmission today.