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Cholera kills fast. The World Health Organization's cholera fact sheet says it can be fatal within hours if untreated, and researchers estimate 1.3 to 4.0 million cases and 21,000 to 143,000 deaths worldwide each year. Robert Koch identified the bacterium in 1884, but for decades nobody could show how it caused the disease. The person who did was a pathologist working mostly after hours in Kolkata. This post covers what Sambhu Nath De found, how he designed the experiments, and what a student can learn from his way of working.
Who was Sambhu Nath De?
According to a biography by his colleagues A. Sen and J. K. Sarkar in Resonance (2012), reprinted from a 1990 special issue of Current Science, De was born in 1915 in Garibati, a village about 30 km north of Calcutta. His family had lost its money, and he studied on scholarships, with a local well-wisher giving him free board in the city. He passed his MB in 1939, joined Calcutta Medical College as a demonstrator of pathology in 1942, and went to University College Hospital Medical School in London in 1947 for a PhD under the pathologist G. R. Cameron.
His London start went badly. His experiments on rats kept failing, and he became so low that he planned to go home. Cameron saw that the "failure", fluid in the rats' lungs, was itself worth studying, and steered him onto it. Cameron later wrote to De's father-in-law that De "passed through a bad period of depression where nothing seemed to go right… he has regained confidence. He is doing excellent work." De finished his PhD in 1949 and returned to Calcutta.
Why had nobody explained cholera before?
In a 1980 lecture quoted by Sen and Sarkar, De pointed out that the toxins of diphtheria, tetanus and botulism were found within a few years of their bacteria, while cholera's took 75 years. His explanation: Koch's authority had decreed that the cholera poison acted on the whole body. So researchers injected cholera preparations into the blood or tissues of many animals, and the effects had nothing to do with the disease. Koch concluded that a true cholera process could not be reproduced in animals at all.
De's idea was simple. Cholera is a disease of the intestine, so test the bacteria where they act.
How did the rabbit loop experiment work?
His first attempts failed in an instructive way. When he put cholera bacteria into the small intestine of rabbits, the animals had no diarrhoea but died within days, and at autopsy the caecum was full of fluid. He reasoned that the fluid was being produced but was trapped in the caecum. So he bypassed it: he tied off a four-inch segment of small intestine with two silk ligatures, put in a loopful of bacteria in one cubic centimetre of medium, and examined the loop the next day. The loop held about 15 cubic centimetres of watery "rice-water" fluid, while a control loop given sterile medium stayed collapsed.
He published this in The Journal of Pathology and Bacteriology in 1953 with D. N. Chatterje. Sen and Sarkar note that De later found an almost forgotten 1915 paper that had used a tied intestinal loop, and he credited it in a footnote of his 1961 book.
What did the 1959 Nature paper show?
The next question was what in the bacteria caused the fluid. Working at the Bose Institute in the evenings and on Sundays, De first broke up washed bacteria and tested the extract. It did nothing. He then grew the bacteria in different liquid media, filtered out every cell, and tried the bacteria-free filtrate. In mid-1958 one medium worked: the sterile filtrate alone made the loop fill with fluid.
He was surprised, because he had expected the cause to be a toxin bound inside the bacterial cell. Sen and Sarkar write that he spent months repeating and checking the result before sending it to Nature in December 1958. The paper, Enterotoxicity of bacteria-free culture-filtrate of Vibrio cholerae (1959), describes exactly how the filtrate was made, from the strains used down to the composition and pH of the peptone medium. That detail is what lets other laboratories repeat a result.
The finding mattered because it located the disease. Cholera does its damage through a substance the bacteria release in the gut, which pulls fluid into the intestine. This is why replacing lost fluid and salts saves lives: WHO says most people with cholera can be treated with oral rehydration solution, and severe cases also need intravenous fluids and antibiotics.
Why was he so little recognised?
De could not finish purifying the toxin, his colleagues record. Around 1963 to 1964 a different strain, the El Tor biotype, replaced the classical strain in Calcutta, and he found no filtrable toxin in it. His old strains lost the ability to make toxin, and he had no equipment to freeze-dry them. The toxin was later purified by researchers abroad. He received no academy fellowship, only the Coates Medal of Calcutta University in 1956. Recognition came late: the Nobel Foundation invited him to its 43rd Nobel Symposium, on cholera and related diarrhoeas, in 1978. He died on 15 April 1985.
What can a student take from De's way of working?
Test the idea where the problem actually is. Others injected cholera into blood because an authority said so. De put it in the gut.
Read a failed experiment as data. The fluid-filled caecum told him what to change, just as Cameron had used his failed rat experiments.
Always have a control. The collapsed control loop is what made the swollen one convincing.
Check before you announce. He spent months repeating his result before publishing it.
Credit others. He pointed readers to the 1915 loop paper himself.
If you are studying how microbes cause disease for biology, our guide to microbes in human welfare and the story of John Snow's cholera map show the same disease from two other angles. For the underlying ideas, such as how water moves across a cell membrane, the Fermi Concept Library covers every concept in biology with its own lesson.
Next time an experiment in your lab or a problem in your notebook "fails", write one line on what the failure tells you before you start again.
