Editing Robert Koch (section)

==Scientific contributions==
===Techniques in bacteria study===
Robert Koch made two important developments in microscopy; he was the first to use an [[oil immersion lens]] and a [[Condenser (optics)|condenser]] that enabled smaller objects to be seen.<ref name=":0" /> In addition, he was also the first to effectively use photography ([[microphotograph]]y) for microscopic observation. He introduced the "bedrock methods" of bacterial staining using [[methylene blue]] and [[Bismarck brown Y|Bismarck (Vesuvin) brown dye]].<ref name=":16" /> In an attempt to grow bacteria, Koch began to use solid nutrients such as [[potato]] slices.<ref name="Brock Biology of Microorganisms"/> Through these initial experiments, Koch observed individual colonies of identical, pure cells.<ref name="Brock Biology of Microorganisms"/> He found that potato slices were not suitable media for all organisms, and later began to use nutrient solutions with [[gelatin]].<ref name="Brock Biology of Microorganisms"/> However, he soon realized that gelatin, like potato slices, was not the optimal medium for bacterial growth, as it did not remain solid at 37&nbsp;°C, the ideal temperature for growth of most human pathogens.<ref name="Brock Biology of Microorganisms"/> Furthermore, many bacteria can hydrolyze gelatin, making it a liquid. As suggested to him by his post-doctoral assistant [[Walther Hesse]], who got the idea from his wife [[Fanny Hesse]], in 1881, Koch started using [[agar]] to grow and isolate pure cultures.<ref name=":7">{{Cite journal|last=Hufford|first=David C.|date=1988-03-01|title=A Minor Modification by R. J. Petri|url=https://academic.oup.com/labmed/article-lookup/doi/10.1093/labmed/19.3.169|journal=Laboratory Medicine|language=en|volume=19|issue=3|pages=169–170|doi=10.1093/labmed/19.3.169|issn=0007-5027}}</ref> Agar is a [[polysaccharide]] that remains solid at 37&nbsp;°C, is not degraded by most bacteria, and results in a stable transparent medium.<ref name="Brock Biology of Microorganisms"/><ref name=":5">{{cite book|author=Koch|first=Robert|title=Robert Koch |chapter=Die Ätiologie der Tuberkulose (1882) |series=Klassische Texte der Wissenschaft |date=24 March 1882|trans-title=The Etiology of Tuberculosis|chapter-url=http://edoc.rki.de/docviews/abstract.php?id=610|journal=Physiologische Gesellschaft zu Berlin/Berliner Klinische Wochenschrift|volume=19|pages=221–30|publisher=Springer |location=Berlin, Heidelberg |doi=10.1007/978-3-662-56454-7_4|isbn=978-3-662-56454-7|quote=From page 225: ''"{{lang|de|Die Tuberkelbacillen lassen sich auch noch auf anderen Nährsubstraten kultivieren, wenn letztere ähnliche Eigenschaften wie das erstarrte Blutserum besitzen. So wachsen sie beispielsweise auf einer mit Agar-Agar bereiteten, bei Blutwärme hart bleibenden Gallerte, welche einen Zusatz von Fleischinfus und Pepton erhalten hat.}}"'' (The tubercule bacilli can also be cultivated on other media, if the latter have properties similar to those of congealed [[blood serum]].  Thus they grow, for example, on a gelatinous mass prepared with [[agar-agar]], which remains solid at blood temperature, and which has received a supplement of meat [[broth]] and [[peptone]].)}}</ref>

==== Development of Petri dish ====
Koch's booklet published in 1881 titled "''Zur Untersuchung von Pathogenen Organismen''" (''Methods for the Study of Pathogenic Organisms'')<ref>{{Cite book|last=Koch|first=Robert|url=https://edoc.rki.de/handle/176904/5146|title=Zur Untersuchung von Pathogenen Organismen|publisher=Robert Koch-Institut|year=2010|location=Berlin|doi=10.25646/5071|orig-year=1881}}</ref> has been known as the "Bible of Bacteriology."<ref>{{Citation|last1=Booss|first1=John|date=2014|url=https://linkinghub.elsevier.com/retrieve/pii/B9780444534880000018|series=Handbook of Clinical Neurology|volume=123|pages=3–44|publisher=Elsevier|language=en|doi=10.1016/b978-0-444-53488-0.00001-8|isbn=978-0-444-53488-0|access-date=2021-04-15|last2=Tselis|first2=Alex C.|title=Neurovirology |chapter=A history of viral infections of the central nervous system |pmid=25015479}}</ref><ref>{{Cite journal|last=Hurt|first=Leslie|date=2003|title=Dr. Robert Koch:: a founding father of biology|url=https://linkinghub.elsevier.com/retrieve/pii/S1068607X02001671|journal=Primary Care Update for OB/GYNS|language=en|volume=10|issue=2|pages=73–74|doi=10.1016/S1068-607X(02)00167-1}}</ref> In it he described a novel method of using glass slide with agar to grow bacteria. The method involved pouring a liquid agar onto the glass slide and then spreading a thin layer of gelatin over it. The gelatin made the culture medium solidify, in which bacterial samples could be spread uniformly. The whole bacterial culture was then put on a glass plate together with a small wet paper. Koch named this container as ''feuchte Kammer'' (moist chamber). The typical chamber was a circular glass dish 20 cm in diameter and 5 cm in height and had a lid to prevent contamination. The glass plate and the transparent culture media made observation of the bacterial growth easy.<ref name=":8">{{Cite journal|last=Shama|first=Gilbert|date=2019|title=The "Petri" Dish: A Case of Simultaneous Invention in Bacteriology|url=https://pubmed.ncbi.nlm.nih.gov/31030894|journal=Endeavour|volume=43|issue=1–2|pages=11–16|doi=10.1016/j.endeavour.2019.04.001|pmid=31030894|s2cid=139105012}}</ref>

Koch publicly demonstrated his plating method at the Seventh [[International Medical Congress]] in London in August 1881. There, [[Louis Pasteur]] exclaimed, ''"C'est un grand progrès, Monsieur''!" ("What a great progress, Sir!")<ref name=":2" /> It was using Koch's microscopy and agar-plate culture method that his students discovered new bacteria. [[Friedrich Loeffler]] discovered the bacteria of [[glanders]] (''[[Burkholderia mallei]]'') in 1882 and [[diphtheria]] (''[[Corynebacterium diphtheriae]]'') in 1884; and [[Georg Theodor August Gaffky]], the bacterium of [[typhoid]] (''[[Salmonella enterica]]'') in 1884.<ref name=":6" /> Koch's assistant [[Julius Richard Petri]] developed an improved method and published it in 1887 as "''Eine kleine Modification des Koch’schen Plattenverfahrens''" (A minor modification of the plating technique of Koch).<ref>{{Cite journal|last=Petri|first=Julius Richard|date=1887|title=Eine kleine Modification des Koch'schen Plattenverfahrens|url=https://archive.org/details/1887-petri-eine-kleine-modification-des-koch-schen-plattenverfahrens-2020-braus-|journal=Centralblatt für Bacteriologie und Parasitenkunde|volume=1|pages=279–280}}</ref> The culture plate was given an eponymous name [[Petri dish]].<ref>{{Cite journal|last=Mahajan|first=Monika|date=2021|title=Etymologia: Petri Dish|journal=Emerging Infectious Diseases|volume=27|issue=1|pages=261|doi=10.3201/eid2701.ET2701|pmc=7774570}}</ref> It is often asserted that Petri developed a new culture plate,<ref name=":0" /><ref>{{Cite journal|last=Zhang|first=Shuguang|date=2004|title=Beyond the Petri dish|url=https://pubmed.ncbi.nlm.nih.gov/14755282|journal=Nature Biotechnology|volume=22|issue=2|pages=151–152|doi=10.1038/nbt0204-151|pmid=14755282|s2cid=36391864}}</ref><ref>{{Cite journal|last1=Grzybowski|first1=Andrzej|last2=Pietrzak|first2=Krzysztof|date=2014|title=Robert Koch (1843-1910) and dermatology on his 171st birthday|url=https://pubmed.ncbi.nlm.nih.gov/24887990|journal=Clinics in Dermatology|volume=32|issue=3|pages=448–450|doi=10.1016/j.clindermatol.2013.10.005|pmid=24887990}}</ref> but this was not so. He simply discarded the use of glass plate and instead used the circular glass dish directly, not just as a moist chamber, but as the main culture container. This further reduced the chances of contaminations.<ref name=":7" /> It would also have been appropriate if the name "Koch dish" had been given.<ref name=":8" />

===Tuberculosis===
[[File:Aetiologie der Tuberkulose.jpg|thumb|Koch's drawing of tuberculosis bacilli in 1882 (from ''Die Ätiologie der Tuberkulose'')]]
During his time as the government advisor with the Imperial Health Agency in Berlin in the 1880s, Koch became interested in [[tuberculosis]] research. At the time, it was widely believed that tuberculosis was an inherited disease. However, Koch was convinced that the disease was caused by a bacterium and was infectious. In 1882, he published his findings on tuberculosis, in which he reported the causative agent of the disease to be the slow-growing ''[[Mycobacterium tuberculosis]]''.<ref name="Brock Biology of Microorganisms" /> He published the discovery as "''Die Ätiologie der Tuberkulose''" (''The Etiology of Tuberculosis''),<ref name=":5" /> and presented before the German Physiological Society at Berlin on 24 March 1882. Koch said,<blockquote>When the cover-glasses were exposed to this staining fluid [methylene blue mixed with [[potassium hydroxide]]] for 24 hours, very fine rod-like forms became apparent in the tubercular mass for the first time, having, as further observations showed, the power of multiplication and of spore formation and hence belonging to the same group of organisms as the anthrax bacillus... Microscopic examination then showed that only the previously blue-stained cell nuclei and detritus became brown, while the tubercle bacilli remained a beautiful blue.<ref name=":2" /><ref name=":3" /></blockquote>

There was no particular reaction to this announcement. Eminent scientists such as [[Rudolf Virchow]] remained sceptical. Virchow clung to his theory that all diseases are due to faulty cellular activities.<ref name=":12">{{Cite journal|last1=Kaufmann|first1=Stefan H. E.|last2=Schaible|first2=Ulrich E.|date=2005|title=100th anniversary of Robert Koch's Nobel Prize for the discovery of the tubercle bacillus|url=https://pubmed.ncbi.nlm.nih.gov/16112578|journal=Trends in Microbiology|volume=13|issue=10|pages=469–475|doi=10.1016/j.tim.2005.08.003|pmid=16112578}}</ref> On the other hand, [[Paul Ehrlich]] later recollected that this moment was his "single greatest scientific experience."<ref name=":9" /> Koch expanded the report and published it under the same title as a booklet in 1884, in which he concluded that the discovery of tuberculosis bacterium fulfilled the three principles, eventually known as Koch's [[postulates]], which were formulated by his assistant Friedrich Loeffler in 1883, saying:<blockquote>All these factors together allow me to conclude that the bacilli present in the tuberculous lesions do not only accompany tuberculosis, but rather cause it. These bacilli are the true agents of tuberculosis.<ref name=":12" /></blockquote>

===Cholera===
[[File:Robert Koch (Deutsche Cholera-Expedition in Ägypten 1884).jpg|thumb|left|Photograph of Koch (third from the right) and other members of the German Cholera Commission in Egypt, 1884]]
[[File:Professors Koch and Pfeiffer working in a laboratory, invest Wellcome L0030175.jpg|thumb|Koch (on the microscope) and his colleague [[Richard Friedrich Johannes Pfeiffer]] (standing) investigating cholera outbreak in Bombay, India ]]
In August 1883, the German government sent a medical team led by Koch to [[Alexandria, Egypt]], to investigate a cholera epidemic there.<ref>{{Cite journal|last=Howard-Jones|first=N.|date=1984|title=Robert Koch and the cholera vibrio: a centenary|journal=British Medical Journal|volume=288|issue=6414|pages=379–381|doi=10.1136/bmj.288.6414.379|pmc=1444283|pmid=6419937}}</ref> Koch soon found that the [[intestinal mucosa]] of people who died of cholera always had bacterial infection, yet could not confirm whether the bacteria were the causative pathogens. As the outbreak in Egypt declined, he was transferred to Calcutta (now [[Kolkata]]) India, where there was a more severe outbreak. He soon found that the river [[Ganges]] was the source of cholera. He performed autopsies of almost 100 bodies, and found in each bacterial infection. He identified the same bacteria from water tanks, linking the source of the infection.<ref name=":0" /> He isolated the bacterium in pure culture on 7 January 1884. He subsequently confirmed that the bacterium was a new species, and described as "a little bent, like a comma."<ref name=":32">{{Cite journal|last1=Lippi|first1=D.|last2=Gotuzzo|first2=E.|date=2014|title=The greatest steps towards the discovery of Vibrio cholerae|journal=Clinical Microbiology and Infection|volume=20|issue=3|pages=191–195|doi=10.1111/1469-0691.12390|pmid=24191858|doi-access=free}}</ref> His experiment using fresh blood samples indicated that the bacterium could kill red blood cells, and he hypothesized that some sort of poison was used by the bacterium to cause the disease.<ref name=":0" /> In 1959, Indian scientist [[Sambhu Nath De]] discovered this poison, the [[cholera toxin]].<ref>{{Cite journal|last1=Nair|first1=G. Balakrish|last2=Takeda|first2=Yoshifumi|date=2011|title=Dr Sambhu Nath De: unsung hero|journal=The Indian Journal of Medical Research|volume=133|issue=2 |pages=127|pmc=3089041|pmid=21415484}}</ref> Koch reported his discovery to the German Secretary of State for the Interior on 2 February, and published it in the ''Deutsche Medizinische Wochenschrift'' (''German Medical Weekly'') the following month.<ref>Koch, R. (20 March 1884) {{lang|de|2=[https://books.google.com/books?id=yY41AQAAMAAJ&pg=PA191 "Sechster Bericht der deutschen wissenschaftlichen Commission zur Erforschung der Cholera"]}} (Sixth report of the German scientific commission for research on cholera), ''{{lang|de|Deutsche medizinische Wochenscrift}}'' (German Medical Weekly), '''10''' (12):  191–192. On page 191, he mentions the characteristic comma shape of ''Vibrio cholerae'':  ''"{{lang|de|Im letzten Berichte konnte ich bereits gehorsamst mittheilen, dass an den Bacillen des Choleradarms besondere Eigenschaften aufgefunden wurden, durch welche sie mit aller Sicherheit von anderen Bakterien zu unterscheiden sind. Von diesen Merkmalen sind folgende die am meisten charakteristischen: Die Bacillen sind nicht ganz geradlinig, wie die übrigen Bacillen, sondern ein wenig gekrümmt, einem Komma ähnlich.}}"'' (In the last report, I could already respectfully report that unusual characteristics were discovered in the bacteria of enteric cholera, by which they are to be distinguished with complete certainty from other bacteria. Of these features, the following are the most characteristic: the bacteria are not quite straight, like the rest of the bacilli, but a little bent, similar to a comma.)</ref>

Although Koch was convinced that the bacterium was the cholera pathogen, he could not entirely establish critical evidence the bacterium produced the symptoms in healthy subjects (following [[Koch's postulates]]). His experiment on animals using his pure bacteria culture did not cause the disease, and correctly explained that animals are immune to human pathogen. The bacterium was then known as "the comma bacillus", and scientifically as ''Bacillus comma''.<ref>{{Cite journal|last1=Winslow|first1=C. E.|last2=Broadhurst|first2=J.|last3=Buchanan|first3=R. E.|last4=Krumwiede|first4=C.|last5=Rogers|first5=L. A.|last6=Smith|first6=G. H.|date=1920|title=The Families and Genera of the Bacteria: Final Report of the Committee of the Society of American Bacteriologists on Characterization and Classification of Bacterial Types|journal=Journal of Bacteriology|volume=5|issue=3|pages=191–229|doi=10.1128/JB.5.3.191-229.1920|pmc=378870|pmid=16558872}}</ref> It was later realised that the bacterium was already described by an Italian physician [[Filippo Pacini]] in 1854,<ref>See:
* Fillipo Pacini (1854) {{lang|it|2=[https://books.google.com/books?id=xdtQAAAAcAAJ&pg=PA397 "Osservazioni microscopiche e deduzioni patologiche sul cholera asiatico"]}} (Microscopic observations and pathological deductions on Asiatic cholera), ''{{lang|it|Gazzetta Medica Italiana:  Toscana}}'', 2nd series, '''4'''(50):397–401; '''4'''(51):405–12.
* Reprinted (more legibly) as a [https://books.google.com/books?id=F9s_AAAAcAAJ&pg=PA1 pamphlet.]</ref> and was also observed by the [[Catalonia|Catalan]] physician Joaquim Balcells i Pascual around the same time.<ref name="Real Academia de la Historia 2018">{{cite web|year=2018|editor=Real Academia de la Historia|editor-link=Real Academia de la Historia|title=Joaquín Balcells y Pasqual|url=http://dbe.rah.es/biografias/18541/joaquin-balcells-y-pasqual|archive-url=https://web.archive.org/web/20190708211444/http://dbe.rah.es/biografias/18541/joaquin-balcells-y-pasqual|archive-date=8 July 2019|access-date=1 August 2020|language=es}}</ref><ref>{{cite web|year=2015|editor=Col·legi Oficial de Metges de Barcelona|editor-link=:ca:Col·legi Oficial de Metges de Barcelona|title=Joaquim Balcells i Pascual|url=http://www.galeriametges.cat/galeria-fitxa.php?icod=EGMM|archive-url=https://web.archive.org/web/20200801110910/http://www.galeriametges.cat/galeria-fitxa.php?icod=EGMM|archive-date=1 August 2020|access-date=1 August 2020|language=ca}}</ref> But they failed to identify the bacterium as the causative agent of cholera. Koch's colleague [[Richard Friedrich Johannes Pfeiffer]] correctly identified the comma bacillus as Pacini's ''vibrioni'' and renamed it as ''[[Vibrio cholerae|Vibrio cholera]]'' in 1896.<ref name=":42">{{Cite book|last=Hugh|first=Rudolph|url=https://books.google.com/books?id=NP7AY38J1hcC|title=Public Health Service Publication|date=1965|publisher=U.S. Department of Health, Education, and Welfare, Public Health Service, Environmental Health Service, National Air Pollution Control Administration|pages=1–4|language=en|chapter=Nomenclature and taxonomy of Vibrio cholerae Pacini 1854 and Vibrio eltor Pribam 1933}}</ref>

=== Tuberculosis treatment and tuberculin ===
Koch gave much of his research attention to tuberculosis throughout his career. After medical expeditions to various parts of the world, he again focussed on tuberculosis from the mid-1880s. By that time the Imperial Health Office was carrying out a project for disinfection of [[sputum]] of tuberculosis patients. Koch experimented with [[arsenic]] and [[creosote]] as possible disinfectants. These chemicals and other available drugs did not work.<ref name=":0" /> His report in 1883 also mentioned a failed experiment in an attempt to make a tuberculosis vaccine.<ref name=":11" /> By 1888, Koch turned his attention to [[synthetic dyes]] as antibacterial chemicals. He developed a method for examining antibacterial activity by mixing the gelatin-based culture media with a yellow dye, [[Auramine O|auramin]]. His notebook indicates that by February 1890, he tested hundreds of compounds.<ref name=":9" /> In one of such tests, he found that an extract from the tuberculosis bacterium culture dissolved in glycerine could cure tuberculosis in guinea pigs. Based on a series of experiments from April to July 1891, he could conclude that the extract did not kill the tuberculosis bacterium, but destroyed (by [[necrosis]]) the infected tissues, thereby depriving bacterial growth. He made a vague announcement in August 1890 at the Tenth [[International Medical Congress]] in Berlin,<ref name=":12" /> saying,<blockquote>In a communication which I made a few months ago to the International Medical Congress [in London in 1881], I described a substance of which the result is to make laboratory animals insensitive to inoculation of tubercle bacilli, and in the case of already infected animals, to bring the tuberculous process to a halt.<ref name=":2" /><ref name=":3" />

I can tell […] that much, that guinea pigs, which are highly susceptible to the disease [tuberculosis], no longer react upon inoculation with tubercle virus [bacterium] when treated with that substance and that in guinea pigs, which are sick (with tuberculosis), the pathological process can be brought to a complete standstill.<ref name=":9" /></blockquote>By November 1890, Koch demonstrated the effectiveness of the extract in treating humans by administering the vaccine through the Bacillus Calmette-Guerin (BCG) technique.<ref name=":10">{{Cite journal|last=Sakula|first=Alex|date=1985|title=Robert Koch: The story of his discoveries in tuberculosis|url=http://link.springer.com/10.1007/BF02938285|journal=Irish Journal of Medical Science|language=en|volume=154|issue=S1|pages=3–9|doi=10.1007/BF02938285|pmid=3897123|s2cid=38056335}}</ref> This absorbs the vaccine through the skin by means of multiple shallow punctures on the skin and many patients and doctors went to Berlin to get Koch's remedy.<ref name=":0" /> While this was effective in humans, his experiments also revealed that when the substance was inoculated into his tuberculosis-infected test guinea pigs, they presented with severe symptoms. This outcome, characterized by an exaggerated immune response, coined the term "Koch’s phenomenon."<ref>{{Cite journal|last=Hunter|first=Robert L.|date=2020|title=The Pathogenesis of Tuberculosis-The Koch Phenomenon Reinstated|journal=Pathogens|volume=9|issue=10|pages=e813|doi=10.3390/pathogens9100813|pmc=7601602|pmid=33020397|doi-access=free}}</ref> This is known as an extreme skin reaction that manifests itself at the BCG vaccination site within a few days after the vaccine is administered to an individual infected with tuberculosis. When a normal guinea pig was inoculated with pure ''tubercle bacillus'', the wound would close rapidly and heal within several days. Afterwards, the site of the injection would open and form an ulcer until the animal died. However, if the same inoculated culture was injected into a guinea pig that was previously infected with tuberculosis, the site of the injection becomes dark, and eventually heals normally and quickly (Moreland, 2024). The uncertainty in the chemical nature coined the term phenomenon in the name "Koch’s phenomenon."

Koch published his experiments in the 15 January 1891 issue of ''Deutsche Medizinische Wochenschrift'',<ref>{{Cite journal|last=Koch|first=Robert|date=2010|orig-year=1891|title=Fortsetzung der Mitteilungen über ein Heilmittel gegen Tuberkulose|url=https://edoc.rki.de/handle/176904/5175|journal=Deutsche Medizinische Wochenschrift|language=de|volume=17|pages=101–102|doi=10.25646/5100|via=Robert Koch-Institut}}</ref><ref>{{Cite journal|last=Koch|first=Robert|date=1891|title=A Further Communication on a Remedy for Tuberculosis|journal=The Indian Medical Gazette|volume=26|issue=3|pages=85–87|pmc=5150357|pmid=29000631}}</ref> and ''[[The British Medical Journal]]'' immediately published the English version simultaneously.<ref>{{Cite journal|last=Koch|first=R.|date=1891|title=A Further Communication on a Remedy for Tuberculosis|journal=British Medical Journal|volume=1|issue=1568|pages=125–127|doi=10.1136/bmj.1.1568.125|pmc=2196966|pmid=20753227}}</ref> The English version was also reproduced in ''[[Nature (journal)|Nature]]'',<ref>{{Cite journal|date=1891|title=Dr. Koch's Remedy for Tuberculosis|journal=Nature|language=en|volume=43|issue=1108|pages=281–282|doi=10.1038/043281a0|bibcode=1891Natur..43..281.|s2cid=4050612|doi-access=free}}</ref> and ''[[The Lancet]]'' in the same month.<ref>{{Cite journal|last=Richmond|first=W.S.|date=1891|title=Professor Koch's Remedy for Tuberculosis|url=https://linkinghub.elsevier.com/retrieve/pii/S0140673602157059|journal=The Lancet|language=en|volume=137|issue=3514|pages=56–57|doi=10.1016/S0140-6736(02)15705-9}}</ref> ''The Lancet'' presented it as "glad tidings of great joy."<ref name=":10" /> Koch simply referred to the medication as "brownish, transparent fluid."<ref name=":4" /> Josephs Pohl-Pincus had used the name tuberculin in 1844 for tuberculosis culture media,<ref>{{Cite journal|last=Caspary|date=1884|title=Angioneurotische Dermatosen|url=https://doi.org/10.1007/BF02097828|journal=Vierteljahresschrift für Dermatologie und Syphilis|language=de|volume=16|issue=1|pages=141–155|doi=10.1007/BF02097828|s2cid=33099318|quote=Pohl-Pincus wrote: Wir werden deshalb alas Tuberculin darzustellen suchen [We shall therefore endeavor to describe it as tuberculin]}}</ref> and Koch subsequently adopted as "tuberkulin."<ref>{{Cite journal|last=Koch|first=R.|date=1891|title=Weitere Mittheilung über das Tuberkulin|url=http://www.thieme-connect.de/DOI/DOI?10.1055/s-0029-1206810|journal=Deutsche Medizinische Wochenschrift|language=de|volume=17|issue=43|pages=1189–1192|doi=10.1055/s-0029-1206810|s2cid=73993276 }}</ref>

The first report on the clinical trial in 1891 was disappointing. By then 1061 patients with tuberculosis of internal organs and of 708 patients with tuberculosis of external tissues were given the treatment. An attempt to use tuberculin as a therapeutic drug is regarded as Koch's "greatest failure."<ref name=":12" /> With it his reputation greatly waned. But he devoted the rest of his life trying to make tuberculin as a usable medication.<ref name=":10" /> His discovery was not a total failure: the substance is now used to test for hypersensitivity in tuberculosis patients.<ref name=":0" />

=== Acquired immunity ===
Koch observed the phenomenon of acquired [[immunity (medical)|immunity]]. On 26 December 1900, he arrived as part of an expedition to [[German New Guinea]], which was then a protectorate of the German Reich. Koch serially examined the [[Indigenous people of New Guinea|Papuan people]], the indigenous inhabitants, and their blood samples and noticed they contained ''[[Plasmodium]]'' parasites, the cause of [[malaria]], but their bouts of malaria were mild or could not even be noticed, i.e. were [[subclinical]]. On the contrary, German settlers and Chinese workers, who had been brought to New Guinea, fell sick immediately. The longer they had stayed in the country, however, the more they too seemed to develop a resistance against it.<ref>{{lang|de|Hugo Kronecker: Hygienische Topographie}} In: A. Pfeiffer (Editor): {{lang|de|21. Jahresbericht über die Fortschritte und Leistungen auf dem Gebiete der Hygiene.}} 1903. Publisher: Friedrich Vieweg und Sohn, Braunschweig, 1905. p. 68</ref>

=== Koch's postulates ===
{{Main|Koch's postulates}}
During his time as government advisor, Koch published a report on how he discovered and experimentally showed tuberculosis bacterium as the pathogen of tuberculosis. He described the importance of pure cultures in isolating disease-causing organisms and explained the necessary steps to obtain these cultures, methods which are summarized in [[Koch's postulates|Koch's four postulates]].<ref name="Bacteriology, historical">Amsterdamska, Olga. "Bacteriology, Historical." ''International Encyclopedia of Public Health.'' 2008. Web.</ref> Koch's discovery of the causative agent of anthrax led to the formation of a generic set of [[wikt:postulate|postulates]] which can be used in the determination of the cause of most infectious diseases.<ref name="Germ theory of disease" /> These postulates, which not only outlined a method for linking cause and effect of an infectious disease but also established the significance of laboratory culture of infectious agents, became the "gold standard" in infectious diseases.<ref>{{Cite journal|last=Tabrah|first=Frank L.|date=2011|title=Koch's postulates, carnivorous cows, and tuberculosis today|journal=Hawaii Medical Journal|volume=70|issue=7|pages=144–148|pmc=3158372|pmid=21886302}}</ref>

Although Koch worked out the principles, he did not formulate the postulates, which were introduced by his assistant Friedrich Loeffler. Loeffler, reporting his discovery of diphtheria bacillus in 1883, stated three postulates as follows:<ref>{{cite journal |last1=Loeffler |first1=Friedrich |title=Untersuchungen über die Bedeutung der Mikroorganismen für die Entstehung der Diphtherie beim Menschen, bei der Taube und beim Kalbe |journal=Mittheilungen aus dem Kaiserlichen Gesundheitsamte (Reports from the Imperial Office of Public Health) |date=1884 |volume=2 |pages=421–499 |url=https://books.google.com/books?id=Y6lQAAAAYAAJ&pg=PA421 |trans-title=Investigations of the relevance of microorganisms to the development of diphtheria among humans, among doves, and among heifers |language=German}}  From 424:  ''"Wenn nun die Diphtherie eine durch Mikroorganismen bedingte Krankheit ist, so müssen sich auch bei ihr jene drei Postulate erfüllen lassen, deren Erfüllung für den stricten Beweis der parasitäten Natur einer jeden derartigen Krankheit unumgänglich nothwendig ist:''<br /> 
''1)  Es müssen constant in den local erkrankten Parteien Organismen in typischer Anordnung nachgewiesen werden.''<br />
''2)  Die Organismen, welchen nach ihrem Verhalten zu den erkrankten Theilen eine Bedeutung für das Zustandekommen dieser Veränderungen beizulegen wäre, müssen isolirt und rein gezüchtet werden.''<br />
''3)  Mit den Reinculturen muss die Krankheit experimentell wieder erzeugt werden können."''<br />
(Now if diphtheria is a disease that's caused by microorganisms, then it must also be able to fulfil those three postulates whose fulfilment is absolutely necessary for the strict proof of the parasitic nature of any such disease:<br />
1)  In the given diseased patients, there must always be shown [to be present] organisms in typical disposition.<br />
2)  The organisms to which one would attribute — according to their behaviour in the diseased parts — a relevance for the occurrence of these changes, must be isolated and cultured in pure form.<br />
3)  The disease must be able to be reproduced experimentally via pure cultures.)</ref>
:1. The organism must always be present in every case of the disease, but not in healthy individuals.
:2. The organism must be isolated from a diseased individual and grown in pure culture.
:3. The pure culture must cause the same disease when inoculated into a healthy, susceptible individual.<ref name=":6">{{Cite journal|last=Weiss|first=Robin A.|date=2005|title=Robert Koch: the grandfather of cloning?|journal=Cell|volume=123|issue=4|pages=539–542|doi=10.1016/j.cell.2005.11.001|pmid=16286000|doi-access=free}}</ref><ref>{{Cite journal|last1=Byrd|first1=Allyson L.|last2=Segre|first2=Julia A.|date=2016|title=Adapting Koch's postulates|url=https://pubmed.ncbi.nlm.nih.gov/26816362|journal=Science|volume=351|issue=6270|pages=224–226|doi=10.1126/science.aad6753|pmid=26816362|bibcode=2016Sci...351..224B|s2cid=29595548}}</ref>

The fourth postulate was added by an American plant pathologist [[Erwin Frink Smith]] in 1905, and is stated as:<ref>{{cite book |last1=Smith |first1=Erwin F. |title=Bacteria in Relation to Plant Diseases |date=1905 |publisher=Carnegie Institution of Washington |location=Washington. D.C. |volume=1 |page=9 |url=https://www.biodiversitylibrary.org/item/60542#page/31/mode/1up}}</ref>

:4. The same pathogen must be isolated from the experimentally infected individuals.<ref>{{Cite journal|last=Hadley|first=Caroline|date=2006|title=The infection connection|journal=EMBO Reports|volume=7|issue=5|pages=470–473|doi=10.1038/sj.embor.7400699|pmc=1479565|pmid=16670677}}</ref>