Robert Koch: Microbe Hunter Conceptual Advances to Come 1. Microbes make people sick 1. Corollary: sterilization can prevent sickness! 2. Specific microbes cause specific diseases 1. Corollary: vaccination can prevent sickness Koch's Work 1. "Control Experiments" 1. Why perform controls? They help to show that your interpretation of the experiment is correct. 2. Controls can be positive or negative 3. If an experiment is designed to show X, causes Y, then: 1. Negative Control Experiment: a similar experiment in which X is selectively removed, and thus Y does not occur. 2. Positive Control Experiment: a similar experiment in which X is still present, and so Y occurs, even though other things might have changed 2. Koch's experiments on anthrax aimed to prove that Bacillus anthracis causes anthrax 1. Experiment: innoculate mice with sliver of wood soaked in blood of animal that died of anthrax. Mice die of anthrax. 2. Negative control: innoculate mice with sliver of wood soaked in blood of healthy animal. Mice live. 3. Koch was able to culture "pure" anthrax bacilli 1. Grown in aqueous humor of ox eye 2. Passaged the bacilli several generations 3. Still caused disease 4. Results published in 1876 --> Koch became famous 4. Also observed anthrax can form spores 1. Spores are hardy and not metabolically active, can resist disinfecting treatments that kill vegetative form 5. Also grew pure cultures on potato slices and Petri dishes 3. The ability to isolate single colonies is a critical technique of microbiologye 1. Does not confuse experiments 4. Staining bacteria 1. Critical to visualize them in tissue 2. TB can be visualized with the acid-fast (Ziehl-Neelsen) stain 1. TB is often V-shaped or crooked in morphology 3. Another staining method: Gram staining 1. Invented by Gram (not Koch!) in 1884 2. Procedure: 1. fix bacteria to glass slide by heating 2. stain with crystal violet (deep purple color) 3. add iodine (promotes adherence of stain) 4. rinse slide with water and ethanol (decolorization) 5. counterstain with safranin or fuchsin (red pink) 3. Gram positive bacteria have different cell wall structures that causes them to retain the crystal violet stain better than gram negative bacteria 1. Gram positive: dark purple/blue 2. Gram negative: pink 4. Gram positive bacteria: Streptococus pyogenes (strep throat), Staphylococcus (MRSA, Toxic Shock Syndrome), Bacillus anthracis (anthrax) 5. Gram negative: Yersinia pestis (bubonic plague), Vibrio cholerae (cholera), Legionella pneumophila (Legionnaires' Disease) 6. Gram variable: Mycobacterium tuberculosis (TB) (ACID FAST) 5. Koch's identification of the bacilli that causes Tuberculosis 1. Isolate bacilli from affected patients only 2. Passaged on blood-serum jelly 3. Reinfected animals develop TB (and aerosolized bacillus capable of causing disease) 4. Now, MTB is handled only under Biosafety Level 3 (out of 4) 6. Koch also deveoloped Tuberculin 1. Tuberculin: prepared from cultures of M. tuberculosis 1. Koch claimed it could cure TB, turned out not to be true 2. But a modified tuberculin (PPD = purified protein derivative) did have diagnostic value: injected into skin, body will make an immune response (delayed-type hypersensitivity reaction) if previously exposed to TB 1. > 10 mm within 48-72h means probably infected with TB (or previously vaccinated) 7. Koch's work on cholera (1883) 1. Cholera is caused by a "comma-shaped" bacterium, Vibrio cholerae 1. Found in Egypt and India in patients and in water, but couldn't transfer disease to new hosts 2. Other theories to explain cholera 1. Electric theory: caused by atmospheric electricity 2. Ozonic theory: caused by shortage of ozone 3. Telluric theory: disease is an emanation from the Earth (advocated by Max von Pettenkofer): did not dispute bacteria were there too, but not primary 1. Pettenkofer drank water contaminated with Vibrio cholerae and did not get cholera - why not? 1. Prior immunity? 2. Vibrios weren't grown in a manner that would make them infective 3. Genetic variation in susceptibility? 4. Stomach acidity not neutralized? 8. Koch's Postulates 1. The microorganism must be found in all organisms suffering from the disease (but not in healthy organisms.) 2. The microorganism must be isolated from a diseased organism and grown in pure culture. 3. The cultured microorganism should cause disease when introduced into a healthy organism. 4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. 9. Asymptomatic Carriers 1. Typhoid Mary: Mary Mallon, asymptomatic carrier of Salmonella typhimurium (the cause of typhoid) 1. Worked as a cook in New York - infected ~ 40 people 2. Forcibly quarantined on an island in New York
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