1. Mic 101: Lecture 3 SST Developments of laboratory techniques, vaccination and chemotherapy

2. .. Limitation of Koch’s Postulates 11. The causative organism might not be present in every stage of the disease.Eg.Vibrio cholerae might not be present at the time of extreme vomiting and diarrhoea because the bacteria release toxin which causes the diarrhoea even in the absence of an organism. 2. Many organisms or entities cannot (at the present time) be grown in pure culture, such as prions responsible for Creutzfeldt–Jakob disease.prionsCreutzfeldt–Jakob disease 3. Not all hosts exposed to an infectious agent will acquire the infection. Non- infection may be due to such factors as general health and proper immune functioning; acquired immunity from previous exposure or vaccination; or genetic immunity. It is ethically unacceptable to expose a host experimentally to prove pathogen- disease relationships. In summary, a body of evidence that satisfies Koch's postulates is sufficient but not necessary to establish causation.

3. Modification of Koch’s Postulates New methods led to revised versions of Koch’s postulates by Fredricks and Relman : A nucleic acid sequence belonging to a particular pathogen should be present in most cases of an infectious disease. Microbial nucleic acids should be found preferentially in those organs or gross anatomic sites known to be diseased, and not in those organs that lack pathology. Fewer, or no, copies of pathogen-associated nucleic acid sequences should occur in hosts or tissues without disease. With resolution of disease, the copy number of pathogen-associated nucleic acid sequences should decrease or become undetectable. With clinical relapse, the opposite should occur. When sequence detection predates disease, or sequence copy number correlates with severity of disease or pathology, the sequence-disease association is more likely to be a causal relationship. The nature of the microorganism inferred from the available sequence should be consistent with the known biological characteristics of that group of organisms. Tissue-sequence correlates should be sought at the cellular level: efforts should be made to demonstrate specific in situ hybridization of microbial sequence to areas of tissue pathology and to visible microorganisms or to areas where microorganisms are presumed to be located. These sequence-based forms of evidence for microbial causation should be reproducible.

4. Definitions Species refers to a group of organisms having same genetic content, morphology, metabolic pattern and are able o reproduce. T D Brock defined microbial species as organisms sharing 100% similarity in DNA sequence. Eg. Bacillus cereus, Escherichia coli, Pseudomonas putida.

5. Microbiological Culture A microbiological culture means the growth of a defined population of microbes under defined set of parameters. Eg. When microbiological sample collected from an environmental, clinical or pathological origin is grown in a specific medium under specific temperature, humidity, pressure for specific time, it is called a culture. Culture media is the physical and nutritional substance on which microorganism is grown. It contains carbohydrates, proteins, pH to support the growth of microorganisms. Culture media can be of 3 types: Solidified agar Liquid broth Semisolid media

6. Examples of Culture Medium

7. Culture: Mixed and Pure When a culture has more than one species present in it, it is a mixed culture. Eg. A sample from soil has all the virus, bacteria, fungi, algae from the soil. Therefore, it is a mixed culture. When a culture has cells from only one species, it is called a pure culture. Eg. If a soil sample is cultured, diluted and processed to isolate every single species, then we have pure cultures of Aspergillus fumigatus, Bacillus subtilis, Bacillus cereus etc. Microbiological isolation refers to the method of selecting cells from only one species to grow in one specific culture. Several different methods for isolating a pure culture from a mixed culture: These include: Serial Dilution to extinction Growing on specific media (SOLID/LIQUID/SEMISOLID)

8. Serial Dilution Serial dilution is stepwise dilution of a substance in solution used to reduce the concentration of microscopic organisms or cells in a sample.

9. Dilution Methods

10. Growing on Specific Media There are some media, which allow only one species to grow. These are called selective media and contain substance which only one species can tolerate and the others can not. Eg. Eosine Methyline Blue agar (EMB) excludes many bacteria from mammalian intestine and allows only E coli to grow.

11. Colony Colony means individual organisms of the same species living closely together, usually for mutual benefit, such as organisms species stronger defense or for nutritional requirements A bacterial colony is defined as a visible cluster of cells, derived from a single ancestor, they are genetically identical. A colony is often called a colony-forming unit (CFU).

12. Inoculation, Growth, Identification Inoculation is introduction of organisms into a defined location. Eg. When a sample is added to a sterile medium, it is inoculation of culture. When organism or protein components are introduced into animal body for protective immunity, it is immunologic inoculation. Growth in microbiology is increase in the number of cells e.g. if we inoculate 2 cells in a culture medium and the cells divide to increase the number of cells, then it is microbial growth. Identification in microbiology means detecting the taxonomy, genetic make-up, metabolic pattern and virulence factor of an organism. Eg. An unknown organism should be grown in culture, observed under the microscope, biochemically tested for metabolic pattern, analyzed by DNA hybridization for genetic match and the virulence factors should be checked by PCR to identify an organism.

13. Microscopic Observation

14. Vaccination A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine can have live, attenuated organism, a purified part from the organism or a modified part from the organism. In 1798 Edward Jenner had observed that milkmaids exposed to cowpox never developed the serious smallpox.

15. History of Vaccination In 1880, Pasteur used Koch’s technique to isolate and culture the bacteria that caused chicken cholera He discovered that bacteria, if allowed to grow old, could become avirulent. Avirulent bacteria could still stimulate something in the host to resist subsequent infection and immunize the host to that disease

16. Pasteur’s Approach and Modern Approach

17. Pasteur’s Preparation of Rabies Vaccine

18. Chemotherapy Use of chemicals to treat a disease. Two types-  synthetic drugs (chemically prepared in laboratory outside biological systems) egsalvarsan  antibiotics ( naturally produced by bacteria and fungi) e.g.- penicillin

19. Criteria of a good antimicrobial chemotherapeutic 1.Selective toxicity: kill the pathogen without showing dangerous side-effect in the host 2.Should not allow development of resistance in the pathogen during time of treatment 3.Should be distributed in the host tissue without difficulty and be metabolized easily

20. Anti-microbial Chemotherapeutic Agents Different action: 1. Microbiocidal can kill microbes (antibiotics) by damaging cell wall, cell membrane, protein, phospholipid and oxidation. 2. Microbiostatic stop the growth of microbes by stopping cell division. (sulfanilamide)