CONVENTIONAL MICROBIOLOGICAL TECHNIQUES
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The document discusses conventional microbiological techniques used in diagnostic microbiology laboratories. It describes how Robert Koch and Ronald Ross helped develop culturing pathogens and the discovery that specific microbes cause diseases. It also discusses how conventional techniques like growing bacteria in broth or on solid media, staining, and microscopy are still important today, but that molecular biology techniques may revolutionize disease diagnosis in the future. Gram staining remains one of the most rapid diagnostic methods for identifying bacteria in clinical specimens.
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CONVENTIONAL MICROBIOLOGICAL TECHNIQUES
- 2. My tribute to those who made life safe 20-02-2016 Dr.T.V.Rao MD 2
- 3. Robert Koch Perfected Culturing the Common Pathogens 20-02-2016 Dr.T.V.Rao MD 3
- 4. A great quotes by Ronald Ross • I have failed in finding parasites in mosquitoes fed on malaria patients, but perhaps I am not using the proper kind of mosquito • “The screws of my microscope were rusted with sweat from my forehead and hands, and its last remaining eye-piece was cracked 20-02-2016 Dr.T.V.Rao MD 4
- 5. Beginning of Diagnostic Microbiology •In the late 1800s, the realization that identifiable microbes caused specific diseases led to pathogens Specific medical diagnosis. Although the time honoured techniques of growing bacteria in broth or solid cultures and staining and examining them under microscopes are still important today20-02-2016 Dr.T.V.Rao MD 5
- 6. A GREAT QUESTION TODAY HOW KEEN WE ARE WITH THE WORK 20-02-2016 Dr.T.V.Rao MD 6
- 8. The Real Good of the Past in Diagnostic Microbiology • In the good old days, the microbiology laboratory used to be a labour intensive place equipped with incubators and microscopes. Microbiologists were patient scientists waiting at least 24 hours before their isolated cultures were grown enough for identification 20-02-2016 Dr.T.V.Rao MD 8
- 9. Where we stand Today • Most neglected and least invested specialty • Reasons can be many 20-02-2016 Dr.T.V.Rao MD 9
- 10. CONVENTIONAL METHOD MEANS •Based on or in accordance with what is generally done or believed: 20-02-2016 Dr.T.V.Rao MD 10
- 11. What is a Technique A way of carrying out a particular task, especially the execution or performance of an artistic work or a scientific procedure 20-02-2016 Dr.T.V.Rao MD 11
- 12. American Society for Microbiology encourages Artistic and Conventional Techniques on Streaking and Culturing 20-02-2016 Dr.T.V.Rao MD 12
- 13. A MATTER OF CONFLICT WHO WILL COLLECT THE SPECIMENS • Proper specimen collection, container labelling, and culture requests are the responsibility of the ordering physician. Technologists in the Clinical Microbiology Laboratory will be familiar with specimens of choice and proper collection techniques 20-02-2016 Dr.T.V.Rao MD 13
- 14. 14 SPECIMEN COLLECTION whose duty it is ? The specimen is the beginning. All diagnostic information from the laboratory depends upon the knowledge by which specimens are chosen and the care with which they are collected and transported. —Cynthia A. Needham 20-02-2016 Dr.T.V.Rao MD
- 15. If you accept the Truth many Suboptimal Specimens are Processed • I should share my experience the ideal collecting of a sample remain with greater challenges, we many times receive suboptimal sample for processing, few posses courage to reject, it just goes 20-02-2016 Dr.T.V.Rao MD 15
- 16. LABORATORY HANDLES THE SPECIMENS • The technologist in the laboratory will directly handle specimens of clinical and environmental source which are received from the Postal Service or hand carried to the laboratory. 20-02-2016 Dr.T.V.Rao MD 16
- 17. Are we Collecting the Right specimens • Select an appropriate sites or organs for sampling. • Use sterile equipment for sampling. • Labelling the sample • Keep and transfer the sample inappropriate medium and condition.20-02-2016 Dr.T.V.Rao MD 17
- 18. Microbiological techniques • Bacteria will grow on practically any source of organic food which provides carbon compounds to be respired for energy, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally provided dissolved in water. 20-02-2016 Dr.T.V.Rao MD 18
- 19. 20-02-2016 Dr.T.V.Rao MD 19
- 20. General View on the Parameters Used in the Process of Microorganism Identification • Before one can proceed to identify a microorganism, the characteristics of that organism have to be determined in details. The major characteristics which are observed 20-02-2016 Dr.T.V.Rao MD 20
- 21. Gram Staining continues to be Most rapid method to diagnose • The rapid Gram stain evaluation is reliable, easy to perform, and well suited for the routine clinical laboratory • Eg – CSF examination in emergencies • Sputum in case of acute pneumonias • Bacterial Vaginitis • Any other transudates • Pleural fluid • Pericardial fluids 20-02-2016 Dr.T.V.Rao MD 21
- 22. Molecular Biology in Infancy • Molecular biology techniques promise to revolutionize the diagnosis of infectious disease—to date a promise still in its infancy 20-02-2016 Dr.T.V.Rao MD 22
- 23. Laboratory Medicine Under threat Microbiology is No exception •Great questions to many Microbiologists How much Time we are spending in the Laboratory Who are doing the Bench work Are laboratories equipped with minimal requirements of infrastructure and Biosafety?20-02-2016 Dr.T.V.Rao MD 23
- 24. The young Microbiologists should know 20-02-2016 Dr.T.V.Rao MD 24
- 25. Use a bright field light microscope • Use a bright field light microscope to view and interpret slides, including • 1. correctly setting up and focusing the microscope • 2. proper handling, cleaning, and storage of the microscope • 3. correct use of all lenses • 4. recording microscopic observations 20-02-2016 Dr.T.V.Rao MD 25
- 26. Properly prepare slides • Properly prepare slides for microbiological examination, including • A. cleaning and disposing of slides • B. preparing smears from solid and liquid cultures • C. performing wet mount and/or hanging drop preparations • D. performing Gram stains 20-02-2016 Dr.T.V.Rao MD 26
- 27. Properly use aseptic techniques • Properly use aseptic techniques for the transfer and handling of microorganisms and instruments, including • A. sterilizing and maintaining sterility of transfer instruments • B. performing aseptic transfer • C. obtaining microbial samples 20-02-2016 Dr.T.V.Rao MD 27
- 28. Use appropriate microbiological Media and Test Systems • A. Isolating colonies and/or plaques • B. Maintaining pure cultures • C. Using biochemical test media • C. Accurately recording macroscopic observations20-02-2016 Dr.T.V.Rao MD 28
- 29. Use standard microbiology laboratory equipment correctly • A. using the standard metric system for weights, lengths, diameters, and volumes • B. lighting and adjusting a laboratory burner • C. using an incubator right Temperature to be monitored20-02-2016 Dr.T.V.Rao MD 29
- 30. Streaking is the Beginning of learning Alphabets in Microbiology 20-02-2016 Dr.T.V.Rao MD 30
- 31. Streaking the Urine specimens is the best way to learn the matters with precision 20-02-2016 Dr.T.V.Rao MD 31
- 32. Streaking is Key to success in Separation of Pathogens from Commensals 20-02-2016 Dr.T.V.Rao MD 32
- 33. KNOW THE MICROBES BEFORE YOU PROCEED • The cultural characteristics of a microorganism usually vary depending on the media used and many other factors. Some experienced microbiologists could have a good guess about the identity of a microorganism just by its cultural characteristics, but this was proven to be a bad technique. Students as well as microbiologists are advised to follow strict procedures for the identification of isolates from clinical specimens. 20-02-2016 Dr.T.V.Rao MD 33
- 34. Macroscopic (colony) morphology •Size •Shape •Colour ( Pigment ) •Surface appearance •Haemolysis 20-02-2016 Dr.T.V.Rao MD 34
- 35. General View on the Parameters Used in the Process of Microorganism Identification • Before one can proceed to identify a microorganism, the characteristics of that organism have to be determined in details In clinical terms, it is the shape, size, colour, elevation and other characteristics of the colony formed on the culture plate. In taxonomy, it includes the nutrient requirements for the growth of the organism and the physical factors such as temperature, pH and the incubation period. These factors are used to identify certain pathogenic species but less commonly used in routine procedures 20-02-2016 Dr.T.V.Rao MD 35
- 36. Is our experience Matches Scientific Approach • Some experienced microbiologists could have a good guess about the identity of a microorganism just by its cultural characteristics, but this was proven to be a bad technique. Students as well as microbiologists are advised to follow strict procedures for the identification of isolates from clinical specimens. 20-02-2016 Dr.T.V.Rao MD 36
- 37. BIOCHEMICAL CHARACTERISTICS • Frequently, the identity of a species requires detailed knowledge of its biochemical activities, since other characteristics are not sufficiently distinctive or differential. For example, the bacterium Escherichia coli, a normal inhabitant of our intestinal tract, is indistinguishable microscopically from Salmonella typhi, the bacterium that causes typhoid fever. However, if these two bacteria are examined for their metabolic (or biochemical) characteristics, they are found to be very different and distinguishable on this basis. 20-02-2016 Dr.T.V.Rao MD 37
- 38. Morphology and Staining • This includes the microscopic appearance of a stained preparation of the organism. Useful information to be taken into account, are the size of the individual cells, cell shape and arrangement and staining reaction if differential staining procedures is used.20-02-2016 Dr.T.V.Rao MD 38
- 39. TILL TO DATE THERE IS NO FASTER METHOD AS GRAM’S STAINING • Some laboratories which have a little facility could give the report of a microbiological examination of a clinical specimen just by stating their morphological characteristics and the sensitivity testing results 20-02-2016 Dr.T.V.Rao MD 39
- 40. LABORATORY HANDLES THE SPECIMENS • The technologist in the laboratory will directly handle specimens of clinical and environmental source which are received from the Postal Service or hand carried to the laboratory. 20-02-2016 Dr.T.V.Rao MD 40
- 41. Gram staining most Rapid method as the Situation warrants • Look up reference images. If you are not certain what a bacteria is, look through a collection of reference images, sorted by shape and result of the gram stain. You can find databases online at the National Microbial Pathogen Database 20-02-2016 Dr.T.V.Rao MD 41
- 42. Disadvantages of Microscopic methods •Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species. • Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator. 20-02-2016 Dr.T.V.Rao MD 42
- 43. Staining is limited Sensitive • Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible. • Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns. 20-02-2016 Dr.T.V.Rao MD 43
- 44. Problems With Traditional Methods • Cultivation-based methods insensitive for detecting some organisms. • Cultivation-based methods limited to pathogens with known growth requirements. • Poor discrimination between microbes with common behavioural features. • Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response. • Visual appearance of microorganisms is nonspecific. 20-02-2016 Dr.T.V.Rao MD 44
- 45. Problems With Traditional Methods • Examples of Failures With Traditional Approaches • Detection and speciation of slow-growing organisms takes weeks • (e.g., M. tuberculosis). • A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus). • Diseases presumed to be infectious remain ill-defined with not detected microorganism (e.g., abrupt fever after tick bite). 20-02-2016 Dr.T.V.Rao MD 45
- 46. Culture of fungi • Fungi specimens obtained from non sterile sites must be inoculated onto media containing antibacterial agents. Specimens should be allowed to grow for 4 wk before being discarded. 20-02-2016 Dr.T.V.Rao MD 46
- 47. Use of Physical Separation Procedures Streak Plate technique •As we have seen in previous labs, single colonies may be achieved by using the streak plate technique 20-02-2016 Dr.T.V.Rao MD 47
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- 49. Most of us Miss the Parasites •Parasites cause hidden epidemics, many practitioners do not even think •Microbiologists evaluate for Bacteriological examination can miss the Parasites and Fungus •Still our society there sare several parasitic infections prevails •Simpler techniques are missed as stool examination and entrusted with most of the times to the technicians without much understanding20-02-2016 Dr.T.V.Rao MD 49
- 50. Can we diagnose the stool parasites by conventional methods with accuracy •Microsporidia •Isospora Belli & Cyclospora •Cryptosporidium 20-02-2016 Dr.T.V.Rao MD 50
- 51. Parasitology Testing Laboratory Services • This test evaluates stool for presence of parasites and levels of beneficial flora, imbalanced flora, possible pathogenic bacteria and possible fungal pathogens. This Comprehensive Parasitology Profile uses the most technologically advanced procedures to accurately identify a wide range of protozoal parasites, including amoebae, flagellates, ciliates, coccidia and microsporidia. This stool test can help reveal hidden causes behind acute or chronic conditions that develop from parasitic infection or dysbiosis. 20-02-2016 Dr.T.V.Rao MD 51
- 52. Empowering conventional techniques with modern technology • Specimens are carefully analysed by highly trained technicians using computer-enhanced video microscopy, new staining procedures, and advanced immunoassay techniques. These accurate detection methods allow for increased detection rates, increasing the awareness of the important relationship between parasitic infection and a broad spectrum of illnesses and diseases 20-02-2016 Dr.T.V.Rao MD 52
- 53. IS THE CONVENTIONAL METHODS CONVINCING THE CLINICIANS YES / NO •I am confident it is certainly no many times tests are ordered without forethought on the existing the clinical diagnosis and it is just fishing in troubled water,20-02-2016 Dr.T.V.Rao MD 53
- 54. Not a one step Diagnosis •Even Today Many Clinicians think Microbiologist diagnosis is one step diagnosis as in Biochemistry as we many steps to be perform coming to minimal conclusions20-02-2016 Dr.T.V.Rao MD 54
- 55. MOVING TO FEATURE NEEDS • In the get-it-done-yesterday environment Hospitals and Microbiology laboratories are finding that the traditional microbiological methodologies and especially sending microbiological samples to outside labs cost them time, money and opportunities Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can significantly speed up the time to results from 7-10 days to 24-48 hours. 20-02-2016 Dr.T.V.Rao MD 55
- 56. Limitations of Conventional Microbiological Testing • Conventional microbiological methods have well-known inherent limitations. These include small test sample volumes, prolonged incubation periods, incompatibilities with membrane filtration, and ambiguity associated with using turbidity as a detection endpoint 20-02-2016 Dr.T.V.Rao MD 56
- 57. India can face many challenges • The increase in worldwide travel, coupled with increasing immigration into the India, contributes to the spread and incidence of parasitic infections. In addition, parasitic infections are commonly transmitted through fecally contaminated food, water, or other materials within this country. 20-02-2016 Dr.T.V.Rao MD 57
- 58. Conventional techniques of Malaria • Current diagnostic methods for malaria include clinical diagnosis, microscopy, serology, molecular diagnosis, and antibody detection. Giemsa staining of the peripheral blood smear is the gold standard for the diagnosis of malaria, but also has limitations. Using microscopy as the only means of diagnosing malaria can lead to false negatives. Another diagnostic tool is Rapid Diagnostic Tests or RDTs also known as dipstick assays. It 20-02-2016 Dr.T.V.Rao MD 58
- 59. Gold Standard for diagnosis of Malaria • The gold standard for the diagnosis of malaria is the Giemsa staining of the peripheral blood smear(Thick and thin smear), but its also has its limitations. However, even under optimal conditions the sensitivity of microscopy is only about twenty parasites/μl of blood, and subjective interpretation and reader errors further reduce the accuracy of diagnosis. 20-02-2016 Dr.T.V.Rao MD 59
- 60. Human dedication matters • Conventional microscopic examination of peripheral thick and thin blood smears remains the gold standard for malaria diagnosis. Although this method requires a trained microscopist, and sensitivity and specificity vary compared with recent technical advances, it is inexpensive and reliable. 20-02-2016 Dr.T.V.Rao MD 60
- 61. Medical Microbiologists losing the working opportunities • While Rapid Microbiological Methods (RMM) offer high degree of automation, significant reduction in time to results, faster product release, ability to employ non-microbiologists to operate the system, and improved control; 20-02-2016 Dr.T.V.Rao MD 61
- 62. My Dear Young Microbiologists • Most people want to do excellent work. Apathy becomes a problem when team members feel there is no solution or they have no voice • I wish you are the future leaders in Microbiology for next 40 years hope you all wish for change to live in comfort, • Assess your role • Purpose of the speciality • Try Impress others with hard work and sincerity • If Medical People do not wish to work some body take you place ? • NEXT WHAT ? 20-02-2016 Dr.T.V.Rao MD 62
- 63. Are we ignoring Anaerobes ? 20-02-2016 Dr.T.V.Rao MD 63
- 64. Tele Diagnosis
- 65. CDC helps in Digital diagnosis ➲The CDC now offers tele diagnosis to help laboratories diagnose malaria and other parasitic diseases. When laboratories are not certain about identifying parasites on a slide, they can e-mail to the CDC images of the suspected parasites. Experts then review the images and discuss findings with the submitting lab within only a few hours, allowing near real-time diagnosis as well as an opportunity for training in microscopic diagnosis. 20-02-2016 Dr.T.V.Rao MD 65
- 66. A long parasite of 15 cm was extracted without damage to the physical structure •An approximately 15 cm long filamentous macroscopic parasite was extracted sent intact in Normal saline, to Microbiology 20-02-2016 Dr.T.V.Rao MD 66
- 67. Histopathology sections and Photomicrograph 20-02-2016 Dr.T.V.Rao MD 67
- 68. As reported from CDC – Atlanta USA • Based on the images, we agree this is a female Dirofilaria (possibly D. repens in India), as indicated by tall, polymyarian musculature, external cuticular ridges, and paired reproductive tubes. 20-02-2016 Dr.T.V.Rao MD 68
- 69. World First for Malaria - mobile phone diagnosis now available • xRapid is a world first in mobile health, providing automatic diagnosis of malaria via an iPhone app. It is the first commercially available mobile app that has the functionality to quickly and accurately diagnose a major disease. 20-02-2016 Dr.T.V.Rao MD 69
- 70. ARE WE READY FOR CHANGE TO AUTOMATION ? 20-02-2016 Dr.T.V.Rao MD 70
- 71. THE FUTURE OF DIAGNOSTIC MICROBIOLOGY IS CHANGING • The physical structure of laboratories, staffing patterns, work flow, and turnaround time have all been profoundly influenced by technical advances. The implementation of nucleic acid amplification- based molecular techniques provides complementary, rapid, and on-demand diagnosis services. These changes will continue, and lead diagnostic microbiology inevitably to a modern discipline, which can face many challenges in the future. 20-02-2016 Dr.T.V.Rao MD 71
- 72. TODAYS PROBLEM WITH DIAGNOSTIC MICROBIOLOGY • We Teach More than what we see, and diagnose • WE Talk More ? • We discuss more ? • However we do less ? • 1Bench work 2 Fail to improve the laboratory skills ? 20-02-2016 Dr.T.V.Rao MD 72
- 73. Laboratories should progress with scientific Developments •Modern science is fast-moving, and no laboratory can exist for long with a program based on old facilities. Innovation and renewal are required to keep a laboratory on the frontiers of science. • Burton Richter 20-02-2016 Dr.T.V.Rao MD 73
- 74. Wish to be A Better Microbiologist • A microbiologist needs to be both brilliant and methodical. An ability to think critically and analytically is a prerequisite, as is an advanced understanding and knowledge of computers. • Microbiologists must be experts at working with statistics and must stay abreast of developments in statistical techniques 20-02-2016 Dr.T.V.Rao MD 74
- 75. How we can improve our Diagnostic Microbiology 20-02-2016 Dr.T.V.Rao MD 75
- 76. 20-02-2016 Dr.T.V.Rao MD 76