Skills in Medical Microbiology

1. SKILLS IN MEDICAL MICROBIOLOY Dr.T.V.Rao MD Dr.T.V.Rao MD

2. Why Microbiologists need Skills • Successful microbiology work demands reliable precision in identifying microbes, observing their characteristics and behavior, applying biological theory to observations and drawing accurate conclusions based on biological data, known facts and concepts, original research and existing research. Dr.T.V.Rao MD

3. SKILLS for MD, MSc in MicrobiologyAt the end of the course the student shall be able to • 1. Plan the laboratory investigations for diagnosis of infectious diseases • 2. Perform laboratory procedures to arrive at the etiological diagnosis of diseases caused bacteria, fungi, viruses and parasites. • 3. Perform and interpret immunological and serological tests. • 4. Operate routine and sophisticated instruments in the laboratory Dr.T.V.Rao MD

4. Bacterial Identification a Part of MD, M.Sc. ( Microbiology ) Examination • The Indian Medical Curriculum for Post Graduates in Microbiology emphasizes with Identification of Bacteria from Pure and a Mixture of Bacterial agents. A student must have minimum skills to Identify, Isolate, Characterize , prove the pathogenic nature and test its Antibiograms Dr.T.V.Rao MD

5. Why We Should Identify a Bacteria • Accurate and definitive microorganism identification, including bacterial identification, is essential for correct disease diagnosis, treatment of infection and trace-back of disease outbreaks associated with microbial infections. Bacterial identification is used in a wide variety of applications including microbial forensics, criminal investigations, bio-terrorism threats and environmental studies, including Epidemiological studies. Dr.T.V.Rao MD

6. Microbiology LaboratorySkills Test: Bacterial Unknown ? • This skills test will examine your ability to i) isolate two different bacterial, or even three from a mixed culture and ii) identify each bacterium using pertinent diagnostic characteristics. Many times you have already performed or are presenting performing these diagnostic tests. Therefore, you should be familiar with how they are conducted. Dr.T.V.Rao MD

7. How to Evaluate • The Teachers should try to evaluate students with a Mixture of One commensal,One truly pathogen, and other can be a opportunistic pathogen, as more understanding can be created in the learners, the Importance of Emerging opportustic bacterial pathogens Dr.T.V.Rao MD

8. A student is given a Mixture of Bacterial agents • The student will receive a mixed culture unknown containing two or three different bacteria. They can be of any combination from among those bacteria listed by your Laboratory when you were trained as student when practicing Dr.T.V.Rao MD

9. Read the Question and create Mind Map for all Procedures • Carefully read the Clinical question provided to you, it guides you what is a probable microbes to be identified. • To begin, read the instructions given by Examiner carefully. Begin working immediately. The “Mixed Culture Unknown Answer Sheet and Flow Chart” and the “Skills Test Log Sheet” must be submitted as a stapled unit to your Examiner by the stated deadline. Dr.T.V.Rao MD

10. All post graduates should be familiar with Isolation, Description and Identification of Bacteria Staphylococcus and Micrococcus; Anaerobic Gram positive cocci. Streptococcus and Lactobacillus. Neisseria, Branhamnella and Moraxella Corynebacterium and other coryneform organisms. Bacillus: the aerobic spore bearing bacilli Dr.T.V.Rao MD

11. Other Pathogens which are always Incorporated in Evaluations • The Enterobacteriaceae • Vibrio's, Dr.T.V.Rao MD

12. Record the findings in the Log Book • You are to keep a log of your activities and observations in this test • Start working with Skill test log Dr.T.V.Rao MD

13. Skills on Day 1Selecting the Specimen for Evaluation • The Examiner will provide a rack of mixed culture unknowns that have been randomly labeled with a code number. Select one tube and immediately record the number of that unknown on the report sheet that will also be provided by your Examiner. Dr.T.V.Rao MD

14. Gram staining – from Mixture you are Provided • Prepare a heat-fixed smear from your mixed culture unknown on a glass slide. Gram stain your smear. Observe and record your results. These observations may give you an idea of what to expect upon isolating your unknown bacteria. Dr.T.V.Rao MD

15. Limitations of Gram Staining • However, it is conceivable that you may isolate two bacteria having the same Gram-stain reaction and morphology, in which case it may be difficult to distinguish between the different isolates. • Eg Two gram negative bacteria, E.coli and Salmonella spp Dr.T.V.Rao MD

16. Hanging Drop Procedure: Dr.T.V.Rao MD

17. Hanging Drop give clues to Basic Identification in Gram Negative Bacteria • 1. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.  If too much Vaseline is used, it will be squeezed toward the center and mix with the drop or squeeze out the edges and get on the objective lens of the microscope. • 2. Place a loopful of the culture to be tested in the center of the prepared coverslip. • 3. Turn the clean concavity slide upside down (concavity down) over the drop on the coverslip so that the Vaseline seals the coverslip to the slide around the concavity. • 4. Turn the slide over so the coverslip is on top and the drop can be observed banging from the coverslip over the concavity. • 5. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives. Dr.T.V.Rao MD

18. Hanging Drop • 6 Turn the objective to its lowest position using the coarse adjustment and CLOSE THE DIAPHRAGM. • 7. Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the edge of the drop is observed as an irregular line crossing the field. • Move the slide to make that line (the edge of the drop) pass through the center of the field. • 9. Without raising or lowering the tube, swing the high dry objective into position (Be sure the high dry objective is clean). • 10. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can be seen as a thick, usually dark line. Dr.T.V.Rao MD

19. Hanging Drop • 11. Focus the edge of the drop carefully and look at each side of that line for very small objects that are the bacteria.  The cells will look either like dark or slightly greenish, very small rods or spheres.  Remember the high dry objective magnifies a little less than half as much as the oil immersion objective. • 12. Adjust the light using the diaphragm lever to maximize the visibility of the cells. • 13. Observe the cells noting their morphology and grouping and determine whether true motility can be observed. Dr.T.V.Rao MD

20. Hanging Drop • 14. Brownian movement should be visible on slides of all the organisms, but two should also show true motility. • 15. Wash the depression slide and after soaking in Lysol or common bleach buckets. Dr.T.V.Rao MD

21. What you choose for Plating and Isolation • Obtain one (1) of each of the following agar plate media: • MacConkey Agar (MAC) Blood Agar • = Mannitol Salts Agar (MSA) • = Tryptic Soy Agar (TSA) [Nutrient Agar (NA) may be substituted for TSA] • Your choice can be wide, but you should be appropriate and optimal choosing other media available in the Laboratory, Dr.T.V.Rao MD

22. Caution in Choosing Media • Too many media will confuse the situation, Leads to unnecessary questioning. • If you choose inappropriate Medium you loose the path of Identification. • Optimal media are few for identification all basic pathogens Dr.T.V.Rao MD

23. Streaking will expose your Skills • Using your very best technique, separately streak a loopful of the mixed culture • Unknown onto the MAC, MSA, and TSA plates, blood agar. Or any other of your choice. Incubate these plates at 35°C- 37°C overnight. Dr.T.V.Rao MD

24. Importance of Streaking • Use your best skills in streaking the culture plates. • The streaked plate indicates your skills, • Examiners can identify you past experience with laboratory bench work. • Best Instructions are well represented in Koneman’s Diagnostic Microbiology. Dr.T.V.Rao MD

25. Streaking the plates is a ARTPerfect It Dr.T.V.Rao MD

26. Begin with Skills Test Log Sheet • Mixed Culture Unknown Number xxx • Date: xx/xx/xxxx • Description of Gram Stain Results of Mixed Culture Unknown: (Optional) To create a systematic reporting Log on to URL - http://www.as.ysu.edu/~crcooper/STLS.pdf Dr.T.V.Rao MD

27. Crucial Reporting • Your crucial reporting of identification starts from 2nd and by 3rd day you will need to submit by the stated deadline (as listed in the laboratory syllabus or as provided by your examiner ) Dr.T.V.Rao MD

28. Day 2 • Observe your streak plates for isolated colonies and record your results. • NOTE 1: MAC medium is a differential and selective growth medium. Only Gram-negative bacteria grow on this medium. In addition, bacteria that ferment lactose appear as pink colonies (occasionally purplish) or as white colonies with a pink center. Non-lactose fermenters appear clear, colorless, or white only. Dr.T.V.Rao MD

29. Mannitol Salt Agar helps in Gram Positive cocci • Composed of NaCl, Mannitol and phenol red as pH indicator • Both selective and differential • Selective since it allows only the growth of high salt or saline loving organisms. • Differential since manitol fermenter organisms change the red pH indicator color (neutral) to yellow (acidic). • Non-mannitol fermenters do not change the color of the media. • Usually allows the growth of Staphylococci especially for differentiating Staphylococcus aureus from others. Dr.T.V.Rao MD

30. Mannitol Salt Agar • Mannitol fermenters includes: Staphylococcus aureus • Non-mannitol fermenters includes: Staphylococcus epidermidis • Positive growth but non-mannitol fermenters includes: Micrococcus luteus • Negative growth includes: Escherichia coli, Pseudomonas aeruginosa Dr.T.V.Rao MD

31. Gram positive – Mannitol Salt Agar • MSA medium is also a differential and selective growth medium. Only Gram-positive bacteria grow on this medium. In addition, those Gram-positive bacteria that ferment Mannitol grow as white colonies and cause the surrounding medium to appear yellow. Gram-positive, non-mannitol fermenters appear clear, colorless, or white only with the exception of Micrococcus luteus. The latter microbe appears naturally as a yellow colon Dr.T.V.Rao MD

32. Use of Tryptic Soya Agar • TSA and NA media are not differential or selective growth medium. • Both Gram-positive and Gram-negative bacteria grow on this medium. Hence, the purpose of this medium is to select bacteria based upon differences in colony morphology. Dr.T.V.Rao MD

33. Tryptic Soya agar • Tryptic soy agar (TSA) inoculated with (A) Staphylococcus aureus, (B) Staphylococcus epidermidis, and (C) Escherichia coli demonstrating growth of all three organisms. TSA is a general purpose medium that will allow for the growth of all three organisms. Dr.T.V.Rao MD

34. Choose the Selective Medium with caution ( TCBS ) • TCBS is a selective isolation medium for culture of pathogenic Vibriospp primarily from clinical samples. On this medium most Enterobacteriaceae in faeces are suppressed for at least 24 hours although slight growth of Proteus spp and Streptococcus faecalis may occur but they are readily distinguished from vibrio colonies Dr.T.V.Rao MD

35. Salmonella Shigella Agar • Salmonella Shigella Agar (SS Agar) is a differentially selective medium for the isolation of pathogenic enteric bacilli, especially those belonging to the genus Salmonella. This medium is not recommended for the primary isolation of Shigella. Dr.T.V.Rao MD

36. Picking the isolates after separation • From your different media, identify two or even three different bacteria. On the underside of your medium, mark the location of two distinct, and well-isolated colonies. Using a loop, pick a small portion of these two colonies and inoculate each onto TSA or NA slants. Incubate the slants at 35°C-37°C. These slants will serve as your stock cultures to inoculate your diagnostic media Dr.T.V.Rao MD

37. With Caution – It is crucial to Identify Gram positive and Gram Negative Bacteria • Depending upon the nature of your mixed culture unknown, two different colony types may not appear on MAC or MSA plates. Remember, these media are selective. If you have two Gram-negative bacteria, they will grow on the MAC plate, but not the MSA plate, Dr.T.V.Rao MD

38. A crucial step • Conversely, if you have two Gram-positive bacteria, they will grow on the MSA plate, but not the MAC plate. If you have one Gram positive and one Gram-negative bacterium in your mixed culture unknown, then you should see the growth of only one colony type on the MAC and only one colony type on the MSA plate. In most cases, two colony types should appear on the TSA (or NA) plate. Dr.T.V.Rao MD

39. Answer Sheet and Flow Chart • Mixed Culture Unknown Number xxx • Identity of Unknown Isolate 1 ……. • Identity of Unknown Isolate 2 …… • Identity of Unknown Isolate 3 ….. • Prepare a flow chart of your work for each isolate.. Be sure to identify which flow chart corresponds to which isolate. Dr.T.V.Rao MD

40. You may now Gram stain your isolated bacteria by one of the following two options: Option 1 : From the location marked on the plates from which you isolated your unknown bacteria, remove some of the colony with a loop and make a heat-fixed smear on a glass slide. Gram stain your smear. Observe and record your results Dr.T.V.Rao MD

41. Option 2 • Incubate your stock cultures for 12-18 hours (i.e., until Day 3), then from each tube separately remove some of the growth of the unknown bacterium with a loop and make a heat-fixed smear on a glass slide. Gram stain your smear. Observe and record your results. • Caution As the Examination is final by 3rd this can be only 2nd option Dr.T.V.Rao MD

42. Biochemical Test for Identification for Identification of Genus and Species • Students should choose the Biochemical tests which are available, familiar in the past, and Rapdily reactive as the Examination is for 2 or 3 days, and should come to maximum conclusions. However should choose few the tests giving late reaction for academic discussion Dr.T.V.Rao MD

43. Biochemical tests help in Identification of several Bacterial isolates EVERYTHING that a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICALFINGERPRINT. That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism Dr.T.V.Rao MD

44. Biochemical Reaction Use of substrates and sugars to identify pathogens: - Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar b- Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface - H2S production: Depends on production H2S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate Dr.T.V.Rao MD

45. Biochemical Reaction (cont.) c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator d- Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Color of medium turns pink (positive) e- Dr.T.V.Rao MD

46. Biochemical Reaction (cont.) f- Oxidase test: Some bacteria produce Oxidase enzyme Detection by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive) g- Catalase test: Some bacteria produce catalase enzyme Addition of H2O2 lead to production of gas bubbles (O2 production) h-Coagulase test: Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method i- Urease test: Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH3 Alklinity of mediaand change color of indicator from yellow to pink Dr.T.V.Rao MD

47. Common Tests To identify Bacterial isolates • Indole • Methyl Red/Voges Proskauer • Citrate • H2S production • Urea hydrolysis • Motility • Lactose fermentation • Sucrose fermentation • Glucose fermentation & gas production Dr.T.V.Rao MD

48. I M Vi C Tests I M Vi C is an acronym that stands for indole , methyl red, Voges-Proskauer , and citrate . To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test), methyl red - Voges Proskauer broth (MR-VP broth), and citrate test. Dr.T.V.Rao MD

49. Triple Sugar Iron Agar Bacteria that ferment any of the three sugars in the medium will produce byproducts These byproducts are usually acids, which will change the color of the red pH-sensitive dye (phenol red) to a yellow color. Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic byproducts of lactose or sucrose fermentation. Many bacteria that can ferment sugars in the anaerobic butt of the tube are enterobacteria. Dr.T.V.Rao MD

50. Reading the results on TSI enables to identify the several pathogens Dr.T.V.Rao MD