Hanging Drop Method_microbiology_KripaRaghunathan.pptx

The Hanging Drop Method PowerPoint visually explains the microbiological technique used for observing live microorganisms, particularly their motility and morphology, in a natural, aqueous environment.

1. Hanging Drop Method MICROBIOLOGY

2. INTRODUCTION Hanging drop method permits the examination of the living microorganisms, which involves fixation of microbial suspension into a drop of fluid over the glass slide.

3. INTRODUCTION Therefore hanging drop method is the process, in which the microorganisms are suspended into a drop of fluid.

4. INTRODUCTION It is a modified version of the wet mount technique . It makes the use of glass slides with one small concave depression towards the centre .

5. INTRODUCTION It is an important tool to determine the intrinsic movement of the microorganisms, and that’s why used to test the motility of different organisms like bacteria, filamentous fungi and yeasts.

6. INTRODUCTION All the bacterial cells will show erratic motion, including both motile and non-motile forms under the microscope, which we call as Brownian motion .

7. INTRODUCTION The   self-propulsion  in bacteria is identified by either of the following mechanisms like flagellar , gliding, corkscrew and bending type of motion.

8. INTRODUCTION In hanging drop motility testing, we can see numerous immortalized cells towards the  edge of fluid , under the microscopic observation

9. history of Hanging Drop Method It was first discovered by a scientist named Robert Koch in the year 1878 .

10. history of Hanging Drop Method Previously , a hanging drop technique was used to examine the  Nocardia sp.  on liquid paraffin droplets.

11. history of Hanging Drop Method On further study, the method has been used extensively to demonstrate the bacterial morphology and motility.

12. history of Hanging Drop Method Later , considering the large drop size in the hanging drop method, scientists started using the  micromanipulation technique  to get control over the drop size .

13. Hanging drop-principle Hanging drop preparation is a special type of wet mount in which a drop of medium containing the organisms is placed on a microscope slide, often used in dark illumination to observe the motility of bacteria.

14. Hanging drop In this method, a drop of culture is placed on a coverslip that is encircled with petroleum jelly (or any other sticky material).

15. Hanging drop The coverslip and drop are then inverted over the well of a depression slide.

16. Hanging drop The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation. This preparation gives good views of microbial motility.

17. Procedure of Hanging Drop Method

18. Hanging drop preparation Slide Preparation Take a clean glass slide and apply a paraffin ring, adhesive-tape ring to make circular concavity. (This step is not needed if a glass slide with depression is available ).

19. Hanging drop preparation Slide Preparation Hold a clean coverslip by its edges and carefully dab vaseline on its corners using a toothpick .

20. Hanging drop preparation Slide Preparation Place a loopful of the fresh broth culture to be tested in the center of the prepared coverslip. Use a light inoculum (not visibly turbid).

21. Hanging drop preparation Slide Preparation Turn the prepared glass slide or 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.

22. Hanging drop preparation Slide Preparation Turn the slide over so the coverslip is on top and allow organisms to “settle” for a minute. The drop can be observed hanging from the coverslip over the concavity.

23. Hanging drop preparation Microscopic Observation 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.

24. Hanging drop preparation Microscopic Observation Turn the objective to its lowest position using the coarse adjustment and CLOSE THE DIAPHRAGM.

25. Hanging drop preparation Microscopic Observation 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.

26. Hanging drop preparation Microscopic Observation Without raising or lowering the tube, swing the high dry objective into position (be sure the high dry objective is clean).

27. Hanging drop preparation Microscopic Observation 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.

28. Hanging drop preparation Microscopic Observation Focus on 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 dark or slightly greenish, with very small rods or spheres.  Remember the high dry objective magnifies a little less than half as much as the oil immersion objective.

29. Hanging drop preparation Microscopic Observation Adjust the light using the diaphragm lever to maximize the visibility of the cells. Observe the cells noting their morphology and grouping and determine whether true motility can be observed.

30. Hanging drop preparation Microscopic Observation Brownian movement should be visible on slides of all the organisms, but there should also show true motility. Wash the depression slide and after soaking in lysol buckets or discard the prepared glass slide.

31. Brownian movement When examining living organisms to determine the characteristic of active locomotion, it’s important to differentiate true mobility, in which the organisms move in different directions and shifts their position within the field.

32. Brownian movement This is different from or passive drifting of organisms moving in the same direction within convectional flow within the fluid, or Brownian movement that is an oscillatory move around the same point by all tiny bodies that are suspended in fluids because of irregularities in the bombardments they receive from the water molecules.

33. Hanging drop preparation Result and Interpretations Directional purposeful motility is a positive test.   Motile organisms change position with respect to one another. Brownian movement (random jiggling or shaking due to molecular bombardment),

34. Hanging drop preparation Result and Interpretations Directional purposeful motility is a positive test.   where the organisms remain in the same relative position with respect to each other, should not be mistaken for true motility .

35. Hanging drop preparation Result and Interpretations Campylobacter   and  Vibrio cholerae   display very active motility  (darting motility)  which appears as tiny dots darting in and out of the field.

36. Results - negative motility For all organisms that are not able to demonstrate motility, after the initial wet mount, Repeat the wet mount following incubation in broth or test via tube method .

37. Results - negative motility Incubate at 30°C for non-fermenting rods that are Gram-negative (24 hours). Incubate enterococci as well as Listeria at 30 degC for 2 hours .

38. Results - negative motility Other microorganisms could be incubated at temperatures suitable in order to grow, typically 35 degrees Celsius.

39. Uses of Hanging Drop Method The spiral morphology is studied specifically in the hanging drop method because its shape changes during the process of heat fixing .

40. Uses of Hanging Drop Method Spirochetes , a type of spiral bacteria, require examination in a live state and their shape as well as their arrangement could be observed under a dark-field microscope .

41. Uses of Hanging Drop Method Mobility or motility in a bacterium can be studied using a hanging drop technique that allows the bacterial cells can freely move within the medium of liquid .

42. Uses of Hanging Drop Method The cytological changes that take place as cells divide, spore development and the germination of bacteria are the processes that require investigation in a living environment or using the hanging drop method .

43. Uses of Hanging Drop Method The cytoplasmic inclusions, such as vacuoles, granules and so on. are easily discernible employing this technique.

44. Advantages of Hanging Drop Method It is an essential instrument to analyze the motility of bacteria as well as the shape, size , and configuration of bacteria. It doesn’t change the shape or arrangement of cells .

45. Advantages of Hanging Drop Method In addition hanging drop, this method offers a clearer image of the motility of bacteria in comparison to the wet-mount method .

46. Advantages of Hanging Drop Method It also aids in the classification of bacteria, determining if they are motile or not .

47. Advantages of Hanging Drop Method Spiral bacteria like spirochetes need to be examined in a  living state , and there shape and arrangement can be well studied under a dark field microscope .

48. Advantages of Hanging Drop Method the wet mount technique dries out more quickly than the hanging drop method. Since the hanging drop method does not dry out as quickly, it allows for longer examination of the microorganism and a more reliable observation

49. Advantages of Hanging Drop Method The Brownian movement is a study method, which triggers erratic motion of bacteria in the view field because of the explosive bombardment by water molecules .

50. Advantages of Hanging Drop Method The petroleum jelly that is used in this technique is used to seal the coverslip onto the slide, which aids in the examination of the specimen tested for several times.

51. Disadvantages of Hanging Drop Method It is not a safe method to use when one is dealing with extremely pathogenic organisms, as humans can easily get an infection. The coverslips, which are one part of the apparatus, are quite fragile, and handling them can pose a challenge

52. Disadvantages of Hanging Drop Method hanging drop technique include the possibility of overlooking a few motile cells among countless nonmotile cells, the observation process can be time consuming while viewing under microscope, to distinguish true and Brownian mov Setting up the apparatus needed for the hanging drop technique takes time, and the cavity slides used are expensive

53. Conclusion The hanging drop method is the best way to study the  different events  of the organism in a living state, the mobility, shape and arrangement of the bacterial cells. Hanging drop method is a kind of  motility test , which is performed by taking the microorganisms from liquid media on the glass slide.

54. Thank you Kripa raghunathan