microbiology - HANGING DROP METHOD .pptx

hanging drop prep basics

1. HANGING DROP METHOD

2. CONTENTS Introduction History Mechanism Microscopy Results 2

3. MOTILITY T he hanging drop method is used to observe the motility of bacteria in a live, unstained state. INTRODUCTIOn

4. How do Bacteria move??? Motion can be achieved by one of mechanisms: 1) by the use of flagella 2) internal flagella known as the axial filament 3) Gliding bacteria secrete copious slime

5. iNTRODUCTIOn Bacteria are suspended in a small drop of fluid, which can be Saline Nutrient broth Distilled water, placed on a cover slip The coverslip and drop are then inverted over a concavity of a specially designed cavity slide.

6. HISTORY OF HANGING DROP METHOD It was first discovered by a scientist named Robert Koch in the year 1878. 6

7. MECHANISM Utilizes the surface tension between the coverslip and slide which holds the drop in place, creating a “hanging drop” Allows differentiation between: True bacterial motility (directional, purposeful movement) Brownian motion (random, passive movement due to molecular collisions) Speed and Direction of bacterial movement Pattern of motility ( e.g., tumbling, darting, or gliding) 7

8. MICROSCOPIC OBSERVATION 8 Move the slide to make the the edge of the drop pass through the center of the field. Under 40 x - o bserve the slide until the edge of the drop can be seen as a thick, dark line.

9. Focus on the edge Concentration of organisms is more at the edge because Aerobic organisms tend to move towards the edge Surface tension is less at edge hence concentration of organism is more Contrast is better and organism are clearly visible

10. differentiate 10 BROWNIAN MOVEMENT Random, vibratory movement Consistent amplitude Displacement <1 cell length Oscillatory, stationary Increases with temperature TRUE MOTILITY Directional movement Varies in speed and direction Path deviation >1 cell length Motility Continues across field of view Temperature independent

11. Result and Interpretations DARTING MOTILITY Campylobacter and Vibrio cholerae display very active motility (darting motility) which appears as tiny dots darting in and out of the field. 11 Directional purposeful motility is a positive test.

12. EXAMPLES 12 NON MOTILE (typically negative in hanging drop test) Klebsiella spp Shigella spp Staphylococcus aureus Streptococcus spp. Enterococcus spp MOTILE (typically positive in hanging drop test) Escherichia coli Salmonella spp Pseudomonas aeruginosa Proteus mirabilis Vibrio cholerae Campylobacter jejuni Helicobacter pylori

13. Advantages Rapid results (minutes) Cost-effective Simple equipment Real-time observation No reagents required Doesn’t change the shape or arrangement of cells. Clearer image of the motility in comparison to the wet-mount method. 13 Limitations Subjective interpretation Requires fresh cultures Limited to certain bacterial sizes Time – consuming for multiple samples Operator skill dependent

14. Clinical Relevance Hanging drop methods can detect motility at dilutions up to 10⁻⁹ Rapid screening tool for bacterial identification Cost-effective in developing countries First-line motility assessment 14

15. Thank you

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17. Speaking engagement metrics Impact factor Measurement Target Achieved Audience interaction Percentage (%) 85 88 Knowledge retention Percentage (%) 75 80 Post-presentation surveys Average rating 4.2 4.5 Referral rate Percentage (%) 10 12 Collaboration opportunities # of opportunities 8 10 17

18. Result and Interpretations Directional purposeful motility is a positive test. 18