Asst Lec Mariam A Ali Asst Lec Iman k kadhim AlMustaqbal University college 2 st Class 1 st Semester Department of pharm ID: 1009574
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1. Medical microbiologyLab 3: The hanging drop slide and bacterial motility; Acid fast staining procedure.Asst. Lec. Mariam A. AliAsst. Lec. Iman. k. kadhimAl-Mustaqbal University college 2 st Class, 1 st Semester Department of pharmacy
2. Bacterial motility Eucaryotic cells can move by means of different locomotor organelles such as cilia, flagella, or pseudopods. Procaryotes move by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Almost all spiral bacteria and about half of the bacilli are motile, whereas essentially none of the cocci are motile.
3. Site of flagellaPeritrichous (E.coli) Monotrichous ( Vibrio cholerae)
4. Site of flagellaLophotrichous (pseudomonas) amphitrichous (Spirillum volutans)
5. Motility testingMotility could be detected by:Wet mount slideHanging Drop technique.Flagella stain.Semi-Solid media Inoculation.
6. 1- Wet Mount slideWhen working with non-pathogens, the simplest way to determine motility is to place a few loopfuls of the organism on a clean slide and cover it with a cover glass. Organisms are observed in a drop that is suspended under a cover glass in a concave depression slide
7. Cover glassesDepression slidesPetroleumJelly (Vasoline)ToothpicksBacterial culture2- Hanging Drop MethodMaterials needed:
8. Step 1: Preparing Cover GlassUsing a toothpick, place a small dab of Vaseline in each corner of a clean cover glass. Handle the cover glass by its edges to avoid fingerprints.Aseptically transfer a loopful of a liquid bacterial culture into the center of the cover glass.
9. Step 2: Attaching Depression SlideVaseline attaching cover glass to depression slideCarefully lower depression slide with depression facing down.
10. Step 3: Invert Depression Slide Quickly invert depression slide. Drop will now be suspended from the cover glass inside the concave hollow.Suspended drop
11. Step 4: Examine under MicroscopeWith 10x objective and using course adjustment knob, focus on the edge of the hanging drop.To increase contrast ,move condenser all the way down and keep light as low as possible by closing iris diaphragm.Drop Edge of drop10x objective
12. With 40x objective, focus on isolated cells near inside edge of drop. Flagella are not visible with light microscope. Motile cells move independently. Observe slide immediately after preparation as motility decreases over time. Also, as drop dries out and recedes, water currents will appear to herd and move bacteria in the same direction – this has nothing to do with motility!Bacteria
13. 3- Flagella staina ferric-tannate mordant and a silver nitrate solution are applied to a bacterial suspension. The resulting dark precipitate that forms on the bacteria and their flagella allows them to be easily visualized under the microscope. This silver-plating technique is also used to stain the very slender spirochetes.
14. 4. Semi-Solid media InoculationIt depends on the ability of motile bacteria to move through semi-solid media.Ordinary solid media contain 1.5-2.0% AgarSemi solid media contain about 0.4% Agar
15. How to Perform Test: Using a sterile bacteriological needle, pick a colony of the test organismStab quickly a tube of semi solid media. (avoid using bent needles).Incubate the semi solid media for 24 hoursProcedure of Motility Test
16. Reading Results: (a) Pattern of growth of a motile organism. The entire medium is turbid with the growth of the organism, which has moved away from the stab line.(b) Pattern of growth of a nonmotile organism. Only the stab line is turbid with growth. a b
17. Semi solid media with tetrazolium chloride (color indicator) A colored indicator can be used to make the results easier to see.+ – +
18. Staining bacteria cells:the acid-fast stain
19. Paul Ehrlich was a German physician. He developed the acid-fast stain in 1882 as a means of staining the tubercle bacillus, Mycobacterium tuberculosis. His original method has undergone modifications by Ziehl and Neelsen that are still used today. History of the Acid-fast stain
20. The acid-fast stain distinguishes different types of bacteria based on the wax content of their cell wall. Bacteria with a high wax content retain the primary stain carbolfuchsin when decolorized with acid-alcohol. These are acid-fast bacteria. Bacteria with a low wax content lose carbolfuchsin when decolorized with acid-alcohol and take up the counterstain methylen blue. These are non acid-fast bacteria.This stain is important in distinguishing acid-fast bacteria of the genus Mycobacterium.
21. Cell wall of Mycobacterium tuberculosis
22. ACID-FAST STAIN PROCEDUREStain with carbolfuchsin……….…5 min. 2. Wash off with tap water4. Decolorizer Acid-Alcohol (3% HCl-Ethanol 95%)5. Wash off with tap water6. Counterstain with methylene blue………2 min.7. Wash off with tap water8. Blot dry with bibulous paper
23. Acid Fast staining of Mycobacterium