Techniques to examine Motility
Flagella are bacterial structures that allow directed movement, called motility. Motility enables bacteria to move towards favorable environments and away from unfavorable ones and is sometimes important in the characterization and identification of bacteria. Arrangement of flagella varies among species. A flagellum may occur singly at one end, or there may be more than one flagella at one or both ends (polar). Flagella may occur in tufts, or they can be arranged all around the cell (peritrichous). Not all motile bacteria have flagella and many bacteria are non-motile. There are different ways to examine motility or motility organelles. You should begin with the motility part of the SIMS test and if that is positive, do a wet mount to confirm motility and/or do a fagella stain to see if you can see flagella.
SIM medium for motility
This special media relies on the ability of motile bacteria to move through a tube of semisolid medium. The growth of motile bacteria in such a tube will produce turbidity throughout the solid medium, whereas non-motile organisms will grow only along the line of inoculation. The media we will use is Sulfur Reduction/Indole Production/Motility media (SIM tubes) It is useful not only to determine motility but also tests for two metabolic pathways. Motility should be assessed first and if the SIM motility test appears positive do a confirmatory wet mount or hanging drop .
Inoculating the SIM tubes involves a technique you have not yet practiced. It is very similar to inoculations using the flame sterilized loop, but you will use an inoculating needle (the wire extending from the handle will not have a loop on the end). After picking up a visible amount of your isolate on the tip of a flame sterilized inoculating needle, you will stab it deeply into the center of the medium in the SIM tube, stopping just before the bottom of the tube or, if you are running out of needle, stab it until the you are almost to the end of the needle.
Withdraw the needle through the same inoculation channel. This procedure is also known as "making a soft agar deep".
Inoculate a positive control organism using the same technique.
Incubate for 24-72 hours (or as appropriate)
Motile organisms will exhibit growth radiating from the stab inoculation line.
Non motile organisms will exhibit growth only along the stab inoculation line.
- Motility detection is possible due to the semisolid nature (low concentration of agar) of the SIM medium. Growth radiating out from the central stab inoculation line indicates that the test organism is motile. The motility test should be assessed first. Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line.
TEST FOR TRYPTOPHASE and SULFUR REDUCTION by SIMS
The ingredients in SIM (sulfate/ indole/ motility) medium enable detection of two other metabolic capabilities that some bacteria have and others lack: digestion of tryptophan by the enzyme tryptophanase to indole and/or sulfur reduction with the production of hydrogen sulfide. These characteristics are sometimes used to differentiate Gram-negative rods of the Enterobactericaea group of enteric bacteria can be differentiated: Sulfur Reduction and /Indole Production. SIM medium contains nutrients, iron, and sodium thiosulfate.
The indole test is used for detecting tryptophanase. Casein peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. Bacteria possessing the enzyme tryptophanase cleave tryptophan, producing indole. Indole combines with dimethyl-aminocinnamaldehyde to form a blue-green compound.
The hydrogen sulfide test relies on the use of sodium thiosulfate and ferrous ammonium sulfate as indicators of hydrogen sulfide production. Ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide, a black precipitate.
- Indole test: Dispense 2 drops of the remel® spot indole reagent into the SIM tube. Observe for the development of a blue color within 1-3 minutes. A pink color indicates a negative test.
Hydrogen Sulfide Test. When hydrogen sulfide gas is produced, the gas reacts with the ferrous ammonium sulfate precipitating as ferrous sulfide. An insoluble black precipitate in the medium indicates the organism produces H2S .
Approximate Formula* Per Liter
Pancreatic Digest of Casein - 20.0 g
Peptic Digest of Animal Tissue - 6.1 g
Ferrous Ammonium Sulfate - 0.2 g
Sodium Thiosulfate - 0.2 g
Agar - 3.5 g
Remel® spot indole reagent: (per liter)
p-Dimethylaminoaminocinnamaldehyde 10,0g (CAS6203-18-5)
Hydrochloric acid 100.0 ml (CAS 7642-01-0
demineralized water 900.0 ml (CAS 7732-18-5)
Vracko, R. and J. C. Sherrie. 1963. Am J Clin. Path. 39: 429-432.
Hanging Drop Wet Mount
In 2011 we do not have the slides for hanging drop technique. However we have a gridded slide that we cna use. If you would like to perform this technique please see your instructor.
Motility in living cultures can be observed by the Hanging Drop Technique
. Use a loopful of an isolate either from a broth culture or from a tube of water into which a small amount of growth on solid medium has been transferred. The presence of directed movement (rather than Brownian motion) implies the presence of flagella. A hanging drop slide is a quick way to examine motility.
Put 4 tiny drops of oil on each corner of a clean dry coverslip.
Aseptically transfer a loop full of culture suspended in a liquid to the center of a coverslip.
Locate a depression slide. (This slide will be thicker than a slide used to make a smear and will have 1 or 2 bowl-like depressions.)
Turn the depression slide upside down so that the bowl is over the drop on the coverslip. Touch the coverslip gently and the oil and coverslip will adhere lightly to the slide.
Flip the slide over and examine the drop using the compound scope at 450x magnification with the diaphragm on the microscope adjusted (most of the source of light blocked) to enhance the contrast of the transparent organisms.
- Non motile organisms will shake back and forth in the drop, true motility is shown when an organisms makes progress through the field of view.
Flagella are fragile protein structures that are too thin to be resolved by the human eye using a brightfield microscope. However, if coated with stain and mordant, the diameter increases, sometimes enough to exceed the resolution limits of the human eye. Because motility is important functionally, it is worthwhile to determine if your bacterial isolates possess flagella. Since the flagella are quite fragile and often fall off the cell during the culture or staining process (or they may not take on enough stain complex to be thick enough to view) a negative flagella stain is not conclusive of non-motility. Use this stain in conjunction with other functional tests for motility such as the hanging drop and/or SIM test.
The advantage to staining the bacterial flagella, rather than simply relying on the functional tests for motility, lies in the stain's ability to reveal number and arrangement of flagella. It is common for a motile species to have more than one per cell. Flagella arrangement is quite variable among bacterial species. The most common arrangements you might observe are peritrichous (many flagella positioned all around the bacterial cell) and polar (one or more found at one or both ends of a cell). There are flagellated bacteria in many genera including: Aeromonas, Proteus, Bacillus and others.
FLAGELLA STAIN Procedure:
(adapted from: Murray, R. G. E., R. N. Doetsch, and C. F. Robinow. 1994. Light microscopy. In P. Gephardt, R. G. E. Murray, W. A.Wood, and N. R. Krieg (ed.), Methods for general molecular bacteriology. American Society for Microbiology, Washington, DC. and MelliesReed, Jay. Bacterial Flagella Stain Protocol Resource Type: ASM Curriculum: Protocol Publication Date: 9/8/2008)
1. Clean a new slide with BonAmi cleanser to remove any film or oils that might be on the slide. You will need 1 cleaned slide for each bacterial isolate you wish to examine. Dry it and run it through the flame of your Bunsen burner to remove any oils from your fingers. Cool with out touching the slide.
2. Using a flame sterilized loop, transfer a small amount of growth from an agar plate or slant cultures into 2ml water in test tube (does not need to be sterile). Mix gently and check that the suspension is only slightly cloudy. Using too much inoculum will cause difficult in visualizing the flagella. Add more water if you need to dilute the innoculum.
3. Pipet 5 μL of the culture suspension to one end of the slide using your P20 micropipet .
4. Spread the suspension gently by holding the pipet sideways over the slide and rolling the pipet tip gently over the liquid once.
5. Discard the pipet tip in the autoclave bag.
6. Air dry thoroughly. Do not heat fix.
Presque Isle Cultures flagella stain
7. Flood the smear area of the slide with Presque Isle Cultures Solution I, the mordant. Incubate at room temperature for 4 minutes.
8. Gently rinse with distilled water. Shake excess water from slide.
9. Flood with Presque Isle Cultures Solution II, the silver stain.
10.Use your clothes pin slide holder to pass the slide over, rather than in, a Bunsen burner flame by moving slide back and forth slowly, just until steam is emitted. Be careful not to overheat sample, as excess heat will destroy the flagella. Incubate at room temperature for 4 minutes.
11.Rinse in a gentle stream of distilled water. Carefully blot dry with bibulous paper or air dry.
12.View using oil immersion, at 1,000x magnification, by brightfield microscopy. Bacteria and flagella will appear golden brown. If too much stain was applied and the microscope slide was not really clean, it is difficult to find the cells and flagella.
The ingredients in the stain and mordant are proprietary reagents of Presque Isle Cultures, P.O. Box 8191, Erie, PA 16505; http://www.picultures.com
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