Motility test- Hanging drop technique

MOTILITY TEST

HANGING DROP TECHNIQUE:

AIM:

 To observe the bacterial motility by following hanging drop technique

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 is used in dark illumination to observe the motility of bacteria.

Hanging Drop Method Preparation

In this method a drop of culture is placed on a cover slip that is encircled with petroleum jelly (or any other sticky material). The cover slip and drop are then inverted over the well of a depression slide. The drop hangs from the cover slip, and the petroleum jelly forms a seal that prevents evaporation. This preparation gives good views of microbial motility.

 Materials required:

1.      Glass slides (glass slide with depression) or Normal glass slide with adhesive or paraffin ring, Paraffin wax, Loop, Coverslip, Microscope, Bunsen burner

2.      Young broth culture of motile bacteria (e.g. Proteus mirabilis)

Procedure:

1.      Take a clean glass cavity slide and hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.

2.      Place a loopful of the broth culture to be tested in the center of the coverslip.

3.      Turn the 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 hanging 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.

6.      Observe the slide and Focus the edge of the drop carefully in the microscope.

Result: The movement of bacteria was observed under the 40x objective with optimum light background.

GRAM'S STAINING

Gram staining

AIM:

To stain the bacterial smear by following Gram’s staining procedure and observe their morphology and arrangements

PRINCIPLE

The differences in cell wall composition of bacteria account for the Gram staining. Gram-positive cell wall contains a thick layer of peptidoglycan. In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of bacteria. The outer mem

brane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counter stain, the positively charged dye safranin, is added.

Materials required:

Inoculation loop, slide, Bacterial culture, Bunsen burner, Gram staining kit

PROCEDURE:

Gram Staining Procedure:

1.      Prepare a smear over a clean slide and heat fixed.

2.      Flood the smear with crystal violet and wait for 1 minute.

3.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

4.      Flood slide with the mordant: Gram’s iodine. Wait 1 minute.

5.      Wash slide in a gentle and indirect stream of tap water for 2 seconds.

6.      Decolorize the smear by Flood with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add decolorizer drop by drop on slide until decolorizing agent running from the slide runs clear.

7.      Flood slide with a counter stain, safranin. Wait 30 seconds to 1 minute.

8.      Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.

9.      Observe the results of the staining procedure under oil immersion (100 x) of a microscope.

Results:

§  Gram-negative bacteria will stain pink/red and

§  Gram-positive bacteria will stain blue/purple.