What is the technique for gram staining test?

Answer 1

the gram staining procedure involves four basic steps;

1.the smear is first flooded wit the primary stain crystal violet dye.in this case the primary stain is the first dye applied in any multicelled staining procedure and it stains all the cells.

2.the smear is rinsed with water to remove any excess crystal violet and then its flooded wit a dilute solution of iodine called grams iodine.iodine acts as a mordant.i.e. the substance that increases the interaction and affinity of cellular components for a dye.this is done so that the cells can stain more strongly.

in this case the iodine complex thereby decreases the solubility of the dye within the cells

3.the stained smear is rinsed again and then 95% alcohol or a mixture of alcohol and acetol is briefly added.this solvent acts as decolorizing agents and they readily remove the dye iodine complex from gram negative but not from gram positive bacteria.

4.a counter stain is then applied to import a contrasting color to the now colorless gram negative bacteria.for this purpose,the red dye safrannin is used.this dye stains gram negative as well as gram positive bacteria but because the gram positive are already stained purple,it impacts little difference

Answer 2

Gram staing involves 4 important steps .

1 , Add crystal violet stain .

2 Add iodine solution .

3 , add , organic solvet e.g. alcohol ,

4 , add counter stain safranine .

Answer 3

After you have marked your slide in order to know where your bacteria is going to be and after you have put a little bit of water on to the slide so that it would be easier to put the bacteria on to the slide and after you have sterilized your tool that you will use to pick the bacteria your next steps are these:

1. prepare a smear of gram - and gram+ bacteria in the same region of the slide

2. air dry smear & heat fix

3. rest the slide (smear side up) on a beaker that has been filled three-quarters with tap water

4. cover smear with Hucker's crystal violet dye for a min and a half

5. rinse of dye

6. cover smear with Gram's iodine for 1 min

7. rinse the slide in beaker of water

8. decolonize with alcohol ( about 8 drops slowly)

9. rinse right away in fresh water

10. counter stain with Safranin for a min and a half.

11. Rinse the slide and gently blot dry

Now you are ready to see the slide under a microscope

Answer 4

PIES: purple, iodine, ethanol, safranin.

heat fix the smear prepared on slide.

add few drops of crystal violet - 20-30 sec. and Wash off with distilled water

Fix with iodine - 1 min. Wash off with alcohol (ethanol)

Rinse in ethanol to differentiate - roughly for 10 seconds. Rinse quickly.

Counterstain in safranin - 30 sec. Wash off with distilled water

observe under microscope under 10X there after under 40X for clear vision....

LOVE YOU DEEKS

Answer 5

To perform a Gram stain, one should obtain two bacteria cultures, for example, Escherichia Coli (E. Coli) and Staphylococcus epidermidis (S. epi). A mixed smear should be prepared with both bacterial species. To prepare the smear, use an inoculating loop and obtain a loopful of each bacteria being sure to sterilize (a flame works well) the loop in between bacterial species. Once the smear is prepared, heat fix the slide by running the slide over a flame several times. Heat fixing kills bacter and helps them adhere to the surface of the slide. The slide should be placed over a sink or area that allows draining of liquids to occur. Cover the surface of the slide with a basic dye, such as Crystal Violet, for 60 seconds and rinse the slide gently and briefly with water, draining any excess liquid left over. Add Gram's Iodine, a mordant, and let it sit on the slide for 60 seconds and then thoroughly rinse the slide. Decolorize the slide for 10-15 (more time may be necessary depending on age of bacterial samples) with 95% Ethanol and immediately wash the slide with water to stop the decolorizing process. Flood the surface with a counterstain, such as Safranin, for 60 seconds and then rinse the slide with water. Blot the slide dry with paper towel or bibulous paper and view the gram stain through a microscope. Gram positive cells will appear purple and gram negative cells will appear pink if the colored stains mentioned before are used.

Answer 6

To Gram stain a sample, there are five steps.

1) The sample must be adhered to the slide. This can be done by passing the slide through a flame or flooding it with methanol.

2) Crystal violet is then added to the slide. This penetates into all the cells.

3) Iodine is then added to the slide. The iodine is a mordant, meaning that it helps the crystal violet stay in the cells by combining with the crystal violet to form a large, complex molecule.

4) Decolorizer is applied. This is a mix of acetone and alcohol. Rinse away all traces of brown and purple. This solvent dissolves part of the lipid in Gram negative bacteria. It also causes the thick peptidoglycan layer in Gram positive bacteria to become dehydrated and shrink, trapping the crystal violet-iodine molecule in the cell. This will leave the Gram positive cells purple and the Gram negative cells as colorless.

5) Safranin is added to the sample as a counter stain. It will dye the Gram negative cells pink so that they can be seen.

At the end of the process, Gram positive organisms will be purple, Gram negative organisms will be pink.

Answer 7

In all bacteria that are Gram stained all the steps are used.

It isn't until the end that you will know if they are Gram+ or Gram-.

In Gram staining: bacterial cells take up crystal violet. (Step 1)
Iodine is then added. (Step 2) It acts as a mordant, a chemical that helps retain the stain in certain cells.
Those structures that cannot retain crystal violet are decolorized with 95% ethanol or an ethanol- acetone solution (Step 3), rinsed (Step 4) and subsequently stained (counterstained) with safranin (red). (Step 5)

Gram+ will be violet while Gram- will be red. The thick cell walls of Gram-positive bacteria retain such stains as the crystal violet-iodine dye in the cytoplasm. Gram- will not.
All samples must be under 24 hours old or you might get an incorrect reading.