Week 7-Assignment：Gram Staining

1.Materials and appliances:

Microscope, slide glass, rubber head dropper, beaker, absorbent paper, inoculating ring

The crystallization of lithospermic acid ammonium solution, stained with Lugol's iodine solution, 95% alcohol, safranin dye.

2.Steps:

(1)Wash the slides and dry with gauze.Draw a small circle in the slide back, (it can help us to determine the position of approximately bacteria liquid in the observations the last).

(2)Drying the slide on the flame to remove the grease.

(3)Smear:

The bacteria we used is in the liquid medium. Hold the liquid tube in left hand, and the right hand hold the inoculating ring. Burn the inoculation ring in the flame. After it comes cool(about 10 seconds) , dip the bacteria from the tube, and spread on a glass slide evenly in the ring we draw before.

The entire process must be operated near the alcohol lamp flame to avoid the interference of bacteria. Finally, do not forget to burn the inoculation ring at the end.

4.Dry: Put the smear in the air to make it dry .

5.Fixed: Let the velum upward, fixed by flame 2-3. To prevent the dyeing rinse wash the bacteria away.

6.Dyeing: Put the fixed smear on the newspaper, plus ammonium oxalate crystal violet liquid, dyed a minute.

7.Wash: Rinse slowly with water, smear on the smear.mordanting with a minute washing.

8.Mordanting：Drip solution and with a minute washing.

9.Decolorization: absorption of residual water, continuous addition of 95% ethanol decolorization 20-30s to the outflow of liquid

purple, wash immediately.

10.redyeing：adding counterstain safranin 3-5min, washing.

Experimental results:

We use a microscope to observe, we can look at the final results.