P20 YOGURT BACTERIA

OBJECTIVES

To reveal bacteria that stay in yogurt such as Streptococcus Thermophilus and Lactobacillus Bulgaricus and to know how to do the Gram Stain in order to reveal them.

MATERIAL

• Crystal violet
• Ethanol
• Distilled water
• Safranin
• Three beakers
• Dropper
• Bunsen burner
• Lighter
• Forceps
• Slide and coverslide
• Dissection needle
• Iodine (Lugol)
• Petri dish

PROCEDURE

Take the dissection needle and burn it in the Bunsen burner and then spread it on the yogurt.

Put the yogurt on a slide, then take a dropper and throw a few drops of Chrystal violet, wait for 1 minute and 30 seconds.  After that clean the slide with some distilled water.

Now do the same with Iodine, throw on it few drops and wait for 1 minute, then clean it with water again.

Wash it well with ethanol.

Now you have to dye the slide with safranin and to put again some water, after that add the coverslide and put it into the microscope. 

RESULTS 
The bacteria that are observed of violet colour belong to the gram positive (G +), whereas those that are observed of pink colour belong to the gram negative (G-).

Streptococcus Thermophilus and Lactobacillus Bulgaricus belong to G+ because they don't have a light pink colour. 

QUESTIONS

EXPLANATION OF GRAM STAIN

The first step in any staining should always be fixing with heat. Then, by adding the violet crystal, it enters all bacterial cells, both Gram positive and Gram negative).

Lugol is formed by I2 (iodine) in equilibrium with KI (potassium iodide), which is present to solubilise iodine. I2 enters the cells and forms an insoluble complex in aqueous solution with the violet crystal.

The alcohol-acetone mixture which is added serves to effect the discoloration, since the I2 / violet crystal complex is soluble therein. Gram-positive organisms do not discolour, while Gram-negative organisms do. To disclose the Gram negative cells a contrast dye is used. It is usually a red dye, such as safranin. After contrast staining the Gram negative cells appear red, while the Gram positive cells remain blue.

OBSERVATION OF G+ OR G- 

The first membrane belongs to Gram + and the second to Gram -.