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 -. 

P19 CROMATOGRAPHY

OBJECTIVES

• To know the bases of chromatographic analysis.
• To learn the procedure for separating the photosynthetic pigments. 

MATERIAL 

• ethanol
• Funnel
• paper 
• spatula
• Cromatography paper
• mortar
• Petri dish
• spinach 
• Calcium carbonate

PROCEDURE

• Wash the spinach and cut them, then dry them.
• Grind the spinach in a mortar and add 50 ml of ethanol and a small amount of calcium carbonate, which fits at the tip of the spatula.
• Filter the mixture into a funnel with filter paper.
• Put a part of the mixture in a Petri dish (3-4 mm high).
• Put a rectangle of paper for chromatography in form V, and wait for the pigments to be differentiated.

RESULTS

The different pigments that are from bottom to top are:

Beta carotene (red - orange)

Chlorophyll alpha (bluish green)

Chlorophyll beta (bright light green)

Xanthophylls (yellow)

QUESTIONS

Why do we add calcium carbonate?

To avoid the degradation of the pigments.

Which is the colour of every pigment

• Beta carotene (red - orange)
• Chlorophyll alpha (bluish green)
•  Chlorophyll beta (bright light green
• Xanthophylls (yellow)

For what purpose it does have different colour pigments?

To know the different solubility of photosynthetic pigments in an organic solvent such as ethanol, which is miscible with water.

Why do they separate in the paper? 

When the alcohol is placed with the pigments dissolved by a chromatography paper, the different pigments are separated as the medium is increasingly poorer in ethanol and richer in water.