Compare and contrast three differential stains discussed in

Compare and contrast three differential stains discussed in lab or class. Include the primary stain, counter stain, and the purpose or use they serve in the field of microbiology. Describe a typical gram negative cell, in terms of structure (internal and external). In your description include the cell wall, motility appendages, and at least three internal structures. Compare and contrast three appendages commonly found in bacteria. Include their appearance and their purpose.

Solution

if we are using more than one dye or stain to identify or differentiate bacterial structures,it is a differential stain. Examples for differential staining are, Gram staining, Ziehl Neelson staining and endospore staining.

Gram staining- helps to differentiate gram positive and gram negative bacteria. Gram positive bacteria has a very thick layer of peptidoglycan but gram negative bacteria has a thin layer of peptidoglycan and a thick layer of lipopolysacharide. In gram staining , primary stain- crystal violet and the mordant-iodine form a complex and colur the bacteria. but the decolouriser - ethyl alcohol, shrinks the cell and removes the crystal violet and iodine complex from the gram negative cell as they dont have peptidoglycan but the complex remains in the gram positive cells. Now the Counter stain- Safranin stains the gram negative bacteria and not gram positive bacteria. So at the end Gram positive bacteria will be in violet or purple colour and gram negative bacteria in red or pink colour.

Ziehl-Neelson staining / Acid fast staining- Helps to differentiate bacteria which has a mycolic accid , waxy layer in the outer surface of the cell wall. Examples are Mycobacteria and Nocardia. In this staininng method, the primary stain- Carbol fuchsin is added to the smear through a blotting paper while steaming. Remove the blotting paper and wash the slide. Acid fast bacilli retains the red colour of carbol fuchsin. Now the decolouriser- acid alcohol will remove the carbol fuchsin from the non acid fast bacilli. Counter stain- crystal violet is added, and it stains the non acid fast bacilli puple.

Spore staining- helps to stain bacterial spore, and differentiate vegetative cell and spores. Add the primary stain - Malachite green and boil this to help penetrate the dye to the spore coat. After rinsing the slide with water add the counter stain - safranin. All the spores appears green in colour and vegetative cell will be in pink colour.

2. GRAM NEGATIVE CELL WALL- It consists of a single layer of peptidoglycan as outer layer and a thick layer of lipopolysacharide, which causes the endotoxicity along with protein and phospholipids. The peptidoglycan in the outer membrane with porins is permeable and has lipoprotein attached with it. there is a periplasmic space between the outer membrane and the inner plasma membrane. This space contains the binding proteins for aminoacids , sugars, vitamins etc. In gram negative bacteria Flagella has four supporing rings compared to gram positive.

3. BACTERIAL APPANDAGES- three major bacterial appandages are glycocalyx, flagella and pili.

Glycocalyx- helps to adhere to surfaces, helps from drying out as it can retain moisture, protects against other virus and antibacterial agents, and it protects from phagocytosis.

Flagella- it is a locomotory organ. it has the flagellin protein arranged spirally. it turns clockwise and anticlock wise and helps the bacteria to move forward. It helps the bacteria to move towards nutrition, oxygen and light. There could be single or multiple flagella.

PILI- Made of protein pilin. it is a tube like protrusion outward to the cell. Helps to recoganise surface receptors, and attachment. It also helps in conjugation to transfer the plasmid and are called sexpili. they play important role in the drug resistance.