cell & molecular biology

Experiment 2

Introduction

The basic principles of histology and cell structure serve as an important background for the study of specific cells, tissues, and organ systems. This laboratory serves as an introduction to the rest of the course – you will use the principles you learn here in every subsequent lab.

Histological Staining Methods

Cells are difficult to see by light microscopy. To help us visualize the structure and features of cells, dyes are used to impart a particular color to cells. These dyes react with different chemical features of proteins, nucleic acids and carbohydrates and can be used to highlight certain cellular structures.

Hematoxylin and eosin (H&E) staining is the standard method of staining in histology. Hematoxylin is a basic dye (positively charged) that binds to negatively charged DNA and RNA and is blue in color. Eosin is pink in color and is an acidic dye (negatively charged) that binds to positively charged particles like the mitochondria and many components of the cytoplasm. Positively charged structures are therefore said to be "eosinophilic." While H&E staining is widely used, it is limited in its ability to differentiate between cytoplasmic organelles and many other tissue components.

The periodic acid-Schiff method (PAS) is useful for staining structures rich in polysaccharides (glycogen), mucopolysaccharides (ground substance, basement membrane, mucous), glycoproteins (thyroglobulin), and glycolipids. In this method, periodic acid oxidizes 1,2-glycols and 2,2-amino alcohols to aldehydes, which are then stained reddish purple by the Schiff reagent.

Osmium staining blackens lipids and stains the Golgi apparatus under the light microscope. It is also used as a fixative for electron microscopy.

The Cell

The cell is the fundamental unit of living organisms. Cells grow, adapt to their environment and reproduce, processes which characterize life. Cells also assemble into groups to form complex structures. Cells and the extracellular material they make comprise the tissues of our bodies. Several different types of tissues then organize to form organs. The cells in an organ communicate and work together to perform the functions of that organ.

The cell is limited by the cell membrane, also known as the plasma membrane. The cell’s content is divided into two main compartments: the nucleus and the cytoplasm that surrounds the nucleus. Cytoplasm is further divided into organelles, cytosol and inclusions. Organelles are assemblies of specific macromolecules organized to carry out complex functions. Many organelles are surrounded by a membrane that separates their internal environment from the cytoplasm. Membrane-bound organelles concentrate enzymes and reactants, increasing biochemical efficiency and isolating harmful proteins and molecules from the rest of the cell. Cytosol is a gel-like substance that contains dissolved macromolecules, organic compounds and ions. In addition, cytosol contains the cytoskeleton (microtubules, actin filaments and intermediate filaments) that organize the organelles and provide mechanical support. Lastly, inclusions are insoluble substances in the cytosol such as glycogen and lipid droplets.

Dimensions of Cells

One of the most important skills that you will take away from this course is the ability to understand the dimensions of cells and their subcellular components. When it comes to size, there are three key facts that you must remember:

• Most eukaryotic cells have a diameter of 7-20 microns (µm), while prokaryotic cells are smaller (0.2 - 5 µm).

• Red blood cells have an average diameter of 7.2 µm, and are a useful reference for size approximation.

• The approximate diameter of a secretory granule is 1 µm.

With the unaided eye, one can only see exceptionally large cells, such as the human ovum, which has a diameter of 100 µm. Therefore, we must use a microscope to visualize cells in a tissue. Microscope images in this course come from the light microscope (magnification up to 400x) and the electron microscope (magnification up to 500000x). The limit of resolution of the light microscope is 0.2 µm, while the practical limit of resolution of the electron microscope is about 1 nanometer (nm). Thus, light microscopes allow one to visualize cells and their larger components such as nuclei, nucleoli, secretory granules, lysosomes, and large mitochondria. The electron microscope is necessary to see smaller organelles like ribosomes, macromolecular assemblies, and macromolecules.

With light microscopy, one cannot visualize directly structures such as cell membranes, ribosomes, filaments, and small granules and vesicles. Using an appropriate staining technique, however, makes aggregates of these smaller structures or the regions they occupy visible by light microscopy. For example, while it is not possible to see the membranes and ribosomes that compose the surface of the rough endoplasmic reticulum, these structures are represented in light microscope slides by clumps of basophilic material in certain regions of the cell. It is important to be able to correlate the appearance of cells at the light microscope level with the structures visible in electron micrographs

Organelles

The cell membrane is about 10 nm thick and cannot be resolved by the light microscope. The limits of the cell can be visualized with the light microscope when there is a heavy concentration of glycoproteins or proteoglycans at the cell surface. The presence of large amount of carbohydrate on the cell membrane makes Periodic acid-Schiff (PAS) an effective method to stain the cell membrane.

The nucleus is limited by a nuclear envelope that consists of a two membrane bilayers and nuclear pores that allow passage of material into and out of the cell. Chromatin, complexes of DNA and protein, is the major component of the nucleus and consists of two histological structures. Heterochromatin is condensed chromatin scattered throughout the nucleus or accumulated along the inner surface of the nuclear envelope. Heterochromatin is considered transcriptionally inactive. In contrast, euchromatin in abundant in cells engaged in transcription. Euchromatin is dispersed and not easily stained.

The nucleus often contains one or more nucleoli that are spherical or oval bodies composed chiefly of ribonucleoproteins. Nucleoli are usually stained with basic dyes because of their high RNA content and are prominent in cells that are actively participating in protein synthesis.

The endoplasmic reticulum (ER) is a system of interconnected membranous sacs, channels, or cisternae in the cytoplasm. It has two subtypes: rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER). The RER is a ribbon-like structure surrounding the nucleus near the base of the cell. Its surface appears rough due to the ribosomes attached to its membrane and it is the first organelle into which membrane-bound or extracellular proteins are inserted. SER lacks ribosomes and participates in lipid synthesis and detoxification.

The Golgi apparatus is a system of membranous cisternae and vesicles arranged in stacks near the nucleus. The Golgi processes and modifies sugar side chains on proteins that are being secreted or destined for the plasma membrane or other membrane-bound organelles like the lysosome. Therefore, the Golgi apparatus is particularly prominent in cells synthesizing large amounts of glycoproteins and proteoglycans, such as goblet cells that produce mucous in the gut epithelium. The Golgi can be stained with osmium or silver stains and appears as a network of black-staining tubules or clusters of granules.

Secretory vesicles or granules usually contain specific substances synthesized by cells that are exported to the extracellular medium. They include zymogen granules, mucous droplets, and mast cell granules.

Mitochondria are organelles that vary greatly in number, size, and shape between different cells. They are unusual in that they contain their own mitochondrial DNA and ribosomes; mitochondrial proteins come from genes in both the nuclear and mitochondrial DNA. These organelles also undergo self-replication. Structurally, two features characterize mitochondria: double bilayer membranes, and cristae, folds that project from the inner membrane into matrix.

Lysosomes also vary in size and shape, but can be recognized as membrane-bound organelles containing granular material. There are more than 40 lysosomal enzymes that are active at acidic pH.

4 considerations before selecting a dye:

1. Consider the membrane permeability of the dye. DAPI has a low membrane permeability and is suitable for staining fixed cells. Hoechst (e.g., 33342) has a higher membrane permeability and can also be used in live cells.

2. Think about your fluorescent wavelengths. For example, if you were identifying a protein of interest at the lipid membrane using a secondary antibody emitting light in the green channel, it is better to choose a lipid dye that emits light in the far-red channel. That way, you can avoid them interfering with each other.

3. Determining the concentration of your dye is crucial. Too low a concentration will not provide a strong visualization, while too high a concentration will be toxic to your samples.

4. Understand exactly how the dye works. Mitochondrial dyes like Rhodamine are dependent on membrane potential. They can only be applied to live cells, making them useful tools to analyze health and cell viability. Lysosome dyes require the acidic environment of this organelle to work properly. They also work best in live cells.

Common stains and their uses are as follows:

1. Iodine: Stains carbohydrates in plant and animal specimens brown or blue-black. Stains glycogen red.

2. Methylene blue: Stains acidic cell parts (like nucleus) blue. Use on animal, bacteria and blood specimens. Can be used as a substitute for Janis B Green.

3. Eosin Y: Stains alkaline cell parts (like cytoplasm) pink. Use on plants, animals and blood. Can be used as a substitute for Congo Red and Carmine.

4. Toluidene blue: Stains acidic cell parts (like nucleus) dark blue. Good to show mitosis in plant cells.

5. Wright’s stain: Stains red blood cells pink/red.

6. Crystal Violet: Stains bacteria purple.

7. Aceto-orcein: Biological stain for chromosomes and connective tissue.

8. Sudan III: Biological stain used as a lipid indicator.

In the following experiment we will discuss about staining of microbial cell using Gram staining method and plant cell by making use of Safranin staining method.

1.Gram staining

Gram staining:

Introduction:

The Gram’s staining is performed to distinguish between two large groups of bacteria depending on their cell wall constituents. These bacterial groups are named as gram-positive and gram-negative bacteria. The presence of a thick peptidoglycan layer in the gram-positive bacteria causes it to appear purple while the thinner peptidoglycan layer in gram-negative bacteria cause it to appear pink after Gram’s staining. The safranin serves as a counterstain in gram’s staining.

Principle

The Gram stain is a differential staining technique most widely applied in all microbiology disciplines laboratories. It is one of the most important criteria in any identification scheme for all types of bacterial isolates. Different mechanisms have been proposed to explain the gram reaction. There are many physiological differences between gram-positive and gram-negative cell walls. Ever since Christian Gram has discovered Gram staining, this process has been extensively investigated and redefined. In practice, a thin smear of bacterial cells is stained with crystal violet, then treated with an iodine containing mordant to increase the binding of primary stain. A decolourizing solution of alcohol or acetone is used to remove the crystal violet from cells which bind it weakly and then the counterstain (like safranin) is used to provide a colour contrast in those cells that are decolourized. Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.In a properly stained smear by gram staining procedure, the gram-positive bacteria appear blue to purple and gram negative cells appear pink to red.

Requirments:

Gram's Crystal Violet (S012)

(Solution A) - Crystal Violet -2.000 gm and Ethyl alcohol, 95%- 20.000 ml

(Solution B) - Ammonium oxalate- 0.800 gm and Distilled Water- 80.000 ml

Solution A and B are mixed and stored for 24 hours before use. The resulting stain is stable.

Gram's Iodine(S013)

Iodine- 1.000 gm, Potassium iodide- 2.000 gm and Distilled water- 300.000 ml

Gram's Decolourizer (S032)

Ethyl alcohol, 95%- 50.0 ml and Acetone 50.0 ml

Safranin,0.5% w/v (S027)

Safranin O-0.500 gm and Ethyl alcohol, 95%- 100.000 ml

Procedure:

1. Prepare a thin smear on clear, dry glass slide.

2. Allow it to air dry and fix by gentle heat.

3. Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of known gramnegative organisms,use less crystal violet).

4. Drain the stain.

5. Flood the smear with Gram’s Iodine (S013). Allow it to remain for 1 minute.

6. Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear. 7. Wash with tap water.

8. Counter stain with 0.5% w/v Safranin (S027). Allow it to remain for 1 minute.

9. Wash with water.

10. Allow the slide to air dry or blot dry between sheets of clean bibulous paper and examine under oil immersion objective.

Results:

2. Safranin staining

Safranin Staining:

Introduction:

The safranin stain is a cheaper and safer-lab stain. It is a certified stain for chromosomes. It can be used to stain animal as well as plant cells for better cytological and histological analysis. It allows easy and rapid detection of the plant's vascular tissue.

In the plant cell, the cell as well as the background is transparent and it is difficult to visualise the cells as such. Safranin is a dye which can be taken up by a cell and it gives a pink colour. safranin staining imparts colour to the cell or its components and enhances its contrast and makes it easier to see the structure of the cells.

Requirements:

Plant tissue (stem/leaves), Blade, 1% safranin solution, glycerol, ditilled water, paint brush, watch glass, slides and cover slips

Procedure:

1. Take a clean and grease free slide and wash it.

2. Collect appropriate plant tissue and wash it.

3. Take a sharp blade and prepare thin sections of plant tissue and suspend them in watch glass containing distilled water.

4. Then, transfer those sections in the watch glass filled with 1% safranin solution. Allow it to stain for 30s to 1 min.

5. Now, collect the stained section and mount it on clean glass slide, add a drop of glycerol to cover tissue and absorb excess of stain using tissue paper. At the end, cover the section with glass cover slip.

6. Finally observe the slide under microscope in 4X and 10X resolution.

Observations:

Reference Material

Gram staining: http://himedialabs.com/TD/K001.pdf

Cell staining: http://vlab.amrita.edu/?sub=3&brch=187&sim=324&cnt=1

Questions

1. Which stain can be used stain animal cell?

2. Give examples of other stains use to stain plant and microbial cell?

3. What is the differnce between gram positive and gram negative microbes?