Project 5: STAINING IN MICROBIOLOGY
The purpose of simple stain is to highlight the entire microorganism so that that cellular shapes and basic structures are visible. In simple stain only one dye is used. Differential stains react differently with kinds of bacteria. This staining technique is used to distinguish between different types of bacteria. Two differential stain procedures are used: the Gram stain and the acid-stain. Using the Gram stain can be classified into 2 groups: Gram positive and Gram negative. The procedure consists of flooding the bacteria with 2 dyes consecutively then attempting to decolorize the bacteria with alcohol-based decolorizing agent, this will give the following result: the bacteria that the purple color will be classified as gram-positive and the bacteria that lost the color as a result of decolorization and became pink will classified as gram-negative. Acid-fast stain is used to identify bacteria that have a waxy material in their cell walls. Mycobacterium tuberculosis, the pathogen that causes tuberculosis and Mycobacterium leprae, the causative agent of leprosy are identified thanks to acid-fast stain. Special stain techniques are used to isolate and view specific parts of the microorganism. The special stain shows cell structures such as flagella, endospores and capsules.
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| Simple stain result |
2. Differential & Special Staining:
a. Why are Special stains not classified as Simple stains?
A simple stain is a single basic dye applied to all bacteria. This method is use to create contrast on the microorganisms to determine the cellular shape and arrangement. Whereas special stains are used to stain and isolate particular parts of microorganisms such as endospores, flagella and reveals the presence of capsules and aid pathologists with diagnosis.
b. Contrast Special Staining to Differential Staining
Special staining results in…
- Negative- used to detect the presence of capsules
- Endospores – used to detect the presence of endospores in the bacteria. Endospores cannot be stained by ordinary methods, it uses a special stain, it uses schaeffer fulton endospore stain.
- Flagella- uses a tedious and delicate staining procedure. Uses a mordant and Carbolfuchsin stain to buildup diameter to make them visible. Number of flagella and arrangements aid diagnosis.
Differential Staining results in…
- Gram Positive and Gram Negative – used to distinguish different kinds of bacteria with crystal Violet stain – which stains bacteria walls purple or bark blue. And Gram Negative which stains with the counterstain Safranin and stains the bacteria red or pink and Acid-Fast, which differentiates species of Mycobacterium from other bacteria. It uses a stain called carbolfuchsin. Mycobacterium include 2 important pathogens Mycobacterium tuberculosis which causes tuberculosis and Mycobacterium leprae, which causes leprosy.
c. Under what conditions can Special Staining be Differential Staining?
When structures are resistant to ordinary methods such as simple staining and gram staining because the dyes do not penetrate within the cell walls, for example endospores.
3. Staining & Structures: (For this Section, refer to Chapter 3)
a. Identify 3 Bacterial Structures for which Special Staining is used.
Three bacterial structures for which Special Staining is used are flagella, bacterial spores and capsules. These and others such as inclusions (organelles inside the cells) are stained using special staining techniques because simple staining techniques only will not improve their visibility under the microscope.
b. Describe what bacteria use each structure for.
1. Flagella is used by bacteria for swimming and movement.
2. Endospores are a mechanism of survival rather than a mechanism of reproduction. Endospore forming bacteria use it to protect themselves against harsh environments.
3. Capsules, which includes "slime layers" and glycocalyx, is used generally for various purposes, among them: for attachment to surfaces; protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients or protection against desiccation.
c. Name the dye used to stain each structure.
Flagella is stained by using the special stain techniques, which will highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Fluorescent protein stain, NanoOrange from Molecular Probes is also used.
Malachite green is used with heat to force the stain into the cells and give them color and the counterstain safranin is added to the non spore forming bacteria to identify the spore forming and the non spore forming bacteria. Also known as the Schaeffer-Fulton method.
Capsules can be visualized by means of two methods; the Positive Capsule Staining or the Negative Capsule Staining method. The PCS or Anthony Method uses as primary stain Crystal violet in combination with a decolorizing agent, copper sulfate. As a result the capsular material absorbs the light blue color of the copper sulfate in contrast to the purple bacterial cell. The NCS method uses acidic stains such as Nigrosin, that will not penetrate the bacterial cells and will create a dark back ground. The bacteria will appear as unstained with a clear area around them, which represents the capsule.
d. Place the dye in 3 (c) into a Chemical Group.
1. Uses a variety of solutions, including an acidic solution
2. Basic
3. Basic for PCS and Acidic for NCS
4. Bacterial Structures and Special Staining: (See Structures in 3a)
a. In what different forms (if any) does each structure exist in bacteria?
In Bacteria, you may find the same structures in different forms. For the first example, we will talk about the capsule. The capsule is different depending on the type of bacteria. Bacillus Anthracis for example is composed of a polypeptide (polyglutamic acis),while in Yersinia Pestis you will find that a capsule is produced by mixed amino acids.
When it comes to Flagella, you will find that not all bacteria are motile. Flagella is what helps bacteria move around. You will also find that some bacteria have more flagella than others. Here are different numbers of flagella, as well as their location.
Flagella arrangements are:
1. Monotrichous - a single flagellum at one pole (also called polar flagellum) E.g. Vibrio cholerae
2. Amphitrichous - single flagellum at both poles. Eg. Spirilla
3. Lophotrichous - two or more flagella at one or both poles of the cell E.g. Spirillum undula
4. Peritrichous - completely surrounded by flagella E.g. E.coli
b. Identify the location of each structure in the Bacteria
c. How would you use location of a structure for bacterial identification?
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| Gram stain |
If you look at question 4a in this presentation you will notice that we wrote about flagella and how the location varies on different bacteria. You will also be able to notice that capsules are different as well. Bacteria as a whole may have very similar structures, but may vary depending on what bacteria it is. some have tougher capsules than others as well as you will see that same may be slimy while some are not.
5. Bacterial Structures & Disease:
(Consider sharing 5 a-d, in this section, among all Group members)
Name structures from 3 (a) that enhance ability of these medically-
important bacteria to infect or cause disease and briefly describe each:
a. Bacillus Anthracis causes anthrax, a disease of cattle, sheep and horses that can be transmitted to humans. It is often mentioned as a possible agent of biological warfare.
S Layer - Part of the cell envelope commonly found in bacteria. A monomolecular layer of protein or glycoprotein. It enhances the ability of the bacteria in the following ways:
-protection against phagocytosis
-resistance against low pH
-barrier against high-molecular-weight substances (e.g., lytic enzymes)
-adhesion (for glycosylated S-layers)
-stabilization of the membrane
-provision of adhesion sites for exoproteins
-provision of a periplasmic compartment in Gram-positive prokaryotes together with the peptidoglycan and cytoplasmic membranes
Capsule- A polymer of amino acids unlike most other bacteria which have polysaccharide capsules. The cell excretes the capsule for protection and virulence. The nontoxic capsule had an important role for infection establishment while the disease phases are mediated by the toxin.
Endospores- Bacillus are rods that produce endospores. Endospores can live in the soil and other inhospitable environments for many years because spores are very resistant to physical and chemical damage. Spores that germinate and grow produce a fatal toxin which enable Bacillus Anthracis.
b. Pseudomonas Aeroginosa- gram negative rod shaped asporogenous and monoflagellated bacterium.
Plasmids- have catabolic enzymes encoded by genes and can use exotic substance like toluene, camphor and hydrocarbons of petroleum as primary carbon and energy sources. Permits survival of microorganism very diverse and challenging environment. Allows resistance to antibiotics and is significant to lifestyle as pathogen.
Flagellum- very important during early stages of infection it allows attachment to and invade tissues of host. Contributes to its ability to adhere to mucosal surfaces and epithelial tissue.
Sideophores (pyochelin and pyoverdin) bind to iron and bring iron into cell. Iron deprivation of infecting pathogen are key part of human defense mechanism.
c. Clostrudium difficile- inhibits intestinal tract and causes serious diarrhea.
5 amino acids (leu lle Pro Trp and Val) allows energy metabolism and glycogen increase. Allows amino acid fermentation in order to create a source of energy that can utilize sugar which expresses toxins and which prioritizes growth over toxic production so you can grow and colonize as much as possible before infection.
Two S-layer most importantly it increases the bacteria to adhere and adapt.
It also ensures:
- protection against phagocytosis
-resistance against low pH
-barrier against high-molecular-weight substances (e.g., lytic enzymes)
-adhesion (for glycosylated S-layers)
-stabilization of the membrane
-provision of adhesion sites for exoproteins
-provision of a periplasmic compartment in Gram-positive prokaryotes together with the peptidoglycan and cytoplasmic membranes
Endospores -lives in the soil and other inhospitable environments for many years because spores are very resistant to physical and chemical damage. Spores that germinate and grow produce a fatal toxin which strengthen Clostridium Difficle.
D. Streptocuccus mutans- gram positive bacteria that causes tooth decay
Cell wall- The cell wall is composed of peptidoglycan (murein) and teichoic acids that prevent osmotic lysis of cell protoplast and confer rigidity and shape on cell
Capsule - S. mutans grows and synthesizes a dextran capsule which binds them to the enamel and forms a biofilm of some 300-500 cells. In the metabolism it is able to cleave sucrose (after consuming carbohydrates provided by the animal diet) into glucose plus fructose. The fructose is fermented as an energy source for bacterial growth. Create dental plaque
Slime layer- attaches to the tooth surface and form a biofilm. It attaches to the surface, produces slime, divides and produces micro colonies within the slime layer, and constructs a biofilm. It adheres specifically to the pellicle of the tooth by means of a protein on the cell surface. The glucose is polymerized into an extracellular dextran polymer that cements. The dextran slime produces lactic acid within the plaque that decalcifies the enamel and leads to dental caries or bacterial infection of the tooth.
References:
http://microbewiki.kenyon.edu/index.php/Bacillus_anthracis
http://microbewiki.kenyon.edu/index.php/pseudomenas_aeroginosa
http://ncbi.nlm.nih.gov/clostridium_difficle
http://microbewiki.kenyon.edu/index.php/Streptocuccus_mutans
