TECHNIQUES Interpret results for the following instruments used in the Microbiol
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TECHNIQUES Interpret results for the following instruments used in the Microbiology Lab ? Microscopy: Name Microscope parts and their functions O Demonstrate how total magnification is calculated O Recall the function of Hemocytometer in Cell count Culture media: Identify plates of Nutrient Agar, Casein Agar, Starch Agar Eosin Methylene Blue, and agar slants O Cultures: techniques for making and keeping cultures, streak plates How to use Spectrophotometer and Microbial Growth CONTROLLING MICROBIAL GROWTH Interpret how the following affect Microorganisms O Heat/Temperature Disinfectants O Antibiotic sensitivity O Radiation: Damage by and protection from Ultraviolet light STAINS & STAINING Recall Stains and Reagents used for Gram Stain Acid-fast stain Negative staining and the Capsule Stain Endospores stainExplanation / Answer
The compound microscope has two systems of lenses for greater magnification, 1) the ocular, or eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object.
Basic parts of the microscope:
Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source used in place of a mirror. If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser.
Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. You would only need to adjust this if you were using very thin slides and you weren't able to focus on the specimen at high power.
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X). I
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.
Haemocytometer-
It is a special type of microscope slide consisting of two chambers, which is divided into nine (1.0mm x 1.0mm) large squares which are separated from one another by triple lines. The area of each is 1mm². Cover glass is supported over the chambers at a height of 0.1mm. Because of that the entire counting grid lies under the volume of 0.9 mm² on one side. The cell suspensions are introduced into the cover glass. The hemocytometer is placed on the microscope stage and the cell suspension is counted.
The glass microscope slide has a rectangular indentation that creates an 'H' shaped chamber at the centre. This chamber is engraved with a laser-etched grid of perpendicular lines. Two counting areas with ruled grids are separated by the horizontal groove of the 'H'. There is also a very flat, reusable cover slip. The glass cover slip is held at 0.1 mm above the surface of the counting areas by ground glass ridges on either side of the vertical grooves of the H shape. The device is carefully crafted so that the area bounded by the depth and lines of the chamber is also known. Because the height is constant, the volume of fluid above each square of the grid is known with precision.
The hemocytometer is used by putting the cover slip on the device, and filling the space with a liquid containing the cells you want to count. There is a "V" or notch at either end which is the place where the cell suspension is loaded into the hemocytometer. The fluid is usually drawn into the space by capillary action. A cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height. In addition, the grid arrangement of squares of different sizes allows for an easy counting of cells. It is possible to identify the number of cells in a specified volume by this method.
Staining Type # 3. Gram Staining:
It is one of the most important and widely used differential staining techniques in microbiology. This technique was introduced in 1884 by Danish Physician Christian Gram.
In the first step the smear is stained with basic dye crystal violet (Primary stain) followed by treatment with iodine solution functioning as mordant.
Iodine increases the interaction between cell & dye so that cell stains strongly. The smear is next decolourized by washing with ethanol or acetone. This step generates the differential aspect of Gram stains. Gram positive bacteria retain crystal violet and become colourless.
Finally smear is counter-stained with a simple basic dye different in color from Crystal violet. Safranin is the most common counter stain which colours Gram negative bacteria pink to red and leaves Gram positive bacteria dark purple.
The differences in staining responses to the Gram stain can be related to chemical and physical difference of cell walls. The Gram-negative bacterial cell wall is thin, complex multilayered structure and contains relatively high lipid contents in addition to protein and mucopeptide.
The higher amount of lipid is readily dissolved by alcohol, resulting information of large pore in the cell wall, thus facilitate leakage of crystal- violet – iodine (CV-I) complex which results in decolorization of the bacterial cell.
Which later take counter stain and appears red. In contrast the cell wall of gram+ve bacteria is thick and chemically simple, composed mainly of mucopeptides. When treated with alcohol, it causes dehydration and closure of cell wall pore, thereby does not allow the loss of (CV-I) complex and cell remain purple.
Staining Type # 4. Acid Fast Staining:
It is another important differential staining procedure. It is most commonly used to identify Mycobacterium spp. These bacteria have cell wall with high lipid content such as mycolic acid -a group of branched chain hydroxy lipids, which prevent dyes from readily binding to cells.
They can be stained by Ziehl-Nulsen method, which uses heat and phenol to derive basic fuchsin into the cells. Mycrobacterium spp. were penetrated with basic fuchsin, not easily decolourized by acidified alcohol (acid alcohol) and thus are said to be acid fast.
Non acid fast bacteria are decolourized by arid alcohol and thus are stained blue by methylene blue counter stain.
Staining Type # 5. Endospore Staining:
Spore formation takes place in some bacterial genera to withstand unfavourable conditions. All bacteria cannot form spores, only few bacterial genera including Bacillus, Clostridium, Desulfotomaculum produce sporulating structure inside vegetative cells called endospore.
Endospore morphology and location vary with species and are valuable for identification Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization.
In the Schaffer-Fulton procedure, endospores are first stained by heating bacteria with malachite green, which is very strong stain that can penetrate endospores. After malachite green treatment, the rest of the cell is washed free of dye with water and is counter-stained with safranin. This technique yields a green endospore with red vegetative cell.
Principle of Capsule Staining
Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained.
Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. India ink is dif?cult to obtain nowadays; however, nigrosin is easily acquired.
A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background.
Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope.
Reagents used for Capsule Staining
Crystal Violet (1%)
Crystal Violet (85% dye content) = 1 gm
Distilled Water = 100 ml
Nigrosin
Nigrosine, water soluble = 10 gm
Distilled Water = 100 ml
Principle of Negative Staining
Negative staining requires an acidic dye such as India Ink or Nigrosin.
India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.
Reagents of Negative Staining
India ink
Nigrosin
Nigrosin 100 gm/L, Formalin 5 ml/L in water
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