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basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains
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The student will Use aseptic techniques. Follow oral and written instructions and manage time in the lab efficiently. Apply correct terminology regarding microbiological techniques, instruments when making observations. Use the bright field light microscope to view microbes under oil immersion, make accurate observations and appropriate interpretations and store the microscope according to lab procedures. Properly prepare a bacterial smear for accurate staining and describe the chemical basis for simple staining and negative staining.
A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. (OpenStax CNX, 2018) This alteration of light as it passes through the specimen and into the lens system of the microscope creates the image. Bacterial cells are so small and thin that they alter the light very little and, consequentially, are virtually transparent or invisible. In other words, the cell lacks contrast with the background. Staining the specimen will artificially create contrast so that cells may be visible. Along with magnification and resolution, contrast is the third element that contributes to the amount of detail that can be observed. Some staining techniques involve the application of only one stain to the sample; others require more than one. In simple staining , a single stain or dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. (OpenStax CNX,
Stains, or dyes, aqueous slat solutions. They contain a positive ion and a negative ion one of which imparts the color. The chromogen (sometimes called the chromophore) is the colored ion. If the chromogen is positively charged, the counter ion is the negatively charged hydroxide ion OH-^ making this type of stain a basic stain. (Here the term basic does not mean elementary. Instead, basic refers to chemical property of having excess hydroxide ions.) Because most cell structures have a negative charge, the positively charged, colored Figure 1 A basic stain has a positive chromogen and the negative hydroxide ion. The colorless cell has a net negative charge.
chromogen will be attracted to negatively charged cell. The result of this interaction is the cell retaining the color and the background remaining uncolored. OpenStax Microbiology calls this a positive stain , a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. (OpenStax CNX, 2018) If the chromogen is negatively charged, the counter ion is H+. This makes the stain an acidic stain. In this case the negative chromogen will be repelled by the negatively charged cell and the cell will remain colorless against a dark background. The exercise, Negative Staining, discusses this type of staining process in greater detail. Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin , crystal violet , malachite green , methylene blue , and safranin typically serve as positive stains. (OpenStax CNX, 2018) Before cells are stained with a basic dye, they must be fixed to the slide so that they do not wash away with the excess stain. The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating ( heat fixing ) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation. (OpenStax CNX, 2018) To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear or emulsion ), and the slide is then briefly heated over a heat source. (OpenStax CNX, 2018) In this lab, you will use the following procedure to heat fix a sample for staining.
how the cell divides and how the culture is grown. After division, daughter cells may completely separate or remain attached. Cells also divide in a characteristic plane. If successive divisions occur parallel to one another and daughter cells remain attached, the result is a chain. If they divide perpendicular to each other, the result may be a tetrad or sarcina form. If the plane of division is random, grape-like clusters may form. A species grown in a broth may appear to have a different arrangement compared to the same species grown on solid media. How well you spread out the cells on the slide also influences the microscopic appearance. You will not be able to correctly determine cell arrangement if the smear is too thick and all you see is an amorphous lump. This is another reason you want a thin, well spread out smear. When recording observations of morphology and arrangement of a pure culture, you will want to form an opinion on the most prevalent arrangement. It is also permissible to record more than one arrangement if that is what you observe. The important thing here is that you record what you actually observe under the microscope. Unless your smear is too thick, you will not be penalized for cells that do not appear arranged as we expect. If, however, your drawings and/or observations do not match what is seen through the microscope lens, your scientific credibility may be questioned.
Making accurate observations is one of the learning objectives of this course. Accurate drawings are an important part of your observations. Follow these guidelines for drawing cells. Because details of the microscopic appearance are important, your drawings will not be to scale. When you make a drawing, turn your attention to the group of cells you are drawing and magnify it in your mind’s eye. Cells should be drawn large enough so that you can show an accurate shape and arrangement. Each cell should measure about 4 mm in diameter on your paper. Your drawing should capture the relative length and width of the cells. Most cocci are not perfect spheres. Figure 3 Common arrangements of cocci (Petersen, 2016) Figure 4 Common arrangements of bacilli (Petersen, 2016) Figure 5 Spirals (Petersen, 2016)
Some are a bit elongated. If this is what you see, try to capture that. Some bacilli are long and skinny and some are short and stubby looking. There should be no pointy corners, no open circles, no extra “tails” and no overlapping rings. Take a little extra time to think about these things when drawing. If you think you are looking at bacillus with a pointy corner or a flagellum, check with the instructor. If this is something real, the instructor does not want to take off if you are making accurate observations. Your instructor also wants to make sure you are looking at cells and not dust or other debris on the slide. Experiment/Exercise
4 microscope slides Safranin stain Methylene blue stain Crystal violet stain
Fresh overnight broth cultures E. coli Staphylococcus epidermidis Cultures on solid media (environmental zig zag plate and/or other pure culture t-streak plates)
Lab Report: Simple Staining Name ______________________________ Lab Section __________
Organism (Be sure to write the name correctly.) Stain used and color Drawing (See section on Cell Drawings for guidelines.) Morphology and Arrangement instr initials N/A N/A N/A