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Simple Staining Lab Report, Lab Reports of Biology

basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains

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SSt Simple Staining
Learning Objectives
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.
Background/Theory
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,
2018)
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.
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SSt Simple Staining

Learning Objectives

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.

Background/Theory

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.

Smear and Heat Fixing Procedure

  1. With a wax pencil, divide a clean microscope slide into sections, one for each sample. Label each section with an abbreviation for each sample. No more than three samples per slide.
  2. To make a smear from a broth culture, aseptically obtain a loopful of the culture from the tube and spread it on the slide over an area about the size of a nickel or larger. The larger the area, the more space you will have to view your cells.
  3. Making a smear from a sample growing on solid media, can be tricky. First, you will need to add some liquid to the slide first so that you have something to spread the cells in. Placing an entire drop of water on the slide, while not technically incorrect, will delay the process considerably. A full drop of liquid from your water bottle, approximately 50 μl, will take a long time to air dry. A loopful, closer to 10 μl, of water is plenty and will dry fairly quickly. Second, students tend to get way too many cells. So many cells that they remain in large clumps. This creates some problems. Stains cannot make contact with all cells equally leading to erroneous results when using a differential staining technique. In addition, one cannot adequately observe morphology and arrangement if the cells form large clumps and individual cells cannot be seen. The following steps are designed to minimize these difficulties. Figure 2 A basic stain result. A colored cell with a colorless background..

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.

Cell Drawings

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

Materials per student pair

4 microscope slides Safranin stain Methylene blue stain Crystal violet stain

Cultures

Fresh overnight broth cultures E. coli Staphylococcus epidermidis Cultures on solid media (environmental zig zag plate and/or other pure culture t-streak plates)

Procedure

  1. Each person will make 2 identical slides as follows.
  2. Either you or your partner will divide a slide in half with a wax pencil. On one side place a smear from the E. coli broth. On the other side place a smear using one of the pure culture t-streak plates or the environmental sample (SI exercise). REMEMBER the procedure is different for taking cells from solid media versus liquid media. Let both smears dry BEFORE heat fixing. See the section Smear and Heat Fixing Procedure. Repeat for the second slide.
  3. The other person will make 2 slides, each with S. epidermidis broth on one side and a different pure culture from a plate on the other side.
  4. Each person pick a different stain and stain ONE of their slides according to the following times:  safranin stain, 1 minute  methylene blue stain, 2 minutes  crystal violet stain, 1 minute
  5. Find cells form each smear under oil immersion using your assigned microscope. Be sure to start with the scanning objective and follow the procedure given in Microscope Theory. (Your partner will view their slide with their microscope.) Figure 6 Drawing Examples

Lab Report: Simple Staining Name ______________________________ Lab Section __________

Data and Observations

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

Post Lab Questions

  1. Critique your cell drawings. Are they large enough? Do they capture the relative length and width of the actual cell? Did you draw pointy corners? Outward tails? Inward tails? Open circles?
  2. The stains used in this exercise are basic stains. What is the counter ion? What charge does the counter ion have? What charge does the chromogen have?
  1. What microbial characteristics can one ascertain from a simple stain?
  2. A student is directed to make a simple stain of E. coli with crystal violet, but got mixed up and used safranin instead. How would their observations be different? Would the information you get be any different?
  3. If you dried your smear by placing it near the incinerator instead of air drying it, how might your observations be different?
  4. How is the procedure different when taking cells from a solid medium compared to taking cells from a liquid medium? Why is it important that your smear be thin?
  5. What is the difference between colony morphology and cellular morphology?