Simple and differential Staining of bacteria
In previous exercises you have observed live bacteria via compound brightfield microscopy. This approach allows one to observe bacteria in terms of their motility and provides some insight on the organism’s overall morphology. However, since the bacterial cell is transparent and motile and therefore somewhat difficult to observe when using the compound brightfield microscope, cells are often fixed and stained to make them more visible. Staining provides a reliable means for observing bacteria in terms of their relative size, morphology and cellular arrangement.
Stains are solutions of a dye that has been dissolved in water or alcohol. The dye is a salt (like Na+Cl-). One component of the dye consists of a negatively charged ion, while the other component of the cell consists of a positively charged ion. The ionic component of the dye that imparts color to the cell is called a chromophore. In the case of basic dyes the chromophore is positively charged. The positively charged component of a basic dye is able to interact strongly with a bacterial cell, because most cells under normal conditions have an overall negative charge. The stains that we will be using in the following exercises, methylene blue, crystal violet, malachite green and safranin (red) are all examples of basic dyes.
You will be observing fixed bacteria subjected to simple staining in the first exercise. In a simple stain a basic dye is added to a population of bacteria that has been fixed to the surface of a microscope slide such that all cells in the population take on the color of the dye used. In the second set of exercises you will be performing a Gram stain (a type of differential stain). Gram staining involves the use of two different basic dyes, one that stains a sub-population of the cells and the other that stains another subpopulation. While the simple and differential stains both provide information with respect to size, morphology and bacterial arrangement, the Gram stain is used to distinguish between bacteria with different cell wall structures and compositions. Bacteria can be divided into two major subclasses with respect to how they react to the basic dyes employed in a Gram stain—Gram positive or Gram negative. Gram positive and Gram negative bacteria both have cell walls that are comprised of peptidoglycan. The cell wall of Gram positive bacteria are very thick (up to 80 nm thick); the peptidoglycan that make up the cell wall of Gram positive bacteria is highly crosslinked in the second dimension and also crosslinked in the third dimension. The cell walls of Gram-negative bacteria are thinner (10 nm or less); the peptidoglycan is not as fully crosslinked in the second dimension and not crosslinked at all in the third dimension. In Gram staining one first stains the fixed bacteria with a primary dye (crystal violet) and adds a mordant (iodine) to generate a crystal violet-iodine complex within the cell. The crystal violet-iodine complex is larger than the crystal violet alone and therefore more difficult to remove in subsequent steps. Acetone is added to remove the crystal violet-iodine complex from Gram negative bacteria. The fact that the peptidoglycan layer of Gram-negative bacteria is thin and not fully crosslinked makes it easy to remove the primary dye, conversely, the peptidoglycan layer of Gram positive cells are dehydrated in the presence of acetone such that the crystal violet-iodine complex is trapped within the cell. As the Gram negative cells are decolorized by the acetone, one then counterstains with safranin such that those cells can be easily observed (Figure 1.)
Simple staining: In the first exercise, you will observe bacteria subjected to simple staining in order to analyze different bacteria with respect to their size, shape and arrangement. To this end you will observe, Escherichia coli, Bacillus subtilis and Vibrio spp. as well as Staphylococcus epidermidis and Streptococcus lactis.
Bacteria have three basic characteristic morphologies. Those are the coccus (cocci pl), the bacillus (bacilli pl) and the spiral shaped bacteria such as the spirillum, spirochetes and Vibrio spp. The cocci are spherical shaped bacteria while the bacilli are rod shaped bacteria. There is a great deal of variety with respect to the morphologies that spiral shaped bacteria assume; the spirillum and spirochetes appear different under observation because of the manner in which they move. When spirillum move the long axis of their bodies remain rigid such that they appear to bend at the ends, when the spirochetes move, the long axis of their bodies are not fixed such that they take on a wavy, “permed hair” appearance. Vibrio spp. are rod shaped bacteria that are bent near the middle such that they are often called the “comma shaped bacilli”. When bacteria grow and divide they can stick together in a manner that aids in their characterization with respect to their genus and species. For example if cocci shaped bacteria divide and stick together to form pairs, they are called diplococci; if they stick together to form long chains they are called streptococci and if they adhere to each other to form “grape like” clusters they are called staphylococci. Certain bacillus shaped species such as Bacillus subtilis can adhere to each other in a chain like formation known as streptobacilli.
Describe in the spaces below how the bacteria look in terms of size, shape and bacterial arrangement at a total magnification of 1000X !!!
Escherichia coliVibrio anquillarum
Staphylococcus epidermidisStreptococcus lactis
Bacillus subtilis
Gram staining: The next part of the exercise involves using a differential staining procedure called Gram staining to distinguish between Gram positive and Gram negative bacteria. To this end you will stain and observe, Escherichia coli and/or Staphylococcus epidermidisand/or Bacillus subtilisin a mixed culture.
Preparing a smear from a liquid culture:
Place a small amount of the culture on a microscope slide as shown by your instructor and heat fix the sample.
Using a wax pen (not Sharpie, Why?) label your slide with your initials. This is the top side of your slide and the side of the slide that will be subjected to all of the staining steps
You will make a smear of the bacterial species, by aseptically transferring bacteria from a culture tube onto the glass slide. To this end, obtain a sterile cotton swab and swab into the test tube containing the mixture of bacteria. Wring out the swab on the inner surface of the test tube. Make a thin smear on the surface of your microscope slide. It is important that you make a thin smear such that it is easier to observe individual bacteria.
Allow the smears to dry completely; the smears should become somewhat cloudy as they dry, it is important that the smears are completely dry as the following heat fixation step will distort the morphology of organisms in a smear that is not completely dry.
Heat fix the bacteria in the smear by quickly passing the microscope slide through a flame 8-10 times.
2. Crystal violet—primary stain: Place the slide containing heat fixed smears on a test tube rack in the sink with the smear facing up. Cover the smear with crystal violet and leave the stain on for 1 minute. (What color will the cells be?)
3. Gram’s Iodine—mordant: Use a wash bottle containing WATER to gently wash BOTH sides of the microscope slide, tip off the extra water and IMMEDIATELY cover the slide with Gram’s iodine. Allow the iodine to remain on the slide for one minute. (What color will the cells be?)
4. Acetone wash--Decolorizing step: Use a wash bottle containing WATER to gently wash BOTH sides of the microscope slide.
5. Rinse the slides with acetone for 3 seconds only. Immediately rinse both sides of the microscope slide with water to remove the acetone and to halt the decolorizing process. It is important that you do not allow the acetone to remain in contact with the specimen for longer than 3 seconds because longer contact will remove the crystal violet-iodine complex from the Gram positive cells as well. (What color will the cells be?)
6. Safranin—counterstain: Place the slide on the test tube rack in the sink (smear side up) and cover the slide with safranin; allow the safranin to remain on the slides for 90 seconds. Use a wash bottle containing WATER to gently wash BOTH sides of the microscope slide and gently blot the slides with bibulous paper as described by your instructor.
7. Save your slides until the next lab period where you will observe your stained bacteria using a microscope.
Describe in the space below how the bacteria look in terms of size, shape and bacterial arrangement AND color at a total magnification of 1000X !!!
Which of the bacteria are Gram positive/Gram negative?
Caveats:
It is important to note that not all bacilli are Gram negative and not all cocci are Gram positive!! Also some bacteria can be Gram variable. A good example of this is Bacillus subtilis that is a Gram positive rod that forms endospores. Older cultures of Bacillus may react to the dyes in the Gram stain such that one may observe what look like Gram positive bacilli and Gram negative bacilli in the same population. When observing bacteria in terms of whether they are Gram positive or negative it is important to work with young cultures.
The instructor has placed a demonstration slide of a Gram stain of a four day old culture of Bacillus subtilis (the south side of the lab). Note how some of the bacilli appear pink (or Gram negative).
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