What is Gram staining and how does it differentiate bacterial species?

Gram staining is a laboratory technique that distinguishes bacterial species based on cell wall composition. Gram-positive bacteria retain crystal violet stain and appear purple, while Gram-negative bacteria are decolorized by ethanol and stain pink with safranin counterstain. This differential staining helps identify bacterial species and morphology under microscopic examination.

How do you prepare a bacterial smear for Gram staining?

To prepare a bacterial smear, place two loopfuls of sterile distilled water on a labeled microscope slide. Using a flame-sterilized inoculating loop, remove a small amount of culture from an isolated colony and mix it with the water. Allow the slide to dry at room temperature, then heat-fix the smear by quickly passing it through a flame to adhere cells to the slide.

What are the main steps in the Gram staining procedure?

Gram staining involves sequential applications: crystal violet for two to three minutes, Gram's iodine for two minutes, 95% ethanol for decolorization, and safranin counterstain for 30 seconds. Each step is followed by gentle rinsing with distilled water. The slide is then blotted dry and examined under a microscope using oil immersion at high magnification for optimal visualization.

What bacterial shapes can be identified through microscopic observation?

Three primary bacterial shapes are identifiable: bacillus (rod-shaped), coccus (spherical), and spirillum (spiral-shaped). These morphologies are observed and recorded in the microscope field after Gram staining. Shape identification, combined with staining results, helps classify and differentiate bacterial species in laboratory cultures.

How does yeast reproduction differ from bacterial reproduction?

Yeast from the Ascomycetes phylum reproduces asexually through budding, visible under microscopy as daughter cells forming on parent cells. Yeast can also reproduce sexually via mitosis and haploid spore formation. In contrast, bacteria reproduce primarily through binary fission, a process distinct from the budding mechanism observed in yeast cells.

What distinguishing features characterize Basidiomycota fungi?

Basidiomycota fungi, such as button mushrooms (Agaricus bisporus), exhibit clearly observable gills beneath the pileus, or cap. The fungal structure includes the stipe, or stalk, supporting the cap. These visible gill structures under the pileus are a defining characteristic that distinguishes Basidiomycota from other fungal phyla when examined under a dissecting microscope.

Why is immersion oil necessary for high-magnification bacterial observation?

Immersion oil is required for high-power microscope objectives to improve optical resolution and light transmission through the specimen. The oil reduces light refraction at the lens-slide interface, allowing clearer visualization of bacterial cells and their morphological features at maximum magnification. This technique produces the best micrographs for accurate bacterial identification and documentation.

Microbial and Fungal Diversity

Concepts Instructor Prep Student Protocol

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Microorganisms are defined as life forms too small to see with the naked eye. But while we don't typically see them, microbes are everywhere, doing good things like helping our digestion or making yogurts and wine, to not-so-good things like causing infection or disease.

Speaking of infections, back in 1928, Alexander Fleming returned to his laboratory from vacation, and noticed a strange fungus contamination on one of his bacterial culture plates. Interestingly, the bacterial colonies immediately around the contamination had been destroyed. This observation led to much further research, eventually culminating in the development of penicillin and other antibiotics that could treat bacterial infections. Yet, although they may live side-by-side in the microscopic world, we know from studying their forms and phylogenies that bacteria and fungi are vastly different and diverse organisms. Let's walk through some of the defining characteristics of these two major groups briefly.

Bacteria are unicellular prokaryotes, meaning that that they are single-celled organisms without a nucleus. All bacteria have a cell wall, but its composition and thickness vary. Gram positive bacteria have a cell wall with a thick peptidoglycan layer, which readily retains crystal violet molecules in gram staining. However, gram negative bacteria have a thin cell wall surrounded by an outer lipopolysaccharide membrane, and they only retain the counterstain, the red-colored safranin. Gram staining allows observation of bacterial shapes as well. Round bacteria are called cocci. Rod-shaped bacteria are called bacilli. And spiral-shaped bacteria are called spirilla. Furthermore, bacterial colonies can also be characterized with the naked eye, using traits such as color, shape and size of the colony.

Now, let's take a look at the fungi. Unlike the bacteria, fungi are eukaryotes, and thus their cells have membrane-bound organelles and a nucleus. They are commonly classified into groups based on their reproductive strategies. For instance, Ascomycota, or sac fungi, which includes morels, penicillium, and truffles, have fruiting bodies called ascocarps. Morels are filled with these structures. If we look at these more closely, they contain many smaller structures called asci, which in turn contain many ascospores formed during sexual reproduction. However, Ascomycota often reproduce asexually by budding. The second major fungal group we will examine is the Basidiomycota, or club fungi. Most edible and poisonous mushrooms in the forest are actually reproductive structures of the Basidiomycota. In this group, what we would refer to as the mushroom cap is technically known as the basidiocarp. In gilled fungi, gills under the basidiocarp are lined with tiny structures know as basidia, and these produce basidiospores, microscopic reproductive spores, during sexual reproduction. These spores are typically disbursed by wind, similar to many plant seeds. Here, we have examined several major bacteria and fungi groups, and the characteristics that define them in a very simplified fashion. However, due to the vast numbers of microbial species, upwards of one trillion by many estimates, scientists often identify and group them using additional methods, including examination of their DNA.

In this lab, you will identify different bacteria species using gram staining, and examine colony characteristics. You will also examine fungi species to identify their reproductive structures.

Print Procedure Steps

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Examining Microbial Colony Structures
- To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
- Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
- As the colonies are identified, fill out the observed colony properties in the appropriate column in the table. Click Here to download Table 1
- Then, repeat the observations for each of the other two bacterial species.
- To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
- To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
- Place two loopfuls of sterile distilled water onto the center of the slide.
- After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
- Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
- Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
- Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
- Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
- Next, cover the slide with Gram's iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
- Decolorize the slide with 95% ethanol until stain no longer washes away.
- Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
- Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
- Gently rinse the slide with distilled water and blot dry with absorbent paper.
- After staining the other two slides in the same manner (steps 6 – 17), observe each with a microscope.
- Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
- After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
- Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria's shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. Click Here to download Table 2
- Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
Examining Fungal Structures
- Remove the S. cerevisiae plate from the incubator and place a drop of water onto a microscope slide.
- Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
- Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
- Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
- Now, draw the cells in the appropriate magnification circles.
- When you are done observing the yeast, replace the S. cerevisiae slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
- Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
- Finally, place a button mushroom, Agaricus bisporus, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
Results
- Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
- Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
- Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as S. cerevisiae, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
- Record any observations of yeast cells budding. NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
- Record any observations of spore or asci formation you observed in the table.
- NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
- Record any fungal species that demonstrated gills under the pileus.