How much nutrient agar is needed to prepare bacterial culture plates?

Add 23 grams of nutrient agar to a 2,000 mL beaker containing 1 liter of distilled water. Place the beaker on a hotplate set to high and stir until the agar dissolves completely. This quantity prepares enough nutrient agar medium for multiple Petri dishes used in microbial culture experiments.

What is the proper procedure for sterilizing nutrient agar before plating?

After the agar dissolves, transfer the beaker to an autoclave and run it at 121°C for 15 minutes. Remove the beaker and allow it to cool to the touch for about 15 minutes without letting the agar solidify. This sterilization process eliminates contaminants and prepares the medium for safe bacterial and yeast culture.

How should you prepare Petri dishes before inoculating them with bacteria?

Pour the cooled liquid nutrient agar into each Petri dish until it is about halfway full. Allow the agar to solidify, then seal the sides with Parafilm. Under a fume hood, use a marker to draw two intersecting lines on the bottom of each plate to divide the agar into four equal quadrants, and label with initials, bacterial species name, and date.

Why is flame sterilization important when using an inoculating loop?

Flame sterilization eliminates microbial contamination from the inoculating loop between samples. Heat the looped end in a Bunsen burner flame for 20 seconds, then allow it to cool for about 15 seconds before use. This prevents cross-contamination between bacterial cultures and ensures pure, isolated colonies on your plates.

What is the streak plating technique and how does it isolate bacterial colonies?

Streak plating involves spreading bacteria across quadrants in a specific pattern where each new streak crosses the previous one only once or twice. This dilutes the bacterial population progressively, allowing isolated single colonies to grow in later quadrants. The technique is essential for obtaining pure cultures and observing microbial colony and fungal diversity in laboratory settings.

How should yeast cultures be plated differently from bacterial cultures?

For yeast plating, scoop S. cerevisiae from a vial onto the agar using an inoculating loop and evenly spread it across the entire plate, rather than using the streak plating method. Label the plate with the yeast strain name, date, and your name. Reseal with Parafilm and store upside down at 32°C until the activity.

What storage conditions are required for bacterial and yeast cultures after plating?

After sealing plates with Parafilm, store bacterial cultures lid down in a 32°C incubator until the day of the activity. Yeast cultures should also be stored at 32°C but positioned upside down. Proper incubation temperature and orientation promote optimal microbial growth while preventing condensation from dripping onto colonies.

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.

At the end of this lab, students should know...

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Under which domains are bacteria and fungi classified? Are they prokaryotic or eukaryotic?

Bacteria, which are prokaryotic organisms, fall under the domain Bacteria. Fungi are eukaryotic and are classified under domain Eukaryota.

By what mechanism does Gram staining distinguish positive and negative species of bacteria?

Gram staining reveals differences in the bacterial cell wall. The crystal violet dye used in Gram staining is retained by proteoglycan molecules in thick bacterial cell walls of Gram-positive bacteria. In contrast, Gram-negative bacteria with thin cell walls only retain the counterstain safranin.

What are the three major shapes of bacteria?

Individual bacteria can be round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilla).

What are Ascomycota and what distinguishes these organisms?

Ascomycota are a type of fungi with a reproductive structure called an ascus. These organisms typically reproduce by asexual budding.

What are Basidiomycota and what distinguishes these organisms?

Basidiomycota are a type of fungi also known as the club fungi. They have a club-shaped reproductive structure called basidium and usually reproduce sexually.