JoVE Lab Manual
Lab Bio
Lab Bio
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Microbial and Fungal Diversity
Microbial and Fungal Diversity
Learning Objectives
At the end of this lab, students should know...
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.
List of Materials
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Inoculating loop5
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Microscope slides20
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Microscope cover slips20
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Bacillus cereus (culture plate)1 per station
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Micrococcus luteus (culture plate)1 per station
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Rhodospirillum rubrum (culture plate)1 per station
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Saccharomyces cerevisiae var. ellipsoideus (culture plate)1 per station
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Apothecium prepared slide with asci1 per station
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Bunsen burner1 per station
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Lighter1 per station
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Crystal Violet1 per station
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Gram’s Iodine1 per station
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95% ethanol1 per station
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Distilled water1 per station
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Safranin1 per station
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Compound microscopeDependent on the lab size
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Dissecting microscopeDependent on the lab size
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Nutrient agarDependent on the lab size
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Petri platesDependent on the lab size
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ParafilmDependent on the lab size
Lab Prep
- Nutrient Agar and Bacterial Culture Plate Preparation
- Add 23 g of nutrient agar to a 2,000 mL beaker containing 1 L of distilled water 4 – 5 days before the activity.
- Place the beaker on a hotplate and set the hotplate to high, stirring the mixture with a stirring rod.
- When the agar has dissolved into the water, use a hot pad to transfer the beaker into an autoclave and set it to run at 121 °C for 15 minutes.
- When the cycle is finished, remove the beaker and allow it to cool to the touch for about 15 minutes without allowing the agar to solidify.
- Then, pour the liquid nutrient agar into a Petri dish until the dish is about halfway full.
- Place the lid onto the top and allow it to cool.
- Once the agar is solidified, seal the sides with Parafilm.
- Prepare enough Petri dishes so that each student or student group will have one per bacterial type and one for the yeast, and then place these into a refrigerator for storage.
- Bacteria and Yeast Plating
- To prepare for plating, place the plates under a fume hood and use a marker to draw two intersecting lines on the bottom of each plate to divide the agar into four equal-sized quadrants.
- Label the bottoms of the plates with your initials and the name or initials of the bacterial species that will be plated along with the date.
- When all of the plates have been labeled, light a Bunsen burner and adjust the gas to be just high enough to sustain a blue flame, and heat the looped end of an inoculating loop in the flame of the burner for 20 seconds.
- After removing the loop from the heat, allow the metal to cool for about 15 seconds.
- Then, place the end of the loop in one vial of bacteria, making sure that the loop is coated with bacteria.
- Gently smear the loop onto one quadrant of agar in one of the plates labeled for that bacteria.
- When the entire quadrant has been filled, flame sterilize the loop as previously described (steps 3 – 4), and then streak the rest of the plate following a pattern similar to this one so that the streak in the second quadrant only crosses the first streak one or two times, the third crosses the second in the same manner, and the fourth crosses the third only once.
- Repeat this streak plating for the remaining two bacteria species (step 7).
- Finally, reseal the plates with Parafilm and store the cultures lid down in a 32 °C incubator until the day of the activity.
- To plate the yeast culture, label the bottom of the remaining plate with the yeast strain name, date, and your name.
- Using an inoculating loop, scoop up S. cerevisiae from a vial onto the agar and evenly spread the yeast across the plate.
- Then, cover the plate with the lid and reseal the plate with Parafilm for upside down culture at 32 °C until the day of the activity.
