Bacteria and Archaea Formal Laboratory Report

Pre-Lab Questions
1. Use the following classifications to determine which organism is least related out of the three. Explain your rationale.

Table 2: Classifications
Classification Level American Green Tree Frog European Fire- Bellied Toad Eastern Newt
Domain Eukarya Eukarya Eukarya
Kingdom Animalia Animalia Animalia
Phylum Chordata Chordata Chordata
Class Amphibia Amphibia Amphibia
Order Anura Anura Caudata
Family Hylidae Bombinatoridae Salamandridae
Genus Tursipops Bombina Notophthalmus
Species cinerea bombina viridescens

2. How has DNA sequencing affected the science of classifying organisms?
3. You are on vacation and see an organism that you do not recognize. Discuss what possible steps you can take to classify it.
Experiment 1: Dichotomous Key Practice
A dichotomous key is an identification tool that starts with a broad defining characteristics and splits into two options until an organism can be identified. In this experiment, you will identify organisms by their binomial nomenclature using a dichotomous key.

Dichotomous Key
Images of Organisms

1. Start by observing organism i (Figure 2). Once you have taken notice of its physical characteristics, use the dichotomous key (Figure 3) to identify the organism.
2. Start at number 1 on the key and decide if the organism has feature “1a” or feature “1b”.
3. Which ever option you choose, follow the dotted line over to the next step that will state “Go to #”.
4. Go to that number and again decide between the two options.
5. Eventually you will wind up at a two name scientific name.
6. Once you have identified organism i record your finding in Table 3.
7. Repeat Steps 1 – 6 for all of the organisms in the dichotomous key (Figure 3).

Table 3: Dichotomous Key Results
Organism Binomial Name
Figure 2: Organisms to be identified
Figure 3: Dichotomous key

Post-Lab Questions
1. What do you notice about the options of each step as they go from number one up?
2. How does your answer from Question 1 relate to the Linnaean classification system?
Experiment 2: Classification of Organisms

Use Table 2 below as well as the “tree” (Figure 4; located at the end of this procedure)

1. Select the first organism from Table 4 (E. coli).
2. Use the “tree” (Figure 4; located at the end of this procedure) start at the base, and answer each question until the organism reaches the end of a “branch”. Write the organisms name in the red box.
3. Repeat this for the remaining organisms.
4. After classification, fill in Table 2 with the correct kingdom for each organism.

Table 4: Key Characteristics of Some Organisms
Organism Kingdom Defined Nucleus Mobile Cell Wall Photosynthesis Unicellular
E. Coli       Yes   Yes
Protozoa   Yes Yes No   Yes
Mushroom   Yes   Yes    
Sunflower   Yes Yes Yes Yes  
Bear   Yes Yes      
Figure 4: Experiment 2 – Classification of Organisms Flow Chart

Post-Lab Questions
1. Did this series of questions correctly organize each organism? Why or why not?
2. What additional questions would you ask to further categorize the items within the kingdoms (Hint: think about other organisms in the kingdom and what makes them different than the examples used here)?
3. What questions would you have asked instead of the ones that you answered about when classifying the organisms?
Pre-Lab Questions
1. Based on the scientific name Streptococcus agalactiae, what morphology would you expect these cells to have?
2. Name at least three animal structures that would be analogous to bacterial flagella.
3. Hypothesize how over-washing of hands can affect the population of “good” bacteria that resides on the human skin.
Experiment 1: Testing the Environment
Bacteria is found in almost all environments. Most are harmless, but there are those that cause disease. In this experiment, you will be test different environments for the presence of bacteria. You may be amazed to see how many different species are present on surfaces you come in contact with every day!

30 mL 8.25% Bleach Solution 4 Cotton Swabs (Sterile) 125 mL Nutrient Agar 60 cm Parafilm® Permanent Marker (4) 9 cm Petri Dishes
*Hot Pad *Microwave or Boiling Water Bath *Refrigerator *Scissors (to cut the Parafilm®) *You Must Provide

Notes About Working With Agar Plates…
· Prepared agar dishes should be stored upside-down in the refrigerator until used. This will prevent condensation from disrupting the growing surface.
· After inoculating, replace the cover on the dish, seal with Parafilm®, and store upside-down in a warm location (not to exceed 37.7 °C or 100 °F).
· You should see growth within a few days. The plate will start to smell once microorganisms are growing.
· Before disposing plates, kill the microorganisms by pouring bleach solution onto the agar surfaces and let sit for 20 minutes.
1. Loosen or remove the cap on the Nutrient agar bottle. Place in the microwave (if you do not have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat until the entire bottle of agar is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
2. If you notice the liquid boiling over, STOP the microwave and let the bottle cool down before handling. With a hot pad protecting your hands, remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!
3. Gently swirl the bottle to mix the solution. Pour enough of the liquefied agar solution into the bottom half of 4 petri dishes so that it covers the entire bottom of the dish. Place the lids onto the dishes and CAREFULLY transport these plates into a refrigerator. Allow the plates to sit for 24 hours.
4. After 24 hours, remove the four agar plates from the refrigerator and allow them to sit at room temperature for at least one hour.
5. After the plates have warmed to room temperature, locate a surface to swab for bacteria. Things such as shoes, a table, teeth, bathroom doors, and shopping carts can be great sources. Try to avoid surfaces that are frequently cleaned with antibacterial detergents and soaps. On the bottom of the plate, write the name of the surface to be swabbed with your permanent marker.
6. Remove the lid from the agar plate. Unwrap one sterile cotton swab and smear the surface, being sure to roll the cotton swab to cover all sides.
7. Carefully streak the cotton swab onto the surface of the gelatinous medium (agar), being sure to start at the top and work down in a zigzag motion.
8. Place the lid onto the agar plate and seal it with a strip of Parafilm (hold one end of the Parafilm firmly against the side of the petri dish and stretch the other side to cover the entire perimeter). Place the plate upside down in a warm area to incubate.
9. Repeat this for two additional surface areas and agar plates.
10. For the last plate, label it “control.” Do not rub your sterile cotton swab on any surface, but rather take it straight from the package and streak it onto the plate. This will test for your accuracy in keeping the plates and swabs sterile while performing the experiment.
11. Let the plates incubate in a warm area for three days and then observe the growth. Answer the questions below.
12. After the experiment and after answering questions 1 – 3, set aside the plate with the most bacterial growth for the next experiment. Pour the bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Let the plates sit untouched for 10 – 20 minutes. Then, seal plates with Parafilm® and dispose appropriately.
Post-Lab Questions
1. In the space provided below, draw your plates and indicate the number of different colonies and identify the colony shape.
2. Which plate grew the most bacterial species? Was this a surprise? Why or why not?
3. Was your control plate free of bacterial colonies? If not, how do you think the swabs were contaminated?
Experiment 2: Measuring Antibiotic Resistance
In this experiment you will look at the ability of common antibiotics and other anti-bacterial agents to kill bacteria commonly found in your environment. You will use the bacterial lawn from the plate that produced the largest quantity of bacteria in the first lab.

1 Ampicillin Disc (marked) 10 mL 8.25% Bleach Solution Forceps 1 Kanamycin Disc (marked) 1 Nutrient agar plate from the previous lab
1 Penicillin disc (marked) Permanent Marker Ruler Parafilm®

Note: Be sure to wear gloves and use the forceps when handling the discs. Always pick them up if spilled.
1. Using one of the plates covered with bacteria from the first lab, draw four even quadrants onto the bottom of the plate. Label them penicillin, ampicillin, kanamycin and control.
2. Wearing your gloves, goggles, and apron, open the plate and place the appropriate disks into the appropriate quadrants using forceps. Do not place anything in the control quadrant. DO NOT TOUCH THE PLATE WITHOUT GLOVES.
3. Place the lid back onto the plate and cover it with Parafilm®. Allow it to incubate in a warm area for 3 days.
4. After 3 days, observe the zone of resistance from each disc (the region surrounding the disks where no bacteria grew). Using a ruler, measure these zones from the bottom of the agar plate.
5. After you are done with the experiment, pour enough bleach solution to cover the surface of the agar. Allow the plate to rest for 10 – 20 minutes. Seal the plate with Parafilm® and dispose appropriately.
Post-Lab Questions
1. Did your bacterial lawn contain only one species of bacteria? If not, why do you think that is? Can you tell?
2. Which antibiotic was most effective in killing the bacterial lawn? Which was the least effective?
3. Each bacterial species shows different antibiotic susceptibility. What can you say about the bacteria that you grew?

Bacteria and Archaea Formal Laboratory Report

Many of you may have written a lab report in high school and are familiar with the typical layout of one. The lab report is just a way to put together the information on a laboratory experience that explains to the reader the who, what, why, and how of that experience. It is set up similarly to a research article that would be submitted to a journal for publication. Although all professional journals have their particular format, the headings you will use in this report will generally be the ones you will see in a journal. Also in the sciences, all references are written in APA format.
One of the issues that is common in student papers is the internal citation. All referenced materials should correspond to an internal cite in the paper. If you do not cite an article, leave it out of your references. You need an internal citation after a piece of information that you took from a published article. If you use that information in two continuous sentences, you may wait until the end of the second sentence for that citations. If you quote an article you need an internal citation. Quotes should be a last resort in writing a lab report. You should read various sources and then combine and rearrange the information into your own words. A longer scientific paper may be appropriate for quotations.

Parts of the Formal Lab Report

1st Page

Title Page

Title of Laboratory Report (you could just use the title of the lab)
Your Name
Your class identification (BIO 105 – section or instructors name)