Question: Laboratory #4 Assignment: Part I: Estimating Microbial Population Size

Laboratory #4 Assignment Template

Part I:  Estimating Microbial Population Size: 5 points

Bacteria are the oldest organisms on earth and yet we have only known about them since the 17^th century when the microscope was invented.  Our ability to see life at a small scale revolutionized our understanding of the world – particularly our understanding of the causes of many human diseases.  Bacteria are also abundant – a recent estimate suggests that the number of bacteria on earth at any given time is five million trillion trillion or 5 x 10 to the 30th power.  And it is estimated that there are almost a trillion different species of microbes – most of which have not yet been discovered! 

Because bacteria are small and abundant – we can’t count them the way we can count larger, less abundant organisms.  Instead, we can use a procedure known as serial dilution to estimate the number of bacteria in a sample.   Serial dilution is used to decrease a bacterial concentration in a stepwise process so that a smaller number of bacteria can be counted, and this count is used to estimate the original population size. 

This is Part I of the laboratory activity – learning how to estimate microbial population sizes by learning how to perform a serial dilution.  Next week, you will use materials from your kit to perform a serial dilution.

The Learning Objectives for Part I of this week’s Laboratory Activity are:

By the end of this laboratory activity, you should be able to:

·        Describe different techniques used to estimate microbial population size.

·        Explain how to perform a serial dilution of a bacterial sample.

·        Determine the number of bacteria in an original culture using a standard plate count.

Before answering these questions, read the document posted in this assignment module titled “Methods of Estimating Bacterial Population Sizes”.   

For another discussion of serial dilution, the following site is helpful: https://www.wikihow.com/Do-Serial-Dilutions

1. (0.5 points) In the image above, the final dilution is 1: 10^-6.

2. (0.5 points) In the image above, the final dilution is 1:_____10^-4_____________.

3. (0.5 points) Which is the correct term for the cloudy haze formed by small particles in a solution?

A.      colonies

B.       colony-forming units

C.       diluent

D.      turbidity

E.       confluence

4. (0.5 points) What are two reasons serial dilution is a useful technique for counting populations of bacteria?

1.       It allows for simpler counting and verification of populations of bacteria

2.       It makes it possible to count concentrations for specific test methods

5. (0.5 points) In the image above, circle the petri dish that is best used to estimate the number of bacteria in the original culture.  Why is this the best plate to use?

It is the best plate as it has a 29 colonies which do not overlap which makes it easy to count.

6. (0.5 points) Using the plate you chose in question #4, estimate the number of bacteria per unit volume in the original culture (you will need to count the number of CFUs on the plate). 

The standard formula: 

Number of CFU = 29

Volume plated = 0.1 mL

Dilution factor = 10^-6

No. of bacteria per unit volume = × 1,000,000 = 290,000,000 CFU/mL

The estimated number of bacteria in the original culture = 2.9× 10⁸ CFU/mL (don’t forget to include the unit).

7. (0.5 points) In a standard plate count, what is the relationship between a colony and a CFU?

The genesis of a colony is the colony-forming unit.

8. (0.5 points) Which is the correct term for continuous microbial growth on an agar plate?

A.      colonial growth

B.       colony-forming units

C.       diluent

D.      turbidity

E.       confluent growth

9. (0.5 points) Suppose you counted 37 colonies on an agar plate made with 0.1 ml of a 10^-4 dilution of the original sample. What was the CFU/ml of the original sample?

A.      3.7 x 10^-4

B.       3.7 x 10^-2

C.       3.7 x 10⁴

D.      3.7 x 10⁶

E.       3.7 x 10¹⁰

Part II: Scientific Reading: 5 points

In this part of this week’s laboratory activity, you will read an article about microbial diversity and abundance in soils planted with a genetically modified organism (GMO canola plants).  The researchers used serial dilution methods to estimate population sizes and research an important question – does the planting of GMO crops impact the soil microbiome?

The Learning Objectives for Part II of this week’s Laboratory Activity are:

By the end of this laboratory activity, you should be able to:

·        describe the structure of a scientific article

·        explain why the research in this paper is important and relevant

·        describe how and why serial dilution was used to support the conclusions of this research

·        use APA format to properly cite the article

After reading the article (Diversity of bacterial communities in the rhizosphere and root interior of field-grown genetically modified Brassica napus), answer the questions below.  For each question provide a brief but complete answer (This is NOT an essay.  Answer each question individually).

You should write in complete sentences and proofread your work for spelling and grammatical errors. Note that the answer may be found in more than one place in the article. Remember that you should write in your own words. Do not copy and paste from the article!!!  THIS ASSIGNMENT MUST BE WRITTEN IN YOUR OWN WORDS – DO NOT CUT AND PASTE FROM THE ORIGINAL ARTICLE OR ANY OTHER SOURCE. Using the words of another writer is plagiarism – a violation of the Penn State Academic Integrity policy. 

1.  What is the “rhizosphere”?  

2. According to the article, what benefits do genetically modified herbicide tolerant crops provide farmers?

3. What are the concerns about these GMO crops as stated in the paper?

4. Why is this research important?

5. What is the hypothesis and/or research goal of the study? Where do you find the hypothesis or goal in the paper (in the Abstract, Introduction, Materials and Methods, Results, Discussion)?

6. What are the major methods used? What tense is used? (The Methods section is often the most difficult for new students of science to understand – discuss the methods in a general sense and limit your answer to 5 sentences or fewer.)

7.  How was serial dilution used in this study?  Why was serial dilution used?

8. What are the major results?

9. What conclusion(s) did the authors make about their hypothesis/goal?

10. What new hypothesis/goal emerges from the results?

11.  How would you properly cite this article in the “References” section of a lab report? (For this class, we will use APA format.  The following site will give you the information you need to properly reference: https://www.mendeley.com/guides/apa-citation-guide)

Part III:  Fungal Diversity Under the Microscope: 10 points

When we think of fungi, many of us think of mushrooms.  But fungal diversity is much greater – fungi can cause animal and plant diseases, synthesize compounds that we use as antibiotics and medicine, and more recently we have explored the use of fungi to make biodegradable packaging materials to replace the use of non-degrading plastics.  Yet, we have so much more to learn about the fungi – both in terms of diversity and their role in the environment.

In this part of the lab, you will be using your microscope to explore fungal diversity.  You will need to obtain two different fungal samples.  They can be mushrooms from the grocery store, yeast for baking and brewing, mold found on food in your kitchen, or fungi gathered from outside.  You should be able to identify the phylum to which each sample belongs.

The Learning Objectives for Part III of this week’s Laboratory Activity are:

By the end of this laboratory activity, you should be able to:

·        prepare a wet mount of fungal samples

·        take high quality photos of your samples using the microscope

·        identify your fungal samples to the phylum level

·        describe the fungal life cycle

·        define and use the terms plasmogamy, karyogamy, dikaryon, mating type, and fungal spore

NOTE:  If you have asthma or a mold allergy – do not use mold for this exercise!

A.  You will photograph each of your samples and look at their morphology at two different magnification levels.  You may want to use the dye (methylene blue solution) included in your kit.  Be sure to review the proper use of the microscope from Lab #3 and ask any questions you may have.

To observe fungal spores, you can set a fresh (not dried) fungal fruiting body on a slide and wait for several hours for the fungus to release its spores onto the slide.  You can also observe the gills, asci and/or hyphae by taking thin slices and putting them on a slide.  You will need to add water to the slide for your observations.  Whatever you choose, have fun and explore!

NOTE:  Be sure to review the video on how to take a cell phone picture through the microscope (http://players.brightcove.net/17907428001/HJWla8X7_default/index.html?videoId=5446853801001)

B. Select one of your samples and diagram the sexual life cycle of this organism (you can sketch the life cycle on a separate piece of paper and insert a photo here):

C.  In your own words, define the following terms:

Plasmogamy:

Karyogamy:

Dikaryon:

Mating type:

Fungal Spore: