BISC209:Project1

Project: Soil Microbial Communities & Diversity

In this series of nine labs you will learn:

1. To think, work, and write as microbiologists
   
2. To use the basic tools and techniques of traditional and molecular microbiology
   
3. To investigate the diversity and identity of soil microorganisms in a habitat of your own choosing
4. To make careful, unbiased observations and to record and analyze them for meaning and importance
   
5. To design controlled experiments and collect data from those experiments to answer questions that arise from your observations
   
6. To show data in effective figures or tables
   
7. To make and articulate conclusions from experimental results
   
8. To write intelligibly in scientific research report format about your investigation and its conclusions, including its significance or implications
   
   

Introduction to the Project

In the 1980's scientists discovered that, despite microbes invisibility to us, the microbial world is as, or more, diverse than the macroscopic world of plants and animals. Traditional measures of diversity relied on physical traits, but such criteria can not be used to assess relationships between microorganisms and macroorganisms because there are so few physical traits common to both. In the 1980's Carl Woese suggested that the deoxyribonucleic acid (DNA) sequences of certain common genes could be used to measure relatedness among radically different organisms. He picked the genes that encode ribosomal RNA (rRNA). Ribosomes, the protein-RNA complexes that are the scaffold on which proteins are synthesized, are common to all cells, both prokaryotic and eukaryotic. Despite differences in size, the sequences of rRNA molecules contain regions that are highly conserved, thus highly similar. Woese chose the intermediate sized rRNA molecule, 16S rRNA in prokaryotes and 18S rRNA in eukaryotes because it was large enough to contain enough information for genetic comparisons but small enough for the gene to be sequenced easily.

Comparing sequences of the gene (16S DNA) that encodes 16S rRNA in different bacteria can be used to identify them. We can also use rDNA sequencing to deduce relationships between different bacteria and among organisms as diverse as bacteria and humans. Woese's ground-breaking work altered the phylogenetic tree of life and showed that the prokaryotic world was evolutionarily much older than expected and much more important.

Recent advances in molecular tools for gene sequencing and in other types of microorganism identification dramatically expanded our knowledge of the contribution of microbes in their (and our) environment. It is estimated that less than 1% of bacterial species have been described, primarily because so many are unculturable by traditional methods. Current culture-independent estimates of the number of bacteria in a gram of soil range from several hundred to close to 9,000 orders of magnitude greater than the number derived from culture-dependent methods. It has been speculated that there might be 10 billion species of bacteria on Earth!

Your goal in this semester long project, is to use both culture and molecular methods of bacterial identification to investigate the diversity of bacteria in soil from a Wellesley greenhouse habitat and to explore the specific role of some of the bacteria in that community.

Molecular Strategy for Identification of Culturable & Non-culturable Bacteria in a Soil Sample

Isolate Genomic DNA from Environmental Samples

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PCR Amplify Genomic DNA from Universal Bacterial Primers

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Clone 16S rDNA PCR products into a plasmid vector and transform E. coli with plasmid
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Pick transformed E.coli colonies and transfer to 96 well plates for sequencing

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Analyze Diversity of Soil Bacterial Community & Relationships

Modeling and assessing microbiological techniques: aseptic transfer, isolation streak plates, personal safety in the lab

Whether you are trying to keep a desired organism from being overgrown by a contaminant, or preventing contamination of yourself, your lab bench, or your lab partner by cultured organisms, being aware of potential sources of contamination in a microbiology lab is critical. Your success in the lab depends on being open to learning and adopting the standard procedures used in microbiology. Today you will practice Aseptic transfer technique and immediately assess your success using a flourescent powder.

Activity 1: Tools of aspetic transfer
If possible watch YouTube video on lab safety. Watch the YouTube video on how to use the bunsen burner and broth to broth Aspetic transfer technique. Follow the demonstration and suggestions of your lab instructor to help improve your technique.

Imagine that an organism is suspended in the stock tube (Tube A) on your bench.
1. Use Tube A and the instructions for aspetic transfer (broth to broth) to transfer a loop of Tube A to a destination tube of broth. 2. Use Tube A and the instructions for isolation streak technique to transfer a loop of Tube A to a destination plate of agar.
3. Use Tube A and the instructions for transfer to a slant to make a slant tube.

Activity 2: Gram Stain technique

Perform a gram stain on a bacterial smear slide of Staph. epidemidis, E. coli and a mix of both cultures.
Make only one slide with 3 separate tiny water droplets.

The morphological characteristics of bacteria, such as size, shape, and arrangement, can be demonstrated by staining a bacterial smear so that individual bacterial cells can be visualized. For most species, these characteristics are genetically determined and thus typical of the species. However, there are some species of bacteria that show considerable variation, or pleomorphism in these characteristics even within a single culture. For example, both Mycoplasma (a bacterium lacking the rigid cell wall of most bacteria) and Arthrobacter (a soil bacterium) show forms ranging from coccoid to rodlike to filamentous. Some pathogenic species such as Mycobacterium tuberculosis and Corynebacterium diptheriae are also pleomorphic.

Bacteria range in size from as small as 0.2 µ to 3.0 µ. They may show the following shapes: spherical (coccus), rod-shaped (bacillus), curved (spiral), or helical (spirochete). They may also assume a characteristic arrangement, based on the way cell division and subsequent separation of the cells occur in that particular species. This typical arrangement shows up better in broth cultures than on a solid medium because in growth on a solid medium the cells are packed together tightly, thus modifying the natural arrangement. There are various types of arrangements: singly, as in most of the Gram-negative rods; in pairs, as found in the bacillus Klebsiella pneumoniae and the cocci Streptococcus pneumoniae and Neisseria: in chains, as in Bacillus and Streptococcus: in regular packets of four or eight as in some Micrococcus; in irregular clumps as in Staphylococcus; and in parallel lines and/or sharp angles as seen in Corynebacterium diptheriae. This last arrangement is called palisade or Chinese character formation. Because of their waxy cell walls, Mycobacterium species are difficult to emulsify and tend to stick together in clumps on the smear; the pathogen Mycobacterium tuberculosis may form long cords of cells in culture. Finally, individual cells may show deviations from the standard form. For example, cocci of Neisseria show flattened sides, making them bean-shaped when occurring in pairs; the rods of Corynebacterium and Mycobacterium often appear club-shaped with swollen ends or knobs. Both groups may show irregular staining. The diplococci of Streptococcus pneumoniae appear slightly elongated and lancet-shaped, with one flattened end and one tapered end.

Making a Bacterial Smear - General directions

1. Place a very small loopful of water on the slide first (use the deionized water bottle on your bench-remove the cover and dip your loop in – sterility is not required for this step).
Note: If the bacterial smear is being made from a broth, simply place a loop or two of the broth culture on the slide and spread as above do not use additional water.

2. Flame the loop, cool and touch it to the colony or slant growth and place the now contaminated loop into the drop. Use large circular motions to spread the drop to about the size of a quarter over the slide.
3. Reflame the loop.

4. Allow the smears to air dry.

5. Heat fix (kill and attach organisms to slide) by rapidly passing the slide (smear side up) through a flame 3 times. Use a slide holder and avoid contact with hot glass.< br>

Gram Stain

1. Prepare a bacterial smear slide Gram Stain of the desired organism(s).
2. Flood the slide with Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) and stain for 1 minute. Crystal violet is the primary stain.

3. Rinse the slide in a very gentle stream of water; drain off excess water by touching the edge of the slide to a paper towel.

4. Flood slide with Gram's Iodine (mordant), let stand for 1 minute, and rinse with a gentle stream of water.

5. Quickly, drip Decolorizing reagent (80% isopropryl alcohol, 20% acetone) down the length of the slide to remove the excess dye. This step is tricky as it is easy to over- or under-decolorize. IMMEDIATELY rinse with a gentle stream of water.

6. Flood the slide with the Counterstain solution (0.6% safranin in 20% ethanol) for 2 minutes; rinse with water.

7. Blot dry using the bibulous paper package in your benches. Do not tear out the pages,just insert your slide and pat it dry. Then observe microscopically with the oil immersion lens.

8. Follow the instructions for using the Microsope properly. Do not use the 45x objective, use only the 10 and 100X objectives and be very careful to avoid getting oil on any objective except the 100x.

Links to Labs in the Soil Microbes Project

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9

We would like to thank Charles Deutsch, Patricia M. Steubing; Stephen C. Wagner and Robert S. Stewart, Jr.; Kyle Seifert, Amy Fenster, Judith A. Dilts, and Louise Temple; and the instructors of the Microbial Diversity Course at the Marine Biological Lab in Woods Hole, MA for their valuable assistance in the development of these labs.