Prokaryotes II – Lab Exercises

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Exercises 1-2 are from the PARE project; SLCC is a participant:

Genné-Bacon EA, Bascom-Slack CA. The PARE Project: A Short Course-Based Research Project for National Surveillance of Antibiotic-Resistant Microbes in Environmental Samples. J Microbiol Biol Educ. 2018;19(3):19.3.97. Published 2018 Oct 31. doi:10.1128/jmbe.v19i3.1603

Please choose one live specimen or prepared slide to image from Exercises 4-6. Take a picture under the microscope and submit via Canvas. Make sure that your picture is appropriately centered, cropped, and focused. Add a scale bar.

Exercise 1: Record data for PARE project

Materials Needed

  • Your soil data collection sheet
  • Your data calculations worksheet
  • Access to the web-based Error-Checking Spreadsheet
  • Access to the PARE Global database

Procedure

  1. For this project to be of value to other students and to the scientific community, each team must accurately record their results into the database.  One entry per team of up to 4 individuals!  The collective sum of results provides a rich overview of antibiotic resistance prevalence and provides an opportunity to perform regional comparisons.  All database information is available to other PARE researchers for analysis, so please use care to ensure that all calculations are correct and that data are entered correctly.
  2. Discuss with your teammates what values should be entered into the database.  Your instructor may ask you to enter your data into an Error-Checking Spreadsheet so that the values can be compared and edited to correct mistakes or outliers (data outside the expected range) prior to entry into the database.
  3. Access the database by following the link below:

http://tinyurl.com/PARE-data-form

  1. Enter your data in the form as directed, being careful with your spelling and accuracy in entering values.  The database does not allow you to edit entries after submission so be sure to enter all information as accurately as possible.  Make only one entry per team.  The form will ask you to provide data for both plate set 1 and 2, as well as information about where the soil was collected.  Working as a group to fill out the form will also help you catch errors and typos.  Pay attention to spelling text entries and the number of zeros on numeric entries.  Enter your instructor’s last name only and be sure to spell it correctly.
  2. At the end of the form, you will receive a message indicating that your data have been submitted.  Your information will not be recorded until you receive the confirmation message: “Your response has been recorded.”  

Exercise 2: Use BLAST database to identify your colony from last week

Copy and paste your sequence into the BLAST database and record the species of your bacteria in the space below:

https://blast.ncbi.nlm.nih.gov/Blast.cgi

Exercise 3: Examine your environmental culture

Look at the plate that you inoculated with an environmental swab last class. 

  1. Count the number of colonies on your plate.
  2. Measure the diameter of the largest and smallest colony on your plate by observing the plate under a stereo microscope and using a ruler.
    1. Largest _____________
    1. Smallest_____________
    1. Range_____________
  • How many different types of bacterial colony do you observe? Describe them. (consider color and colony morphology – form and margin)
ColorFormMarginSize (diameter)
    
    
    
    
    
    
    
  • Was your prediction from last week correct?

Exercise 4: Make wet mounts and examine living cyanobacteria.

Materials:

  • Cultures of Nostoc, Gloeocapsa, Oscillatoria, Merismopedia, and Gloeotrichia
  • Microscope slides
  • Coverslips
  • Methylene blue
  • India ink

Exercise 4.1: Gleocapsa: Consists of small, circular cells.  Recently divided cells each form their own sheath, but tend to remain associated within the sheaths of the mother cells.  For this reason, it is common to see small groupings of cells (“incipient colonies”) arranged in three dimensions within a common sheath.  These soon separate by fragmentation.

Use India ink to negatively stain a group of cells to observe the sheaths.

Observe and draw a group of cells.

Exercise 4.2: Merismopedia: Consists of a two dimensional colony of small, round cells that are united by their common sheath.  The cells divide in only two dimensions resulting in a flattened colony whose members are in regular rows and files.  New colonies are formed by the fragmentation of larger and older colonies.

Observe and draw a portion of a colony.

Exercise 4.3: Oscillatoria: Consists of unbranched filaments, each of which is a strand of uniform cells (referred to as a “trichome”) within a common sheath.  Cell division is in one dimension, resulting in elongation of the filament.  Cells are more or less cylindrical.  Filaments change their orientation or position by gliding.  The genus name arises from the oscillation of the filament that may be observed during movement.  Filaments reproduce by a type of fragmentation that occurs at points where cells (called necridia) have died (apoptosis).  This results in the formation of small segments from the original filament (hormogonia) that are able to disperse by gliding.  Oscillatoria does not form heterocysts or akinetes.

Observe and draw a filament.

Are necridia and hormogonia present?

Observe movement of a filament, if possible.

Exercise 4.4: Nostoc: Consists of a filament of rounded cells surrounded by a common sheath that gives the appearance of a beaded necklace.  If growth conditions are favorable, these may form a slimy macroscopic mass that is sometimes called “witches’ butter” or “star-jelly.”   Some cells may develop into heterocysts or akinetes, depending upon environmental conditions.  Fragmentation of the filaments helps disperse this species.  Nostoc is a widespread and common species and is a major component of pond scum.

Observe and draw a filament.

Are heterocysts or akinetes visible?  If so, draw one of each

Nostoc may form mutualistic partnerships with plants, animals, or fungi.  

Exercise 4.5: Observe the “pre-coralloid” roots of a cycad. (This is a plant at the back of the room – ask if you are not sure which one).  These roots are negatively geotropic (i.e. they grow upwards out of the soil) and are specialized to form a mutualistic relationship with nitrogen-fixing cyanobacteria such as Nostoc.  If colonized, they will develop into coralloid roots.  The cycad provides an environment favorable for growth of the cyanobacteria, and the cyanobacteria provides a source of fixed nitrogen that is favorable for growth of the cycad.

Draw one (or several) coralloid roots:

Exercise 5: Examine live culture of Halobacterium

Halobacterium sp. is an archaeon that inhabits extremely salty environments, including the Great Salt Lake. 

Materials:

  • Halobacterium culture
  • Microscope slide
  • Coverslip
  • Toothpick or inoculating loop
  • Immersion oil

Make a wet mount and observe Halobacterium under the microscope.

  1. Put a drop of salt water (25%) on a slide and add a tiny smear of Halobacterium using a toothpick or inoculation loop. Mix gently.
  2. Add a coverslip.
  3. Observe under 63x and oil immersion (100x).

Draw a Halobacterium cell. Are the cells moving?

Exercise 6: Examine prepared slides of prokaryote diversity

Materials:

  • immersion oil
  • prepared slides of:
    • mixed Archaea (slide may say Archaebacteria)x`
    • Escherichia coli
    • Neisseria gonorrhoeae
    • Staphylococcus aureus
    • Streptococcus pneumoniae
    • Bacillus anthracis
    • Borrelia burgdorferi
    • Helicobacter pylori
    • Treponema sp.

Important: Please clean the immersion oil off the slides when you are finished. Return the slides to their tray.

Exercise 6.1: Examine prepared slides of Proteobacteria

Examine prepared slides of E.coli, Helicobacter pylori, and Neisseria gonorrhoeae. Examine the slides under 100x (oil immersion). You may wish to work as a group so that you have fewer slides to clean. Record the shape and draw a few cells of each.

Shape_____________________                             Shape_____________________                   Shape_____________________

Exercise 6.2: Examine prepared slides of Gram-positive bacteria

Examine prepared slides of Staphylococcus aureus, Streptococcus pneumoniae, and Bacillus anthracis. Examine the slides under 100x (oil immersion). Record the shape and draw a few cells of each.

Shape_____________________                             Shape_____________________                   Shape_____________________

Exercise 6.3: Examine prepared slides of spirochetes

Examine prepared slides of Treponema and Borellia burgdorferi. Examine the slides under 100x (oil immersion). Record the shape and draw a few cells of each.

Exercise 6.4: Examine prepared slide of Archaea

Examine prepared slides of mixed Archaea. Examine the slides under 100x (oil immersion). Record the shape and draw a few cells.