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Objectives

Students should be able to:

  1. Distinguish the key features of prokaryotic cells.
  2. Recognize the diversity of prokaryotes
  3. Understand structural forms of bacteria, including colony morphology, reproduction, growth patterns, and cellular shapes and structure.
  4. Employ aseptic techniques in performing bacterial smear and streaks.
  5. Use simple and Gram staining techniques, and describe the staining results obtained.
  6. Distinguish bacterial groups using differences in their metabolic characteristics

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Introduction

Modern systematists have placed living organisms into three large taxonomic (biological classification groups) called Domains. They are the Archaea, Eubacteria, and Eukarya. Two of these Domains namely, Archaea, and Eubacteria, or simply Bacteria are also described as prokaryotes. Relative to the Eukaryotes, members of the prokaryotes are characterized by simple cellular structure that has limited, or is lacking in membrane-bounded compartments known as organelles. Archaea, such as methanogens and halophiles, have been associated with extreme environments like marshes and deep salts. Although they have the same shape, size, and appearance, and are diverse and widely distributed as members of the more common Bacteria, Archaea are distinguished by a number of chemical differences, including that of the cell wall composition. This group is not discussed further in this exercise.

Much diversity also exists amongst members of the Domain Bacteria. They are of immense economic importance on account of their ability to cause human and livestock diseases, as well as carry out vital biogeochemical reactions that support all ecosystems.

Lab Safety

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You will be using various types of bacteria in this lab. One needs to practice caution when working with bacteria that are POTENTIALLY PATHOGENIC.
 

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The laboratory tables will need to wiped down with disinfectant before and after the lab. The instructor will demonstrate this procedure. Gloves and eye protection will also be necessary
 

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You will also be working with glassware, Bunsen burners, stains and microscopes.

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Pre-lab Activities

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Pre-Lab Activity #1 - Bacterial Shapes

Using resources available to you find imagery of the following bacterial types:

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Coccus

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Spirillum

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Bacillus

Print these out for use during lab. You will also attach them to your post-lab questions.

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Pre-Lab Activity #2 - Cyanobacteria

Using resources available to you, find imagery of the following members of cyanobacteria.

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Anabena

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Oscillatoria

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Gleocapsa

Print these out for use during lab. You will also attach them to your post-lab questions.

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Pre-Lab Activity #3 - Bacterial Diseases

Using resources available to you, describe a disease caused by each of the three types of bacteria, coccus, spirillum and bacillus. Write this on a separate sheet of paper and attach it to your Pre-Lab Questions.

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Pre-Lab Activity #4 - Data Collection Sheet

Download and print out

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Data Collection Sheet for Prokarya

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Colony Morphology (Set printing for Landscape)

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Pre-Lab Questions (html version)    Pre-Lab Questions (Word version)

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The Laboratory Activities and Data Collection

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Lab Activity #1 - Bacterial Colony Morphology


Materials:

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Plates of different species of bacteria (I - IV)


Procedures:

  1. Follow the procedures as outlined on the Data Collection Sheet

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Lab Activity #2 - Bacterial Cellular Shapes / Modifications


Materials:

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Prepared Slides (V - IX)


Procedures:

  1. Follow the procedures as outlined on the Data Collection Sheet

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Lab Activity #3 - Bacterial Identification by Simple Staining


Materials:

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Disinfectant

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Glass slides

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Distilled water (Squirt bottle)

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Toothpick or cotton swab

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Bunsen burner

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Staining tray

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Crystal violet stain

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Methylene blue stain

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Bibulous paper

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Clothes pin

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Tongs

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Compound light microscope

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Immersion oil
 

Procedures:

Bacteria are ubiquitous, taking residence in virtually all types of habitats, including organs and surfaces of other organisms. Many of these bacteria exist in symbiotic relationships with man, and are referred to as human normal flora. In this exercise, you will isolate and examine some of these bacterial species. 

  1. Put aside a clean, sterile slide.
  2. Transfer a drop of distilled water over the center of the slide’s surface.
  3. Use a toothpick or cotton swab to scrape the inside surfaces of your cheek or the gaps between the teeth.
  4. Transfer the scrapings onto a drop of distilled water on the center of the slide.
  5. Using the toothpick or swab smear the mixture evenly over a small area of the slide about the size of a dime.
  6. Discard the swab into a disinfectant-containing beaker or some other container designated for that purpose.
  7. Allow time for the smear to air dry.
  8. Fix the smear by passing the slide through a Bunsen burner flame a few times. This procedure helps kill the bacteria and make the cells adhere to the glass surface.
  9. Transfer the slide to a staining tray.
  10. Irrigate the smear with several drops of Methylene blue and allow it to rest for about a minute. You may also use Crystal violet in place of Methylene blue.
  11. Rinse off the stain drops of water from a squirt bottle, and carefully remove excess moisture with bibulous paper.
  12. Examine your preparation under the microscope, initially using the scanner, low power, and high power objectives in that order.
  13. For best viewing results, now place a small drop of oil immersion on the slide and examine with the oil immersion objective for best results.

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Lab Activity #4 - Bacterial Identification by Gram Staining


Materials:

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Disinfectant

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Glass slides

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Wax Marking Pen

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Distilled water (Squirt bottle)

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Inoculating loop

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Bunsen burner

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Selected bacterial plate or broth cultures (Gram+ and Gram-)

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Staining tray

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Gram iodine stain

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Decolorizer (Acid/Alcohol)

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Safranin stain

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Bibulous paper

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Clothes pin

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Tongs

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Compound light microscope

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Immersion oil
 

Procedures:

Gram stain technique provides one of the means by which to distinguish bacterial types. The stain results separate bacteria into two categories based on the thickness of the peptidoglycan layers, the chemical component in the cell wall. Gram-positive bacteria constitute the group with thick cell wall, hence are able to retain the crystal violet iodine complex described in this procedure; they stain purple or bluish. Gram-negative species have relatively thinner cell wall, which accounts for their loss of crystal violet during destaining but counterstained red or pink with Safranin.

You will be making a slide in this procedure that will have both types of bacteria. One of a Gram+ bacterium and one of a Gram- bacterium.

  1. Prepare the slide by placing three small drops of water in two places on the slide. See the diagram below.


     
  2. Flame sterilize the wire inoculating loop using the Bunsen burner (Follow the procedure outlined by the instructor). If you are using sterile disposable loops then this procedure is not necessary.
  3. Aseptically transfer one loopful of each bacterial culture (Gram+ and Gram-) to each of the separate water droplets on the slide. Flame sterilize the loop in between transfers. If you are using a disposable loop, discard it and use a new one for the second transfer.
  4. Using the loop, smear the mixture evenly over a small area of the slide about the size of a dime.
  5. Flame sterilize the loop. If you are using a disposable sterile loop place it in a disinfectant-containing beaker or some other container designated for that purpose.
  6. Allow time for the smear to air dry.
  7. Fix the smear by passing the slide through a Bunsen burner flame a few times. This procedure helps kill the bacteria and make the cells adhere to the glass surface.
  8. Transfer the slide to a staining tray.
  9. Add drops of Crystal violet onto the smears, and wait for a minute or two.
  10. Wash off the Crystal violet with a gentle stream of water.
  11. Add a few drops of Gram iodine and wait for about a minute.
  12. Wash off the iodine with a gentle stream of water.

    Iodine forms a complex with crystal violet that is retained below the cell giving the cells a blue or purple color, except when the cell wall has started to degenerate as it does in old culture.
     
  13. Rinse the preparation with Gram Decolorizer (Acid/Alcohol). Continue rinsing until the solution draining is faint violet.

    Only the Gram-negative bacteria are destained, while the gram- positive bacteria will retain the Crystal violet, thus staining blue to purple.
     
  14. Gently wash off with water. This way you will avoid continued destaining.
  15. Irrigate the preparation with Safranin, and allow it to stand for half a minute.
  16. Carefully rinse off the Safranin with water and blot with bibulous paper.
  17. Examine your preparation under the microscope, initially using the scanner, low power, and high power objectives in that order.
  18. For best viewing results, now place a small drop of oil immersion on the slide and examine with the oil immersion objective for best results. Note: The gram-negative bacteria usually stains pinkish to red.
  19. Identify Gram + and Gram - bacteria.

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Lab Activity #5 - Turbidimetry Determination of Bacterial Numbers


Materials:

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Disinfectant

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Biohazard container (for used pipets)

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6 Cuvettes

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Sterile water

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5 Sterile culture tubes

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Pipettor

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1mL pipets

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Spectrophotometer

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Trypticase Soy Agar (TSA) broth cultures of selected bacteria
 

Procedures:

Bacterial Growth

Growth in bacteria means increased number of independent cells because the organisms are unicelled. A mature cell simply undergoes DNA replication and the cytoplasm divides into two in a process called Binary fission. In those species where Generation time or duration of a life cycle is short, bacterial growth can be dramatic as cells multiply in a geometric fashion.

Turbidimetry Determination of Bacterial Numbers

There are a few methods available to determine bacterial numbers in a medium. The following procedure guides you on making that determination.

Serial Dilution

  1. Set up five sterile tubes numbered 1through 5 on a test tube rack.

  2. Using your pipettor, transfer 2 ml of sterile broth into tubes 2, 3, 4, and 5.

  3. Using your pipettor, add 2 ml of bacterial broth culture to tubes 1 and 2.

  4. Using your pipettor, aseptically transfer 2 ml of the mixture from tube 2 to 3. Discard the pipette into a disposal bag or disinfectant-filled beaker.

  5. Repeat the above procedure doing a serial dilution, i.e., from tube 3 to 4, and from tube 4 to 5.

Setting up and standardizing your Spectrophotometer

There are slight variations in the design of spectrophotometers. Follow instructions provided in the manual and perform the following:

  1. Select absorbance or % transmittance mode.

  2. Select 550 nm wavelength (or one assigned by the instructor)

  3. Label a cuvette with the letter B. Place the letter at the top of the cuvette so it does not interfere with the spectral beam.

  4. Fill the cuvette marked B, with sterile broth. This tube is referred to as the calibration blank. Its absorbance should read zero (100% transmittance), or be adjusted to zero. 

  5. Label 5 cuvettes (1 - 5) Place your number at the top of the cuvette so it does not interfere with the spectral beam.

  6. Fill each of the 5 cuvettes containing bacterial dilutions (tubes 1-5)

  7. Repeat step 3 (calibration of the spectrophotometer with the blank) between experiments to ensure that settings have not changed.

  8. Mix each bacterial dilution, with the vortex mixer, just before reading absorbance values.

  9. Using the chart provided, determine the number of cells per milliliter of each dilution.

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Post-lab Activity and Data Analysis

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Results and Analysis

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Fill in your Data Collection Sheet with the information requested. Turn it in to the instructor.

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Return all lab materials to the area indicated by the instructor

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Wipe down your lab table with disinfectant.

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Answer the Post-Lab Questions, and turn it in to the instructor.

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Post-Lab Questions (html version)        Post-Lab Questions (Word version)

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