3.2: Procedure

Principles of Biology II (Lab Manual) [BIS 151] · LibreTexts

0%

Contents

    Text

    Size
    100%
    Theme
    Typeface
    Line length
    Spacing

    From LibreTexts, under CC BY 4.0, as the source states it; its equations are written in TeX, which the PDF shows typeset. This page at LibreTexts.

    Part I. Bacterial Cultures and Colony Morphology

    Obtain 2 nutrient agar plates and four sterile swabs. Use a sharpie marker to label the outside of the bottom of the plates (where the agar is located) with your name and the date and draw a line to divide the agar plate in half.

    Identify 4 locations (e.g., kitchen sponge, cell phone, bottom of shoe, dogs’ mouth, etc.) that you would like to swab. Use a sterile swab and moisten with water prior to rubbing that swab gently across the surface of choice. Next, gently rub that swab onto one half of the agar plate to transfer the bacteria. Make sure you label the bottom of the petri dish to reflect the location you have swabbed. If you are swabbing a location that is already moist-such as the mouth of your pet cat-there is no need to pre-moisten the swab prior to collecting the sample.

    Place the agar plates into a Ziploc bag but do not completely seal the bag. Place plates in a safe location that will not be disturbed until the next class period. Discard the swab.

    You will bring the inoculated agar plates to the next class period and will place them in the incubator. This will provide an opportunity for any bacteria present in your sample to grow. You may see colonies begin to form prior to incubation as well.

    During the following class period, you will remove your agar plates from the incubator and observe the growth patterns. You will select one of your plates to sketch and describe the using the space provided below. Be sure to include the various features of your colonies such as their form, elevation, margin, size, texture, and optical characteristics in your descriptions (see illustration and descriptions of these colony characteristics in Figure 1).

    Once you have finished your observations, set the plate aside to be used for microscopic observations as described below.

    Part II. Microscopic Observations

    Prepare and Observe Slide from Environmental Sample

    1. Identify a single colony from your agar plate that you would like to view microscopically.
    2. Obtain a clean glass microscope slide and add your initials with a sharpie to one end. Next, add a small drop of water to the center of the slide.
    3. Using a sterile loop, collect a small sample from a colony of your choice by gently touching the end of the sterile loop to that colony. After collecting your sample, return the lid to the plate.
    4. Carefully use the loop to spread the sample back and forth in the drop of water you previously placed in the center of the glass slide. You will want to move the loop back and forth across most of the slide to spread out the cells in your sample as evenly as possible.
    5. Set the slide on the slide warmer to “fix” the bacterial cells to the slide in preparation for staining. Depending on how much water you initially added to your slide, this can take anywhere from a few to more than 15 minutes.
      Note

      While you are waiting for this step, you should move on to view the Bacterial Types slide as described below.

    6. Once your slide is completely dry, you will transfer the slide to a staining tray. Use the tweezers provided to carefully transfer the slide as it will be hot.
    7. Next, you will apply a drop of methylene blue stain to the smear, allowing the excess stain to run into the staining tray. After a moment, rinse the slide for 2-3 seconds with the bottles of distilled water by gently applying water to the entire slide.
    8. Tap off the excess water and use the blotting paper provided to gently blot dry. Take care not to wipe the slide when drying or you may remove the cells. Rather than rubbing the slide with the blotting paper, you will simply place your slide in between the pages of the booklet and press gently.
    9. Place a small drop of immersion oil onto the region of the slide you wish to view (no cover slip is needed for this slide). Use the 4x (scanning power lens) to locate the specific region under the microscope where the bacterial cells are located.
    10. Next, position the 100x objective lens into place using the rotating nosepiece.
      Caution
      Please note that you should never use the 10x or 40x lens while you have oil on the slide as getting oil onto these lenses can ruin them so you will want to turn the nosepiece in a counterclockwise direction to move directly from the 4x to the 100x lens. DO NOT MOVE THE 10X OR 40X LENS INTO PLACE WHEN THERE IS OIL ON THE SLIDE!
    11. Provide a sketch and a detailed description of notable features for your specimen in data table 3.1 using the 100x objective lens.
    12. Once you have viewed your slide, please place it in in the used slide container provided.
    13. You should also use a kim-wipe to remove the oil from the 100x objective lens. It is important to understand that you should never use anything other than a kim-wipe when cleaning the lenses of a microscope as these other materials can scratch the surface of the lens.
      Caution
      ONLY USE LENS CLEANING WIPES WHEN CLEANING THE MICROSCOPE LENS.

     

    Observe Slide of Bacterial Types

    1. Obtain a slide of mixed bacteria. Locate the three different regions of the slide which include bacteria of different types.
    2. Place a small drop of immersion oil onto the region of the slide you wish to view. Use the 4x (scanning power lens) to locate the specific region under the microscope where the bacterial cells are located.
    3. Next, position the 100x objective lens into place using the rotating nosepiece.
      Caution
      Please note that you should never use the 10x or 40x lens while you have oil on the slide as getting oil onto these lenses can ruin them so you will want to turn the nosepiece in a counterclockwise direction to move directly from the 4x to the 100x lens. DO NOT MOVE THE 10X OR 40X LENS INTO PLACE WHEN THERE IS OIL ON THE SLIDE!
    4. Provide a sketch and a detailed description of notable features for each of these specimens in data table 3.1 using the 100x objective lens.
    5. Repeat steps 2-4 for the remaining 2 regions of the slide where the different bacterial samples are located, being careful to never bring the 10x or 40x lens near the immersion oil.
    6. Once you have viewed all 3 regions of the mixed bacteria slide remove the slide from the stage and use a kim-wipe to remove the oil from the slide prior to returning the slides to the slide box.
    7. You should also use a kim-wipe to remove the oil from the 100x objective lens.
      Caution
      It is important to understand that you should never use anything other than a LENS CLEANING WIPE when cleaning the lenses of a microscope as other materials can scratch the surface of the lens.
    8. Before returning the microscope to the microscope cabinet, return the 4x lens into position and wrap the power cord using the Velcro power cord band. When placing the microscope inside the cabinet be sure to match the number on the microscope with the number on the shelf.

    This page titled 3.2: Procedure was last modified on Tue, 09 Dec 2025 17:47:23 GMT and is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Beth Cliffel.