9: Microbial Microscopy
- Page ID
- 124422
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Research Project Week 4: Microbial Microscopy
Microbes Under the Microscope
In the 17th century, Robert Hooke invented the compound microscope. In fact, Hooke coined the term "cell" because the cell walls of plant cells (specifically those in cork) reminded him of the rooms/ cells in monasteries. This magnificent new tool revolutionized the field of Biology as it allowed scientists to see microscopic organisms for the first time. Antoine Van Leeuwenhoek, inspired by Hooke's work examined, carefully illustrated, and reported on the microorganisms in water as well as those present on his teeth!
Although 21st century light microscopes are much more powerful than those from the 17th century, there are physical limitations to the amount of detail that we can observe. We can only tell apart objects that are more than 0.2 μm (micrometers, 1 × 10–6 or microns) apart. More advanced techniques such as electron microscopy open the window for observing even smaller structures such as viruses and subcellular components, but these microscopes are incredibly expensive and very difficult to use.
Bacterial Cell Morphology
Bacterial cells come in a variety of shapes and sizes (though all are incredibly small). There are three major categories of shape 1) cocci - round ball shaped cells; 2) bacill - longer rod shaped cells; and 3) spirilli - even longer spiral shaped cells (Figure 1). Within these broad groupings, bacterial cells may be connected to each other in different ways depending on how they divide.
Bacterial cells come in a variety of shapes; the shape of a bacterial cell is characteristic of the species. Two of the most common shapes are cocci (spheres) and bacillus (rods). Additional shapes include spiral, stalked, and filamentous cells (Figure 1).
Figure 1. Examples of bacterial cell morphologies. Bacteria can range in size from 0.5µm to 5µm. From LadyofHats, Public domain, via Wikimedia Commons
The morphology (shape) of bacterial cells can be determined by looking at them under the microscope. However, since bacteria are translucent they are impossible to see on their own. In order to better see these bacteria, they need to be stained before microscopic analysis. Stains bind to the cellular components especially cell membranes and cell walls and thereby enhance our ability to see them under the microscope. The staining protocol can be found in Figure 3.
Protocols
Protocol 1: Streak Your Favorite Bacteria for Single Colonies
After observing and recording the results of last week’s experiment, choose your favorite bacteria. This favorite strain will be the one that you plan to fully characterize and identify using DNA sequencing next week. If you are undecided, you can select up to three strains and then decide for sure next week.
- Media plates of choice
- Master plates from last week
- Sterile sticks
Protocol
- Obtain your master plate(s) and as many new sterile plates as the strains you want to streak.
- Label your new plates with the same information as last week:
- Group Name
- Date
- Media Type
- Temperature
- Plate number
- and mark orientation on your plate
- Using one sterile toothpick, pick up a VERY small amount of the patch. Just touch the patch, you don’t need to see the bacteria on the toothpick and you’ll still have thousands!
- Streak this stick in one line across one quarter of the plate. This is your primary streak.
- With a NEW sterile stick, start by touching the primary streak and then color the next quarter of the plate in a back and forth motion (Figure 2). This is your secondary streak.
- With another NEW sterile stick, start by touching the secondary streak and then color the next quarter of the plate in a back and forth motion (Figure 2). This is your tertiary streak.
- Finally, with another NEW sterile stick, start by touching the tertiary streak and then color the last quarter of the plate in a back and forth motion (Figure 2). Be careful not to overlap the primary streak as you do this. This is your final streak.
- Repeat this for each of the strains (up to 3) that you may wish to test next week.
- Choose the temperature you wish to grow at, record your decision in your notebook, and place your plates upside down at that temperature (food side up).
Figure 2. Streaking plate method. To isolate individual bacteria to grow single colonies, we use a quadrant streaking technique. (1) a stick is used to touch the initial colony on the patch plate and is gently brushed back and forth on one quarter of the plate. (2) a clean sterile stick is used to overlap the initial streak in the first quadrant and fill in a second quadrant rather than being used to collect more bacteria from the initial culture. (3) The process from step 2 is repeated in the third quadrant with a new sterile stick. (4) Finally step 4 pulls from zone three. From Microbiology Manual (Heartline) shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline
Figure 3. Streaking plate method results. This image shows a successful streak plate, where there are many isolated colonies in the third and fourth quadrants of the plate. From Microbiology Manual (Heartline) shared under a CC BY-NC-SA 4.0(opens in new window) license and was authored, remixed, and/or curated by Rosanna Hartline
Protocol 2: Repeat Your Screen of Any Interesting Strains
- After observing and recording the results of last week’s experiment, identify any of your microbes that are interesting.
- If you have an antibiotic producer:
- Retest each of these interesting microbes against the same ESKAPE Pathogen relative(s) that you tested last week. This will help you confirm the antibiotic production ability of your strain.
- Retest them against all of the ESKAPE relatives. This will help you determine whether your antibiotic has a broad or narrow spectrum of activity (whether it can kill all bacteria or only a small subset).
- Maybe test them under any other conditions (different medium, different temperatures, different testing protocols. The decision is yours, you have plenty of time to test things, but don’t go overboard!
- For each of these tests, use the protocol from Week 3. If you do NOT have an antibiotic producer yet:
- Retest all of your isolates against at least 4 more ESKAPE Pathogen relatives. This will hopefully help you find an antibiotic producer.
- Maybe test them under any other conditions (different medium, different temperatures, different testing protocols. The decision is yours, you have plenty of time to test things, but don’t go overboard!
- For each of these tests, use the protocol from Week 3.
Protocol 3: Simple Stain
Before bacterial cells can be stained, they must first be attached to a glass slide in a thin layer called a smear. Some people find it helpful to draw a circle on the slide where they will put the bacteria to make them easier to find later. To spread out your bacteria, it is useful to start with a small drop of water on the slide (~2 μL) to help move the bacteria around. As you do this, be especially careful not to transfer too much bacteria or the cells will be clumped together and difficult to distinguish (this is a common challenge for beginning microbiologists). Allow the bacterial smear to air dry completely.
Now that you have prepared a smear of bacterial cells on the slide, you need to fix the cells. Fixing has two purposes. First, it kills the cells quickly and in a way that minimizes the cells shrinking and changing shape so that you can identify the shape accurately. In this process, cellular enzymes are denatured so they cannot damage or destroy the cells after they have been fixed. In addition, fixing glues the cells to the slide so that they will not wash off during the staining process. There are several ways cells can be fixed. One possibility is chemical fixation, which is accomplished by putting a chemical fixative such as 95% methanol on the cells. Instead, we will be using heat fixation. To heat‐ fix the bacteria on your slides, pass the slide through the flame of a Bunsen burner. Be sure to hold the slide carefully with a slide clamp or clothespin so that you don’t burn your fingers. Your heat‐fixed smear is now ready for staining.
Simple Staining
Staining procedures that use only one type of stain are called simple stains. They increase the contrast between the bacterial cells and the slide background, allowing you to see the cells and their morphology easily. Many different dyes can be used as simple stains; today we will use methylene blue. Methylene blue is a basic dye and binds to negatively charged cellular components, including the cell wall.
To stain a heat‐fixed smear, hold the slide over a beaker. Using a dropper, put several drops of the dilute stain solution directly on top of the fixed bacterial smear. The circle you drew around the area of the smear will help guide you to the location of the bacteria. Let the stain sit for about a minute, then tilt the slide over the beaker and rinse the stain off using the dH2O wash bottle. Rinse for 10 seconds or more, until the water running off the slide is clear.
Gently blot the slide dry between Kimwipes. Don’t wipe the slide or even the fixed bacteria might wipe off. The simply stained bacteria are now ready for microscopic observation. To view your sample, place a coverslip over the stained sample; any residual water on the slide will help hold the coverslip in place. The purpose of the coverslip is to help prevent contamination of the microscope with any of the bacterial sample.
Figure 3. Staining is required to increase the contrast between the microbes and the slide.
- Glass microscope slides
- Inoculating loop
- Clothespin
- Methylene blue
- dH2O
- Kimwipes
- Beaker
- Lens paper
Protocol
- Draw a dime‐sized circle on a clean glass slide and label the slide with the identification information of your “interesting” microbe.
- Sterilize the innoculating loop by passing it through the flame until red hot and then allow to cool.
- Use the sterile loop to pick up a drop of water. It should look like a bubble wand with bubble solution in it. Put this drop of water inside the circle on your labeled slide.
- Sterilize the innoculating loop again and use either this or a sterile stick to transfer a very small amount of bacteria from the patch on the agar plate to water drop on the glass slide.
- Spread the bacteria out across the circle.
- Allow the bacteria and water to dry completely.
- Heat‐fix the smears by passing the slide through the flame of a Bunsen burner several times
- Stain the slide with 1–2 drops of methylene blue; let sit for about 1 minute.
- Rinse off the stain with dH2O.
- Carefully blot slide dry between Kimwipes.
- Observe the bacterial morphology using the 40x dry objective. (Ask your instructor).
- Once you know that your slide it good, bring it up to the instructor microscope to take a picture of your bacteria using the 40x dry or 100x oil objective.
- In your lab notebook, be sure to label your drawing with the name of the sample, the total magnification, and the cellular morphology of that species.
- Repeat the simple stain procedure for each of your interesting microbial strains.
Using the Microscope Video
Simple Stain Video
When you are finished streaking, retesting, and examining your bacteria under the microscope, please put your new plates food side up at the appropriate temperature. You should have >1 newly made streak plate and >4 antibiotic test plates.
Stack your master plates from last week and put them in a plate bag with the old plates. Label the correctly colored tape with your group name and the date and use this to seal your bags. There is no reason to store your antibiotic test plates, so after taking good photos of these plates, you can discard them in the biohazard plate waste tub.
The used sticks should go in the appropriate container to be sterilized for re-use. Any tubes, tips, and cotton swabs that you used can be thrown in the red biohazard bin. Please put the used ESKAPE pathogen tubes in the labeled area and return all reagents and tools to the place that you got them.
Using the 70% ethanol sprayers on your bench, spray down your bench space to disinfect it and wipe it clean with a paper towel.
References
Biology 105 Lab Manual
Hernandez, S., Tsang, T., Bascom-Slack, C., Broderick, N., & Handelsman, J. (2018). Tiny Earth: A Research Guide for Student Sourcing Antibiotic Discovery. XanEdu Publishing Inc. Print
Perry et al., Lab Manual for Majors General Biology, 1st edition. Brooks Cole. 2008
Powell and Sullivan. MyMicrobe Project – A Guided Research Project Academex. 2015
Slonczewski, J., & Foster, J. W. (2011). Microbiology: An Evolving Science (2nd ed.). New York: W.W. Norton & Co.