American University of Health Sciences Bacterial Cell Walls Lab Report

Observe your results

Now that you know how to do a Gram stain, it’s time to go on to reading your results. Usually this is the longest lab period of the term. Gram staining is a lot of work, and new students often have to try the technique a few times before they get it right.

All of the slides you are about to look at were prepared with the Gram stain technique, just as I demonstrated in the video above. All were observed with the 100x immersion oil objective in place, and the iris diaphragm wide open.

Why did I set my iris diaphragm wide open to observe these slides?

E. COLI

The first slide you should look at is E. coli. Probably the most famous bacterium of all, E. coli is fast growing, easy to work with and model for bacterial genetics and cell structure. (And was one of the two example organisms given in the lecture for models of the cell wall and peptidoglycan structure.)

Observe the following slide, prepared with the Gram stain and viewed with the oil immersion objective.

  • Can you pick out the Gram reaction and cell shape?
  • Does E. coli demonstrate a typical arrangement? Or are the cells randomly arranged or single?

When looking for arrangement, don’t be tricked by clumps! Any bacterium can form clumps on the slide. Or strange patterns that have more to do with how the loop was stirred around on the slide. When looking at arrangement, make sure that you look closely to see what the individual cells are actually doing. Do they remain attached after division? Or do they separate?

STAPHYLOCOCCUS EPIDERMIDIS

Our next control organism for the Gram stain is S. epidermidis. In the lecture, you’ll learn about it’s close relative Staphylococcus aureus as our second example of peptidoglycan structure and cell wall type. We used to use S. aureus for this experiment, but because S. aureus is a pathogen, we switched to its non-pathogenic relative S. epidermidis a few years ago.)

  • Thinking to what you already know about hierarchical classification (ie. taxonomic rankings), do you expect two species from the same genus to have the same cell wall type? Why or why not?

Observe the following Gram stain of S. epidermidis. This time you should definitely be able to pick out a characteristic arrangement(in addition to the Gram reaction and cell shape.)

BACILLUS SUBTILIS

Bacillus subtilis is yet another important lab bacterium. It’s used to study cell shape and structure, and is a model organism for study of the limited cell differentiation occasionally observed in prokaryotes (thanks to its ability to form endospores, which you’ll learn more about in the next lab.)

Members of the genus Bacillus are sometimes referred to as “Gram-variable”. Not because they have both cell wall types (they don’t!) But because they are particularly prone to giving incorrect results when the culture is old or unhealthy. Typically, the best Gram stains come from overnight cultures. This means that the microbiologist inoculates a fresh culture (from a single colony) before he/she leaves the lab for the evening. That culture is then ready to use upon return to the lab first thing the next morning. (ie. a culture that has been incubated overnight.)

  • How long do most of our model lab organisms take to form colonies on a plate, when incubated at 37°C?

The following picture shows a Gram stain prepared from an overnight culture of B. subtilis, observed with the oil immersion objective.

  • Notice that most cells have stained the same color. What is the Gram reaction for B. subtilis?
  • However, there are some very faint objects on the slide, and they’ve stained a different color. These might be remains of dead bacteria, and demonstrate why older cultures of B. subtilis can be problematic for Gram staining. (And also shows why we should always start experiments with young vigorous cultures. ie. Overnight cultures, or even fresher.)

After you’ve observed the results from the pictures above, use them to fill in the information on pages 49 and 50 of the lab manual.

YOUR PET BACTERIUM

Once you’ve looked at the pictures above and you know what to look for in a Gram stain, it’s time to examine the Gram stain of your Pet bacterium. A good Gram stain will be an absolute requirement if we’re going to identify these bacteria at the end of the course, so make sure you observe the slides carefully, and make complete notes on what you observe (including the Gram reaction, cell shape, and the arrangement, if there is one.)

Pet bacterium Gram stain results (use the same Pet bacterium number you used last week!)

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KOH “STRING” TEST

In real life, performing Gram stains can be pretty difficult. Luckily, there are other ways to determine what kind of cell wall a bacterium has. The KOH string test is a simple ‘hack’ to figure out a bacterium’s Gram reaction without even using a microscope. It works under the principle that some bacterial cells will lyse when exposed to a base like KOH (potassium hydroxide). If they lyse, their cell components will be released. One of those cell components is a long polymer (perhaps you’ve heard of it… it’s called DNA!) If the DNA from millions of lysed cells is mixed together, the strands will tangle and form long strings, and these strings will be evident when you lift your loop off the slide.

  • Of the two kinds of bacterial cell wall you’ve learned about, which type of cell do you think is more likely to be lysed by a base like KOH? The Gram positives, with their thick layers of peptidoglycan? Or the Gram negatives, with their thinner peptidoglycan surrounded by an outer membrane? (What material is it that gives strength and rigidity to the cell wall anyway?)

Watch this short video to see how you can perform a KOH string test. In the video Ankoor is working with E. coli, so if you remember what kind of cell wall E. coli has, you should be able to answer the questions above.

After you’ve watched the video, it’s time to figure out how your pet bacterium behaves in the KOH string test. Use the table below to look-up your pet bacterium’s unknown number to see the results of the KOH test.

Note: the video above shows the KOH string test performed on E. coli. Unfortunately, we didn’t get a chance to perform this test with the other ‘known’ organism from the lab, S. epidermidis. For that one, we’ll just have to trust the results of the Gram stain.

If you want more practice observing KOH string tests, you can always click on the other unknown numbers to see how they behave. (The unknowns assigned were a mix of Gram positives and Gram negatives.)

Pet bacterium KOH string test

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What’s next?

When you’re done working your way through this lab, it’s time to complete the Lab 3 assignment. Fill in the work sheet with the results from this lab, and then submit the assignment back into the drop box.

The assignment is due at the end of the week that this lab is scheduled, and it should be marked about a week after your submission.

Remember, your teaching assistants are here to help! If you have any questions about this lab don’t hesitate to email them!

 

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