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Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au(111). Toluene vapor is a common fixative.

  • What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy?

Resolution

From the results of the Gram stain, the technician now knows that Cindy’s infection is caused by spherical, gram-positive bacteria that form grape-like clusters, which is typical of staphylococcal bacteria. After some additional testing, the technician determines that these bacteria are the medically important species known as Staphylococcus aureus , a common culprit in wound infections. Because some strains of S. aureus are resistant to many antibiotics, skin infections may spread to other areas of the body and become serious, sometimes even resulting in amputations or death if the correct antibiotics are not used.

After testing several antibiotics, the lab is able to identify one that is effective against this particular strain of S. aureus . Cindy’s doctor quickly prescribes the medication and emphasizes the importance of taking the entire course of antibiotics, even if the infection appears to clear up before the last scheduled dose. This reduces the risk that any especially resistant bacteria could survive, causing a second infection or spreading to another person.

Go back to the previous Clinical Focus box.

Microscopy and antibiotic resistance

As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant. Strains of bacteria such as methicillin-resistant S. aureus ( MRSA ), which has developed a high level of resistance to many antibiotics, are an increasingly worrying problem, so much so that research is underway to develop new and more diversified antibiotics.

Fluorescence microscopy can be useful in testing the effectiveness of new antibiotics against resistant strains like MRSA. In a test of one new antibiotic derived from a marine bacterium, MC21-A (bromophene), researchers used the fluorescent dye SYTOX Green to stain samples of MRSA. SYTOX Green is often used to distinguish dead cells from living cells, with fluorescence microscopy. Live cells will not absorb the dye, but cells killed by an antibiotic will absorb the dye, since the antibiotic has damaged the bacterial cell membrane. In this particular case, MRSA bacteria that had been exposed to MC21-A did, indeed, appear green under the fluorescence microscope, leading researchers to conclude that it is an effective antibiotic against MRSA.

Of course, some argue that developing new antibiotics will only lead to even more antibiotic-resistant microbes, so-called superbugs that could spawn epidemics before new treatments can be developed. For this reason, many health professionals are beginning to exercise more discretion in prescribing antibiotics. Whereas antibiotics were once routinely prescribed for common illnesses without a definite diagnosis, doctors and hospitals are much more likely to conduct additional testing to determine whether an antibiotic is necessary and appropriate before prescribing.

A sick patient might reasonably object to this stingy approach to prescribing antibiotics. To the patient who simply wants to feel better as quickly as possible, the potential benefits of taking an antibiotic may seem to outweigh any immediate health risks that might occur if the antibiotic is ineffective. But at what point do the risks of widespread antibiotic use supersede the desire to use them in individual cases?

Key concepts and summary

  • Samples must be properly prepared for microscopy. This may involve staining , fixation , and/or cutting thin sections .
  • A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining , capsule staining , endospore staining, and flagella staining .
  • Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
  • Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.

Fill in the blank

Ziehl-Neelsen staining, a type of _______ staining, is diagnostic for Mycobacterium tuberculosis.

acid-fast

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The _______ is used to differentiate bacterial cells based on the components of their cell walls.

Gram stain

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Short answer

How could you identify whether a particular bacterial sample contained specimens with mycolic acid-rich cell walls?

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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