Lab Segment 2

Other than morphology, what else could you learn from the use of a simple stain?

A simple stain can help you discern the size of the organism, the quality of the smear preparation, and can also help indicate whether or not contaminating organisms are present.

What is a correct association regarding the difference between an acidic dye and a basic dye?

Acidic = Negatively charged chromogen; Basic = Positively charged chromogen

It is necessary to dilute the bacterial inoculum when transferring from what type of culture media?

Any solid (agar-based) media

What is the best way to hold the upper slide when using it to spread the inoculum across the slide being stained?

At a 45 degree angle relative to the slide being stained

Smear preparation from a slant culture differs from smear preparation from a broth culture in what way?

Bacteria from a slant culture must be mixed with a loopful of water. Because the slant culture is solid, not liquid, water is required to make the smear a uniform thickness. This video demonstrates how to add water to your slide.

Why are basic stains attracted to the bacteria itself?

Cell wall components carry a negative charge and the chromogen has a positive charge; opposite charges attract one another.

Which of the following describes streptobacilli?

Chains of rod-shaped cells

Which of the following describes streptococci?

Chains of spherically shaped cells

Which of the following describes staphylococci?

Clusters of spherically shaped cells

What is the purpose of allowing the bacterial smear to dry once it has been placed onto the surface of the slide?

Drying helps remove excess water to ensure optimal heat fixation.

Arrange these steps for preparing a smear from a broth culture in their correct order.

Flame loop and cool. Suspend broth culture. Remove cap and pass tube through the flame Obtain loopful of culture from tube Pass mouth of tube through flame & re-cap Spread culture on slide. Let air-dry Flame loop

After you've removed a loopful of broth culture from the culture tube, what's your next step in preparing a smear?

Flame the opening of the tube. You should flame the mouth of the test tube before and immediately after removing a loopful of culture, as shown in this video. This helps prevent airborne contaminants from entering the culture.

You're preparing a smear from a broth culture. You've labeled a clean slide. You've flamed the inoculating loop and let it cool. What's your next step?

Flick the culture tube In preparing a smear from broth, you want to be sure enough cells make it onto your smear. Cells in broth cultures tend to settle to the bottom of the tube. This leaves very few cells in the top layer, where you'll be inserting the loop. Flicking the tube resuspends the cells evenly in the broth.

A student is observing a stained smear that was prepared from a slant culture. This micrograph shows what was observed. Assuming he placed culture on the slide during preparation of the smear, which of these errors most likely explains the observed result?

He forgot to fix the slide. One of the purposes of fixation is to make the cells stick to the slide. If the student had forgotten to fix the slide, the cells probably washed off during the staining procedure.

What is the purpose of heat fixation?

Heat fixation adheres the cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria

Why is it important not to use thick or dense bacterial smears?

In a thick smear the bacteria will be too concentrated, reducing the amount of light passing through the slide, the stain may not penetrate adequately, and it will be difficult to visualize individual cells.

Which of the following is an example of a basic stain?

Methylene blue

Why doesn't nigrosin penetrate bacterial cells?

Nigrosin has a negatively charged chromogen and is therefore repelled from bacterial cells.

These stained bacteria are from a heat-fixed smear created from a slant culture. Which of the following would produce an improved smear?

Obtain a smaller amount of culture for the smear. When making a smear from a solid culture, you need only a tiny amount of bacteria. If you obtain too much culture, the cells will appear crowded, making their arrangement difficult to determine. Note the difference in this micrograph of a smear that was prepared using a much smaller amount of culture.

Which of the following describes a proper fixation technique?

Pass the slide two or three times through a flame. This method gently heats the slide, which denatures the proteins within the bacteria. Much like frying an egg, this causes the bacteria to become more rigid, preserving their natural shape. It also causes the bacteria to adhere to the glass slide.

During heat fixation, it is important that the slide be passed only 2-3 times through the outer portion of the Bunsen burner flame to prevent overheating. Overheating can cause which of the following to occur?

Plasmolysis of the bacterial cell wall, distorting the cell morphology

What two characteristics are used to describe the cellular morphology of a microscopic organism?

Shape and arrangement

What is the difference between spirilla and spirochetes?

Spirilla are rigid, while spirochetes are flexible.

What is the definition of a basic stain?

Stain with a positively charged chromogen

Under which of the following conditions would you be most likely to prepare (and use) a smear?

Staining bacterial cells to observe under the microscope. Smears are prepared by spreading bacteria on a microscope slide. Once the sample is spread out and stained, the characteristic shape and arrangement of the bacteria can be seen under the microscope.

What would you expect to happen if you failed to heat fix the slide?

The bacteria are likely to get washed away during the staining procedure

A student prepares a smear from a broth culture; air-dries it, and fixes it. He then remembers that he forgot to flick the broth culture tube before removing the bacteria. What is the most likely consequence of this error?

The bacteria may be difficult to find on the slide. Broth cultures should be resuspended so that bacteria are evenly distributed throughout the broth. Otherwise, you could end up with very few bacteria in the smear. With very few bacteria in the smear, finding bacteria will be difficult. You'll waste a lot of time searching.

Why does the presence of grease or dirt on a glass slide result in a poor smear preparation?

The grease and dirt can create artifacts that interfere with accurate visualization of the organisms.

A student properly flames her loop but then forgets to let it cool. Instead, she immediately inserts the hot loop into her broth culture. What is the most important issue associated with this mistake?

The hot loop may create aerosols when it touches the culture. When the hot loop touches the liquid broth culture, it will cause some of the broth (and bacteria) to briefly boil, creating a bacteria-containing aerosol. These airborne bacteria could enter the student's respiratory tract or land on her skin.

A student prepared a smear from a broth culture, air-dried it, and heat-fixed it. This was followed by a staining procedure that colored the cells purple. Which of the following errors most likely caused the poor appearance the student's micrograph, shown here?

The slide was not cleaned completely. The white, circular spaces on this micrograph are areas where oily residue remains on the slide. When liquid culture was added to the slide, the culture did not penetrate the oily regions, and cells "lined up" along the edges of these regions (indicated by arrow). This gives the impression that the cells are arranged in chains, but this is a false arrangement caused by the oily patches.

Why is it necessary to stain microorganisms with biological stains in order to visualize them with a microscope?

The stain provides necessary contrast between the cells and the background.

What would the slide look like under the microscope if methylene blue is used instead of nigrosin?

The stain would stain the cells rather than the background

What can you conclude about the pictured specimen?

This is a picture of a negative stained specimen The bacteria would not have been heat fixed in the preparation of this slide The pictured bacteria are bacilli

What can you conclude about the pictured specimen?

This is a picture of a negative stained specimen The bacteria would not have been heat fixed in the preparation of this slide The pictured bacteria are cocci

What is the purpose of holding the slide parallel to the stream of water during the final rinsing step?

To avoid dislodging the organisms from the surface of the slide

What is the purpose of applying a stain to a bacterial smear?

To provide contrast between the organism and the background.