Chapter 3 Homework

Which waxy molecule, found in the cell wall of acid-fast bacteria, prevents these cells from being Gram stained? Acid-alcohol Mycolic acid Methylene blue Carbolfuchsin

Mycolic acid

Which of the following pairs is mismatched? basic dye - negative stain acidic dye - capsule stain nigrosin - negative stain methylene blue - simple stain crystal violet - simple stain

basic dye - negative stain

You are performing a Gram stain on gram-negative bacteria and you stop after the decolorizer step. What is the appearance of the bacteria at this point? purple red colorless brown

colorless

What does resolution mean? the ability of a microscope to stay in focus when the objective lenses are moved from low power to high a measure of the ability of a medium to bend light the total magnification of the visual image seen the ability of a microscope to distinguish fine details and differentiate between two very close objects

the ability of a microscope to distinguish fine details and differentiate between two very close objects

A microorganism measures 5 μm in length. Its length in mm would be 500 mm. 0.5 mm. 50 mm. 0.005 mm. 0.05 mm.

0.005 mm.

Why is a specimen smaller than 200 nm not visible with a light microscope? Visible light is only good at wavelengths below 390 nm. It is too easy to lose on the stage. Anything smaller than 200 nm cannot interact with visible light. The lenses only go to 100 X magnification power.

Anything smaller than 200 nm cannot interact with visible light.

MicroCareers: Nice to See You

Complete Review As the resident microbiologist in the imaging core for the university, it is your job to aid the researchers in preparing photographic data and representative figures for publication. You prepare samples for staining and set up the computers and microscopes to photograph and measure the cells. Due to their small size and the fact that most do not contain any pigments, these cells must be colorized using chemicals or compounds. Generally, dyes or charged stains are used to colorize the cell and allow for visualization. Before bacterial cells may be stained, they have to be "fixed" to the glass slide or they will be rinsed away with the stain. To "fix" a bacterial cell to a glass slide, a drop of the culture must be air dried and then heated in the flame of a Bunsen burner to drive off any water molecules that may be trapped between the bacterial cell and the surface of the glass. The heating also denatures some proteins and sugars on the cell surface that will stick to the surface of the glass and aid in retention of the bacterial cells during any wash steps, some of which may be stringent and would wash away any cells not adhered to the glass. Following the fixation of the bacteria to a glass slide, the cells may then be stained using many different methods and dyes. Simple stains are used for visualization of the cells' general morphology without any distinction between possible species present. Simple stains are composed of a single dye, such as crystal violet, safranin, or methylene blue and are labeled as "simple" because after soaking the smear in the dye for 30-60 seconds the slide is rinsed off with water.

A student from one of the research labs is having trouble preparing a slide for examination and photographing. The bacterial slide that he has brought to you was prepared using a commercially purchased stain. He has asked for your help in determining what he is doing wrong so that he can change the lab protocols and continue on with his project. After examining the slide under oil immersion, you determine that no bacteria are present even though the student is able to show you the culture he used to make that slide that has visible growth in the liquid medium. Which of the following statements does NOT explain the fact that there are no bacteria present on the student's slide? Rinsing with alcohol during the washing step stripped the bacteria off the glass slide. Overheating during the fixation step boiled the water within the bacterial cells and resulted in the cells bursting. By not allowing a glass slide to completely air dry before heat fixation, the flame will cause the surrounding water to boil and this will damage the bacterial cell. Insufficient heating of the slide did not drive out the thin layer of water and this resulted in minimal bonding between the bacteria and the glass slide.

Rinsing with alcohol during the washing step stripped the bacteria off the glass slide.

You volunteer to help the student with his fixation technique and in choosing a proper stain for the project he is working on. After watching and helping the student correct any problems with his fixation technique, you now need to determine which stains to use. The single criterion for the project is to be able to determine cell shape and size in a pure broth culture after some treatments. No differentiation between cell types is required (i.e., Gram-positive or Gram-negative), so you want to convince the student that a simple stain would be his best option. The charged dyes used in simple staining will penetrate the bacterial cell and will be retained after rinsing the slide with water to remove surplus dye. Which staining procedure would be best to use to stain the slides required for this student's project? Why? An acidic stain such as nigrosin will stain the cells a dark black coloration for easier viewing under the microscope. Malachite green and heat will colorize the cell for easier visualization and is a simple process. Using crystal violet followed by a rinse and Gram's iodine will stain the cells a dark blue with large crystals. The positively charged methylene blue will be attracted to the negatively charged components of the cell wall and will be retained.

The positively charged methylene blue will be attracted to the negatively charged components of the cell wall and will be retained.

This activity asks you to sort items according to the most appropriate method for viewing. For each of the following items, indicate which viewing method is most appropriate-the unaided eye, light microscopy, scanning electron microscopy, or transmission electron microscopy-by dragging it into the correct bin.

Unaided eye examining your cat for the presence of fleas Light microscope examining a clinical specimen, such as a sputum smear, for the presence of bacteriaexamining a blood smear for the presence of malarial parasites Scanning electron microscope viewing the three-dimensional shape of a protozoan and the arrangement and distribution of cilia on its surface determining the three-dimensional shape and arrangement of bacterial cells Transmission electron microscope confirming the 9 + 2 microtubule arrangement in a eukaryotic flagellumviewing a cross section of poliovirus (30 nm)viewing ribosomes (20 nm) within a bacterial cellviewing the layers of the gram-negative cell wall in cross section

A new project has come up in the core center - a researcher wants to study bacterial biofilms and will need you to document the process of biofilm formation. Bacterial biofilms are colonies of different species of bacteria that interact and allow the bacteria to grow in a new environment. Since this will be a mixed culture, you will need to use a staining protocol that allows for differentiation between bacteria based on cell wall properties. The Gram staining procedure uses a series of stains and alcohol decolorization to differentially color different species of bacteria that may be present in the growing biofilm. This differential colorization will allow for determination of bacterial morphology and give some insight into the bacterial cell wall composition. Put the following descriptions in order for the staining reactions in the cells of a bacterial smear during the Gram staining procedure.

Unstained cells (start of process) All cells are stained purple. Dye molecules are rendered insoluble in presence of mordant; dye is rinsed away. Cell wall of Gram-negative cells is broken down; Gram-positive cells remain intact. Clear cells are stained a red or pink whereas the color of other cells remains unchanged.

What is the total magnification of a specimen viewed with a 10x ocular lens and a 45x objective lens? 450x 10x 45x 4.5x 100x

450x

Which of the following pairs of terms is properly matched? Acid-alcohol—decolorizing agent Carbolfuchsin—decolorizing agent Methylene blue—decolorizing agent Steam heat—decolorizing agent

Acid-alcohol—decolorizing agent

A virus measures 100 nm in length. What is its length in μm? 0.001 μm 0.1 μm 10 μm 0.01 μm 1 μm

0.1 μm

Which of the following is NOT equal to 1 mm? 0.1 cm 10-3 m 0.001 m 106 nm 100 μm

100 μm

You have been asked to lead a demonstration for the undergraduate microbiology lab course about the uses of negative staining when studying bacteria. A "negative" stain does not stain the bacterial cell itself but stains the space between cells. Under magnification, the acidic (negatively charged) nature of the stain will be repelled by the negatively charged bacterial cell wall and will leave the cell colorless in a stained background. Negative stains are used primarily to reveal the presence of negatively charged bacterial capsules; therefore, they are also called capsule stains. Encapsulated cells appear to have a halo surrounding them. The negative stain procedure does not require heat fixation, which limits any chances of alteration in bacterial cell shape and size. The bacterial suspension is added to a drop of stain, such as nigrosin or eosin, and drawn across the glass slide using a coverslip. Nigrosin staining-not safranin staining-of Klebsiella pneumoniae will allow for the visualization of the cell shape and the determination of the presence of a capsule. True False

True

Which of the following measurements is correctly matched with microorganisms of that size? Viruses; 10 micrometers Yeast; 1 centimeter Bacteria; 2 micrometers Bacteria; 10 nanometers

Bacteria; 2 micrometers

What is meant by light rays being divergent? It is spreading out It is coming together to a focused beam It is heading upwards

It is spreading out

Acid-fast staining is used to detect members of which bacterial genus? Streptococcus Mycobacterium Clostridium Staphylococcus

Mycobacterium

In a typical brightfield microscope (seen in the animation), at which point does magnification begin? The stage The condenser lens The ocular lens The lamp The objective lens

The objective lens

What happens to the light rays when they hit the specimen? They are focused into a small area towards the objective lens. They are absorbed by the stage. They are reflected, refracted, or absorbed by the specimen. They are diverted to the ocular lens.

They are reflected, refracted, or absorbed by the specimen.

What is the role of the ocular lens? To focus the light to a high intensity in a small area To do the bulk of the magnification To recreate the image in the viewer's eye To adjust the wavelength of light

To recreate the image in the viewer's eye

Which of the following signs and symptoms is NOT associated with active pulmonary tuberculosis? Chest pain Weight gain Fever Cough with blood

Weight gain

Which microscope achieves the highest magnification and greatest resolution? electron microscope compound light microscope fluorescence microscope phase-contrast microscope darkfield microscope

electron microscope

Which of the following correctly traces the path of light through the compound microscope? condenser; light source; specimen; ocular lens; objective lens light source; condenser; specimen; objective lens; ocular lens light source; condenser; objective lens; specimen; ocular lens light source; specimen; condenser; objective lens; ocular lens condenser; light source; specimen; objective lens; ocular lens

light source; condenser; specimen; objective lens; ocular lens

The purpose of a mordant in the Gram stain is to make the bacterial cells larger. remove the simple stain. prevent the crystal violet from leaving the cells. make the flagella visible. make gram-negative cells visible.

prevent the crystal violet from leaving the cells.

What structure does light pass through after leaving the condenser in a compound light microscope? ocular lens objective lens specimen illuminator

specimen