3/31/2019 Chapter-04 Homework

Chapter-04 Homework
Due: 1:59pm on Wednesday, January 23, 2019
You will receive no credit for items you complete after the assignment is due. Grading Policy

Microbiology Animation: Microscopy and Staining: Overview

Click here to complete this activity.

Then answer the question.

Part A
What is the role of lenses in microscopy?

ANSWER:

Lenses increase the contrast to determine structural differences in stained specimens.

Lenses are only used to magnify specimens that are smaller than whole red blood cells.

Lenses focus either light or electrons to create a magnified image of a specimen.

Correct

MicroCareers: Nice to See You

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.

Part A - Fixation
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?

Select all that apply.

Hint 1. Why Fix?

Even after the slide is dry, some water molecules are still present and are trapped between the bacteria and the surface of the glass. This layer of
water inhibits the bacterial interaction with the glass.

Hint 2. What’s the worst that could happen?

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3/31/2019 Chapter-04 Homework
Without heat or chemical fixation, the bacteria you are attempting to stain will be rinsed away during a washing step.

ANSWER:

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.

Overheating during the fixation step boiled the water within the bacterial cells and resulted in the cells bursting.

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.

Correct

Part B - Simple Staining

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?

Hint 1. Why so simple?

“Simple” refers to the fact that these staining procedures use a single stain and water to rinse the slide. The stain penetrates the cell and some is
retained during the single wash step.

Hint 2. Basic stain

Due to the inherent properties of the stain, the stain’s positive charge will be attracted to the negatively charged proteins and DNA in the cell.

ANSWER:

Malachite green and heat will colorize the cell for easier visualization and is a simple process.

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

An acidic stain such as nigrosin will stain the cells a dark black coloration for easier viewing under the microscope.

Using crystal violet followed by a rinse and Gram’s iodine will stain the cells a dark blue with large crystals.

Correct

Part C - Gram Staining

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.

Hint 1. Gram stain components

The components of the Gram staining procedure are the primary stain, mordant, decolorizing agent, counterstain, crystal violet, Gram’s iodine,
alcohol, and safranin.

Hint 2. Overview of the Gram stain

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3/31/2019 Chapter-04 Homework

ANSWER:

Reset Help

Unstained cells (start of process) Stained cells (end of process)

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

The correct ranking cannot be determined.

Correct

Part D - Negative Staining

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.

Hint 1. What gets stained?

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3/31/2019 Chapter-04 Homework
The stain carries a negative charge, so it is unable to penetrate the cell wall. This stain is used to colorize the empty or negative space (hence the
name) between the cells.

Hint 2. Use of negative stains

ANSWER:

True

False

Correct

Microbiology Animation: Dichotomous Keys: Practice

Click here to complete this activity.

Then answer the questions.

Part A
Why is the unknown in this example not Pseudomonas?
ANSWER:

It is a Gram-negative cell.

It can tolerate oxygen.

It ferments lactose.

It is a rod-shaped cell.

Correct

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Part B
What test result indicates that the unknown can utilize citrate as its sole carbon source?
ANSWER:

The medium turns blue.

It turns the medium black.

The culture becomes turbid.

The medium turns pink.

Correct

Part C
How would the results be different if this organism was Salmonella?
ANSWER:

It would be a rod.

It would not ferment lactose.

It would produce hydrogen sulfide.

It would not tolerate oxygen.

Correct

Part D
How is fermentation of lactose detected?
ANSWER:

The drop in pH turns the indicator dye yellow.

The acetoin produced turns the medium pink.

The cells form a black precipitant.

The medium become turbid when exposed to air.

Correct

Part E
How many questions are needed in this dichotomous key to determine if the unknown is Bacteroides?
ANSWER:

Three

Two

Four

Five

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Correct

Microbiology Animation: Light Microscopy

Click here to complete this activity.

Then answer the questions.

Part A
Why is a specimen smaller than 200 nm not visible with a light microscope?

ANSWER:

The lenses only go to 100 X magnification power.

It is too easy to lose on the stage.

Visible light is only good at wavelengths below 390 nm.

Anything smaller than 200 nm cannot interact with visible light.

Correct

Part B
What happens to the light rays when they hit the specimen?
ANSWER:

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

They are absorbed by the stage.

They are diverted to the ocular lens.

They are focused into a small area towards the objective lens.

Correct

Part C
What is the role of the ocular lens?

ANSWER:

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3/31/2019 Chapter-04 Homework

To adjust the wavelength of light

To focus the light to a high intensity in a small area

To recreate the image in the viewer’s eye

To do the bulk of the magnification

Correct

Part D
What is meant by light rays being divergent?

ANSWER:

It is heading upwards

It is spreading out

It is coming together to a focused beam

Correct

Part E
In a typical brightfield microscope (seen in the animation), at which point does magnification begin?

ANSWER:

The ocular lens

The objective lens

The lamp

The stage

The condenser lens

Correct

Chapter 4 Pre-Lecture Quiz Question 5

Part A
Which of the following is NOT true of the work of Woese and Fox?

Hint 1. Classification and Identification of Microorganism

Woese and Fox spend years sequencing ribosomal RNA.

ANSWER:

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3/31/2019 Chapter-04 Homework

Their classification system included two domains of prokaryotic cells: Archaea and Bacteria.

They determined that shape and biochemical tests provided enough information to categorizemicroorganisms into their correct taxonomical
classification.

They proposed a three-domain taxonomy system.

They discovered that there are three basic types of ribosomes.

Correct

Score Summary:
Your score on this assignment is 100%.
You received 2 out of a possible total of 2 points.

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