lab #3 3-4

Why are bacterial cells difficult to see with a standard light microscope?

Their cytoplasm is essentially transparent, so there is little contrast between the cells and the background.

What is the purpose of a simple stain?

To make bacterial cells more visible so their morphology, size, and arrangement can be determined.

What are the three basic bacterial morphologies?

Cocci, bacilli, and spirilla.

What are cocci?

Spherical bacterial cells.

What are bacilli?

Rod-shaped bacterial cells.

What are spirilla?

Spiral-shaped bacterial cells.

What is a stain?

A solution containing a solvent and a colored molecule called a chromogen.

What is a chromophore?

The portion of a chromogen that gives it its color.

What is an auxochrome?

The charged portion of a chromogen that allows the dye to interact with cells.

What is a basic stain?

A stain whose chromogen has a positive charge and is attracted to negatively charged bacterial cells.

Why do basic stains attach to bacterial cells?

Most bacterial cell surfaces have a negative charge, which attracts the positively charged chromogen.

What are three common basic stains?

Methylene blue, crystal violet, and safranin.

What color is crystal violet?

Purple/violet.

What color is safranin?

Red/pink.

What color is methylene blue?

Blue.

Why are basic stains applied to heat-fixed smears?

Heat fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins, making the cells more visible.

What is one disadvantage of heat fixing?

It can distort bacterial cells to some extent.

What is a bacterial smear (emulsion)?

A thin preparation of bacteria spread across a microscope slide for staining and observation.

What should a properly prepared bacterial smear look like after air drying?

It should be slightly cloudy.

How large should a bacterial smear generally be?

Approximately the size of a dime.

Why should you avoid spattering the bacterial emulsion?

To prevent aerosol production and contamination.

Why must a smear be air-dried before heat fixing?

The smear needs to dry before heat fixing so the preparation is not overheated or disrupted.

How is a bacterial smear heat-fixed?

The dried smear is passed through the upper portion of a flame two or three times.

What can happen if a smear is overheated?

Cells can be distorted and aerosols may be produced.

What should you do after heat fixing before staining?

Allow the slide to cool.

What three stains are used in Exercise 3-4?

Crystal violet, safranin, and methylene blue.

How long is crystal violet applied in this exercise?

30–60 seconds.

How long is safranin applied?

Up to 1 minute.

How long is methylene blue applied?

30–60 seconds.

What should you do after applying the stain?

Gently rinse the slide with distilled water and then blot it dry.

Why should you blot rather than rub the slide?

Rubbing can disturb or remove the smear.

Which microscope objective is used to observe the stained bacterial slides?

The oil-immersion objective.

What characteristics should you record when examining the stained bacteria?

Cell morphology, arrangement, and size.

What happens if a smear is overstained?

The cells may become too dark, making morphology and other details more difficult to distinguish.

What happens if a smear is understained?

The cells may be too faint, making them difficult to see clearly.

What is the main difference between a simple stain and a differential stain?

A simple stain generally uses one basic dye to visualize cells, while a differential stain uses multiple reagents to distinguish between different types of cells or structures.

What bacterial arrangements can be observed with simple staining?

Arrangements such as pairs, chains, clusters, tetrads, palisades, and angular arrangements can be observed.

What is a tetrad arrangement?

Four cocci arranged together.

What is a streptococcus arrangement?

Spherical cells arranged in chains.

What is a staphylococcus arrangement?

Cocci arranged in clusters.

What is a streptobacillus arrangement?

Rod-shaped cells arranged in chains.

What is a palisade arrangement?

Rod-shaped cells stacked side-by-side.

What is an angular arrangement?

A pair of rods that are bent where the cells join.

What causes the palisade and angular arrangements seen in Arthrobacter?

Snapping division, in which cells divide lengthwise and remain more or less attached.

Why is determining bacterial morphology important?

It is an important first step in identifying a bacterial species.