Guiding Question Four

How can a stain (dye) be defined chemically?

A colored chemical compound containing a chromogen that gives the dye its color.

What does the ability of a stain to bind to cellular components depend on?

The electrical charge of the stain and the cellular component.

What charge does an acidic stain have?

A negative charge.

What charge does a basic stain have?

A positive charge.

What happens when an acidic stain encounters bacterial cells?

The negatively charged stain is repelled by the negatively charged bacterial surface.

What happens when a basic stain encounters bacterial cells?

The positively charged stain is attracted to negatively charged bacterial components.

What does an acidic stain bind to?

It is repelled by the bacterial cell and stains the background instead.

What does a basic stain bind to?

Negatively charged components of the bacterial cell.

Compare acidic and basic stains by charge.

Acidic stains are negative; basic stains are positive.

Compare acidic and basic stains by what they stain.

Acidic stains color the background; basic stains color the bacteria.

What does a positive/simple stain look like?

Colored bacteria on a mostly clear background.

What does a negative stain look like?

Clear/unstained bacteria against a colored background.

Compare simple and differential staining.

Simple staining uses one stain to visualize bacteria; differential staining distinguishes different types or characteristics.

What can simple staining be used to visualize?

Bacterial morphology, arrangement, presence, and number.

When should bacterial smears be prepared?

Before staining and examining a bacterial specimen.

What are the four main rules for preparing a bacterial smear?

Prepare the slide, label it, prepare the smear, and heat-fix it.

How should the glass microscope slide be prepared?

Make sure it is clean; clean with 70% ethanol if necessary.

Why must the microscope slide be clean?

Oils or grease can prevent the smear from spreading properly.

How should you hold the microscope slide?

Hold it by the edges.

How should the microscope slide be labeled?

Label one end of the slide with the bacterial organism’s name.

Why should the slide be labeled before preparing the smear?

To identify which organism is on the slide.

When do you add water when preparing a bacterial smear?

When using bacteria from a solid culture, slant, or Petri dish.

When do you NOT add water?

When using a broth culture.

Why is water unnecessary for a broth culture?

The bacteria are already suspended in liquid.

How much water should be added to a solid-culture smear?

A small drop.

What happens if too much water is added?

The smear takes longer to air dry.

How should bacteria be transferred from a solid culture?

Gently tap a sterile loop onto a colony; do not use the loop like a scoop.

How should the bacteria be spread on the slide?

Gently spread them in a circle about the size of a quarter.

Why should cell clumps be gently dislodged?

To spread the cells evenly so morphology and arrangement can be seen.

What should you do after spreading the smear?

Allow it to air dry completely.

Should you blow on or wave the slide to dry it faster?

No.

How is a bacterial smear heat-fixed?

Pass the completely dry slide quickly through the heat three times.

Why is heat fixation important?

It kills bacteria, makes them stick to the slide, and coagulates proteins to make cells more visible.

What happens if you overheat the smear?

Cells can burn and aerosols can be created.

Why is creating a proper bacterial smear clinically important?

It allows accurate observation of bacterial morphology and arrangement.

What happens if a smear is too thick?

Too many cells overlap, making morphology and arrangement difficult to see.

What happens if a smear is too thin?

There may be too few cells to see clearly.

What does a good bacterial smear look like?

A slightly cloudy circle that is evenly spread and still allows you to read text through it.

What is the purpose of a simple stain?

To visualize bacteria and determine their morphology, arrangement, presence, and number.

How many reagents are used in simple staining?

One stain/reagent.

What type of stain is used for simple staining?

A basic stain.

Why is a basic stain used?

Its positive charge is attracted to negatively charged bacterial components.

What basic stains are used in this lab?

Crystal violet, safranin, methylene blue, and carbol fuchsin.

What color is crystal violet?

Purple/blue.

What color is safranin?

Red/pink.

What color is methylene blue?

Blue.

What color is carbol fuchsin?

Pink/fuchsia.

What three clinical applications can a simple stain have?

Determining bacterial presence, morphology/arrangement, and number.

Are simple stains differential or diagnostic?

No. Simple stains are not considered differential or diagnostic.

What equipment is needed to prepare a bacterial smear?

Bacterial culture, clean microscope slide, inoculating loop/needle, and water bottle.

When should water be added to the slide?

When using bacteria from a solid culture.

When should water NOT be added?

When using a broth culture.

What amount of water should be added?

A small drop.

What happens if too much water is added?

The smear takes longer to air dry.

How long should a properly prepared smear take to air dry?

The slides say it should air dry completely, but they do not give a specific time.

What are three common mistakes when preparing a bacterial smear?

Using too much water, using too many cells, and overheating the slide.

How can you avoid using too many cells?

Tap the loop on the colony instead of scooping it, then spread the cells evenly.

How can you avoid overheating?

Quickly pass the slide through the heat three times instead of holding it in the flame.

What materials are needed for simple staining?

Heat-fixed smear, staining tray/rack, basic stain, water bottle, and bibulous paper.

How many drops of stain are used for simple staining?

The slides do not specify a number of drops; they say to flood the smear with stain.

What happens after the smear is flooded with stain?

Gently rinse it with water.

How should the stained slide be dried?

Blot with bibulous paper; do not wipe.

What are the basic stains and their exposure times?

Carbol fuchsin: 30–60 sec; methylene blue: 1–2 min.

What are three common mistakes during simple staining?

Over/under-staining, washing too forcefully, and wiping the smear instead of blotting.

How can you avoid losing bacteria during rinsing?

Hold the slide parallel to the water stream and rinse gently.

How should you dry the slide after rinsing?

Blot it with bibulous paper; do not wipe.

What type of stain does negative staining require?

An acidic, negatively charged stain.

Why does the acidic stain stain the background instead of the bacteria?

The negative stain is repelled by the negatively charged bacterial surface.

What does negative staining look like?

A colored/dark background with clear, unstained bacteria.

What stain is used for negative staining in this lab?

Nigrosin.

What are two practical applications of negative staining?

Determining bacterial morphology/arrangement and determining accurate cell size.

Why is negative staining useful for delicate bacteria?

It does not require heat fixation.

Why can negative staining show a more accurate bacterial size?

Heat fixation can cause cells to shrink.

What is the main clinical application of negative staining?

Determining morphology and arrangement of delicate bacteria without heat-fixing them.

Does negative staining require heat fixation?

No.

Why should you not heat-fix the negative stain initially?

The bacteria need to remain unaltered so their morphology and size can be observed accurately.