Micro Lab Practical 2

What are 3 goals in preparing a smear?

1. Adhere cell to slide 2. Ensure shrinkage of cells doesn't occur during staining 3. Prepare thin smears

Why is the slide with the bacteria passed through the flame?

The heat is used to make sure the cells adhere to the slide.

How is the 1st step in preparing a bacteriological smear different when the bacteria are growing in a liquid medium v.s. when the bacteria are growing on a solid media?

Liquid-start by placing 1 or 2 loop fulls of the liquid directly on the slide
Solid media- place 1 or 2 loopfulls of water on the slide and then use the innoculating wire to disperse the organism in the water.

What is the effect of water when making a smear from solid media?

Liquid makes the transferring & spread of smear a lot easier

What 4 things to avoid during smear preparation?

1. Avoid producing aerosols 2. Do not spatter the smear as you mix it. 3. Do not blow on or wave the slide to speed up air drying 4. do not overheat when heat fixing

How many stains are used in a simple stain?


What are 3 commonly used dyes for simple staining?

1.Methylene blue 2.Crystal Violet 3. Safranin

What is the color-bearing ion of the dye called?


What is the charge on basic dye?


Is a basic dye cationic or anionic?

Basic dye is cationic

What is the charge on an acidic dye?


Is an acidic dye cationic or anionic?


What is the typical charge of a bacterial cell?


Why does a basic dye bind to the bacterial cell?

bacterial cells are negative and attract to the positvely charged basic dyes

Why does the acidic dye not bind to the bacterial cell?

Both the acidic dye and the bacterial cell are negatively charged & same charges repel

What factor determines how long the dye should remain on the smear in simple staining?

Weak stain leave on longer. Long stain don't leave on as long. It also depends on the affinity of the dye.

What is a possible consequence of leaving a stain on the bacterial smear too long (over staining)?

Becomes too intense.

What is a consequence of not leaving a stain on the smear long enough (under staining)?

It does not create enough contrast.

What 3 facts about the bacteria can be determined or demonstrated by using the simple stain?

1.Morphology 2. Size 3. Arrangement of the cell

What 2 facts about the bacteria can be determined by using the negative stain?

1. Morphology 2. Cellular arrangement of bacteria

What are 2 reasons for not heat-fixing bacteria used in the negative stain?

Heat fixing would kill and make the live bacteria possibly dissappear.

Are the microbes used in the negative stain alive or dead?


Why is the negative stain useful for observing spirochetes?

Spirochetes are so thin they get distorted by heat-fixing. When determining the size accurately a negative stain can be used because it produces minimal shrinkage.

What type of dye, acidic or basic, is used in the negative stain?


Name 2 stains used in the negative stain?

Nigrosine ink & india ink

Is the background or the bacterial cell stained in the negative stain?

The background is stained and the cell is transparent.

How is the cell wall of Gram + bacteria different than gram - bacteria?

Gram positive bacteria will have a thick layer of peptidoglycan. Gram negative has an outer membrane covering a thin peptidoglycan on the outside

4 gram reagents in order of usage

1. Primary Stain 2. Mordant 3. Decolorizer 4. Counterstain

What is the name and function of the dye used as the primary stain?

Crystal violet colors all cells purple

What is the name and function of the chemical used as the mordant?

Iodine solution binds primary dye to the cell

What two chemicals may be used as the decolorizer?

Acetone & alcohol

What is the function of the decolorizer?

Removes dye from stained cell

What dye is used as the counterstain?


What is the function of the counterstain?

Colors decolorized cell a different color.

What color are gram negative bacteria?

Pinkish red

What color are gram positive bacteria?


Why is the gram stain more valuable than simple stain?

It not only allows determination of cell size, morphology, and arrangement, but info about other features as well. Helps determine differences in the cell wall

Why is gram stain typically the first differential test run on a specimen brought into the lab?

By helping to determine the cell wall you could run a series of tests to identify the microbe when you find out if it is gram + or gram -

Factors that affect gram staining

1. Thickness of smear 2. Length of heat fixing 3. Freshness of reagents 4. Age of culture 5. Washing consistency 6. Decolorization

What is the most crucial step in the gram staining process and why?

Decolorization is the most crucial so you can decide whether the cell is gram + or gram -

How does the age of the culture affect results?

Some gram positives lose their ability to retain the crystal violet in as little as 24 hours.

Why is the spore stain useful?

It detects the presence and location of spores.

How should a smear appear after air drying?

The stain should appear slightly cloudy.

Why should you avoid overheating in a smear preparation?

Aerosals may be produced if overheated.

Would you want to know if these potentially deadly microbes were present in food?

Yes, because they could potentially harm you.

What is used to destroy endospores?


Why is heating the malachite green necessary?

The vegatative cell and spore mother cell can be decolorized w/water while the spore cells retain it and it counter stains with the safranin.

What's the name of the protein that is in the outer covering that makes spores difficult to stain?


Why are cultures of Bacillus older than 1 day used in spore stain technique?

Older bacteria are under greater nutritional stress so there will be more endospores present and able to visualize

What is needed to preform the spore stain technique?

Adequate ventilation, eye protection, a lab coat, and gloves

What colors will endospores and vegetative cells stain using the schaeffer-fulton techniques?

Spores are green and vegetative and spore mother cells are red.