Micro Practical 1: Staining Information

Modified version of unknown practice based on given study guide

What is a simple stain? What is its purpose?

Staining by use of one stain solution  

• Its purpose is to highlight the shape and arrangement of an organism

What is the procedure for simple staining? Include an example.

Stain utilizing an appropriate stain solution for one minute. Then rinse with tap water.

• Ex. Methylene blue, malachite green, crystal violet, safranin

What is a differential stain? What is its purpose?

Staining by use of more than one stain solution

• It allows us to differentiate bacteria or identify specfic structures 

This refers to Gram Stain, Endospore Stain, and Acid-Fast stain

Which organism is shown in this image?

Bacillus subtilis

Which organism is shown in this image?

Staphylococcus epidermidis

Which organism is shown in this image?

Escherichia coli

Which organism is shown in this image?

Neisseria sicca

Which organism is shown in this image?

Mycobacterium smegmatis

List the characteristics of Bacillus subtilis

Gram reaction: Gram positive

Arrangement: Chain

Shape: Bacillus/rod shape

Acid fast reaction: Acid fast negative 

Endospore Stain: Contains Endospores

List the characteristics of Staphylococcus epidermidis

Gram reaction: Gram positive 

Arrangement: Chain/Cluster

Shape: Coccus/spherical

Acid fast reaction: Acid fast negative

Endospore stain: No endospores

List the characteristics of Escherichia coli

Gram reaction: Gram negative

Arrangement: Single

Shape: Bacillus/ rod shape

Acid fast reaction: Acid fast negative

Endospore stain: No endospores 

List the characteristics of Neisseria sicca

Gram reaction: Gram negative

Arrangement: In pairs

Shape: Coccus/spherical

Acid fast reaction: Acid fast negative

Endospore Stain: No endospores 

List the characteristics of Mycobacterium smegmatis

Gram reaction: Gram positive 

Arrangement: Cluster

Shape: Bacillus/rod shaped 

Acid fast reaction: Acid fast positive 

Endospore stain: No endospores

Which organism requires a gram stain?

Each of the given organisms are to be gram stained

• If you receive a bacterium with a coccus shape, no other tests are required

When should you you complete an endospore stain and a acid fast stain?

If your organism is bacillus (rod) shaped, an endospore stain should be conducted after your gram stain. 

• This narrows it down between Bacillus subtilis, Escherichia Coli, and Mycobacterium smegmatis

An acid fast stain should only be conducted if the result of the endospore stain leaves you unsure

• This should narrow it down further, indicating either Escherichia Coli or Mycobacterium smegmatis

How can you remember the stains for each organism from our uknown?

For positive gram stain remember:

Buy my stuff:

• Bacillus Subtilis 

• Mycobacterium Smegmatis 

• Staphylococcus

Everything else is negative for gram staining 

For endospore stain remember:

• Bacillus Subtilis  

For acid fast staining remember:

• Mycobacterium Smegmatis 

List the procedures for Gram staining

1. Use the inoculating loop to smear the specimen and heat fix it by passing the slide 3-5 times over the burner 

2. Flood the smear with crystal violet and leave it for 1 minute 

3. Rinse the excess stain 

4. Flood the smear with iodine and leave it for 1 minute

5. Rinse the excess stain

6. Tilt the slide into the basin and spray with acetone - alcohol for about 5-10 seconds

7. Rinse the slide 

8. Flood the smear with safranin and leave it for 1 minute to 90 seconds

9. Rinse the slide 

10. Use bibulous paper and blot the slide dry gently

Make sure the smear is not too thick, try to spread it out a bit.

What two genera are endospore producing? Give examples.

bacillus

• Bacillus anthracis, the cause of anthrax

and clostridium

• Clostridium tetani- causes tetanus

  Clostridium botulinum- causes botulism

  Clostridium perfringens, causes gas gangrene

What three genera are acid fast positive

Remember LMN

Legionella ← may or may not be on exam

Mycobacterium

• Mycobacterium tuberculosis- the cause of tuberculosis
  

• Mycobacterium leprae- the cause of leprosy

Nocardia

• causes respiratory disease called nocardiosis

List the procedures for acid fast staining

1. Use your inoculating loop to smear the specimen onto the slide and heat fix it 

2. Place filter paper directly onto the smear

    

3. Flood with carbolfuschin and warm over coolest part of your Bunsen burner for five minutes (don’t let it dry)

4. Rinse the slide and remove the filter paper and dispose it in the biohazard bin

5. Rinse the slide with water until the stain stops coming off

6. Decolorize with acid alcohol for 30 seconds

7. Continue to rinse with water

8. Counterstain with Loeffler's methylene blue for 1 minute. 

9. Rinse with water

10. Blot excess moisture from the slide and air dry. 

What does heat and carbol-fuchsin do to the specimen during acid fast staining?

They help to penetrate the mycolic acid layer of the bacterium.

List the procedures for endospore staining

1. Use your inoculating loop to smear the specimen onto the slide and heat fix it 

2. Place filter paper directly onto the smear

    

3. Flood with Malachite green and warm over coolest part of your Bunsen burner for five minutes (don’t let it dry)

4. Rinse the slide and remove the filter paper and dispose it in the biohazard bin

5. Continue to rinse the slide with water for 30 seconds

6. Counterstain with safranin for 90 seconds

7. Rinse with water

8. Blot excess moisture from the slide and air dry. 

Why do we heat the specimen during endospore staining?

It helps penetrate the spore-coat and the cortex containing dipicolinic acid