Micro lab test 2

Who invented the Gram stain and when?

Hans Christian Gram in 1884

What is the primary purpose of a Gram stain?

To differentiate between Gram-negative and Gram-positive bacteria

What makes up the cell wall of Gram-positive bacteria?

A thick layer (80nm) of peptidoglycan, along with Teichoic Acid and Lipoteichoic Acid

What makes up the cell wall of Gram-negative bacteria?

A thin layer (1-11nm) of peptidoglycan and an outer 3rd layer of Lipopolysaccharides (LPS)

What is Step 1 of the Gram stain mechanism?

Primary Stain using Crystal Violet, which penetrates everything

What is Step 2 of the Gram stain mechanism?

Mordant (Fixative) using Iodine, which creates a CV-I (Crystal Violet-Iodine) complex

What is Step 3 of the Gram stain mechanism?

Decolorization using 95% ethanol, which only removes color from Gram-negative bacteria

What is Step 4 of the Gram stain mechanism?

Counter Stain using Safarin (Safranin), which only dyes Gram-negative bacteria

In a completed Gram stain, what color is Staph and why?

Staph is purple. It is Gram-positive, so its thick (80nm) peptidoglycan wall retains the Crystal Violet.

In a completed Gram stain, what color is E. coli and why?

E. coli is red. It is Gram-negative, so the ethanol removes the primary stain from its thin wall, allowing it to be dyed red by the counterstain.

What causes "Gram Variability" (when a Gram stain doesn't work properly)?

Using old cultures.

What is the ideal age of a culture for an accurate Gram stain?

18-24 hours old

proceedure for gram stain vairability with times fpr each action 13 steps

• Aseptic Technique

• Two slides: 1 labeled e. coli, the other staph

• Air dry and heat fix

• Flood w/ Crystal Violet (~1 min)

• Wash w/ H2O

• Flood w/ Iodine (~1 min)

• (Gently) Wash w/ H2O

• [Starred] 3-5 drops of 95% ethanol (decolorizer) (~20 sec)

• Wash w/ H2O

• Flood w/ Safarin (~40 secs)

• Wash w/ H2O

• Blot dry w/ paper

• oil immersion

What are the two categories of bacteria regarding spore staining?

Spore formers and non spore formers

What is the purpose of spores?

Protection and survival, not reproduction

What happens to a vegetative cell in adverse conditions?

The bacteria packages important genes into endospores. The cell disintegrates, leaving only the spore and spore coat.

What happens to a spore when it is back in a good condition?

It turns back into a sporangium.

According to the notes, how old can bacteria survive as spores?

250 million years old

What are three examples of spore-forming bacteria and the diseases they cause?

1. Bacillus anthracis (anthrax)

2. Clostridium tetani (tetanus)

3. Clostridium botulism (Botulism)

What specific bacteria was used as the example for the Spore Staining mechanism?

Bacillus megaterium

What type of media is used to grow spore formers?

AK media

How do you obtain the bacteria for the spore stain mechanism?

Take a loopful grown in an AK slant (solid in a test tube) from the surface of the agar.

What liquid must you not forget to add when prepping the bacteria on the slide?

H2O (Water)

Why is heating and boiling required in the spore staining mechanism?

It allows the stain to penetrate the spore.

What are the 6 complete steps for the Spore Staining Procedure?

1. Aseptic Technique

2. Label slide bacillus

3. 1 drop H2O + culture

4. Air dry and heat fix

5. Flood w/ Malachite green

6. Boil slide and stain at medium heat (5 inches away, until there are bubbles and steam)

What are the 14 comprehensive steps for the Spore Staining Procedure?

1. Aseptic Technique

2. Label slide bacillus

3. 1 drop H2O + culture

4. Air dry and heat fix

5. Flood w/ Malachite gree

6. Boil slide and stain at medium heat (5 inches away, until bubbles and steam)

7. Let the slide cool (40 secs) and repeat 5 total times

8. Wash w/ H2O

9. 4 drops of decolorizer (20 secs)

10. Wash w/ H2O

11. Flood w/ Safarin (40 secs)

12. Wash w/ H2O

13. Blot dry

14. Oil immersion

When looking at a completed spore stain under the microscope, what two things do you see and what colors are they?

1. Endospore (Green)

2. Vegetative cell (Red)

What type of bacteria is the Acid Fast Stain used for, and what do they lack?

Mycobacterium; they don't have a peptoglyceride (peptidoglycan) layer.

What makes up the outer coating of Mycobacterium?

A waxy lipoidal coating made of Mycolic acid (fatty).

Because of the mycolic acid coating, what physical step must be done during the staining process?

It must be heated.

What is the composition of the specific decolorizer used in the Acid Fast Stain?

98% Alcohol + 2% HCl

How do Acid Fast bacteria (Mycobacterium) appear if a standard Gram stain is performed?

They look Gram-positive (GP).

What is the medical relevance of Mycobacterium (what diseases do they cause)?

TB (Tuberculosis) and leprosy.

How do medical professionals test for TB?

They use tuberculin protein to see if antibodies form.

What are the four specific classifications of bacteria listed in the side notes, and what do they mean?

1. Gracilicutes -> Gram-negative (GN)

2. Firmicutes -> Gram-positive (GP)

3. Tenericutes -> No pepto (peptidoglycan layer)

4. Mendosicutes -> Different

What specific species of Mycobacterium is used in the lab procedure?

Mycobacterium gordinae

What are the 13 comprehensive steps for the Acid Fast Stain procedure?

1. Aseptic Technique

2. 2 slides (Myco)

3. Take 5 loopfuls of e.coli + 1 loopful of Myco, mix together

4. Air dry and heat fix

5. Flood w/ carbol Fuschin

6. Boil on medium heat, cool, repeat 5x

7. Wash with H2O

8. Use TB decolorizer (5 drops at most) (20 secs)

9. Wash with H2O

10. Counter stain w/ Methylene Blue (Flood + 40 sec)

11. Wash with H2O

12. Blot dry

13. Oil Immersion

In a completed Acid Fast Stain, what color are Mycobacterium and E. coli?

Myco is red; E. coli is blue.

What is the purpose of Wet mounting?

To test if bacteria are motile or non-motile.

What are two examples of motile bacteria from the notes?

1. E. coli

2. Proteus vulgaris

What is an example of a non-motile bacteria from the notes?

Klebsiella pneumonia

What are the 4 comprehensive steps for the Wet Mounting procedure?

1. Label Slide E. coli

2. 2 drops of E. coli from dropper

3. Place coverslip on top w/ no air bubbles (45° angle)

4. Oil Immersion on cover slip