Microorganisms through a Microscope Flashcards
Differentiate b/w an acidic dye and a basic dye, and give some examples of the commonly used dyes
(Why do basic dyes stain bacterial cells? Why don’t acidic dyes stain bacterial cells?)
- Acidic Dye: Positive ions – Includes eosin, acid fuchsin, & nigrosin.
- - Not commonly used because bacteria are slightly negatively charged at pH 7, and acidic dyes are not attracted to most types of bacteria
- - The stain colors the background instead - Basic Dye: Negative ions – Includes crystal violet, methylene blue, malachite green & safranin
Name the 3 kinds of staining techniques
- Simple
- Differential
- Special
Explain the simple staining technique. What’s the primary purpose of simple stain?
- Simple staining technique uses an aqueous or alcohol solution of a single basic dye
- The primary purpose: to highlight the entire microorganism so that cellular shapes and basic structures are visible
What is the chemical called when added to intensify a staining solution?
Mordant
What are the two common differential stain techniques?
- Gram Stain
2. Acid-fast Stain
Explain the Gram Staining procedures in 4 major steps
- Application of crystal violet (purple dye)
- - After a short time, wash off the purple dye with water - Application of iodine (mordant)
- - After a short time, wash off the iodine with water - Alcohol or alcohol-acetone solution wash (decolorization)
- - After a short time, rinse off the alcohol - Application of safranin (counterstain, red dye)
- - After a short time, wash off safranin with water and blot dry before examination
What color does Gram (+) bacteria appear? And Gram (-)?
Gram (+): Blue/Purple
Gram (-): Red/Pink
Why can Gram (+) bacteria trap crystal violet stain, but not Gram (-)? Hint: cell wall structure / composition
- Gram (+) bacteria: Thicker Peptidoglycan cell wall inhibits the Crystal violet-Iodine (CV-I) complex to be washed off by alcohol
- - Peptidoglycan: disaccharides + aa’s - Gram (-) bacteria: Thin Peptidoglycan and they contain a layer of lipopolysaccharide (LPS), which is disrupted by alcohol wash, so the CV-I complex is washed out through the thin layer of peptidoglycan, appearing colorless until counterstained with safranin.
Are Gram (+) or Gram (-) bacteria easily killed by Penicillin and Cephalosporin? Why?
- Gram (+) bacteria are killed easily by PCN and Cephalosporin
- Gram (-) bacteria are more resistant to these antibiotics because they can’t penetrate the LPS layer.
Which genus of bacteria usually use the acid-fast staining technique? And give examples of the pathogens.
- Mycobacterium, including Mycobacterium tuberculosis and Mycobacterium leprae.
- Nocardia
Explain the acid-fast staining procedures in 3 major steps
- Application of red dye carbolfuchsin, and heat for several mins
– Cool and Wash off stain with water - Application of acid-alcohol (decolorization)
Note: red stain is removed is NOT acid-fast (colorless) - Application of methylene blue (counterstain)
– Appear blue if not acid-fast
How does acid-fast stain work?
Acid-fast stain is more soluble in the cell wall lipids (waxy material) than in the acid-alcohol, therefore, retaining the red color
When is the special staining used? Give some examples
To color and isolate specific parts of microorganisms, such as Endospores, Flagella, and Capsules.
Explain the Capsule Stain procedure in 2 major steps (capsule stain is also called negative stain)
- Mix the bacteria in color solution: India ink or nigrosin
- - Providing a dark background - Stain the bacteria with a Simple Stain, ex. safranin
- - The Capsule doesn’t accept the dye, but the bacterial cell does. Thus, unstained halos appear microscopically
Explain the Endo(spore) Staining procedure in 3 major steps. What color do we see under the microscope after the staining?
Schaeffer-Fulton endospore stain:
- Application of Malachite green and steam for 5 mins
- - Heat helps the stain penetrate the endospore wall - Wash with water for 30 secs
- Application of safranin (counterstain)
- - Stain bacterial cells other than the endospores.
Note: the endorspores appear green w/i red or pink cells