Chapter 4- Staining and Classification

Question 1

Stains are dyes that _____ light and dark ____ by ____ selectively to microbial cells

Answer 1

increase
contrast
binding

Question 2

What must be done before staining (coloring)? What’s the purpose of this?

Answer 2

The microorganisms must be fixed

What’s the purpose of heat fixing the slide?

1. It attaches the bacteria to the slide
2. Kills the bacteria

Question 3

What is the process of fixing the microorganisms?

1. A thin layer of microorganisms is ____________. This is called a ______.
2. The _____ is allowed to _____
3. The smear is then _________________________

Both the ______ and _______ fix the microorganisms to the slide

4. ____ is then applied to the slide

Answer 3

1. A thin layer of microorganisms is spread over the surface of a slide. This is called a SMEAR.
2. The smear is allowed to air dry.
3. The smear is then fixed to the slide by passing it over the Bunsen burner several times

Both the airdrying and heating fix the microorganisms to the slide

4. Stain is then applied to the slide

Question 4

Stains are ___ composed of a ____ and a _____ ion

Answer 4

salts

positive and negative ion

Question 5

Chromophore

Answer 5

name for the color found in the dye

Question 6

If the color is in the positive ion, these stains are known as

Answer 6

basic dyes`

Question 7

If the color is in the negative ion, these stains are known as

Answer 7

acidic dyes

Question 8

Bacterial cells are slightly negatively charged. Based on this information, what type of dye is best used to view bacteria?

Answer 8

Basic dyes

Question 9

Since acidic dyes carry _____ electric charges, and bacteria cells are also ____, they are not ____ to bacterial cells. As a result, it can’t ________________________ and instead stains the ________

Answer 9

negative
negative
attracted
stain the bacterial cells
background

Question 10

When determining what kind of dye to use, it’s important to select the ______ of the bacteria charge. So if there’s a negatively charged bacteria, you should use the color associated with a _____ charge (which is a ____ dye). If there’s a positively charged bacteria, you should use the color associated with a _____ charge (which is a ____ dye).

Answer 10

opposite

positive, basic dye

negative, acidic dye

Question 11

Simple stain

Answer 11

Single basic dye

It’s purpose is to highlight the entire microorganism so that the cell shape/basic structures are visible
ex: crystal violet, methylene blue

Question 12

Differential stains

Answer 12

Uses more than one dye

Purpose is to differentiate bacteria into distinctive groups

ex: the Gram stain, Acid-fast stain, and special stains

Question 13

The procedure of a gram stain
1. _______(a basic dye w purple color) is applied and is referred to as a _____
2. Rinsed off the slide with water
3. Slide is then covered with ____, which is a ______ (purpose is to _______)
4. Next, the slide is washed with ____, a _____ ____. This breaks down the ____ of gram ______ cells.
5. It’s then rinsed off the slide with water, and the slide gets stained with ____ (a basic dye with red color). _______ is referred to as a _________ because it has a contrasting color to the primary stain.

Answer 13

1. CRYSTAL VIOLET (a basic dye w purple color) is applied and is referred to as a PRIMARY STAIN
2. Rinsed off the slide with water
3. Slide is then covered with IODINE, which is a MORDANT (purpose is to INTENSIFY the color of the primary stain)
4. Next, the slide is washed with ALCOHOL, a DECOLORIZING AGENT. This breaks down the cell wall of gram-NEGATIVE cells.
5. It’s then rinsed off the slide with water, and the slide gets stained with SAFRANIN (a basic dye with red color). SAFRANIN is referred to as a COUNTERSTAIN because it has a contrasting color to the primary stain.
6. The slide is rinsed with tap water, blotted dry, and observed under the microscope.

Question 14

The alcohol used in a gram stain only rinses the slide for..

Answer 14

10-30 seconds

Question 15

After the primary stain (_____) and the mordant (____) are used…

Answer 15

(crystal-violet)
(iodine)
all cells are stained purple

Question 16

After the decolorizing agent (____) is used, it removes the _____ ___ from ONLY the ______ ____ _____, making the _____ ______ become ______

Answer 16

alcohol
purple color
gram-negative bacteria (2x)
become colorless

Question 17

After staining with a counterstain (_____), it turns the ______ gram-____ bacteria ____. Since the gram-____ bacteria retained the original _____ color, they are ___ _______ by the counterstain.

Answer 17

After staining with a counterstain (SAFRANIN), it turns the COLORLESS gram-NEGATIVE bacteria PINK. Since the gram-POSITIVE bacteria retained the original PURPLE color, they are NOT AFFECTED by the counterstain.

Question 18

Concept behind the gram stain:

The __ _____ determines what color is seen.
The Gram stain also is important in __________________

Answer 18

cell wall

diagnosing and treating bacteria infection

Question 19

Acid-Fast Stain

Answer 19

Used for bacteria that have waxy cell walls

Ex: Mycobacterium and Nocardia

Question 20

Acid-Fast Stain Procedure:

1. ________ (a ___ dye) is applied to a fixed bacterial smear on the slide. ___bacterial cells stain ____ at this stage.
2. The slide is rinsed with tap water
3. The slide is then washed with _____ ______ (a ________ ____), which removes the red dye only from _______. So now, ______ are ________, and the acid-fast bacteria will ____ ____ ____ ____.
4. The slide is rinsed with tap water
5. It’s then treated with a ______ (________ _____)
6. so the colorless non-acid fast bacteria appear ____ after the counterstain whereas the acid-fast bacteria appear ____

Answer 20

1. CARBOLFUCHSIN (a RED dye) is applied to a fixed bacterial smear on the slide. ALL bacterial cells stain RED at this stage.
2. The slide is rinsed with tap water
3. The slide is then washed with ACID-ALCOHOL (a DECOLORIZING AGENT), which removes the red dye only from NON-ACID FAST BACTERIA. So now, NON-ACID FAST BACTERIA are COLORLESS, and the acid-fast bacteria will RETAIN THE RED COLOR.
4. The slide is rinsed with tap water
5. It’s then treated with a COUNTERSTAIN (METHYLENE BLUE)
6. so the colorless non-acid fast bacteria appear BLUE after the counterstain whereas the acid-fast bacteria appear RED

Question 21

Special stains

Answer 21

Used to color, detect, and demonstrate the presence of structures in bacteria such as:
Endospores
Flagella
Capsules

Question 22

Taxonomy

Answer 22

A way of categotizing or naming organisms

Helps make predictions

Question 23

What characteristics did Linneaeus look for when placing organisms into a group?

Answer 23

Microscopic appearance
Staining characteristics
Biochemical evaluations

Question 24

Strains are

Answer 24

populations of cells that arise from a single cell that differ from other strains and evolve as a group

Question 25

Linneaus determined that those who could sucessfully _______ were ________________. The idea of interbreeding is not as useful for microorganisms due to ______ reproduction.

Answer 25

interbreed placed into the same species asexual

Question 26

Classification is based on _______ _____, so additional information can potentially be _____ and _____

Answer 26

current knowledge reclasssified and renamed

Question 27

2 tests used for classification of microorganisms

Answer 27

Biochemical tests Nucleic acid sequencing

Question 28

The use of a dichotomus key:

Answer 28

Series of paired statements that direct the user to another pair or statement or provides the organism name Either or questions