STAINING TECHNIQUES

Question 1

-> organic or aqueous preparations of dyes that impart a variety of color to tissue or microorganisms

• Differentiate or identify structures and characteristics of bacteria

Answer 1

Dyes or stains

Question 2

• ion that imparts the color

Answer 2

Chromophore

Question 3

The chromophore is a positive ion -›____
• methylene blue, crystal violet, carbol fuchsin, malachite green, safranin

Answer 3

BASIC STAIN

Question 4

• The chromophore is a negative ion -› _______
  eosin, nigrosin, picric acid

Answer 4

ACIDIC STAIN

Question 5

• -> ion not giving off the color

Answer 5

Counterion

Question 6

• directly stains bacteria and bacterial structure
bacteria absorbs the stain

Answer 6

DIRECT STAINING

Question 7

• No special structures are seen
Stain evenly dyes the bacteria
The stain is flooded across the smear

Answer 7

Simple Staining

Question 8

• Only one type of dye is used
• No special structure of a cell is distinguished
• eg. methylene blue

Answer 8

Simple Staining

Question 9

• Can visualize bacterial structures
• Can isolate specific parts of bacterial structure

Answer 9

Selective/Special Staining

Question 10

-> Not easily colorized, not easily decolorized
-> Impenetrable

Answer 10

Endospore Stain or Schaeffer-Fulton stain

Question 11

Differential Staining
• Two Types:

Answer 11

Gram Stain
Acid Fast Stain

Question 12

-> The first stain used in the procedure
-> A stain that will impart the color

Answer 12

Initial/Primary Stain

Question 13

•
-> allow stains to stick to the bacteria
- for a bridge between cell and stain

Answer 13

Mordant

Question 14

-› no chemical eg. heat or cold
-› ferrous sulfate, iodine, tannic acid

Answer 14

Physical Mordant

Chemical Mordant

Question 15

-> Removes color
-> Bacteria in question retain the color
-> Bacteria without the specific structure or composition in them will not retain the color

Answer 15

Decolorizer

Question 16

• Bacteria that were decolorized will absorb the color

Answer 16

Counterstain/Secondary Stain

Question 17

• Negative/Relief Staining
• The background is stained instead for contrast
- Demonstrate the HALO EFFECT
• DRY INDIA INK/NIGROSINE STAIN

Answer 17

INDIRECT STAINING

Question 18

TYPES OF IONIZABLE DYES USED IN STAINING BACTERIA

Answer 18

Basic dyes
Acidic dyes

Question 19

▪ Commonly used in bacteriology
▪ These are cationic dyes with positively charged groups that adhere to negatively charged molecules.

Answer 19

BASIC DYES

positive

Question 20

Basic dyes

Answer 20

Includes:
METHYLENE BLUE
MALACHITE GREEN

BASIC FUCHSIN
CRYSTAL VIOLET
SAFRANIN

Question 21

▪ These are anionic dyes with negatively charged group that bind to positively charged cell structures

Answer 21

ACIDIC DYES

Question 22

ACIDIC DYES

Answer 22

EOSIN
ACID FUCHSIN
ROSE BENGAL

Question 23

▪ Example: METHYLENE BLUE, IODINE

Answer 23

SIMPLE STAINING

Question 24

▪ Directed towards the forms and shapes of the cells

Answer 24

SIMPLE STAINING

Question 25

▪ ***Basic dyes*** have greater affinity for nuclei; ***Acid dyes*** have greater affinity for cytoplasm

Answer 25

SIMPLE STAINING

Question 26

▪ Application of single staining solution

Answer 26

SIMPLE STAINING

Question 27

▪ Example: GRAM STAINING, ACID FAST STAINING

Answer 27

DIFFERENTIAL STAINING

Question 28

▪ Makes use of primary stain, mordant, decolorizer and counterstain

Answer 28

DIFFERENTIAL STAINING

Question 29

▪ Application of 2 or more stains

Answer 29

DIFFERENTIAL STAINING

Question 30

▪ Example: INDIA INK, NIGROSIN STAIN

Answer 30

▪ NEGATIVE STAINING

Question 31

▪ Results the bacteria appearing as light colored bodies against the dark background

Answer 31

▪ NEGATIVE STAINING

Question 32

GRAM STAINING ▪ Named after________ (Danish Scientist), worked with______ in City hospital morgue in Berlin.

Answer 32

HANS CHRISTIAN GRAM CARL FRIEDLANDER

Question 33

▪ PRINCIPAL STAIN in the laboratory for microscopic examination of bacteria

Answer 33

Gram stain

Question 34

▪ Divides bacteria into two large groups: GRAM POSITIVE and GRAM NEGATIVE.

Answer 34

Gram stain

Question 35

Gram staining PRINCIPLE: Based on the differences in composition between gram positive cell walls which contain thick_____ with NUMEROUS_______ and gram negative cell walls which consist of______.

Answer 35

peptidoglycan; TEICHOICE ACID CROSS-LINKAGES THINNER LAYER OF PEPTIDOGLYCAN

Question 36

If the______ is rinsed too vigorously prior to the application of iodine, it will not be retained and will leave the gram negative bacteria unstained

Answer 36

crystal violet dye

Question 37

If the decolorization is prolonged, the ———-will be removed and the gram positive bacteria will not be stained.

Answer 37

Gram – positive complex

Question 38

If the decolorization is insufficient, the organism may_______

Answer 38

falsely appear as gram positive cells.

Question 39

If the______ is left applied for more than a minute, the ________will be eliminated from the previously stained cells

Answer 39

safranin dye Gram positive complex

Question 40

Failure to leave the safranin dye for a sufficient time will result in ______and background materials.

Answer 40

unstained gram negative bacteria

Question 41

All cocci are gram – positive except for

Answer 41

Neisseria Moraxella Veilonella

Question 42

All bacilli are gram – negative except for

Answer 42

Actinomadura, Arcanobacterium, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Gordonia, Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Tropheryma whipplei, and Tsukamurella. All Awesome Biologists Collect Cool Exotic Germs Knowing Lab Microbes Need Real Scientific Testing and Training. Actinomadura Arcanobacterium Bacillus Clostridium Corynebacterium Erysipelothrix Gordonia Kuthria Listeria Mycobacterium Nocardia Rhodococcus Streptomyces Tropheryma whipplei Tsukamurella

Question 43

REASONS WHY GRAM – POSITIVE BACTERIA BECOME GRAM NEGATIVE BACTERIA

Answer 43

1. Removal of MgRNA 2. Aged, dying, and autolyzing cells 3. By using acidic iodine during staining 4. Due to technical error or the wrong use of stains

Question 44

EXCEPTIONS IN GRAM STAINING

Answer 44

1. Organisms that exist almost exclusively within host cells (Chlamydia) 2. Organisms that lack cells walls (Mycoplasma and Ureaplasma) 3. Organisms with insufficient dimension to be resolved by light microscope (Spirochetes)

Question 45

Acid fast ▪ Heat is applied as a mordant in the ______while tergitol is used in the______

Answer 45

Ziehl Neelsen Method Kinyoun Method

Question 46

▪ In this procedure, the cell wall of acid – fast bacteria resists the acid – alcohol (hydrochloric acid – ethanol mixture) decolorization step.

Answer 46

ACID FAST STAINING

Question 47

▪ It utilizes carbolfuchsin as the primary stain and methylene blue or malachite green as the secondary stain.

Answer 47

ACID FAST STAINING

Question 48

▪ It is used to stain bacteria that have high lipid contents in their cell walls.

Answer 48

ACID FAST STAINING

Question 49

Acid fast ▪ It utilizes______ as the primary stain and _____ or _____ as the secondary stain.

Answer 49

carbolfuchsin methylene blue or malachite green

Question 50

Acid fast principle

Answer 50

PRINCIPLE: • The primary stains binds to mycolic acid in the cell walls of acid fast bacteria, and is retained after decolorizing with acid alcohol. • Acid Fast Bacilli (AFB) retain the primary stain and deep-pink or red-colored while non – AFB are either blue or green colored.

Question 51

– Hot staining method

Answer 51

Ziehl Neelsen

Question 52

– Cold staining method

Answer 52

Kinyoun

Question 53

– differentiates Mycobacterium smegmatis from Mycobacterium tuberculosis.

Answer 53

Pappenheim Method

Question 54

– differentiates Mycobacterium leprae from Mycobacterium tuberculosis.

Answer 54

Baumgarted Method

Question 55

– selective for the cell wall of AFB.

Answer 55

Auramine – rhodamine Method

Question 56

WAYS TO FACILITATE ACID – FAST STAINING

Answer 56

1. The use of heating or steaming process for five to seven minutes to temporarily remove the mycolic acid, while the smear is flooded with stain. 2. By increasing the concentration of dye and phenol in the staining reagent. 3. A prolonged contact of the specimen with the primary stain. 4. The addition of wetting agent like tergitol