Module 06: Differential Staining

Question 1

Punctiform are usually how large in mm diameter?

Answer 1

1mm in diameter

Question 2

What are some common colony shapes?

Answer 2

(1) Round
(2) Filamentous
(3) Irregular

Question 3

What are some common colony margins?

Answer 3

(1) Smooth (entire)
(2) Curled
(3) Wavy
(4) Lobate
(5) Filamentous

Question 4

What are some colony surface characteristics?

Answer 4

(1) Smooth
(2) Concentric
(3) Wrinkled
(4) Contoured

Question 5

What are the general forms of media?

Answer 5

(1) Broth culture
(2) Agar slant patterns

Question 6

What are different forms of agar preparations?

Answer 6

(1) Agar slant
(2) Agar deep
(3) Agar plate

Question 6

What are some selected broth culture properties?

Answer 6

(1) Turbidity or cloudiness
(2) Pellicle formation
(3) Sediment formation

(Note that growth is located at the bottom of the tube. The rest of the broth is clear)

Question 7

What are some agar slant patterns?

Answer 7

(1) Filiform (even)
(2) Echinulate (pointed)
(3) Arborescent (branched)

Question 8

What are some margin (edge) forms?

Answer 8

(1) Curled
(2) Entire
(3) Filamentous
(4) Lobate
(5) Undulate
(6) Serrate

Question 9

What are some whole colony forms?

Answer 9

(1) Circular
(2) Granular
(3) Punctiform
(4) Filamentous
(5) Irregular
(5) Rhizoid

Question 10

What are some surface properties?

Answer 10

(1) Concentric
(2) Contoured
(3) Radiate (ridges)
(4) Rugose (wrinkled)
(5) Smooth

Question 11

What are some forms of elevation?

Answer 11

(1) Convex
(2) Pulvinate
(3) Umbonate
(4) Flat
(5) Raised
(6) Subsurface (as in pour plates)

Question 12

What are some agar slant stroke cultures?

Answer 12

(1) Arborescent (branched)
(2) Beaded
(3) Echinulate (pointed)
(4) Filiform (even)
(5) Rhizoid (rootlike)
(6) Spreading

Question 13

This type of agar slant stroke culture is characterized to be branched.

Answer 13

Arborescent

Question 14

This type of agar slant stroke culture is characterized to be pointed.

Answer 14

Echinulate

Question 15

This type of agar slant stroke culture is characterized to be even.

Answer 15

Filiform

Question 16

This type of agar slant stroke culture is characterized to be rootlike.

Answer 16

Rhizoid

Question 17

Cite the steps of differential staining.

Answer 17

(1) Before staining
(2) After primary staining (Crystal violet)
(3) After mordant, iodine
(4) After decolorizer, alcohol or acetone or alcohol
(5) After counterstain, safranin

Question 18

Under differential staining, this is known as the primary staining.

Answer 18

Crystal violet

Question 19

Under differential staining, this is known as the mordant.

Answer 19

Iodine

Question 20

Under differential staining, this is known as the decolorizer.

Answer 20

Alcohol or acetone alcohol

Question 21

Under differential staining, this is known as the counterstain.

Answer 21

Safranin

Question 22

How should we perform differential staining?

Answer 22

(1) Most stains used in microbiology are differential stains.
(2) Use more than one dye so that different cells, chemicals or structures can be distinguished.

Question 23

How should one perform the ziehl-neelson stain kihyoun modification in differential staining?

Answer 23

(1) A small amount of an organism is suspended in a saline solution fixed on a slide.
(2) Slide is flooded using Carbol Fuschin and phenol for three (3) minutes and gently rinsed with water.
(3) Slide is decolorized with 3% HCl in70% alcohol until color appears to be removed (approx. 2 mins) and rinsed with water.
(4) Slide is flooded with methylene, which is a blue counterstain for 30 seconds, and is rinsed with water and air dried.

Question 24

This distinguishes between two large groups of microorganisms name gram positive and gram negative.

Answer 24

Gram staining

Question 25

Under gram staining, this is known as purple staining.

Answer 25

Gram positive cells

Question 26

Under gram staining, this is known as pink staining.

Answer 26

Gram negative cells

Question 27

This sis a chemical used to fix the staining reaction.

Answer 27

Mordant

Question 28

What is the difference between gram positive and gram negative cells?

Answer 28

The structure of the thinner cell walls of Gram negative bacteria cannot hold the dyes previously used, once the decolorizer is applied.

Question 29

Cite the steps of gram staining.

Answer 29

(1) Crystal violet (1 min) : rinse (2) Iodine (1 min) : rinse (3) Acetone Alcohol (10–15 sec) : rinse (4) Safrinin (1 min)

Question 30

Enumerate the steps in performing gram staining.

Answer 30

(1) Cover the smear with crystal violet reagent for 1 minute. Add more stain if drying occurs. (2) Rinse the slide (front and back) in slowly running water for 5 sec. (3) Rinse the smear with iodine reagent and pour-off the excess. Cover the smear again with the iodine and allow it to remain for 1 min. (4) Rinse the smear with running water as in step 02. (5) Apply the alcohol decolorizer dropwise and slowly, Continue to add this reagent until dye no longer runs off from the smear. (6) Rinse with running water again in step 02, to prevent additional decolorization. (7) Cover the smear with safranin for 1 min. (8) Rinse again with water. (9) Blot dry but do not rub. Observe as directed.

Question 31

This form of staining is used to stain the cells of the genera.

Answer 31

Acid-Fast Stain

Question 32

These are known to cause many diseases in humans, including tuberculosis, leprosy, and other lung and skin infections.

Answer 32

(1) Mycobacterium (2) Nocardia

Question 33

Why do mycobacterium and nocardia use acid fast staining?

Answer 33

Cells of these bacteria have large amounts of waxy lipid in their cells walls, so the Gram stain and other water-based stains don’t work well on them.

Question 34

How does gram staining work?

Answer 34

Cells are determined to be acid-fast or not, based on whether they retain their primary stain after decolorization.

Question 35

Cite the steps of acid fast staining.

Answer 35

(1) Create a smear of the organism that you are testing. Cover the smear with a strip of blotting paper. (2) Saturate paper with Ziehl’s carbolfuchsin and heat for 3 – 5 minutes. Remove blotting paper. (3) Wash slide with tap water and then decolorize the smear for 10 - 15 seconds with acid alcohol. Rinse again with tap water. (4) Apply crystal violet for 1 minute, wash, blot dry.

Question 36

Enumerate the materials used for acid fast stain.

Answer 36

(1) Blotting paper : Ziehl's carbolfuchsin (3 – 5 min heat) : rinse (2) Acid Alcohol (10 – 15 sec) : rinse (3) Crystal violet (1 min)

Question 37

These are characterized to be dormant, highly-resistant cells inside the cytoplasm of the bacteria, that can survive environmental extremes (desiccation, heat, harmful chemicals)

Answer 37

Endospores

Question 38

What are some environmental extremes that endospores can survive?

Answer 38

(1) Desiccation (2) Heat (3) Harmful chemicals

Question 39

What are some of the most notable genera that undergo endospore staining?

Answer 39

(1) Bacillus (2) Clostridium

Question 40

Why can't endospores be stained?

Answer 40

Endospores cannot be stained by normal staining procedures because their walls are practically impermeable to all chemicals.

Question 41

How does endospore staining work?

Answer 41

(1) Endospore stain uses heat to drive the primary stain, malachite green into the endospore. (2) After cooling, the sample is decolorized with water and counter stained with safranin. (3) Results in green stained endospores and red-colored vegetative cells.

Question 42

Enumerate the materials needed for endospore staining.

Answer 42

(1) Malachite Green (5 min heat) : rinse (2) Safrinin (20 sec)

Question 43

Enumerate the steps of bacterial smear preparation.

Answer 43

(1) Grasp the inoculating instrument with your thumb and index finger as if it were a pencil or pen. (2) Insert the inoculating instrument at a 60 degree angle into the upper cone (hot portion) of the Bunsen burner flame. Heat the entire wire portion to redness. (3) Pick up a tube of culture with your free hand. With broth (liquid) cultures, shake from side to side to bring the organisms into suspension. (4) Remove the plug or cap of the tube with free fingers of the hand holding the inoculating loop. (5) Flame the tip of the tube by passing it quickly through the upper cone of the flame. (6) Insert the sterilized inoculating loop and remove a small amount of culture (Note: Do not be disturbed by the sizzling noise) (7) Flame the tube again and replace the plug or cap. Put the tube in a rack. (8a) With liquid cultures, place a loopful of broth on a slide, spread the broth over the small area, and allow the smear to dry. (8b) With agar cultures, place a small amount of growth in a loopful of water already on the slide mix, spread over a small area and allow to air dry. (9) Flame the inoculating instrument and put it on a metal surface (10) Pass the smear through the hot portion of the flame, do not overheat

Question 44

This is the inoculating instrument used for liquid cultures.

Answer 44

Inoculating loop

Question 45

This is the inoculating instrument used for solid medium.

Answer 45

Inoculating Needle

Question 46

Enumerate the steps of simple staining.

Answer 46

(1) Apply only enough stain to cover the smear and allow to remain for 30 seconds. (2) Pour off the stain. Rinse with slowly running water. (3) Dry the slide by placing it between pieces of blotting paper. Do not rub.