Gram Stain

Steps of Gram Stain

1. Drop of Water

2. Mix Bacteria

3. Air Dry

4. Heat Fix

5. Apply Primary Stain (Crystal Violet) for 1 min

6. Rinse

7. Apply Mordant (Gram’s Iodine) for 1 min

8. Rinse

9. Apply Decolorizer (Acetone) for 30 Seconds

10. Rinse

11. Apply Secondary Stain (Safranin) for 1 min

12. Rinse

13. blot with bilious paper

What color will gram positive bacteria be

Purple

What color will gram negative bacteria be

Red / Pink

Is B. cereus gram positive or negative

Gram positive

Is E. Coli gram positive or negative

Negative

Difference between Gram positive and Gram negative bacteria

gram positive bacteria have a single, thick cell wall made out of peptidoglycan and a single plasma membrane
gram negative bacteria have a very thin peptidoglycan layer that is between an inner membrane and an extra outer lipid membrane

Is b. Megaterium gram positive or negative

gram positive

is M. smegmatis gram positive or negative

gram positive

is s. marcescens gram positive or negative

gram negative

is S. Epidermis gram positive or gram negative

Gram Positive

Morphology: B. megaterium

• Bacillus

• Singly, in pairs, or short to long chains

Morphology: e. coli

• Bacillus

• Singly or in pairs

Morphology: M. Smegmatis

• Bacillus

• Single

Morphology: m. luteus

• Coccus

• Tetrad & irregular clusters

Morphology: S. marcescens

• Bacillus

• Singly, pairs, or short chains

Morphology: s. epidermidis

• Coccus

• Pairs, Tetrads, or short chains

Acid Fast Stain: M. Smegmatis

Positive

Acid Fast Stain: S. epidermidis

Negative

Spore Stain: B. Megaterium

Positive

Capsule Stain: K. pneumoniae

Positive

Capsule Stain: s. epidermidis

Positive

Name these parts of the microscope

1. Ocular Lens

2. Objective Lens

Name these parts of the microscope

1. Slide Holder

2. Arm

3. Stage

Name these parts of the microscope

1. Mechanical Stage Controls

2. Coarse Focus

3. Fine Focus

Name these parts of the microscope

1. Iris Diaphragm Lever

2. Base

3. On/Off switch

If you are viewing a specimen using the high-dry objective, what is the total magnification of the image?

400x

List IN ORDER the exact sequence of steps you should perform in order to view a prepared specimen with a compound microscope:

1. Clean All Lenses

2. Place slide on stage

3. rotate 10x objective into place

4. Move stage up (looking from side) close to objective lens

5. Move specimen under objective lens using stage control knobs

6. Move stage down with course adjustment knob wile looking in ocular lens until image is seen

7. Fine focus with Fine adjustment knob

8. Add oil and rotate directly to the 100x objective

9. Fine focus until image is clear

Agar Slant Terms: Abundance of Growth

1. None

2. Slight

3. Moderate

4. Large

Agar Slant Terms: Pigmentation

Used to describe the pigment of bacterial growth

Agar Slant Terms: Optical Characteristics

1. Opaque: No light transmission

2. Translucent: Partial light transmission

3. Transparent: full light transmission

Agar Slant Terms: Form

1. Filiform

2. Echinulate

3. Beaded

4. Effuse

5. Arborescent

6. Rhizoid

Agar Slant Form: Filiform

Continuous, threadlike growth with smooth edges

Agar Slant Form: Echinulate

Continuous, threadlike growth with irregular edges

Agar Slant Form: Beaded

Nonconfluent to semiconfluent colonies

Agar Slant Form: Effuse

Thin, spreading growth

Agar Slant Form: Arborescent

Tree like growth

Agar Slant Form: Rhizoid

Rootlike growth

Agar Slant Terms: Consistency

1. Dry

2. Buttery

3. Mucoid

Agar Slant Consistency: Dry

Free from moisture

Agar Slant Consistency: Buttery

Moist and shiny

Agar Slant Consistency: Mucoid

Slimy and glistening

Agar Plate Terms: Size

1. Pinpoint

2. Small

3. Moderate

4. Large

Agar Plate Terms: Pigmentation

Color of colony

Agar Plate Terms: Form / Shape of colony

1. Circular

2. Irregular

3. Rhizoid

Agar Plate Form: Circular

Unbroken, peripheral edge

Agar Plate Form: Irregular

Indented, peripheral edge

Agar Plate Form: Rhizoid

Rootlike, spreading growth

Agar Plate Terms: Margin

1. Entire

2. Lobate

3. Undulate

4. Serrate

5. Filamentous

Agar Plate Margin: Entire

Sharply defined, even

Agar Plate Margin: Lobate

Marked indentations

Agar Plate Margin: Undulate

Wavy indentations

Agar Plate Margin: Serrate

Toothlike appearance

Agar Plate Margin: Filamentous

Threadlike, spreading edge

Agar Plate Terms: Elevation

1. Flat

2. Raised

3. Convex

4. Umbonate

Agar Plate Margin: Flat

Elevation not discernible

Agar Plate Margin: Raised

Slightly elevated

Agar Plate Margin: Convex

Dome-shaped elevation

Agar Plate Margin: Umbonate

Raised, with elevated convex central region

Broth Terms

1. Uniform fine turbidity

2. Flocculent

3. Pellicle

4. Sediment

Uniform fine turbidity

Finely dispersed growth throughout

Flocculent

Flaky aggregates dispersed throughout

Pellicle

Thick, pad-like growth on surface

Sediment

Concentration of growth at the bottom of broth culture may be granular, flaky, or flocculent

List IN ORDER the exact sequence of steps you should perform in order to prepare a specimen for simple staining from a broth culture:

1. Flame the loop

2. Flame the mouth of the broth tube

3. Loop full of bacteria

4. Put bacteria on slide

5. Air dry

6. heat fix

7. Apply Crystal violet - 1 min

8. Rinse

9. Blot with bibulous paper

6. How does the simple stain procedure differ when preparing a specimen from an agar culture?

1. Will need to put a loop full of sterile water on a slide

2. Flame loop and get sample of bacteria

3. Mix bacteria into drop of sterile water

List the 3 steps of the acid-fast staining procedure

1. Drop of Water

2. Mix Bacteria

3. Air Dry

4. Heat Fix

5. Apply Primary Stain (Carbol Fuchsin) Over Steam - 5 min

6. Rinse

7. Apply Acid Alcohol - 30 seconds

8. Rinse

9. Apply Secondary Stain (Methylene Blue) - 1 min

10. Rinse

11. Dry with bibulous paper

What color will acid fast positive bacteria appear

Red / Pink

What color will acid fast negative bacteria appear

Blue

What is the difference in structure between an Acid-fast positive and Acid-fast negative species?

Acid Fast Positive: Have a thick, waxy outer layer rich in mycolic acids and glycolipids that prevents the carbol fuschin from being washed out by acid alcohol
Acid Fast Negative: Do not have a thick, waxy outer layer

List the steps of the spore staining procedure.

1. Drop of Water

2. Mix Bacteria

3. Air Dry

4. Heat Fix

5. Apply Primary Stain (Malachite Green) over steam - 5 min

6. Rinse

7. Apply Secondary Stain (Safranin) - 1 min

8. Rinse

9. Dry with bibulous paper

What are endospores?

Contains a copy of the genetic material of a cell and is surrounded by a tough spore coat; produced when the bacteria undergoes tough conditions like lack of nutrients are high heat

Describe the procedure for doing a capsule stain.

1. Drop of Primary Stain (Congo Red) on one end of slide

2. Mix in bacteria

3. use another slide to spread the red stain/bacteria mixture across the slide

4. Air dry

5. Flood slide with maneval’s stain - 1 min

6. Rinse

What is a capsule?

a thick, gelatinous outer layer that surrounds the cell wall of some bacteria that helps with evading immunity, helps adhesion, and prevents drying