Gram staining & Negative vs Simple stains

Cell Morphology

Reffers to shape of conglomerate of millions/billions of cells

• Size

• Shape

• Color

• margins(edges)

• texture/appearance

• elevation

Growth Characteristics

• growth/no growth- colonies on solid media (agar)

• Turbidity on broth (cloudiness)

Contamination

• unexpected growth

• incorrect species?

Staining purpose

• Cells are naturally colorless in bright field microscope

• helps to provide information on cell size, shape and arrangement

Cell Morphology (specific) 

• Coccus- Round

• Bacillus- rod

• Vibrio- curved rods

• Spirochete- Spiral 

• Spirilla (two sperms joining) 

Diplococci

Two round

Tetrad

4 coccci

Sarcina

Cube of cocci

Staphylococci

Bunches of round

Streptococci

Lined up cocci

Diplobacilli

2 rods

Streptobacilli

Line of rods

Palisades

THINK: roman columns next to each other- rods touching at each end 

Coccobacilli

Rounder rod

Simple stain

• White backround- bacteria color of the stain

• Heat fixed so cells(cytoplasm) tend to shrink

• Stain has positively charged chromogen (attracted to cell wall) 

Negative Stain

• India ink

• Dark background, bacteria look white

• Not heat fixed, better for seeing morphology since cells remain true size

• Stain has negatively charged chromogen (repels cell wall)

Negative stain procedure

1. Small drop india ink near one end

2. Small amount of culture and disperse in drop of stain without spreading the drop

3. Use second slide to spread drop of stain + evenly

4. Rest one end of clean slide on center of draw at an angle until it spreads along edge then push slide toward the clean slide

5. let air dry and DO NOT heat fix

Simple staining specifics

• Enhance visibility; stains cell allowing for contrast with white background

• Morphology: cocci, spirilla, bacilli

• Arrangement: isolated, clustered, or in chains

• Non differential- does not distinguish between bac.

• + charged chromogen= binds to negatively charged lipid membrane (bacteria takes up color)

Gram staining purpose

• Most important differential stain

• First test run on specimen for identification (cell size, shape, arrangement)

• helps doctors determine right antibiotics for infections

Gram- negative

• Structure- thin peptidoglycan, outermembrane of lipopolysaccharides

• Decolorized by alcohol

• Color: pink

• “Sandwich”

Gram-positive

• Structure: thick peptidoglycan, no outer membrane of lipopolysaccharides

• Resistant to decolorization 

• Color: purple 

Gram staining stages

1. cells transparent prior to staining

2. Crystal violet stains both. Iodine = mordant (intensifies and locks in color if g+)

3. Decolorization (alcohol/acetone) removes crystal violet (if gram -) 

4. Safranin counterstains gram - 

Gram stain procedure

1. mixed smear, air dry, heat fix 10 sec

2. Crystal violet 60 sec

3. Iodine 60 sec

4. Decolorizer (ethanol) for 3 second

5. Safranin (counterstain) 60 sec

6. Blot dry w/ bibulous paper

Common mistakes

Over decolorization= redish g+ cells (false negative)

Under decolorization= purple G- cells (false positive

Too much bacteria= thick smear, hard to see individual cells