Gram Staining

Gram Staining

• Gram +

• Gram -

Cell Wall

Gram-Positive Bacteria• Take up the crystal violet

stain used in the test, and

then appear to be purple-

colored when seen

through a microscope.

• This is because the thick

peptidoglycan layer in the

bacterial cell wall retains

the stain after it is washed

away from the rest of the

sample, in the

decolorization stage of

the test.

Gram-Negative Bacteria• Does not retain violet stain

after decolorization.

– Their peptidoglycan layer

is located under a layer of

fat.

– The alcohol used in

decolorization degrades

the outer lipid layer making

the cell wall incapable of

retaining the crystal violet.

• This causes bacteria to take

up the counterstain (safranin)

and appear red or pink.

Gram Staining

Gram Staining

Process:

Can be done with either:• A Liquid Medium or• A Solid Medium

Removing Inoculum from

a Liquid Medium

• Flame the lip of the culture tube. This creates a convection current which forces air out of the tube and prevents airborne contaminants from entering the tube.

• Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculum.

• Again flame the lip of the culture tube.

• Replace the cap.

Transfer a sample of the

medium to the slide.

• Allow the medium to air dry.

Removing Inoculum from a Solid

medium• Place several loopfuls of tap water on a

slide.

• Sterilize the inoculating loop.

• Allow it to cool before moving on.

• Scrape off a small amount of the organisms

and close the lid.

• Transfer a small sample of the colony to the drop, and emulsify (mix with inoculating loop).

1

3

2

Fix Slide

• Fix the dried film by passing

it briefly through the Bunsen

flame two or three times

without exposing the dried

film directly to the flame.

Gram Staining

Flood for 1 minute with Crystal Violet

The Crystal Violet is

gently washed off the

slide with running

water.

Don’t use so much water

that you wash the

bacteria off of your slide

as well.

Gram Staining

Flood with Grams Iodine for 1 minute

Gram Staining

Decolorize with 95% ethyl alcohol.Caution: Do not over-decolorize. Add reagent drop by drop until alcohol runs almost clear, showing only a blue tinge.

Gram Staining

Counterstain with safranin for 45 seconds.

Gram

Staining

Gram staining is one type of

DIFFERENTIAL Stain.

•Crystal (Violet)•Iodine•Alcohol {Ethanol}•Safranin

Shape

• Cocci

– round

• Rod

–Bacillus

• spiral

Shape

• Cocci

– round

• Rod

–Bacillus

• spiral1

2

3

Cocci Morphology

Gram-Positive Bacteria• Gram-Positive cocci

• Clusters: usually characteristic of

Staphylococcus spp., such as S. aureus.

Gram-Positive Bacteria• Gram-Positive cocci

• Chain: usually characteristic of Streptococcus

spp., such as S. pneumoniae, B group

streptococci

Gram-Positive Bacteria• Gram-Positive cocci

• Tetrad: usually characteristic of Micrococcus

spp.

Gram-Positive Bacteria• Gram positive bacilli

• Thick : usually characteristic of Clostridium

spp., such as C. perfringens, C. septicum,

• C. tetanomorphum

•

• Thin: usually characteristic of Listeria spp.

Gram-Positive Bacteria• Gram positive bacilli

• Branched: usually characteristic of

Actinomycetes and Nocardia , such as A.

israelii

Gram-negative bacteria• Gram-negative cocci

• Diplococci: usually characteristic of

Neiseria spp., such as N. meningitidis

Gram-negative bacteria• Gram-negative cocci

• Coccobacilli: usually characteristic of

Acinetobacter spp., which can be either Gram-

positive or Gram-negative, and is often Gram-

variable.

Gram-negative bacteria• Gram-negative bacilli

• Thin rods: usually characteristic of

enterobacteriaceae, such as E. Coli

Gram-negative bacteria• Gram-negative bacilli

• Coccobacilli: usually characteristic of

Haemophilus spp., such as H. influenzae

Gram-negative bacteria• Gram-negative bacilli

• Curved: usually characteristic of Vibrio

spp.; Campylobactor spp., such as V.

cholerae

Gram-negative bacteria• Gram-negative bacilli

• Thin needle shape: usually characteristic of

Fusobacterium spp.

Gram Negative

1 2

Gram-Negative Diplococcus

12

Gram-Positive Coccus Chains

1

2

Gram Positive TetraCoccus

12

Gram Negative Sarcinae

Gram Positive Cocci Clusters

1

2

Gram-Negative Rod

12

Gram Positive Club Shaped Rod

12

Branching Rods

12

Gram negative Comma form rods

12

Gram Positive Spore-forming Bacilli

Bacillus megaterium

Gram Positive Spore-forming Bacilli

Bacillus megaterium

Spirillum 1

2

3

4

Spirillum volutans

What shape are these bacteria?

Are they Gram + or

Gram - ?

Bacillus megaterium

Enterococcus faecalis

Lactobacillus

acidophilus

Micrococcus luteus

Rhodospirillum rubrum

Sarcina

aurantiaca

Staphylococcus epidermidis

Gram + Bacilli

Actinomyces

Lactobacillus

Listeria monocytogenes

Gram - Bacilli

Bacteroides fragilis

Campylobacter

Fusobacterium nucleatum

Pseudomonas aeruginosa -

encapsulated

Gram + Cocci

viridans streptococci

Pneumococci

Staphylococcus

Streptococcus