Experiment 4: The Gram Stain

Who was the Gram stain technique devised by?

Christian Gram in 1884, danish guy

How is the Gram stain diff from simple staining?

Gram is used to differentiate types of bacteria dependng on their ability to retain dyes

∴ differential staining technique

Bacteria divided into 2 main groups

1. Gram +

2. Gram -

what is the Gram stain based on?

based on cell wall structure; most bact have peptidoglycan in their cell walls which surrounds and protects the cell.

what type of cell wall do Gram + cells have?

THICK peptidoglycan, rigid structure

what type of cell wall do Gram - cells have?

THIN peptidoglycan; only a few layers

Steps of Gram stain technique (7)

1. stain fixed smear w primary stain (crytsal violet)

2. rinse xs CV with water

3. apply Gram’s iodine (mordant); binds to CV and forms complex thats hard to remove in Gram +

4. rinse xs Gram’s iodine w water

5. wash with decolourizing agent (most imp step) —> 95% ethyl alcohol —> removes basic dyes like CV

6. rinse xs alcohol w water

7. apply counterstain (safranin, red)

Gram + after staining

not decolourized by alcohol so its purple

bc of thicc peptidoglycan

Gram - after staining

decolourized by alcohol and absorbs the red safranin

bc thin peptidoglycan

Exp 4 learning objectives

1. understand and perform Gram stain

2. diff and classify bact as Gram +/-

3. asses cell wall structure based on stain outcome

exp 4 materials (individual)

1. Staphylococcus epidermis (12-18h)

2. Serratia marcescens (12-18h)

3. staining solns

   1. huckers CV (primary)

   2. Grams iodine (mordant)

   3. 95% ethyl alcohol (decolourizing)

   4. safranin

4. demo slides of Sm Se

how to take samples from SOLID cultures

1. place 2-3 loopfuls of water onto slide

2. put culture on the water

no need for water if taking from broth culture

prepare smears for gram staining (9)

1. Three dime sized circles on slide and put each microorganim on each cricle and last circle is for pure borth of choice (small amt of cells) —> create emulsion w solid and water

2. lets smears air dry. heat fix smear side up

3. cover w 1-3 drops of CV for 1 min and do not allow stain to dry (add more drops if need be)

4. rinse xs w water.

5. 1-3 drops of Grams iodine. sit for 1 min. wash w water

6. decolourize w 95% alc. 10-30 secs until run off liq is clear. 4-5 drops is good.

7. rinse xs alc w water.

8. counsertrain w 1-3 drops of safranin. sit fot 1 min

9. rinse xs w water. blot dry w paper towel. let xs moisture air dry

Errors in this technique (7)

1. thick smears —> decolourizing takes too long = false -ves

2. overheat fixing —> cell wall ruptures = false -ves

3. over decolourzing = false -ves

4. consistent washing and drying (excess water = dilute mordant)

5. dont let stains on for more than stated

6. age and pH (acidity/alkanity) affects (older cultures = false -ves/variable) vv

7. underdecolourzing = false +ves

Results: Staphylococcus epidermidis

staphylococcus

gram +ve, purple

Results: Serratia marcescens

streptobacillus, diplobacillus

gram -ve, pink

Results: Staphylococcus epidermidis (PURE broth)

staphylococcus

gram +ve, purple