Lab 2-Gram Staining Flashcards
What are the reagents used in gram staining?
- Crystal violet
- Gram iodine
- Decolonized
- Gram safarin
What is the use of the crystal violet in the gram staining process?
- primary stain
- violet colored, stains all microorganisms
What is the use for using Gram iodine in gram staining ?
- mordant
- violet colored, stains all microorganisms
What is the use of the Decolorizer in gram staining?
- Acetone + Methanol
- Removes crystal violet iodine complex from thin peptidoglycan layers
- Dissolves outer layer of Gram negative organisms
How many cm in a meter?
100 cm= 1 m
How much um in a meter?
1,000,000 um = q m
How many no in a meter?
10 ^9 / billion nm in a meter
What purposes would a cell need to be alive for visualization under a microscope ?
- motility
- cell behavior
- short term
For what purpose would a cell need to be killed for visualization under a microscope?
Physical appearance of cells
Longer term storage possible
Describe briefly the functionality of positive stains
Positive stains: sticks to specimen
Basic
Describe briefly the functionality of the negative stain
Negative stain settles around specimen to form a silhouette
Acidic
What is differential staining?
Differentiates based upon the bacterial cell wall composition
Name an example of differential gram staining
Hans Christian Gram
How does the Hans Christian Gram stain differ between gram positive and negative bacteria
Gram positive: purple/blue
Gram negative : red/pink
What is the effect of the primary stain in Hans Gram stain?
Primary stain- crystal violet
Trapped by both cell walls: purple
What is the purpose of iodine in Hans gram’s stain?
Shrinks both types of cell walls
“mordant”
What are the reagents used in gram stain?
- Crystal violet
- Gram iodine
- Decolorizer
- Gram safarin
What are the steps in gram staining?
- Make bacterial smear from an agar plate
- Preparing bacterial smear from a broth culture- water not required
- Apply the primary stain
- Rinse with water to remove excess iodine and drip Decolorizer (80% methanol and 20% acetone). The effluent should appear pale or clear)
- Flood the slide with counter stain, let stand for 2 mins
- Rinse with water to remove extra stain, dry and observe under oil immersion
What. Is Decolorizer?
Acetone-20%
Methanol-80%
What is the purpose of Decolorizer?
- Removes crystal violet iodine complex from thin peptidoglycan layers
- Dissolves outer layer of Gram negative organisms
Describe the crystal violet
-violet colored, stains all microorganisms
Describe Safarin
- counter stain
- red colored
- Stains thin walled gram negative organism
- pus cells cytoplasm and lobes of nuclei also stain red
How should a bacterial smear be made from the agar plate?-step 1A
- on the back of the slide, draw a line to separate the slide in half
- label one slide+ and one-
- the + and - should still be visible, and will be used as your guide
- place a Loop full of water onto the regions labelled + and -
- suspend some of the material to be stained in a drop out of water on a microscope slide, spread the drop to create a thin film(smear) on the slide
- allow air to dry
- Heat fix by gently warming
How should a bacterial smear from a broth culture-step 1b?
Water is not required
- suspend some of the broth to be stained on a microscope slide, spread the drop to create a thin film (smear) on the slide
- allow to air dry
- Heat fix by gently warming
How to apply the primary stain?-step 2
Flood the smear with crystal violet
Allow to stand for 1 min
Rinse with water to remove excess stain
How to apply the mordant ? -step 3
- flood the smear with iodine solution
- allow to stand for 2 mins
What are steps 4 and 5?
Rinse with water to remove excess iodine
Drip decolorizer (80% methanol +20% acetone) across the slide about 5 sec
The effluent should appear pale or clear
How is counter stain applied?
Flood the slide with Safarin solution
Let stand for 2 mins
What is the final step ?
Step 7-rinse dry and observe under the microscope
- rinse with water to remove excess stain
- blot dry
- observe under oil immersion
What should be done before applying a primary stain?
-Allow the slide to air dry
-once it is dry you pass it through the flame of the alcohol burner a couple of times
NO NOT HOLD IT TO THE FLAME
The slide should be warm but not too hot to touch
Once the side is cool it is now ready to be stained
