Laboratory Experiments in Microbiology-Exercise 7
What is the Gram stain useful for?
It is a differential stain that allows you to classify bacteria as either gram negative or gram positive.
Who discovered the gram staining technique?
Hans Christian Gram
What are the gram staining techniques?
1.Apply primary stain- (crystal violet) all bacteria are stained purple by this basic dye
2. Apply mordant Gram's iodine- the iodine combines with the crystal violet in the cell to form a crystal violet-iodine (CV-1 complex)
3. Apply decolorizing agent (ethanol). The primary stain is washed out of some bacteria (decolorized) and some are unaffected.
4. Apply secondary stain or counterstain (safranin) this basic dye stains the decolorized bacteria red
What is the most important determining factor in the procedure of Gram staining?
bacteria differ in their rate of decolorization
The bacteria that decolorize easily are called?
The bacteria that decolorize slowly and retain stain are?
Why do bacteria stain differently?
Chemical and physical differences in their cell walls
Bacterial cell walls are complex lattice structures composed of layers of ______.
Gram-positive cell walls contain multiple layers of peptidoglycans making them __________.
Gram-negative bacterial cell walls contain a__________ layer of peptidoglycans surrounded by an outer layer of lipoproteins, phospholipids and lipopolysaccharides.
Crystal violet is picked up by the __________.
Iodine react with the dye in the ___________to form CV-1 that is ________ than the crystal violet that entered the cell.
The alcohol decolorizing agent ________________ the cell wall of the ___________ cells.
True or false. The CV-I cannot be washed out of the gram-positive bacteria.
Safranin stains the decolorized bacteria __________.
Gram stain is most consistent when used on young cultures less than _______________.
24 hours old
Why are cells less than 24 hours better?
because when the bacteria dies their cell walls degrade and may not retain the primary stain- which would give inaccurate results.
What are the gram staining reagents?
What do you rinse the stain with?
Preparation of Gram staining:
prepare slide with circle on back of each slide
label the slides for each culture
smear with the inoculated loop
air dry smear
fix the slides
Gram staining procedure:
1. starting with fixed smear cover the smear with crystal violet for 1 minute
2. gently wash off crystal violet with water until it runs clear
3.cover the smear with Gram's iodine for 30 seconds
4.gently wash smear with water
5. decolorize with ethanol
6.gently wash off ethanol
7.cover the smear with safranin for 1 minute
8. wash the smear with water
9. blot it dry
What are some common sources of Gram staining error?
a. loop too hot
b. excessive heat during heat fixing
c. the decolorizing agent (ethanol) left on too long
d. the culture was too old
e. the smear was too thick
What color will a gram-negative cell turn?
A gram positive cell?
Which organism is a coccus?
Bacteria: Staphylococcus epidermidis
name the morphology and arrangement and gram stain result
Can iodine be added before primary stain in gram stain?
Considering you can't identify bacteria from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic?
To identify which type of antibiotics to use. There are antibiotics that are gram specific and there are broad spectrum antibiotics that are effective against both gram negative and gram positive