lab 4 gram stain/endospore Flashcards
Gram ___________ bacteria have a cell wall that’s composed of between 60 - 90% ____________
B. Positive ; peptidoglycan
-thick call wall
what is gram neg
-thinner cell wall also mase of peptidogylcan which is surrounded by an outer membrane with high lipid contect
what is the primmary stain for gram
crystal violet
what is the mordant for gram and what does it do
iodine, causes crystal violet to form large crystals within the cell wall
what is the decolorizer for this lab
alcohol/acetone solution
what does the decolorizer do
dissolves the cyrstal violet and at the same timw dehydrates the thick cell wall of the gram pos (which makes it difficult for the stain to leave the cell wall)
what color is gram pos
purple
what color is gram neg
clear bc of the decolorizer reaction
what is the counterstain for gram
safranin
what makes gram pos pink ?
safranin
explain the procedure…
- start with bacteria smear
2 cover the smear with crystal violet stain for 1 min
3 gently and thoroughly rinse the slide with water
4 cover the smear with gram iodine for 1 min
5 gently and thoroughly rinse the slide with water
6 add decolorizer dropwise until runnoff is clear (20-30)
7 gently and thoroughly rinse the slide with water
8 cover the smear with a safranin stain for 1 min
9 gently and thorughly rinse.. done
what was the result of e coli for this lab…
gram neg
color-pink
shape- rod
arrangemnet- none
what was the result of staphyloccus aureus
gram-pos
color-purple
shape- sphiricle
arramgment- cluster
How many steps are in the gram stain procedure
4
What is the first step in the gram stain procedure?
The heat-fixed smear is flooded with primary stain crystal violet
[for 1 minute]
What is the second step in the gram stain procedure?
adding mordant [gram’s iodine]
[for 1 minute]
What is the third step in the gram stain procedure
[Remember to hold slide at an angle when washing]
(Wash the slide with water [for 5 seconds])
Then wash with the decolorizer 95% ethanol until no more crystal violet washes off the slide.
(Wash the slide with water [for 5 seconds])
What is the fourth step in the gram stain procedure?
Counter-stain the bacteria with safrinin [for 1 minute]
(Then wash the slide with water [for 5 seconds]
Why do we use mordant in the gram stain procedures?
It fixes the primary stain [Crystal Violet] to the cell wall
True OR False?
Safrinin does not affect the color of the gram positive cells
True
Why do we use decolorizer [95% ethanol]?
It dissolves the outer membrane in gram negative organisms [which removes the Crystal Violet stain]
The ____________ stains cells that have become colorless [from the decolorization step]
safrinin
True OR False?
It’s best to perform the gram stain on young, actively growing cultures.
True,
because older bacterial cultures do NOT show typical gram stain results
What bacteria did we use for the gram-stain procedure?
1) Staphylococcus aureus [Staph. A}
2) Escherichia Coli [E Coli]
What are endospores
dehydrated cells with thick cell walls
survive extream heat, lack water
what is the main stain for endospore
malachite green
what is the counterstain stain for endospore
safranin
what is the color of the endospore
green and the surrounding vegetative cell pink
The spore stain is a ______________________ stain
differential stain
Endospores are produced by
Bacillus and Clostridium
Endospores are resistant to
desiccation, heat, chemicals (disinfectants, acids, and staining reagents)
Bacteria produce endospores in response to
hostile environments conditions
(heat, cold, radiation,etc.)
Endospores are a
Protective mechanism
In the proper environment, an endospore will germinate to produce a new ____________ cell, which can grow & reproduce
vegetative
The endospore first develops within the mother vegetative cell when nutrients such as ___________ & ____________ become limiting
carbon & nitrogen
________________ are not metabolically active and are resistant.
spores
Any __________ cells will decolorize with water
vegetative cells
Step 1 of Schaffer-Spore stain
Prepare a heat-fixed smear of Bacillus Subtilis
Step 2 of Schaffer-Spore stain
Place slide on top of beaker of steaming water
Step 3 of Schaffer-Spore stain
Saturate smear with malachite green & steam for 3 minutes
Step 4 of Schaffer-Spore stain
Remove from water bath, set on staining tray, and cool it
Step 5 of Schaffer-Spore stain
Gently rinse with squeeze bottle
Step 6 of Schaffer-Spore stain
Counterstain with safrinin [for 30 seconds]
Step 7 of Schaffer-Spore stain
Step 7 of Schaffer-Spore stain
Step 8 of Schaffer-Spore stain
Examine slide under oil immersion lens
It takes _____________ hours to incubate and see endospores
72 hours
m. smegmatis + s. aureus results (acid fast stain)
shape-rod
color-pink
acid fast- positive
