Hans Christian Gram
is a Danish bacteriologist who named this stain and developed this method in 1884.
How thick is peptidoglycan layer?
Up to 80 layers
Why are most Gram-negative bacteria naturally resistant to large, bulky antibiotic molecules, even if those molecules are very toxic?
The Porins in the outer membrane act as filters. If an antibiotic molecule is physically larger than the porin channel, it simply cannot enter the cell to do its job.
Why do we use "Iodine" if it isn't actually a dye? What is its job?
it links with Crystal Violet to make the molecules too big to escape the cell wall
Some bacteria can turn into an "Endospore." If you boiled an endospore for 10 minutes, would it die?
No (they are highly resistant to heat)
It is an asymmetrical bilayer found only in Gram -
The Outer Membrane
What is "Bouncer" system is consist of?
Porins & Efflux pumps
This molecule consists of three parts: Lipid A, Core Polysaccharide, and O-Antigen.
Lipopolysaccharide (LPS)
Why does the pink Safranin dye show up in Gram-negative cells but not in Gram-positive cells?
Both actually take up the pink dye, but in Gram-positive cells, the dark purple crystal violet is so intense that it "masks" or hides the lighter pink color.
If you were a bacterium living in the human gut, where you are constantly attacked by bile, would you prefer to be Gram-positive or Gram-negative? Why?
Gram-negative, because the outer membrane protects against bile
Lipid A (The Endotoxin)
When the bacteria die and burst, Lipid A is released into the human bloodstream. Our immune system "overreacts" to it, leading to fever, inflammation, and septic shock.
Name three serious illnesses caused by Gram-positive bacteria
Pneumonia, Anthrax, Diphtheria, or Botulism
If a Gram-negative bacterium is alive and healthy, is it releasing Lipid A into your body?
No. Lipid A is part of the physical structure of the outer membrane. It is only released as an "endotoxin" when the cell dies and the membrane breaks apart
If a doctor suspects a patient has a brain infection (Meningitis), which specific body fluid will they send to the lab for a Gram stain?
Cerebrospinal Fluid (CSF)
Based on the "Goals" of the lesson, what are the two main things a student should be able to do by the end of class?
Identify features of G+ and G- bacteria and know the procedure of bacterial staining
A gel-filled gap between the inner and outer membranes in G -
The Periplasmic Space
Why does a Gram-positive cell wall act like a "sponge" during the staining process?
Its thick, porous peptidoglycan traps the Crystal Violet-Iodine complex inside.
If a student forgot to add the decolorizer and went straight from Iodine to Safranin, what "false" result would they see for an E.Coli sample?
It would appear purple (Gram-positive) because the crystal violet was never washed out.
Which group of organisms is "Gram-Neutral" because their cell walls don't contain any peptidoglycan at all?
Archaea
Why would a Gram-negative bacterium have a harder time surviving "dehydration" compared to a Gram-positive bacterium?
The thick peptidoglycan of a G+ cell helps retain moisture better than the thin G- scaffold.
Teichoic Acids
These are long, acidic polymers woven through the peptidoglycan. They hold the thick wall together
Go to the board and draw a G+ cell wall, include at least 3 terms

Create a "Human Porin"
Two students must form it and decide if other students are allowed to pass through.
Amazing performance
If you forget to add Safranin, what will you see when you look at a slide that only contains E. coli?
Nothing/Clear cells (because they were decolorized and never recolored)
If "Gram-negative" bacteria have better protection (two membranes), why haven't "Gram-positive" bacteria gone extinct?
G+ bacteria are cheaper to build (less energy), can form endospores, and their thick wall is better for physical survival in soil/dryness