Lab exercise 14

Why is the Gram stain considered a differential stain

- The Gram stain is called a differential stain because it helps scientists tell two types of
bacteria apart based on how they look under a microscope. Some bacteria turn purple
(Gram-positive) and others turn pink (Gram-negative) after the stain is applied. This color
difference is because of differences in their cell walls. Gram-positive bacteria have thick
cell walls that hold onto the purple stain, while Gram-negative bacteria have thinner
walls that don't hold onto the purple stain but pick up a pink one instead. So, the Gram
stain helps us see which bacteria have thick walls and which have thin walls.

how do gram positive and gram negative bacteria differ in cellular structure and how does this
contribute to their differential staining properties?

- Gram-positive bacteria have a thicker wall that keeps the purple stain, while Gram-
negative bacteria have a thinner wall and an outer membrane that makes them pick up
the pink counterstain.

How does the age of a culture affect the gram stain reaction what is an optimum culture age for a
valid gram reaction?
- The age of bacteria really matters when you're doing a gram stain. When they're just starting out
in the first day they're growing and their cell walls are in good shape for staining. As time goes on
they slow down and their cell walls change making the stain less accurate. After a while they start
to die off and their cell walls can breakdown completely. For best results it's best to do the
staining when the bacteria is still young and active within the first day of growing. That's when
the cell walls are in the best condition.
Which step in the gram stain procedure is the most prone to error? If done incorrectly how might
that step affect the end result?

- The most error-prone step in the Gram stain procedure is decolorization. If done
incorrectly, it can lead to inaccurate staining results. Over-decolorization can remove the
stain from both types of bacteria, causing Gram-positive bacteria to appear Gram-
negative. On the other hand, under-decolorization may not remove the stain from Gram-
negative bacteria, leading them to appear falsely Gram-positive. Getting the
decolorization step right is essential for accurate identification of bacteria.

What is the function of a mordant in which reagent serves this purpose in the gram stain
procedure?

- The mordant (iodine) in the Gram stain helps the primary stain (crystal violet) stick better
to the bacterial cell wall. This makes it harder for the stain to wash away during the next
step, so Gram-positive bacteria keep their purple color.

List the reagents of the gram stain technique in order and their general role in the staining process
 -
In the Gram stain technique, the reagents are applied in a specific order. First, crystal
violet is used to stain all bacteria purple. Then, iodine is added to help the purple color
stick to the bacterial cells. Next, alcohol or acetone is applied to remove the color from
some bacteria, but not others. Finally, safranin is used to add a pink color to the bacteria
that lost the purple stain. This sequential application of reagents allows for the
differentiation of bacteria into Gram-positive and Gram-negative groups based on their
cell wall properties.

in what type of cell gram positive or gram negative would you find lipopolysaccharide the
saccharide in its cell wall where is it found and why is it clinically relevant

- Lipopolysaccharide is found in the outer membrane of Gram-negative bacteria. It's

important because it can trigger a strong immune response in the body, leading to
inflammation and severe infections. LPS also makes these bacteria harder to treat with
antibiotics, making infections more difficult to cure. It's clinically relevant because it
affects how infections caused by Gram-negative bacteria are diagnosed and treated.

conducting additional research why are beta lactams more effective on gram positive infections

- Beta-lactam antibiotics, like penicillins and cephalosporins, work better against Gram-
positive bacteria because they have a thicker cell wall, which the antibiotics can easily
penetrate. On the other hand, Gram-negative bacteria have a thinner cell wall and an
extra outer membrane, making it harder for these antibiotics to get through and kill the
bacteria. Beta-lactam antibiotics are more effective against Gram-positive infections
because they can reach and damage their cell walls more easily.

what additional compounds are found associated with the cell walls of gram positive bacteria but
are absent in gram negative bacteria

- Gram-positive bacteria have compounds called teichoic acids and lipoteichoic acids in
their cell walls, which Gram-negative bacteria don't have. These compounds help give
Gram-positive bacteria their thick cell wall structure and contribute to their ability to
resist environmental stresses. They also play roles in bacterial functions and interactions
with the immune system.

Lab exercise 15
what are the functions of endospores and bacteria?
- Endospores are like shields that bacteria make when things get tough. They help bacteria survive
tough conditions like heat, dryness, and chemicals by protecting their important parts. Bacteria
are tiny living things that grow fast by splitting in two. They act like little chemical factories,
breaking down different substances for energy and growth, and helping recycle nutrients in
nature. Some bacteria are good buddies with plants, animals, and humans, helping out with things
like giving nutrients or keeping away bad germs.
What external structure on the endospore acts as a protective barrier? What is its composition?
- The external structure on the endospore the axis is a protective barrier is the Spore coat. It is
composed of keratin like proteins. This layer provides protection to the endospore helping it from
harsh conditions from environment.
Compared to a vegetative cell how much less water is present in an endospore?
- An endospore has less water compared to a vegetative cell. 10 to 30%
What is the Mordant and the endospore stain?
- With endospore staining he is the Morden helping the primary stain malachite green penetrate the
endospores layer outer. The endospore stain highlights endospores. Malachite green colors the
endospores green and safranin is used as a counter stain to color the surrounding vegetative cells
pink or red.
What is the stimulus for the endospore production in bacteria?
- The stimulus for endospore production is factors that threaten the survival of the endospore like
nutrient depletion or environmental factors. When this happens they have a process called
sporulation which involves the formation of endospores they are highly resistant for survival until
conditions improve.
What conditions are necessary to destroy endospores? In what device are these conditions
achieved?
- To destroy endospores high temperature pressure and moisture. The process of destroying them is
called sterilization which puts them in a condition of high temperature and pressure for a long
period of time. Autoclaves are used to steam under high pressure killing the endospores and other
microorganisms present.
What is the color of endospores after gram staining? after Schaeffer Fulton endospore staining?
- After gram staining endospores appear colorless because they do not take up the primary stain
used in the procedure in the Fulton endospore stain they appear green because of the malachite
green as the primary stain to target the endospores.
What is the secondary stain in the schaeffer Fulton endospore stain?
- The secondary stain used is safranin
-

What is the color of the vegetative cell after the Schaeffer Fulton endospore stain?
-
- After the Fulton endospore staining the color of the vegetative cell is pink or red.

Of these three genera of bacteria what does not produce endospores: Clostridium, Myo bacterium,
or bacillus?
 - Mycobacterium does not produce endospores.
Based on the sporulation cycle are bacterial endospores reproductive structures? Explain why or
why not.
- Endospores aren't reproductive structures because they don't lead to new bacteria instead they are
protective pods formed by some bacteria when conditions are tough.
List and briefly describe 3 examples of diseases caused by endospore forming bacteria and the
names of the specific bacterial agents involved
Anthrax-bacillus anthracis, tetanus-clostridium tetani, botulism- clostridium botulinum.

Lab exercise 16
What makes mycobacterium resistant to staining?
- It's tough to sustain because of its cell walls waxy layer made of lipids
Name another acid-fast bacterial genus
- norcardia
what is the primary stain in kinyoun acid fast staining method? How does this differ from the
primary stain in the Ziehl Neelsen method?
-
In the Kinyoun acid-fast staining method, the primary stain is carbolfuchsin, while in the
Ziehl-Neelsen method, it's basic fuchsin. Both stains color acid-fast bacteria red, but
carbolfuchsin doesn't need heat, making it simpler to use than basic fuchsin, which does
require heating.

What is the secondary stain in both acid fasting methods?

- The secondary stain used as methylene blue
What is the Ziehl neelsen acid fasting what is the mordant?
- The heat
What is the advantage of the Kinyoun staining procedure over the Ziehl Neelson method?
- Kinyoun procedure is simpler and safer because it doesn’t require heat like neelsen.
If you considered an acid fast stain on a sputum sample from a person with tuberculosis what
would you expect to see on the slide?
- Red colored bacteria against blue backround.