Gram, Gram And C. Gram

Gram, Gram And C. Gram
Gram's Stain was discovered in 1882, but published in 1884 by a Danish Bacteriologist Doctor by
the name of Hans Christian Gram. Gram stumbled upon this method while he was examining some
lung tissue from patients that had died of pneumonia. While examining this lung tissue Gram
discovered that certain stains were more favorable and retained by the bacterial cells. It was only a
few years later that Gram produced a staining procedure that he divided into two groups. The two
groups divided almost all bacteria into what he called Gram positive (purple) and Gram negative
(pink).
The Gram's staining procedure is still used today and is the method that forms the basis for
identifying bacteria. In order to perform this procedure, you will need a Bunsen Burner or a Tirrill
burner, alcohol– cleaned microscope slide, and water. You will also need The following reagents:
Crystal violet, Gram's iodine solution, 95% alcohol, and safranin. These stain reagents are important
because they are used to determine the Gram reaction for microorganisms' identification. Crystal
violet will stain the bacterial cell, and Iodine will bind the stain. The alcohol differentiates bacteria
retaining or not the crystal violet within the cell wall, and the safranin will be used as a counterstain
to stain the bacteria. You are now ready to start you process.
First you will need to light your burner to the hottest part of the flame before starting your gram
stain. After the burner is lit you will move to
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Gram Negatives
When I first did my streak plating I could only find two microbes and I was panicked. I thought the
third one was a fastidious bacterium. It turned out that the third one was there all along; the growth
was just too vague. I was able find it the very next day. I gram stained my bacteria and I found out I
had one gram positive and two gram negatives. I called my gram positive U1 and my gram negative
U2 and U3. My gram positive was rods so I was suspected it to be a Bacillus species. I waited for a
couple of days for my gram positive to grow old then I endospore stained them to confirm my
suspicion. The gram positive was indeed a Bacillus species because of the endospores. At first I
thought my gram positive was Bacillus thuringiensis because ... Show more content on
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I got exact the same results as the previous tests so I thought to myself "maybe I happened to have
two bacteria with identical metabolic capabilities". Based on the initial results I got, I thought my
gram negative bacteria was Citrobacter freundii and Klebsiella terrigena. To confirm my bacteria, I
did the KIA tests. If they were Citrobacter freundii and Klebsiella terrigena then I was expected to
see black precipitation in one of the KIA tests only, but both of the KIA tubes had black precipitate
in it. It didn't make any sense at all. I thought my gram negative pure cultures was old so they
weren't producing the proper results so I started everything from scratch. I grew new pure cultures,
did all the tests again and still got the same results. I had no idea what was going on; the
morphology of the two bacteria was completely different from one another. They were clearly two
different bacteria but how comes they kept producing the same results for all of the tests. Well, it
turned out that my U3's pure culture contained U2 bacteria. My U3's

Gram Staining Essay
A gram stain is a simple way to begin differentiating an unknown bacterial sample. Bacteria may
either be gram–negative or gram–positive. Staining makes bacteria cells visible, and this particular
staining method allows us to narrow down whether the bacteria in gram–negative or gram–positive.
Gram–negative stains purple and gram–positive stains pink.
Gram staining is used by students in advanced placement biology high school courses and in college
microbiology courses. Gram staining is conducted in a laboratory, whether it is a research lab or a
classroom lab. Instructors and professors will have the following items on hand for you.
CAUTION: Open flame or heat may be used, follow proper precautions to avoid a fire or injury.
Staining technique ... Show more content on Helpwriting.net ...
Staining technique uses dyes that are non–toxic but produce difficult to remove stains from skin and
clothes, wear gloves to avoid staining on hands.
Once you have gathered all your materials, you may start with the procedure. Before starting with
the actually staining, wash your hands and wipe down your work area to avoid contamination. After
that is done turn on your source of heat. Often times the source of heat will be a Bunsen burner, so
in order to turn a Bunsen burner, use a gas hose to connect the burner to the gas line. Once
connected, turn the gas line all the way on and place the fire striker close to the opening and strike it
to start a flame. Then minimize the flame by slightly closing the handle. After your source of heat is
ready to go, you can start the staining process.
1. Get one of the microscope slides and place it on a flat surface. Add about 6 drops of distilled
water to it
2. Sterilize the metal loop in flame until it turns orange. Make sure only the wire loop part is being
heated so you avoid the whole tool getting hot and burning yourself. Remove the loop from the
flame and let it cool for about three

A Summary Of Gram-Negative Bacteria
Introduction
Every winter the snow falls, and people get sick. Both of these items have components in common.
Both the snow that falls, and the bacteria that gets people sick may be small, but when allowed to
accumulate, characteristics of them become visible. While snow is made from frozen water, with
flakes differentiating in the structure of them, bacteria is like that, to a sense. Certain bacteria
families may have similar genomes, they do have slight differences that make up what they are. Just
like snow being made of frozen water, bacteria is made up of the same four genomes. They're just in
a different order, which makes them unique
One way to tell the difference is to see if the bacteria is gram positive or gram negative. Gram–
Positive bacteria contains peptidoglycan (which is a polymer of amino acids and sugars), while
Gram–Negative Bacteria does not have as much. (Holbrook, 24)
Because Gram–Positive contain peptidoglycan, they do not form string in the KOH test. The
opposite is true for Gram–Negative– it will form a string due to its chemical makeup. The Gram–
Positive bacteria uses the peptidoglycan to act as a wall, so the KOH will not react with it. However,
the cell membrane on the Gram–Negative bacteria reacts with the KOH, which creates a slight polar
charge on the bacteria, which causes it to form a string. If too much force was added by pulling on
the toothpick, this causes the string to break. Therefore, when applying the KOH string test, one
must be

Biochemical Tests On The Gram Negative And Gram Positive...
Microorganisms have the ability to live everywhere and can survive when put together with another
not of its kind. Expectations for this experiment would to successfully obtain credible information
behind the mixed unknown and be able to isolate both the gram–negative and gram–positive
bacterium. In order to obtain the correct test results and to ensure the right gram mixed unknown is
identified there had to be a series of eight biochemical tests performed on the gram–negative
bacterium and five biochemical tests performed on the gram–positive bacterium.
Introduction
The sole purpose behind this conduction was having the ability to understand just how the gram–
negative and gram–positive bacteria can be distinguished and identified when mixed together into
one broth. Understanding the basic characteristics as to why each bacterium has the ability to dye
certain colors and retain those colors. The gram–positive bacteria has the ability to retain the crystal
violet color and remain purple as for gram–negative bacteria, they cannot hold the purple color and
decolorizes when the ethanol hit its. The gram–negative bacterium does have the ability to retain the
counterstain, which is a pink to red color. This happens because of the thickness of the
peptidoglycan layer, in the gram–positive the layer is 20–80 nm thick and have a multiply amount of
layers but for the gram–negative they only have a thin 1–3 nm thick one layered layer (Badon,
2014).
Biochemical tests for the

Gram-Negative Bacteria Lab Report
Title Page
Introduction
The purpose of this lab is to use several techniques to determine the identity of an unknown
bacterium. Many of these tests involve the differences between Gram–positive and Gram–negative
bacteria. Gram–positive bacteria have a thick peptidoglycan wall while Gram–negative bacteria
have a much thinner peptidoglycan wall. This leads to a number of differences in what media each
kind of bacteria can grow on and their physical properties.
Techniques used in this experiment include growth tests on different media, microscopy of a wet
mount, PCR amplification and purification, gel electrophoresis, the catalase test, MSA plates, the
KOH string test, and cycle sequencing. The Oxidase test is another test used to differentiate between
Gram–negative bacteria but was not used in this experiment.
Materials and Methods ... Show more content on Helpwriting.net ...
Acinetobacter is a non–fermenting, Gram–negative bacteria. It was found that there have been some
cases of acinetobacter acidifying mannose which mannitol is derived from. The acidification of the
mannitol would result in the phenol red turning yellow. Acinetobacter baumannii was found to be
vancomycin sensitive because it was more porous than Gram–negative bacteria usually are, which
could also be the case for Acinetobacter johnsonii. The KOH string test was not very reliable
because false positives and false negatives can occur if the ratio of KOH to liquid culture of bacteria
is incorrect and the string that is formed is very thin and hard to see. The oxidase test would have
been used but the bacteria appeared, for all means and purposes, to be Gram–positive in preliminary
findings. The band that was found in the negative control lane is likely either the result of an issue
when loading the wells or as a result of contamination of the control sample. Acinetobacter
johnsonii has been found to grown on PEA very well because of its stiff

Why Do Gram Stains
After performing the gram stains, I needed to outline a plan of action for my metabolic testing. I
employed the help of a dichotomous key, which allows you to make successive choices between two
alternatives. This key would provide a clear cut path for my metabolic testing in order for me to
successfully identify my two unknown bacteria. First, I divided the key into gram positive and gram
negative bacteria. Then I divided the positive half of my key by morphology, rods and cocci. The
other negative half was divided by morphology as well, into rods and cocci, but since we were not
using any Gram negative cocci, that path was a dead end. Gram positive rods consisted of Bacillus
subtitles, and Corynebacterium. Gram positive cocci consisted of Micrococcus luteus,
Staphylococcus aureus, and Streptococcus faecalis. ... Show more content on Helpwriting.net ...
I did not have a gram positive rod as one of my unknowns but in order to complete the key fully, I
examined which metabolic test would distinguish between Bacillus subtitles and Corynebacterium
xerosis. I chose an Amylase test. Bacillus subtitles would produce amylase, breaking down the
starch into glucose, generating a positive result. On the other hand, Corynebacterium xerosis would
not produce amylase, generating a negative result. With that test I would be able to identify which
one could have been the unknown bacteria. However, I did have a gram positive cocci as one of my
unknown bacteria. I first decided to do a lactose fermentation test because a negative result would
identify my bacteria as Micrococcus gluteus, and a positive result would indicate that I was working
with either Staphylococcus aureus or Streptococcus faecalis. If the results were positive, I would
have to pick another test to distinguish between

Gram Staining Lab Report
Gram staining is a way of classifying bacteria into two groups: gram–positive and gram–negative.
The experiment conducted allowed for distinguishing of two specimens, Bacillus subtilis and
Escherichia coli, using several dyes to distinguish the two. The experiment revealed the culture of B.
subtilis to be a Gram–Positive organism and E. coli to be a Gram–Negative organism. Introduction
Cell staining is a method that can be used to view cells and cell components with a microscope. The
structure of the organism's cell wall determines whether the organism is gram positive or negative
when stained. In addition, staining also allows for one to identify cell size, shape and arrangement.
Gram–positive cells have a thicker peptidoglycan cell wall to which the stains are ... Show more
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coli / B. subtilis, glass slides, transfer loops, a Bunsen burner, gram's stains and alcohol) to
successfully complete the experiment. Aseptic technique was used and maintained during the entire
experiment by utilizing the Bunsen burner. I began by labeling two slides and adding a small drop of
water to each slide. Immediately after adding water and maintaining aseptic technique, I smeared
samples of bacteria to each slide, one of which received a sample of B. subtilis and one that received
a sample of E. coli. My next step was to begin staining the slides with a primary stain such as
Crystal Violet for 2 minutes then rinsing it with water afterward. Next, I placed the mordant (gram
stain dye) on the slides for 1 minute and rinsing with water afterward. Subsequently, I used a
decolorizer (95% ethanol) for 10 seconds to remove the dyes from each slide, followed by rinsing
with water immediately and patting each slide dry. Finally, I added a counterstain(safranin) to help
distinguish the slides although this step is not a necessity for gram staining. After conducting the
staining techniques, the slides were now ready for

This week in Lab 3 we will be discussing and practicing simple and gram stains. Staining is the
process of making bacteria visible to isolate and experiment with. Staining helps to show the
bacteria in a color so it is no longer clear by increasing the contrast of the specific organism.
Staining is used in the medical field not just to look at bacteria and cultures but also for diagnostic
purposes. It can be used to highlight certain tissues and even specific parts within a tissue. There are
many different types of staining, including: Acid fast staining ( Ziehl – Neelson Technique),
Acridine orange stain, Auramine – Rhodamine technique, Calcofluor white stain, Capsule stain,
Cytoplasmic inclusion stain, Endospore stain, Giemsa stain, Flagilla ... Show more content on
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The smear concentration will directly affect the gram results by making a false positive or false
negative gram positive stain. Take the slide smears and put them in crystal violet solution for one
minute. Rinse all of the stain off with water. After all the water has been removed from the slide,
place it in the mordant which is Grams iodine solution. A mordant is an inorganic oxide that
chemically reacts with the dye and affixes to the cell. Rinse the solution off with water the same way
we did before and then place the slide in a decolorizing agent for 10–12 seconds. As not to wait for a
long time, rinse this off with water as well. Waiting to wash the decolorizing solution off will clear
all the dye of off the previously dyed cells. Put the slide in the counter stain safranin for one minute
and rinse with water. Place the slide on bibulous paper and wipe an access water of the slide being
careful not to disrupt the bacterial culture. Now that the slide is done, put it under the microscope
with an oil immersion to view under 100x magnification. Gram positive bacteria is blue or purple
and gram negative bacteria is red or

The result of this experiment was prevented growth of any microorganism in nutrient agar out of
aseptic technique and that what we expected and we achieved to our goal of this lab. Another section
of this lab, we using another technique known gram staining. Gram staining is a common technique
used to differentiate two large groups of bacteria based on their different cell wall constituents4.
This technique helps to identify among Gram positive and Gram negative groups by coloring these
cells pink or violet. Gram positive bacteria stain violet due to the presence of a thick layer of
peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with.
Alternatively, Gram negative bacteria stain pink or red, which is attributed to ... Show more content
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In this lab, we sued Sterile toothpicks, a gram positive bacterial sample growing in nutrient broth
(Staphylococcus aureus), a gram negative bacterial sample growing in nutrient broth (E. Coli), Gram
Stain sets, glass slides, Light Microscopes with immersion oil. The first stride was Resuspended the
culture tubes by tapping, then, making a (triple slide) on a glass slid. The triple sild was divided a
slide to three section, place a drop small loopful of Staphylococcus aureus on one end in the slid,
then, place drop of small loopful of the E. coli in the other end, after that, added a drop mix among
Staphylococcus aureus and E. coli in the center of slide. After completely dry slid, it should start the
Gram–Staining Protocol. the Gram–Staining Protocol was used four types of stains those are Crystal
violet, Iodine, decolorize, and safranin, each of stain was stayed about 60S and the rinsed with
water. Finally, drying the slide by pressing the slid among the pages of the Bibulous table and put
the slide under the

Lab Report For The Purpose Of Gram Staining
Gram Staining
Formal Lab Report
Miranda King
Intro Microbiology
Dr. Hendrickson
13 September 2017
Purpose
For the purpose of the gram staining experiment was to learn this specific type of staining technique,
as well as learn how distinguish the differences between Gram positive and Gram negative stains.
This technique uses crystal violet stain, Gram's Iodine, Ethyl Alcohol, and Safranin. These dyes are
used in order to distinguish between the different types of cells. For example, Gram positive staining
will result in a purple color, and Gram negative staining will result in a red or pinkish color
(University of Central Oklahoma department of biology, 2016). When a bacteria results in a purple
stain it is categorized as a gram–positive stain because the alcohol that was used to decolorize the
bacteria was not successful. Its' cell wall is made with a thick layer of peptidoglycan and holds the
crystal violet color (Case, Funke, & Tortora). When a bacteria results in a red or pinkish color, this
means that it loses the crystal violet dye color after being decolorized and keeps the Safranin dye. It
would then become categorized as a gram–negative stain (Case, Funke, & Tortora). A gram–
negative bacteria has a cell wall made of a thin layer of peptidoglycan and a thick layer of
lipopolysaccharides thus holding onto the red dye (Case, Funke, & Tortora). Bacteria have different
shapes and these shapes determine what family they belong in. There are

21 Grams
In 2003, 21 Grams was released. This American drama film was directed by Alejandro González
Iñárritu and written by Guillermo Arriaga. The writer and director did a wonderful job in bringing
each character to life. In the film you can see that the plot wasn't the main concern, but the
characters were. There are three main characters that appear to be very different, living different
lives, but have been connected from a tragic car accident. As the story goes on you can see the
similarities unfold within the three characters. The connections being made between these characters
are easily being broken. They are dealing with their different emotions and internal conflicts such as
addiction, guilt, and uncertainty.
Smoking cigarettes can be considered ... Show more content on Helpwriting.net ...
With each character there was a different lighting for their scenes because color and lighting can
communicate different emotions in a film. Majority of Paul's scenes were shot with whites and blues
because they wanted to illustrate that he was sick, dying, and sad about his future, furthermore, his
guilt. His wasn't angry about this guilt but sad about it. He felt guilty that he made a connection with
Cristina and had her dead husband's heart. Jack on the hand had angry guilt. The guilt was eating
him alive and he was angry about it. His scenes were shot in reds and oranges. These colors
definitely expressed his anger and confusion but it also showed happy times with his family and at
his birthday party. When it comes to Cristina's color schemes, there's a mixture because it was all
dependent on her mood. During her good moments where she is with her family and swimming with
her sister, the lighting is bright and clear. During her moments when she is grieving and doing drugs
the lighting is dim and low with whites and blue

Gram Staining
Title
Elizabeth Huynh
November 16, 2014
Jason Atkins
Unknown #7
Purpose
The purpose of this study was to identify the
There are a group of tests used specifically to differentiate bacteria in the Enterobacteriaceae family.
These
Results
Table 1 Microscopic Data of Differential Gram Stain
Gram Stain (A) Gram–negative Cell Results (B) Gram–positive Cell Results
Before Staining Transparent Color Transparent Color
After Crystal Violet Stain Purple Color Purple Color
After Iodine Stain Purple Color Purple Color
After Decolorization with Alcohol Transparent Color Purple Color
After Safranin Stain Pink/Red Color Purple Color
Table 2 Physiological Tests Conducted to Identify Shigella flexneri
Biochemical Tests Results Symbol
Phenol ... Show more content on Helpwriting.net ...
The Bacteria A cells that were pink/red in color after the addition of the Safranin stain were the
Gram–negative cells. Gram–negative cells have higher lipid content in their walls; therefore they
lose the primary stain color after the decolorization step. After the Gram–negative cells were
counterstained with Safranin, they turned pink or red, whereas Gram–positive cells remained purple.
After the isolation of the Gram–negative bacteria, a variety of tests were performed to identify the
unknown bacterium as Shigella flexneri. Of the three Phenol Red broth tests, the PR Glucose broth
test was the only one whose results were positive as indicated by the change in broth color from red
to yellow and by the presence of bubbles in the tube. These results indicate that the bacterium was
able to ferment glucose with acid and gas end products. The bacterium was unable to ferment
sucrose or lactose as the broth in both tubes remained red and no bubbles were present. Just from the
Phenol Red broth results, the unknown bacterium could either be Salmonella typhimurium or

Shigella flexneri. The results from the Methyl Red and Voges–Proskauer tests show that the
bacterium was able to perform mixed acid fermentation but could not ferment glucose where its acid
products would quickly convert into 2,3–butanediol and

Escherichia And Gram Staining
Most bacteria are classified as either gram positive or gram negative. Gram positive is a purple stain
and gram negative will appear pink or red. Gram negative can cause many kinds of infections. One
type of bacteria is Escherichia which is a rod shaped bacteria. A Gram stain is used in lab setting to
identify what type of bacteria is present. A sample is placed in a nutrient media and incubated. This
media will encourage bacteria growth present. This process allows for further testing and to identify
what kind of bacteria is present to allow for appropriate treatment.
Bacillus are rod shaped, prokaryotic cells, they form spores (gram positive and gram negative) on
gram staining. Escherichia are rod shaped, prokaryotic cells, however they

Gram-Negative Staining Lab Report
Tiffany O'Connor
9/2017
Gram–positive and Gram–negative stain
Purpose: Staining is done to differentiate between two varieties of bacteria, gram–positive and gram
negative.
Theory and Background: Gram–positive and Gram–negative represents whether a cell wall retains
the primary dye color in the staining process after decolorization. The difference in the structure of
the cell wall determine if this happens.
In Gram–positive cells the walls are much thicker than Gram–negative. Gram–positive cell walls
consist of several layers of peptidoglycan and groups of molecules called teichoic acids. The
teichoic acids line up perpendicular to the many layers of peptidoglycan. Gram–negative cells walls
have only one layer of peptidoglycan surrounded by an outer membrane. The outer membrane has
porins, or proteins that act like pores and allow the diffusion of molecules. The outer membrane also
has lipopolysaccharide or endotoxin, This helps maintain the integrity of the wall, it is also a toxin
but only released when the cell disintegrates.
Gram–positive cell wall: Gram–Negative cell wall:
http://textbookofbacteriology.net/structure_5.html=positive ... Show more content on
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The key to the process is in the second step, this is when iodine is applied to the specimen. The
iodine causes the dye to form crystals that get trapped in the cell walls. In Gram–positive cells the
walls are thicker and the layer of dye saturates the cell wall more extensively. The third step in the
process is to apply alcohol which dissolves the lipids in the outer membrane of the Gram–negative
cells. This allows for the violet dye to wash out, leaving these cells colorless. Step four is to add a
counterstain. In this case it was safranin. This is done so the Gram–negative cells will be seen in a
different

Gram Negative Bacteria
Gram Positive Bacteria: After picking test tube #2, I inoculated a CNA plate to isolate my Gram
positive bacteria. The colistin nalidixic acid in the media inhibits the growth of Gram negative
bacteria, the acid affets the outer membrane of the Gram negative so they are unable to reproduce.
This test is performed to determine if the Gram positvie bacterium growing on the plate has the
ability to break down sheep red bloold cells. After the CNA was incubated in a candle jar, I saw
alpha hemolysis, this means incomplete hemolysis. The organism growing on the plate prduced
methemoglobulin, leaving a greenish–brownish cloudy zone around the colony. After seeing the
results from this test, my bacteria could etiher be Streptococcus pneumoniae or Steptococcus mitis
because those are the only ones the show alha ... Show more content on Helpwriting.net ...
Then I decided I was going to do a P disc sensitivity test, but I when I went to get my new media I
asked for the wrong one. I realized that I needed another CNA plate and a P disc to do the test, but it
was too late and I performed a lawn procedure on the T–soy plate I had asked for from bacteria from
the T–soy inoculation on the first day. What I think happened was that when I looked at my
dichotomous key I saw catalase test and I knew I couldn't do it with bacteria from the CNA plate
and that is why I asked for another T–soy plate but my organism was a alpha hemolyser not a beta.
On the third day, I was able to perform the P disc sensitivity test. I asked for the correct media,
which was a CNA plate and a

Gram Staining Procedure Lab Report
Purpose:
The purpose of this lab is to identify a given unknown based on the previous experiment and lab
techniques that we have learned during this course. It was achieved, by preparing a gram staining
procedure, to help us identify the morphology of the bacteria, and to prepare a various of different
test, such as the starch test, oxidase, urease, citrate, MSA, Nitrate Reduction and MP/VP to help us
identify if the bacteria is able to produce the specific catabolism that it being testes for.
Procedure:
Gram Staining Gram Staining is a procedure used to distinguish between gram–positive and gram–
negative bacteria, where one of the main differences is the cell wall. Gram–positive bacteria contain
multiple layers of peptidoglycans and gram–negative lacks of peptidoglycans in the cell wall.
Overall, is a short procedure that consists of the following steps: first apply the primary stain
(crystal violet), second apply the mordant (Gram's iodine), third apply decolorizing agent (ethanol or
ethanol acetone) and finally apply secondary stain (safranin). Another purpose of gram staining is to
determine the bacteria's morphology. In gram–positive bacteria, the stain retains the dye ... Show
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According to (Case, 1946) urea is a waste product of protein digestion in vertebrates and is excreted
in the urine. The presence of urea liberates ammonia from urea which is helpful in identifying
bacteria. The pH of a medium urea is about 6.8 or below, if tested positive, the pH will have an
increase to about 8.4 or above causing the solution to go from a yellow color to a hot pink color, this
is mainly due to the ammonia produced. This experiment is done by following the lab of
"Preparation of Smears." Unknown 3, showed a change from a yellowish solution to a hot pink
color, helping to concluded that it tested positive to having a pH of 8.4 or above and able to liberate

Gram Staining Procedure
Bacterial identification procedures are extremely important in the clinical laboratory.
Microorganisms need to be classified, and identified in order to distinguish one species from
another. Identification can isolate organisms that are disease agents, and enable physicians to make
diagnosis's. It also enables physicians to choose the best treatment/antibiotics to address that specific
organism. For example, some gram negative bacteria can produce endotoxins when they die, which
would be of concern to a doctor prescribing antibiotics. Identifying a specific bacteria as a source of
a disease is the first step in moving towards discovering how it can be transmitted and preventing
reoccurrence. Species can be distinguished by morphology and ... Show more content on
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This staining procedure was developed in the 1882, by a Danish bacteriologist, Hans Christian
Gram. This technique identifies differences in a bacteria's cell wall. It utilizes crystal violet (primary
stain), Gram's iodine (mordant), and safranin (counterstain). Most bacteria fall into two categories,
Gram positive or gram negative. Gram positive bacteria have a thick layer of peptidoglycan, and
techoic acids in their cell walls. This thick peptidoglycan layer retains the crystal violet stain, and
displays the cells as a purple color under the microscope. Gram negative bacteria cell walls have
significant differences from gram positive bacteria. Gram negative cells have a thin peptidoglycan
layer, and do not have techie acids. They also have an outer membrane that is similar to the
phospholipid bilayer of a cell membrance. These differences allow the crystal violet stain to rinse
away from them, then they retain the safranin stain and appear reddish/pink under the microscope.
Although it is very useful, it is not the only tooled needed to identify a bacteria. Some bacteria, are
gram–variable, displaying both positive and negative reactions. Other bacteria, such as
Mycoplasmas, do not have a cell wall, rendering this test ineffective. Finally, it does not provide
enough information alone to identify a

A Summary Of Gram-Negative Bacteria
Introduction:
Every winter the snow falls, and people get sick. Both things have components in common. The
snow that falls, and the bacteria that get you sick may both be small, but when you allow them to
accumulate, you can see characteristics of them. While snow is made from frozen water, with flakes
differentiating in the structure of them, bacteria is like that. Certain bacteria families may have
similar genomes, they do have slight differences that make up what they are.
One way to tell the difference is to see if the bacteria is gram positive or gram negative. Gram–
Positive bacteria contains peptidoglycan (which is a polymer of amino acids and sugars), while
Gram–Negative Bacteria does not have as much. (Holbrook, 24)
Because Gram–Positive contain peptidoglycan, they do not form string in the KOH test. The
opposite is true for Gram–Negative– it will form a string due to its chemical makeup. The Gram–
Positive bacteria uses the peptidoglycan to act as a wall, so the KOH will not react with it. However,
the cell membrane on the Gram–Negative bacteria reacts with the KOH, which creates a slight polar
charge on the bacteria, which causes it to form a string. If too much force was added by pulling on
the toothpick, this causes the string to break. Therefore, when applying the KOH string test, one
must be careful, and to do it slowly.
Another way to figure out if the sample is Gram–Positive or Gram–Negative are to see if it grows on
certain mediums. If the sample grows on

What Is Gram Staining?
Gram Stain
In 1884, a Danish pathologist, Christian Gram, discovered a method of staining bacteria with
pararosaniline dyes. By using two different dyes, he discovered that bacteria fall into two different
groups. The first group, gram positive, retains the primary color, crystal violet. The second group,
gram negative, when washed in a decolorizing solution, becomes colorless and takes on the color of
the counterstain.
The reaction of the Gram–stain in a bacteria is determined by the biochemical composition within
that bacteria. Gram–positive cell walls are composed of tightly linked peptidoglycans which traps
the iodine complex, thus retaining the violet color after decolorization is complete. Gram positive,on
the other hand, have a

Gram Staining Essay
Introduction
This report discusses the technique of Gram Staining in order to characterize bacteria. Gram–
staining is a process in which the cells are immersed in crystal violet, iodine, ethanol, and safranin.
Based on bacteria's cell wall, most common bacteria are either Gram–positive or Gram–negative.
The Gram–positive cell wall are composed of multiple layers of peptidoglycan layers, whereas the
Gram–negative cell wall has one layer of peptidoglycan. Through the technique of Gram–staining,
the Gram–positive cells will turn purple do to the cells multiple layers of peptidoglycan.
Materials and Methods
The area being used underwent an aseptic technique to ensure the culture would not be
contaminated. The surface being used was wiped with alcohol and the lab member's hands were
washed. All non–flammable instruments were flamed. Two slides were then sterilized by wetting the
slide and then drying it.
Before preparing the Gram–staining, the technique of making a bacterial slide with heat fixation was
practiced. On one slide, a small drop of water was placed. Using a sterile inoculating loop, cell
material from a slant stock cultural was placed into the water droplet and mixed. The slide was then
left to air dry. Once the slide was dry, the slide was picked up by a non–flammable tong and ... Show
A drop of water was added to two slides, cleaned using the aseptic technique, by an inoculated loop.
The inoculated loop was sterilized, by placing the loop over the gas flame, and then placed into a
test tube containing Escherichia coli. The Gram–negative was then transferred to the drop of water
on one of the slides. The same method was used with Gram–positive Staphylococcus epidermidis,
transferring the cell material to the other drop of water. The two slides were then left to air dry.
Then, each slide was passed through the cool part of the gas flame three times as previously

The Powers And Functions Of The Gram Panchayat System In...
Introduction: The Constitution (Seventy–Third Amendment) Act, 1992 was enacted to reform the
Panchayat System in India. The Legislature of Indian States were given powers to determine the
powers and composition of Gram Sabha and Gram Panchayats. Hence, the powers, functions and
composition of Gram Panchayats are determined by the State Governments in accordance with the
local needs.
Gram Sabha
Gram Sabha has been envisaged as the foundation of the Panchayati Raj system. A village having
population not less than 1500 forms Gram Sabha and every adult of the village is member of Gram
Sabha.
However, in some states, a Gram Sabha may be formed even if the population is less than 1500. If
the population of several villages are less than ... Show more content on Helpwriting.net ...
Functions: The Gram Sabha performs such functions as the States prescribes from time to time.
They play a vital role by electing the right candidates as members (Panch) of Gram Panchayats,
The keep a check on the activities of Gram Panchayat and influence their decisions for the welfare
of the village.
They conduct general meetings. Beyond the prescribed minimum number of meetings, they can
conduct such meeting as per the needs.
Gram Panchayat
Gram Panchayat is the organization of elected panchas by the members of Gram Sabha of the
village. It is a self–government organization. The number of members in a Gram Panchayat depends
upon the population of the village.
Generally, the number of elected panchas in a Gram Panchayat varies between seven and seventeen
members. However, it may vary from state to state. There is provision for reservation of Scheduled
Castes, Scheduled Tribes and Women candidates. The head of the Panchayat is known as
"Sarpanch".
Functions: The functions of Gram Panchayat includes:
implementation of welfare plans,
social justice and development,
upliftment of women,
economic development,
development of infrastructure such as roads, waterways,

Gram Staining Paper
Background:
Bacteria is a single celled prokaryote microorganisms, rapid mutations causes bacteria can be found
just about anywhere.Gram staining helps to classify two groups of bacteria, Gram–positive and
Gram–negative. Gram–positive bacteria's cell wall has a stronger attraction for crystal violet,
because of more peptidoglycan. Gram–negative is a group of bacteria that doesn't retain the crystal
violet stain used in the Gram staining method, making positive identification possible. These are the
many characteristics used to describe the morphology of a bacterial colony; size, shape, color,
texture, height, and edge. Bacteria can be prokaryotic organisms, they lack a nucleus and
membrane–bound organelles. Most are unicellular, but some species live ... Show more content on
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Use sterile cotton swabs, and swab any surface in classroom.
Touch your finger gently to this circle and then clean your finger with an alcohol pad and touch it to
this sector.
Draw each plate, showing how colonies are spread across the agar surface. Pick several colonies on
your plates and describe them using the terms above.
Place petri dish in incubator and wait.
Place a drop of water on a clean slide.
Heat the inoculating loop until it glows red. Let it cool then remove a small amount of culture from
the agar surface; touch it several times to the drop of water until it just turns cloudy.
Burn the bacteria from the loop and allow the loop to cool, use the loop to spread the suspension
over the surface of the slide to form a thin film.
Allow suspension to air dry.
Hold the slide with a clothespin and then heat fix the bacteria on the slide by passing it over the
flame 3–4 times. Do not overheat the slide, it should feel warm but not

Gram Negative Unknown Lab Report
Gram Negative Unknown Lab Report
April Smith
August 1, 2014
Unknown 20
Abstract
The bases of this experiment was to discover the identify of the unknown from three possible
specimens: Klebsiella pneumonia, Escherichia coli, and Enterobacter aerogenes. Utilizaing the T
streak technique, the bacteria was isolated into pure colonies for further study. The Gram Stain
method was used to identity the morhphology of the bacteria such as the shape and whether the
bacteria was Gram positive or Gram negative. Biochemical test were also used to help identify the
unknown bacteria. The biochemical test used was the Triple Sugar Iron Agar, Sulfur Indole Motility
test, Methyl Red test, Voges–Proskauer test, Citrate test, Urease test, and the Gelatin test. After
observing the morphology of the bacteria using the Gram Stain method and utilizing all the possible
biochemical test, the bacteria was identified to be Enterobacter aerogenes.
Introduction
Gram negative and gram positive bacteria differ from each other in many ways especially in the
composition and size of their cell walls. Unlike Gram–positive bacteria, Gram–negative bacteria
have a thin peptidoglycan layer surround by an outer membrane. This outer membrane contains
many proteins one of them being lipopolysaccharides (LPS), which contributes to the bacteria's
negative charge. One part of this protein is a lipid, called Lipid A, which is considered an endotoxin
because this lipid triggers an immune response stimulating fever

Gram-Negative Unknown Lab Report
Abstract
The objective of this experiment was to determine a Gram–negative unknown using multiple
differential biochemical tests. Differential media have indicators in them to determine if certain
processes and reactions are occurring within the cell. Each of these processes' results is unique to a
different species of bacteria. A streak plate was used to make isolated pure colonies that can be used
in all of the tests performed. A Gram stain was used to determine the morphology of the bacteria and
to confirm that the bacteria were Gram–negative. After all of the differential media were inoculated,
incubated and observed, unknown bacteria number 20 was determined to be Klebsiella pneumoniae.
Introduction
When bacteria are obtained from the environment, one needs to perform many differential tests on
the bacterial species to identify it. First, the bacteria need to be obtained in pure colonies. To do ...
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This result implies that the bacteria are positive for the ability to rapidly convert urea into ammonia
and carbon dioxide using the enzyme urease. The indicator phenol red was used which is yellow or
orange at any PH below 8.4 and pink or red above 8.4. This explains why the medium was red –
ammonia was produced resulting in increased alkanality. Alkaline production increases the pH of the
broth and is detected by phenol red turning the urea broth red instead of yellow or orange like it
would in an acidic environment (Leboffe 187 – 189).
The Gelatin test was observed after being chilled for an hour at the end of the eight days incubation
period. The medium was solid, indicating that the bacteria do no possess the enzyme gelatinase,
which would have hydrolyzed that gelatin to make the medium unable to solidify at cooler
temperatures (Leboffe 192 – 193).
Table 1. Summary of results of the biochemical tests conducted on the unknown

What Is The Characteristics Of Gram Positive Or Gram...
Introduction:
There are two major classes of bacteria that contain peptidoglycan, Gram–positive and Gram–
negative. Gram positive bacteria will stain dark purple due to their thick peptidoglycan cell wall,
while Gram negative will stain pink due to their thin peptidoglycan cell wall. Many Gram positive
and negative bacteria are considered pathogens because they are capable of causing disease in
humans. In order to treat properly individuals who are infected with a pathogenic Gram positive or
Gram negative bacteria, the identity of the bacteria must be found. In this experiment numerous,
differential and selective media, as well as chemical test, were used to evaluate the growth patterns
and characteristics of clinically relevant bacteria. Data collected from the specific results was used
to create a chart that was used to identify an unknown Gram–positive and Gram negative bacteria.
The genus Staphylococcus is a Gram–positive cocci that is commonly arranged in grapelike clusters.
They are universally present in large numbers on the mucous membranes and skin of humans and
other warm–blooded animals. They are non spore forming, nonmotile, facultative anaerobes. Many
species of staphylococcus can be pathogenic. Staphylococcus aureus is the most common pathogen.
It can be recovered from skin lesions, abscesses, and wound infections. It can move from these sites
to the bloodstream and urine, where it then has the possibility to cause more serious conditions in
various organs.

Gram Positive Bacteria
The first unknown test tube P found to be a gram positive bacteria. The biochemical that I performed
that were positive were catalase and blood agar. Catalase has the presence of an enzyme in the test
isolated detected using hydrogen peroxide. Bacteria possess catalase with a small number of
bacteria; isolation is added to hydrogen peroxide when bubbles occur. When the bubbles occurred
on gram positive bacterium it was a conformation of being positive. Also, blood agar plates is useful
for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism.
There are three types of hemolysis, such as, beta– hemolysis, alpha–hemolysis, and y–hemolysis.
Beta–hemolysis breaks down the red blood cells and hemoglobin completely. ... Show more content
on Helpwriting.net ...
The biochemical has allowed me to find the bacterium, which was identified as Streptococcus
pneumonia. In 1881 Streptococcus pneumonia was isolated by Louis Pasteur, the species was known
as pneumococcus due to its role in the disease, pneumonia. In 1926 it was termed diplococcus
pneumonia due to its propensity to exist in pairs of cells, but in 1974 it was renamed Streptococcus
pneumoniae due to its formation of chains in liquid. It also played a key role in history molecular
genetics. It has shown the significant increase in antibiotic resistance because it is due to its use of
natural transformation system used for genetic exchange and can develop resistance to antibiotics
through mutation and natural selection. Streptococcus pneumoniae is known to cause pneumonia, a
disease of the upper respiratory tract that causes illness and death all over the world. The symptoms
of pneumoniae is when you cough greenish or yellow mucous, fever, chills, shortness of breath and
chest pain. The bacteria enter the body by inhalation of small water droplets. To treat bacterial
infections cause by Streptococcus pneumoniae is the treatment

Gram Staining Research Paper
Bacteria cells are examples of prokaryotes (simple cells). They have no nucleus thus, their DNA are
clumped in an area without a nucleus or membrane. Although prokaryotes do not have organelles
such as Chloroplasts, mitochondria or nuclei, bacteria cells still have ribosomes (Radar, Andrew).
Bacteria reproduce and duplicate by the process of binary fission. Binary fission is the processes in
which a single cell divides into two identical daughter cells each with identical DNA to the parent
cell.
Gram staining, also known as Gram's method, is the most important and universally used staining
technique in the microbiology and bacteriology laboratory. It is almost always the first test
performed in the identification of bacteria (Xu, George).The ... Show more content on
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These two types of bacteria have distinct and consistant differences in their cell wall constituents
(Rollins, D.M). Gram positive bacteria stain violet under a microscope due to the presence of a thick
layered wall of peptidoglycan. These bacteria dye violet do to the that fact that the thick
peptidoglycan walls retain the crystal violet. While gram negative bacteria appear red/pink due to
only a thin layered wall of peptidoglycan which does not retain the crystal violet during the
recolouring process with alcohol, usually, acetone or ethyl (Bruckner, Monica).
There are three main types of bacteria.They can be determined and distinguished by their cell
morphology. Cell morphology is the identification of the shape, structure, size of the cell and its
form. It pertains to the shape of bacteria. For instance, Coccus (cocci) bacterium are spherical and
generally round shaped. Bacillus bacterium are generally rod shaped and are a genus of gram
positive bacteria. Finally, the third common type of bacterial morphology is the spirillum bacteria.
Spirillum bacteria s generally a spiral shaped bacterium and are a genus of the Gram

Gram Stain: A Summary
Summary: Microbe 1D was received and a series of tests was done. A Gram Stain was done and the
sample was gram positive. A streak plate revealed that the microbe was circular, convex, yellow,
buttery and opaque. The next test was the oxidase test. The microbe was oxidase negative. The next
test performed was the catalase test. The microbe was catalase positive. The last test performed was
a MSA streak plate. The MSA plate turned yellow. This was the final indicator that the unknown
microbe 1D was Staphylococcus aureus.
Procedure 1 Gram Stain: Purpose: The Gram Stain reveals whether a microbe is either gram positive
or gram negative. It also reveals the shape and arrangement of the microbe (Harley, 2014). Of the
twelve possible microbes, ... Show more content on Helpwriting.net ...
The following catalase negative microbe was eliminated: Sarcina aurantiaca. The remaining catalase
positive microbes remained: Staphylococcus aureus and Staphylococcus epidermidis. The next test
performed was the MSA Streak Plate.
Error: A possible source of error for the catalase test is that the hydrogen peroxide is too old and
would result in a false negative for the catalase test because the peroxide would not react well with
the microbe. This could be prevented by making sure the hydrogen peroxide is not too old (Brady,
Personal Communications).
Procedure 5 MSA Streak Plate
Purpose: The MSA streak plate test is used to differentiate between different microbes by their
reaction on the specific media. The remaining microbes are Staphylococcus aureus and
Staphylococcus epidermidis. On the MSA plate, Staphylococcus aureus turns the MSA plate yellow.
Staphylococcus epidermidis turns the MSA plate a darker red (Harley, 2014).
Procedure: The MSA steak plate test was performed using the procedure on pages 186–187 and
some information on page 191 of Microbiology, Selected Labs (Harley,

Introduction: Background: This lab report will discuss the identification of an unknown microbial
organism by utilizing Bergey's Manual , the scientific method as well as the necessary laboratory
experiments prescribed by the dichotomous key for the specific bacteria type that is determined.
Purpose: To utilize Bergey's Manual along with other prescribed processes in order to satisfactorily
grow, collect data on and successfully identify an unknown given culture. Materials and Methods:
1. Gram Staining: In order to determine the type of bacteria on hand by using the dichotomous key
within the laboratory manual the first test to be utilized would consider whether the microbe
presented is gram positive or gram negative. In order to perform a gram stain you must utilize the
steps listed in figure 7.1 of the laboratory manual as well as the four basic steps. These steps are:
applying the primary stain (crystal violet), applying the mordant (gram's iodine), utilizing a
decolorizing agent (ethanol) and finally applying the secondary stain (safranin). When the process is
completed, look at the slide(s) under the microscope. If the microbe is pink it is ... Show more
content on Helpwriting.net ...
The gram staining when viewed appeared to be gram negative in nature while presenting a bacillus
(rod) shape. When grown on the streak plate the culture seemed to be flat in elevation, entire within
its' margin, circular in appearance and pinkish/ red in color. The agar slant utilized displayed an
entire growth pattern while also seeming pinkish in color. The oxidase testing showed to be
negative, the culture never turned blue or black the pigment presented by the colonies appeared to
stay red at 25 degrees C. Therefore, based on the results and data obtained within the experiments
prescribed, I have determined that my unknown microbe, #80, is Serratia

Gram Stain Lab Report
Purpose: The purpose of a Gram Stain procedure is to distinguish bacteria into two fundamental
groups of cells. There are the Gram positive cells and the Gram negative cells. By staining the cells
either violet or red, we can see the two types and their arrangements on the smear. Theory and
Background: 1. After swabbing some microbes from my cheek, I spread the bacterial smear on the
slide. In order to make them stick to the slide, I hold it near a heat source so they can attach. Then I
flood crystal violet on the smear. The color of violet binds to the Gram positive cell membranes and
to the outer membrane of Gram negative. After waiting about a minute for the dye to be stain both
cells violet, iodine is added to form a strong bond ... Show more content on Helpwriting.net ...
Some of them were in short chains. I also notices that the Gram negative cells, which were stained
red, are rod–shaped. They were spread out across the slide and did not seem to cluster. Conclusion:
1. After viewing the end result of the Gram Stain, I saw that the Gram positive bacterial cells were
stained violet, while the Gram negative cells were stained red. In the mixed culture under the
microscopes, I was able to notice that the Gram positive cells, Enterococcus durans, were in clusters
of short chains. The Gram negative, Proteus mirabilis, were in rod–shaped forms that were spread
across the slide. These cells did not seem to cluster. 2. I came to this conclusion of which cells were
on my slide after I compared my microbes to the physical and chemical characteristics of the
microbes in the textbook. The Gram positive characteristics in the Gram stain matched up with those
of an Enterococcus durans cell. There were some diplococcic or short chains across the smear. These
cells also had the same circular shape. I figured the Gram negative cells were Proteus mirabilis
because of the rod–shaped cells that were spread across the slide

Gram Test Lab Report Essay
Hypothesis: If the bacteria turns out pink/red then the gram test is negative.
Null Hypothesis: If the bacteria turns out pink/red then the gram test is not negative.
Purpose: Identify the three morphological types of bacteria and use the Gram's test to examine the
bacteria infection by the stain technique. Differ between plant and animal cells and the different
cellular components of eukaryotic cell. Describe the cell theory and explain its significance and the
difference between prokaryotic and eukaryotic.
Procedure:
1. Identify which cells are Gram–negative and Gram–positive.
2. Heat fix a culture of bacteria sample to prevent loss of bacteria during rinsing in the procedure
and identify the bacteria on the agar. Sterilize an inoculating loop and place a water droplet of tap
water on a clean slide.
3. Heat the loop again and let it cool and then open a culture tube and heat the neck of the bottle.
4. Lightly move the loop across the top of the agar to collect the bacteria. Flame the neck again and
replace the cap.
5. Use the loop to mix the bacteria with the ... Show more content on Helpwriting.net ...
The first we waited too long at the alcohol stage and it stripped away the bacteria. The second trial
however worked and we saw that the culture for Mary Farmer (Culture A) was pink rod–shaped
bacteria. However on the second trial for Culture B, we accidently blotted the petri dish too hard and
it took off the bacteria, making that trial invalid. After a few more trials, we finally managed to get
both bacterium correct. We also saw that the Culture B was round purplish–pink color and was
round looking bacteria. For the antibiotic test, we found the results of seven antibiotics and their
effectiveness against these two bacterium. The following graph will show the diameter for the zone
of inhibition for the bacteria and antibiotics. We found the antibiotics Chloramphenicol and
Tetracycline were the most effect against the two

Gram Negative Lab Report
I. The most difficult part of the experiment was separating the Gram positive from the Gram
negative. I knew my Gram negative was rod–shaped and my Gram positive was coccus–shaped. I
had to spend 2 days streaking many plates trying to isolate my two bacteria. The morphology of
both were very similar and it was difficult to tell the difference until I was able dilute the colonies by
streaking TSA plates multiple times.
II. I achieved an isolated Gram negative bacteria because I modified the four–quadrant streak style
and time. I believe that my Gram negative bacteria were a fast grower and would over grow the
Gram positive. On the 4th day I was able to successfully isolate my Gram negative, and proceeded
to run the SIM test and the TSI test as instructed. Both tests produced black precipitants, indicating
sulfur reduction. This narrowed down my possible Gram negative bacteria to four bacteria. As I
continued to analyze the SIM test the results showed motility, which was confirmed by my TA and
indole production. With this test I was able to narrow it down to 2 bacteria. I think did another Gram
stain to confirm no contamination, once confirmed, I decided to do ... Show more content on
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From one of my modified streak plates I was able to successfully isolate my Gram positive bacteria.
By my 6th plate, there was a distinction between my Gram positive and Gram negative in
morphology. To make sure it was absolutely pure I continued to streak it two more times. Once I had
a pure plate, I Gram stained it twice to confirm that it was a pure Gram positive coccus. Once it was
pure I proceeded to do the catalase test, the bacteria produced bubbles, indicating a positive result.
From there I was able to narrow down my unknown to a possible of 4 bacteria. By analyzing my
matrix I was able to determine that by doing an Oxidase test, it would eliminate Micrococcus luteus.
However, the oxidase test turned positive, allowing me to confirm that my unknown Gram positive
bacteria was Micrococcus

Gram Red Cell Walls
We were trying to determine the cell walls structure of organisms and based on the observation of
the cell walls, we can decide if they have gram negative or gram positive. My hypothesis was the
unknown would a gram negative cell wall. Experimental Methods In this lab, we used the procedure
from Differential and Special Stains. This experiment was similar to the Simple Stain experiment
but only a few changes were made to it. After we had gone with the heat–fixing the slide, we
covered the slide with crystal violet, and we left it on fore 30 seconds. We rinsed the slide gently
with a wash bottle water and we also covered the slide the slide with iodine for 12–60 seconds. We
rinsed the slide again with a wash bottle water and then we held ... Show more content on
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Gram–positive can sometimes appear Gram–negative when Gram–positive lose their ability to keep
their crystal violet when they are old and also when they are incubated longer. Another reason may
occur when a lot of alcohol were used on Gram–positive organisms, and it would result in loss of
crystal violet stain. The purpose for using a control was to be sure the known gram–negative and
positive organisms stain correctly. If they were not correctly dye, the result would be incorrect. I
improved on the mistake I made when I was doing the simple stain. The only area I think need to
improve on would be to remember that I have to put the slide in focus in order to see the organism
clearly. My unknown was Gram–positive and the shape was rod. This was different than the shape
and the grouping that I saw on the simple stain experiment. My unknown was Bacillus subtilis. I
excluded other organisms because E. coli, staphylococcus, streptococcus, and micrococcus all of
them grew. I came up with this conclusion because my unknown never seem to show growth, even
the media type lab that I did had no

Gram Stain Essay
All media incubated at 37°C, except gelatin.
Slant or gelatin media used a needle to inoculate the microbe.
Day1
Professor handed out one unknown organism to each of the students. Students had to record their
own unknown ID. Used the aseptic techniques to perform the gram stain and inoculate the nutrient
media(nutrient broth, nutrient agar slant, and nutrient agar plate). Only for the nutrient agar plate
media used four quadrant streak method. Gram stain was distinguishing the gram–positive or gram–
negative cells. Preparing of the heat–fixed slide was taking some times while air dries it. Therefore,
inoculated the media of growth during the waiting time. Additionally, placed the media in the "to be
incubated area." The gram stain required four important stains and solution: Crystal violet, iodine,
95% ethanol, and safranin. Crystal violet was for the primary stain. Iodine was the mordant to form
the crystal violet–iodine complex. Used the 95% ethanol to decolorize the stained slide. Safranin
was the counterstain to colorize the gram–negative cell. One had to use the DI water to rinse every
stain between these procedures. It was very hard to decolorize the stains, because it may ... Show
Inoculated the "B12" on the blood agar plate, mannitol salt agar(MSA), phenol red mannitol/glucose
broths, nutrient gelatin tube, MRVP broth, and urea agar. And then, placed everything in the "to be
incubated" area. Blood agar plate helped to detect the hemolytic ability. MSA was for the salt
tolerance and mannitol fermentation. Phenol red broths were for the mannitol and glucose
fermentation. Nutrient gelatin tube was the test for the presence of gelatinase. Gelatin melt at 28°C;
therefore, it incubated at 25°C for a week. MRVP broth incubated for 48 hours at 37°C to test for the
capability of performing a mixed acid fermentation. Furthermore, it determined the ability of
glucose fermentation. Urea test was for the presence of the

Lab Report : The Gram Stain
Lab Report 1–The Gram Stain
Eric Zuberi
Lab section 1
February 8, 2015
This report represents my individual effort. I did not receive or offer aid to anyone when performing
this assignment, nor did I plagiarize any material.
Signed: _____________________________________________ Eric Zuberi I. Introduction
In all areas of biology, it is easy to see that structure is related to function. This statement holds true
in microbiology as well, the study of microorganisms, including bacteria. One characterizing feature
of bacteria is the cell wall, which can generally (although not in all situations) be categorized into
one of two categories: either Gram positive or Gram negative. Gram positive bacteria's cell walls are
composed of a large peptidoglycan layer (up to 90% of their cell wall). Within this large
peptidoglycan layer, one can find techoic acids, which contribute to the maintenance of cell wall
structure, and lipotechoic acids, which attach to membrane lipids. Gram positive bacteria that act as
pathogens can also potentially release exotoxins, which can have very dangerous effects on humans.
Gram negative bacteria, on the other hand, have a very small layer of peptidoglycan in their cell
wall, which is surrounded by an outer membrane. Within the outer membrane, one can find the
lipopolysaccharide layer, which is one of the most distinguishing factors of Gram–negative bacteria.
It is important to note that Gram negative bacteria fail to possess techoic

Gram Stain Lab Report
Introduction
The Gram stain is a technique used to differentiate between two major groups of bacteria based on
the composition of their cell wall. It was developed by Hans Gram in 1882. There are three
primordia steps. The cells are first stained with crystal violet (followed by a mordant), then they are
decolorized with 95% alcohol, and finally counterstained with safranin. Gram positive cells have a
thick peptidoglycan layer that entraps the crystal violet and prevents decolorization. Gram–negative
cells have a thin peptidoglycan layer and the crystal violet is readily removed during the
decolorizing step. When safranin is added gram–negative take up the stain, but not Gram positive
cells since they have already taken up the crystal violet. ... Show more content on Helpwriting.net ...
This media contains peptone, glucose, and a phosphate buffer. The MR test detects organisms that
metabolize through mixed–acid fermentation pathway. The metabolic activities of such organisms
lowers the pH. Addition of the pH indicator Methyl red after 48 hours yields a red color is the acids
from mixed–acid fermentation are present. MR stays red for PH below 4.4, and turns yellow at a pH
6.2 and above.
The Vogues–Porskauer test is to detect organisms that ferment glucose, but convert the product to
2,3–butanediol. The VP test detects the precursor for 2,3–butanediol, acetoin. For this purpose,
Reagents A (a–naphtol) and B (potassium hydroxide), are added after an incubation period of 48
hours. A red result indicates a positive result for the production of acetoin, and therefore, 2,3–
butanediol.
The citrate test is a differential test that employs Simmons citrate agar ( a defined medium), to test
the ability of a microorganisms to use sodium citrate as its sole carbon source, and ammonium as its
sole nitrogen source. Only bacteria that possesses the enzyme citrate permease will be able to
survive and grow in this media.

Gram Staining
In the medical field, certain bacteria need specific antibiotic treatments and to find out what class
the bacteria belongs to (whether if it is gram negative or positive) the specimen would have to
undergo differential (Gram) staining. Hans Christian Gram was the Danish bacteriologist behind the
gram staining technique. His Gram stain technique led to the discovery that Typhus bacillus did not
retain the stain. Another procedure like differential staining is called simple staining. The main
difference with this procedure is its simplicity and use of just one dye while the differential staining
is more complex, uses more staining and helps shows cellular components of the bacteria. There are
3 bacteria that will be in use for the Gram staining. ... Show more content on Helpwriting.net ...
Their cell walls usually contain mycolic acid which does not stain well with conventional staining
methods. So, for acid fast staining dyes that react strongly to the mycolic acid are used like the
Carbolfuchsin. Carla Lamanna described Carbolfuchsin as, "a mixture including phenol, water, and
a dye (basic fucshin) much less soluble in water than in phenol" (Lamanna, 1946). This part of the
lab is very important because certain bacteria families like mycobacteria have that mycolic cell
walls and can cause serious illness. Also, bacteria from the mycobacteria family require certain type
of antibiotics and a specific dosage to combat it. Staining in general has been the backbone of
fighting against infectious bacteria. Acid fast staining is also referred to as Ziehl–Neelsen stain.
They are the founder of the acid stain and are German doctors. They are Friedrich Neelsen and
Franz Ziehl. The lab we are doing, with regards to the acid–fast staining, is in similarity with the
experiment these doctors did to identify acid fast organisms. The Mycobacterium in this experiment
is expected to have a pinkish reddish stain while having a rod type

Gram Stain Essay
The Gram stain is the first step in identifying the unknown microbe on the blood agar plate sent in
from Khokana. It is important to do this first because the outcome of it directs the path of further
testing necessary for proper identification. Gazing upon the fixed and stained slide under the
microscope revealed it to be a series of circular shaped cells in a chain–like pattern. This, along with
the purple color, deemed it to be Gram–positive streptococci. The negative catalase test confirmed
the streptococci shape and arrangement seen under microscopy and indicated that a coagulase test
would be futile as that is a determinate for staphylococcus. The gamma hemolysis noted on the
blood agar plate narrowed the field down to two choices, it was either Streptococcus bovis or
Enterococcus faecalis. A positive bile esculin agar and salt tolerance test finally gave the unknown a
name, Enterococcus faecalis. Now the question became whether or not this could be the pathogen
that is responsible for causing the epidemic of diarrhea, bloody stool, and sepsis amongst the
villagers of the Khokana outbreak. E. faecalis was once thought to be ... Show more content on
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faecalis has a low pathogenicity (scilo) and is non–spore forming, it is a virulent, opportunistic
pathogen to be reckoned with and is thought of as a super–bug with its thick cell wall for protection,
ability to conjugate its resistance with fellow enterococcus cells, and its affinity for creating biofilms
(microbe wiki). It can also grow and adapt in many different environments. It can thrive in a wide
range of temperature with no regard as to whether salt or oxygen are present or not (hence the
positive salt tolerance test), or whether the pH is basic or acidic. Its ability to live and survive on dry
surfaces for up to 4 months at a time make it a perfect candidate to transmit within the hospital
setting, whether that be from person to person contact or via a contaminated surface or instrument
(Public Health Agency of

Gram, Gram And C. Gram

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