Friday, May 31st.
The results of our last tests came in today. The EMB (Eosin Methylene Blue) agar plate was used to test for the presence of E.Coli. For a positive result the bacteria would produce a metallic green color, and since our bacteria did not produce such a result we determined that our bacteria was not E. Coli.
Albumin Test: There was no results because there was no discoloration between the holes which we would have thought would have occurred between the Bovine Albumin and the Goat Anti-horse Albumin. We therefore think that we had a bad batch of albumin.
Our Phenol-Ethyl agar plate that we prepared two days ago came back as a negative test. The phenolethyl agar differentiates between gram-positive and gram negative bacteria because only gram-positive bacteria grow on the plate. Our bacteria did not grow and therefore are gram-negative, as we have seen before in some of our other tests.
With all of our data collected from our tests we were able to determine that our bacteria is Serratia marcescents.
Saturday, June 1, 2013
Friday, May 31, 2013
The Eve of the Finale
Thursday, May 30, 2013
Immunodectection test: deals with antigens. Antigens
generate antibodies. Know it is a bacterial virus, take antibodies and they
will bind to the antigens on the virus and you will know you have a virus.
You have to look at the proteins. Proteins are different. Take hamburger
extract, then look for hamburger proteins. It will have: bovine albumin, goat
anti-horse albumin, goat anti-bovine albumin, goat anti-swine albumin.
In our bodies the outside of our cells are lined with a
specific set of proteins and carbohydrates that identify our cells as belonging
to our body. When the immune system
finds a foreign or harmful cell within the body it creates antibodies to
destroy the potential threat. When
injected with foreign cells all organisms with immune systems produce the same
kind of antibodies to fight against them.
This is good for humans because we can collect and purify antibodies
from other animals and use them to assist our own immune systems and to test
for what kind of cells may be in our food or other organic substance.
In this experiment we are going to test how different
anti-bodies will detect certain antigens (the special markers which cover the
outside and label cells) react in the agar.
We first create a set of holes in the agar and place certain
anti-body solutions within them.
1.
Bovine Albumin
2.
Goat Anti-horse Albumin
3.
Goat Anti-bovine Albumin
4.
Goat Anti-swine Albumin
The Anti-bodies are labeled “Goat” because these particular
anti-bodies came from goats. The term
“Anti-“ tells us what the antibodies are produced to find and mark. In the case of Anti-swine, it will create
antibodies to mark the antigens of swine cells.
When you inject the goat with anti-horse albumin, it will
produce antibodies against the horse albumin.
Same idea with Rh factor. Make an antigen to block the Rh
factor in pregnant mothers. The Antibodies
bind to the stray cells from the baby which have different proteins than the
mother’s cells, and then mark the cells with antigens so that the mother’s
antibodies will not find the cells, mark them as foreign and then create
anti-bodies against the baby. The
injected anti-bodies are only inserted into the mother’s bloodstream and not in
a high enough dose to actually harm the baby, where as if the mother’s immune
system were to find the baby’s different cells, it would mark them as foreign
and create antibodies to eradicate the foreign cells and therefore harm the
baby.
Inject a snake bite victim with horse anti-snake antibodies
because these will cover the snake venom antigens faster than the human’s
antibodies against the snake venom and therefore not allow the venom to hurt
the body.
Exercise 2: ANTIBODY-ANTIGEN REACTION IN AGAR
We took a petri dish and made four wells in it and filled
them with the following albumin: 1-bovine albumin, 2-goat anti-horse albumin,
3-goat anti-bovine albumin, and 4-goat anti-swine albumin. The function of this
experiment is to show you which antibodies will target which antigens.
Serum conversion - Searching for Antigens to determine
whether a person has been exposed to the pathogen. For example: a physician might not be able to
find HIV virus within the patient if it is latent. But if the patient has been exposed to HIV,
then the body will have produced antibodies for the virus which should still be
in the blood.
First add the purified antigen. This antigen will bind to antigens produced
by the immune system to fight the disease we are testing for (i.e. HIV). Next add a serum sample which represents the
blood sample extracted from the patient.
If the specific antigen of our disease is present within the serum, then
our antibodies cultivated to bind to the pathogen antigen will bind to the
pathogen antigen. Next add an enzyme. This enzyme will bind to the primary antibody
if it is connected to the pathogen. Then
enzyme will produce a specific color when it binds, indicating that the
pathogen is present.
We were given a mystery serum, which we are to test for the
presence of a specific pathogen antigen.
We will test it with a Positive control, a negative control, a secondary
antibody that has the enzyme attached and then the substrate.
Procedure:
1.
Add purified disease antigen to the wells of a
microplate strip. Inoculate for 5 minutes
to allow the proteins to bind to the plastic wells via hydrophobic
interaction. This is called an immunosorbent assay because proteins absorb or bind to the plastic wells.
2.
Wash out the wells to remove anything that has
not bound to the plastic walls of the wells.
3.
Add the serum sample and control samples to
wells and incubate. The Serum contains
millions of different types of antibodies, but only if your serum contains
antibodies that were produced in response to the disease will antibodies bind
to the antigen in the wells.
4.
Detect the serum or primary antibodies with
HRP-labeled secondary antibody. If serum
antibodies have bound to the antigen, the secondary antibodies will bind
tightly to the serum antibodies.
5.
Add enzyme substrate to the wells, wait 5
minutes and then evaluated the assay results.
If the primary antibody was present in your serum sample, the wells will
turn blue. This is a positive diagnosis. If the wells remain colorless, the primary
antibody was not present in your serum sample, and the diagnosis is negative.
Our sample had a negative diagnosis, indicating that the
primary antibody was not present in it.
Bacterial concoction
At the end of lab today, Dr. Pathakamuri took bacteria from
every person in micro lab and mixed them up in a beaker of water. He brought
out a UV radiation device that blasts bacteria with UV rays and kills them
after a certain amount of time. To show this, he took samples of before and
after radiation and spread them onto plates and incubated them. We will see
next time if the radiation really worked!
Yummy yogurt
Dr. Pathakamuri took regular milk, boiled it, and placed a
small amount of yogurt in it and incubated it. This was to demonstrate how a
live culture (in the yogurt) would colonize the milk and produce more yogurt.
Also, we had to examine samples and measure the zone of inhibition in the Kirby-Bauer test.
·
Penicillin: not sensitive
·
Neomycin: 20mm
·
Tetracycline: not sensitive
·
Erythromycin: sensitive, intermediate
·
Chloramphenicol: sensitive, intermediate
Staph aureus does not use mannitol.
Ours grew in the mannitol and no color change.
Nose swab: right now all negative. Maybe positive for
Elizabeth
On blood agar unknown bacteria: no hemolysis, therefore no
staph
Blood agar throat: not strep
Eosin Methylene Blue (EMB) Agar – to isolate and differentiate
gram-negative enteric bacilli. The EMB
agar plate contains eosin and methylene dyes and sugars lactose and
sucrose. The dyes will inhibit growth of
many gram-positive, which makes this a selective medium. EMB is also a differential medium that will
distinguish between the bacteria that ferment lactose and/or sugar. If bacteria ferment lactose and/or sucrose
then they will produce acid. The lower
pH will cause the dyes to precipitate on the colonies. If there is a significant amount of acid produced
then the bacteria will be a dark bluish color with a green-metallic sheen. Low acid production results in pink colored
colonies. No fermentation will result in
colorless colonies on the agar surface.
To prepare the petri dish we inoculated an EMB agar with our
bacteria and let it grow overnight in the incubator at 24 degrees Celsius.
Tuesday, May 28, 2013
Lab Tuesday, May 28, 2013
Tuesday, May 28, 2013
Howdy all. To start
off, we checked up on several tests.
 |
| Our TSI agar test shows an alkaline slant with an acid butt. This shows that only glucose was fermented. |
 |
| Our litmus milk reactions. |
Prepare tests:
Blood Agar Plate – to
isolate and support the growth of fastidious bacteria and to differentiate
among bacteria based on their ability to lyse red blood cells or
hemolysis. There are several types of
lysis: alpha-hemolysis (green zone), beta-hemolysis (complete lysis), and gamma
hemolysis. Group A, B, C, and D are important in Lancefield classified
streptococci.
MacConkey Agar Plate –
to determine if bacteria is gram-negative or positive bacteria by their ability
to grow on the medium and ferment lactose.
Phenylethyl Alcohol
(PEA) Agar - to isolate the
gram-positive bacteria from a sample of mixed negative and positive bacteria
Testing Antibacterial
Medicines: Kirby-Bauer Technique
To determine the
sensitivity of our mystery bacterium five different anti-bacterial
medications. Paper discs with each of
the different anti-bacterial medicines are evenly spaced on an inoculated nutrient
plate. During incubation, the medicine
will diffuse out from the discs and create a bacteria-free circle around the
disc where the medicine has had an effect.
The sensitivity of the bacteria to each medicine is determined by the
size of the ring around the disc called a zone
of growth inhibition.
Anti-bacteria:
1.
Tetracycline: inhibit protein synthesis, tRNA
2.
Erythromycin: inhibit protein synthesis,
ribosomes
3.
Penicillin: break down cell wall
4.
Neomycin
5.
Chloramphenical
Procedure:
Take bacteria and
inoculate whole plate – swab easier to spread
Then take forceps and
dip in alcohol– light on fire, let flame die.
After cool use forceps to transfer antibacterial discs to inoculated
plate. Place each disc in a different
spot: one in middle and four in equal quadrants around the middle.
Testing for streptococcus and staphylococcus:
Throat swab is to get the tonsils and to
test for streptopyrogenes. You use the blood agar plate for this sample. The
nose swab is to test for MRSA. A wet swab (dipped in saline solution) is used
for the nose swab. You use the mannitol salt agar when testing for staph in the
nose.
 |
| Miss Hana gets her throat tested. |
 |
| Add the throat sample to the blood agar plate. |
 |
| Hana expertly collects bacteria. |
Monday, May 27, 2013
Lab from Wednesday, Thursday, and Friday. May 22-24, 2013
May 22-23 , 2013
In the past couple of days we have
learned quite a bit about our very own bacteria. Check it out!
- Our bacteria is aerobic: the new sample of bacteria in the broth culture formed near the top; therefore that means it is aerobic.
- Our bacteria can digest fat.
Postive result for othe Glycoside test - Our bacteria can excrete caseinase which is the enzyme used to digest casein molecules.
- Negative test for starch hydrolysis.
 |
| Postive result for Casein test |
 |
| Before... |
 |
| After Iodine. Negative result. |
- Need more time incubating to determine if there is a positive reaction for the litmus milk reaction.
- Our bacteria is motile.
- At this point, it appears as if our bacteria has a positive gelatin test, but we are going to wait a couple more days to see what happens.
 |
| Still liquid even after a 15 min. chill in the fridge indicates a positive result of the gelatin test |
Test# 1: Fermentation of Carbohydrates (Burham Tube) to test
for Sugars: determine ability of some bacteria to ferment a particular
carbohydrate
Heterotrophic bacteria often use sugars to obtain energy by
fermentation pathways. Organic acids,
alcohols, and gases accumulate as waste products. These waste products will vary depending on the
specific bacteria. In this test, acid
production is identified by a change in the color of the pH indicator, phenol red,
which is included in the medium. In the
presence of acid the medium will turn yellow.
In order to collect the gases, an inverted smaller tube, called a Durham
tube, is placed in the medium.
To prepare test: A tube of phenol red containing sugar broth
and a Durham tube is inoculated with our specific mystery bacteria using
aseptic technique. The test tube is then
incubated for 24 hrs. If the media turns
yellow that will indicate a positive result for acid production and
fermentation. And if a gas bubble is
trapped in the Burham tube, that is a positive result for gas production. This will indicate that our bacteria is
capable of fermenting digested carbohydrates.
 |
| Results of glucose tests: positive for sucrose |
The products of
mixed-acid fermentation will include significant amounts of organic acids. These acids lower the pH of medium to
<5. The pH indicator methyl red is
added to the medium and will remain red as long as the pH is 4.5 and below. This indicates a positive test. At higher pH values or when less acid is
present (indicating that the bacteria did not ferment the glucose) the color ma
change to orange or yellow and therefore denote a negative result.
To prepare the test: Our bacteria is inoculated into a tube
of MR-VP broth and incubated for 48 hrs.
 |
| Variable result for test - more pinkish color |
Test#3: Voges-Proskauer Test (Butanediol Fermentation):
Determine the ability of our bacteria to ferment glucose via butanediol
fermentation. If the bacteria does use
butanediol fermentation, then Voges-Proskauer (VP) agents react with acetoin (a
precursor for neutral alcohol 2,3 butanediol) in the presence of oxygen to form
a red product.
To prepare the
test: Inoculate a MR-VP tube with our
bacteria using aseptic technique and allow to incubate for 48 hrs.
 |
| Adding acetoin to the test tube |
Test#4: Citrate Utilization Test: determine if bacteria can
utilize citrate as its sole source of carbon and energy. In order for bacteria to be able to do this
it must have the membrane –associated transporter citrate permease. These cellular enzymes convert citrate in the
cell’s cytoplasm into pyruvate and carbon dioxide. The pyruvate is used for energy and the
Carbon dioxide can combine with NH4+(which is provided in the medium) to produce NaCO3,
an alkaline compound. This compound will
change the pH which will be detected by the pH indicator bromothymol blue. The color of the medium will change from green
to blue and denote a positive test.
 |
| Citrate test |
Test#5: Indole (Tryptophan Degradation) Test: determine the
ability of bacteria to split amino acid tryptophan into indole and pyruvic
acid. Tryptophan can be used as an
energy source by degrading the amino acid into pyruvate. Indole is a byproduct not used by the
bacteria.
 |
| Indole test - negative |
Test#6: Nitrate Test: determine if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. Basically there are two reactions. The first is where the nitrate ion is reduced by the enzyme nitrate reductase into nitrite ions. The second is where the nitrate is reduced completely into molecular nitrogen in the process called denitrification.
Test#7: Urease test: determine the ability of a bacterium to hydrolyze urea. Urease-producing bacteria grow in a medium containing urea, accumulates ammonia, and changes the medium to be more alkaline. The pH change is measured with phenol red which will turn to a bright pink if the pH is alkaline.
Test#8: Motility test: determine if a bacterium is motile. The presence of a flagellum are what causes the bacteria to move around. When motile bacteria is stabbed with an inoculating needle into the semisolid agar, the bacteria will swim away from the stab, showing a cloud of growth. Another way to test for motility is to prepare the hanging drop slide.
Test#9: TSI agar test: differentiate among the gram-negative enteric bacilli as to their ability to ferment glucose, lactose, and sucrose and to produce H2S. If a bacterium ferments any of the sugars present in the TSI agar (lactose, sucrose, glucose) the resulting acids cause the pH to drop.
So far, we
have performed these experiments on our bacteria and watched for the reactions to
occur. The following list is incomplete but is slowly but surely directing us
onward in our discovery. This has been quite an adventure for every one of us,
and we each eagerly anticipate discovering just what kind of bacteria we have!
TESTS RESULTS
Lactose Negative
all
Sucrose Positive
Mannitol Positive
(gas)
Gelatin liquefaction Positive
Starch Negative
Casein Positive
Fat Negative
Indole Negative
Methyl Red Variable
(pink)
Voges-proskair Negative
Citrate utilization Positive
Nitrate reduction Positive
Urease Negative
Another Aerobic test is the Hydrogen-peroxide test. Organisms that breath oxygen can break down Hydrogren-peroxide into Hydrogen and Water. The visual observation is a bubbling of the liquid as the oxygen is released. Anaerobic bacteria are unable to convernt toxic Hydrogen-peroxide and therefore die when treated with Hydrogen-peroxide, which makes this substance a good way to disinfect a live or inaminate area of anaerobic bacteria. Our bacteria is once again an aerobe because of the bubbling result we observed.
 |
| Bubbles! |
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