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.  

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.

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.



Postive result for Casein test

• Negative test for starch hydrolysis.



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

Test#2:  Methyl Red Test (Mixed Fermentation): determine the ability of bacteria to ferment glucose via mixed-acid fermentation.

  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

 Glucose                                                                                               Positive

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



Bubbles!

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.

Friday, May 24, 2013

Today, we prepared the hanging drop slide which is used to determine motility. To do this, we needed the following tools: depression slide, 5nm syringe, coverslip, vaseline, microscope, and broth culture of our bacteria. 

To begin, we put Vaseline on each of the four corners of the cover slip. Then we used the syringe to gather a very small amount of the bacteria to place onto the center of the cover slip. Then we put the cover slip onto the depression slide and placed it under the microscope with the oil immersion.

Our results were that our bacteria is motile. We were able to positively determine that because we actually saw our little bacterium swimming around.What an exhilarating discovery!

-Cecelia