Lab 8 Instructions: See Lab Manual

Lab 8 Instructions: See lab manual

SOIL PROJECT RESULTS

1)After the lab tech emails me the DNA sequence, I will email it to you. Then go to the BLAST website and enter the DNA code I sent you to find out what genus your organism is. Also look at the list of possible species they give you. Blast Instructions are posted on my lab unit 2 website.

2)Use Bergey’s Manual to compare the possible species with your physiological tests that you ran. See if you can determine the species.

UNKNOWN ORGANISM PROJECT

Check the results of your cultures last week (see below for instructions) and compare all of the results to the unknown organism chart to see which organism you had. Not all of your test results will match all of the characteristics on the chart, so guess your organism name by the one that matches most closely.

Chapter 13

Starch agar plate (add iodine; all black with no clear zones is negative)

OF-glucose deeps with and without mineral oil (yellow is positive)

Controls

E. coli (O/F, G)

Pseudomonas aeruginosa (O)

Chapter 15

Urea tubes (pink is positive, yellow is negative)

Gelatin tubes (liquid is positive). Place the tube in ice for 5 minutes to be sure it can stay liquid.

Controls

Pseudomonas aeruginosa (+)

Chapter 16

Phenylalanine slants (add 5 drops of 10% ferric chloride; green is positive)

SIM media (Black is positive for H2S, add 5 drops of Kovac’s reagent; red ring is positive for indole)

Controls

E. coli (H2S and indole +)

Enterobacter aerogenes(H2S and indole neg)

Examine colors of ornithine, arginine, and lysine decarboxylase broths (yellow is positive for protein catabolism, purple is negative)

Chapter 51: Enterotubes

Use the form on the next page to record whether each well was positive or negative. If the first well was yellow, that is positive for glucose, so go to the bottom of the form (see below image) and circle the number “2” under glucose. If that same well has a large gas bubble in it, circle the number “1” for gas. Continue to circle the numbers at the bottom of the form for all test results that were positive. Skip well #4’s indole test until you record the rest of the results. Then use a toothpick to poke a hole in well #4 and add a drop of Kovak’s reagent. If it turns red, it is positive. Dispose of the tube in the biohazard bucket, being careful not to tip it or the Kovak’s reagent will leak out. Now go back to your results form below.

Add all of your circled numbers (positive results) together, but just the ones that correspond to the shaded empty boxes below. The first box is the total from well #1. The second box is the total from wells #2, 3, and half of #4. The third box is the total from half of #4, and #5, 6. Ignore the VP result…no number there. Continue until you have a 5-digit code number written on the bottom of your form. Check the manual to see which organism you have. Sometimes the book says you need the VP result (+ or -) to determine species. Page 3 of this document shows you which numbers go into which box.

To perform the indole test: Add 3 - 4 drops of Kovacs‘ reagent to the H2S/indole compartment.

Allow reagent tocontact the surface of the medium. A positive test is indicated by development of a red color in theadded reagent within 10 sec.

To perform the Voges-Proskauer test:

Add 12 drops ofBarrits’sA reagent (alpha napthol, a carcinogen!) and 3 drops of Barrett’s B reagent(potassium hydroxide; KOH, a very caustic base, found in draino). Put the test tube cap back on and shake. TAKE OFF CAP and allow to stand exposed to air for 15 minutes. A rust color (brownish red) is positive.

ELISA TEST

Background information

Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of a particular antigen in a sample. If the particular antigen is present, we know that the substance we are looking for is also present. An unknown amount of antigen (such as HIV) or antibody (such as a person with autoimmune antibodies) is affixed to a surface, and then a secondary antibody is applied over the surface so that it can bind to the antigen (or to the primary antibody). Since this reaction will not cause any color change, we then link the secondary antibody to an enzyme called horseradish peroxidase, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

ELISA tests could also be used on an antibody instead of the antigen. In this case, there will be two sets of antibodies, so we call them primary and secondary antibodies. The primary antibodies will be attached to the plastic plate, and then the secondary antibodies will attach to the primary antibodies. The secondary antibodies will then be conjugated to the enzyme, horseradish peroxidase, which will create a color change when a substrate is added. An ELISA test can tell us whether or not particular antigens or antibodies are present in the sample (so it is qualitative). However, we cannot measure how many antigens or antibodies are present (which would be quantitative) unless we perform a serial dilution.

For this exercise, you will receive two fluid samples that you pretend are from two patients. One sample is positive for an antigen (we are pretending it is positive for HIV). The other sample is negative. We are not really working with HIV antigens; it is just a non-pathogenic antigen that will turn color if positive.

Summary of the Procedure

Once your group has added their fluids to their assigned wells, let them incubate for 5 minutes and wash it with a buffer that will wash out any unbound antigen. Then put in a primary antibody (since we are pretending the antigen is HIV, the primary antibody would have to be an anti-HIV antibody). Let it incubate for 5 minutes to allow it to bind to the antigen if the antigen is present. Wash again with buffer, which will wash away any unbound antigen. Then add a secondary antibody. These are anti-human antibodies; antibodies against human antibodies. These secondary antibodies also have a horseradish peroxidase (HRP) enzyme attached to them. Allow the tray to incubate another 5 minutes, then wash with the buffer. Now add the substrate, which will bind to the HRP if HRP is present. When HRP comes in contact with the substrate, the color changes to light brown (substrate 1) or green (substrate 2). If the color appears, it means that the substrate found HRP to bind to. If HRP is present, the secondary antibodies must be present. If the secondary antibodies are present, that means the primary antibodies are present. If the primary antibodies are present, that means the antigen is present, so a color change is positive for the antigen (which we are pretending is HIV).

Labeling the Microtiter Plate:

1)Place the Microtiter plate vertically

2)Mark the plate with your lab group number at the bottom of the

plate and circle your group number so you do not confuse it

with a row number.

3)Use a Sharpie pen to label the plate with row numbers 1-4

4)Each row has three wells so you are performing each test in triplicate

to help eliminate experimental errors.

5)Pay attention to each line of instructions, and be careful!

Instructions: This experiment takes about 90 minutes from start to clean-up

1)Pick up these supplies:

• Microtube rack (place the next 3 tubes in this rack)
• HIV tube
• Ab-1 tube
• Ab-2 tube
• Regular test tube rack
• PBS tube for buffer solution (put in regular test tube rack)
• 10 disposable plastic pipettes
• Small waste beaker

2)Use a Sharpie pen to label the 10 transfer pipettes as follows:

3)Add 3 drops of HIV to rows 2, 3, and 4 (NOT to row 1).

4)Incubate at room temperature for 5 minutes.

5)Remove the liquid with the HIV pipette, dispose of it into the waste beaker.

6)Add PBS solution, filling to the top of each well in rows 2, 3, and 4.

7)Remove the PBS solution, using the properly labeled ROW pipette for each row.

8)Add 3 drops of 1Ab to rows 1, 2, and 4 (NOT to row 3).

9)Incubate at room temperature for 5 minutes.

10)Remove the liquid with the 1Ab pipette, dispose of it into the waste beaker.

11)Add PBS solution, filling to the top of each well in rows 1, 2, and 4.

12)Remove the PBS solution, using the properly labeled ROW pipette for each row.

13)Add 3 drops of 2Ab to all rows

14)Place the rack in the incubator at 37 degrees for 5 minutes.

15)Remove the liquid with the 2Ab pipette, dispose of it into the waste beaker.

16)Add PBS solution, filling to the top of each well in all rows.

17)Remove the PBS solution, using the properly labeled ROW pipette for each row.

18)Add 5 drops of Sub 1 to rows 1 and 2.

19)Add 5 drops of Sub 2 to rows 3 and 4.

20)Place the rack in the incubator at 37 degrees for 5 minutes.

21)Check the colors.

• All the wells in row 1 should be colorless (negative control). Why? It does not have HIV
• All the wells in row 2 should have a light brown color (positive, using the first substrate).
• All the wells in row 3 should be colorless….why? It had the HIV, but it was missing the 1 Ab. It might turn light green if sitting out for a while.
• All the wells in row 4 should have a green color (positive control, using the second substrate).

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