Microbial Diagnosis Lecture Sep 12

Question 1

What is the first important step in diagnosis?

Answer 1

an accurate history and exam

Question 2

What are some stains you can use for direct microscopic examination of clinical specimens?

Answer 2

gram stain

acid fast

india ink in the CSF for cryptococcus neoformans

KOH treatment for fungal forms

Fluorescent antibodies

Question 3

What exactly is stained for in diptheria infections?

Answer 3

you stain to see the specific storage granules

Question 4

What does fluoescent microscopy utilize?

Answer 4

Fluorescent microscopy uses fluoescently-tagged antibodies against specific antigens or or antibodies so they can be visualized

Question 5

What does a direct fluoescent antibody test look for?

What does an indirect fluorescent antibody test look for?

Answer 5

Direct: looks for antigens

Indirect: looks for antibodies

Question 6

What are five tests once can use for identification of serum antibodies?

Answer 6

1. complement fixation
2. Hemagglutination and hemagglutination inhibition
3. Latex agglutinatoin
4. ELISA
5. Western blot

Question 7

What can’t serum antibody ID test differentiate between?

Answer 7

past and present infections.

Question 8

How does complement fixation work?

Answer 8

1. A fixed amount of antigen is added to test serum
2. If antibody is present in the serum, immune complexes will form
3. Complement is added to the mix. If the complexes are present, they will fix the complement and sequester it.
4. RBCs and subagglutinating amount of anti-erythrocyte antibody are added.
5. If the complement has not been bound by the complexes, anti-erythrocyte antibody plus complement will lyse the cells.
6. If the complement has been bound by the immune complexes, there will be insufficiency complement to lyse red cells.

So the test is positive if the RBCs survive.

Question 9

How does hemagglutination inhibition for antibody detection work?

Answer 9

1. You add antigen to serum.
2. If antibody is present, it will bind to the antigen and form an Ab-An complex
3. If the antibody is not present, you won’t get any complexes
4. THen you add the RBCs
5. If the antibodies are bound up in complexes, there will be no hemagglutination and the test is positive
6. If the antibodies are not sequestering the antigens, the antigens will cause hemagglutination and the test is negative.

Question 10

How does a western blot work?

Answer 10

Western blots use antibodies to determine what viral proteins are in the serum

1. You run the serum along a gel, which splits up the proteins based on size
2. you transfer this onto a nylon membrane
3. you use preformed, tagged antibodies that are specific to each different viral proteins
4. If the proteins are present, you’ll be able to see the bands in the appropriate places

Question 11

What’s used to detect microbrial proteins?

What’s used to detect microbial DNA?

What’s used to detect microbial RNA?

Answer 11

proteins: Western blot and ELISA

DNA: Southern blots

RNA: Northern blots

SNOW

DROP

Question 12

What is a PCR used for?

Answer 12

It’s used to amplify DNA sequences in clinical specimens

1. If you know the sequence of what your’e looking for, you can design primers to it.
2. You heat the serum to denature the DNA strand
3. You add primer, nucleotides, and tag polymerase to the serum
4. You cool down the serum so the primers will anneal to their assigned sequences
5. You heat it back up so the polymerase will work faster and synthesize the new strand
6. Then you repeat the steps until you hav sufficiency amplified the sequence you’re looking for
7. Then you run it in a gel and use a tagged antibody and see if you have a band where you expect the DNA sequence to be.