Microbial Detection Methods

Question 1

Direct Samples

Answer 1

Collected from Normally Sterile tissues and bodily fluids.
–Highest Quality samples
–May carry some risks
–Methods include Needle Aspirations and Surgical Biopsy
–The results are always useful:
Positive findings are Diagnostic.
Negative findings can exclude infection.

Question 2

Indirect Samples

Answer 2

Specimens of inflammatory exudates:

• -Expectorated sputum
• -Voided urine

Site of origin is Usually Sterile

Assessment of the Normal Flora

Convenient, but carry the risk of Misinterpretation.

Question 3

Samples from Normal Flora Sites

Answer 3

Primary infection is commonly in sites known to be colonized with many orgs (e.g. pharynx, large intestine)

Exams are selectively known to cause infection that are not normally found at the site:

• -Salmonella, Shigella, Campylobacter (stool specimens)
• -Beta-hemolytic Streptococci (throat)

Question 4

Direct Examination by Microscopy

Answer 4

Useful for Larger organisms:

• -Bacteria,
• -Fungi

Standard optical or light microscope

Bright-field, Dark-field, Fluorescence (UV light).

Question 5

Electron Microscopy

Answer 5

Greater resolution than light microscopes.

Using negative staining techniques, direct examination of fluids and tissues to view VIRUSES

Question 6

Acid-Fast Stain

Answer 6

Stain: Carbol fuchsin
Counter stain: Methylene blue
--Red cells are Acid-Fast
--Blue cells are Non-acid-fast
Example: Mycobacterium tuberculosis

Question 7

Stab Culture

Answer 7

Some bacteria grow inside the culture nutrients: stab culture.

Test tube filled with agar and nutrients. Sterile wire is dipped into sample and stabbed into tube.

Example: Legionella pneumophila (Legionnaire’s)

Question 8

Culturing a Clinical Specimen

Answer 8

A clinical specimen is cultured for microbes known to thrive in a particular environment and associated with certain clinical symptoms.

Fecal Samples (Diarrheas):
Enteric pathogenic bacteria
--Salmonella
--Shigella
--Campylobacter
--Yersinia
--E. coli O157:H7
--Vibrio

Respiratory Samples:

• -Streptococcus pneumoniae
• -Bordetella pertussis
• -Haemophilus influenzae
• -Legionella
• -Mycobacterium

Cervical, Vaginal, Penile:

• -Neisseria gonorrhoeae
• -Herpes

Question 9

Virus Culture

Answer 9

Viruses need living cells to reproduce, so often grown in tissue culture (derived from growing cells or tissues)

May be tested by nucleic acid-based methods or viewed under Electron microscope.

Question 10

Seroconversion

Answer 10

A particular Antibody has a low Acute Titer during the acute phase of the infection, but then has a higher titer later and can be detected (convalescent titer).

This indicates infection.

4-fold rise in titer also indicates recent infection.

Question 11

Serology

Answer 11

Compare blood samples.
Evaluates antibodies.

May be useful:

• -when pathogen is not easily detected in other types of samples
• -when source of exposure has been eliminated with no remaining sample to test

Limitations:

• -not useful for a rapid intervention
• -often difficult to obtain a blood sample, even once, let alone twice.

Question 12

Hemagglutination and Hemagglutination Inhibition

Answer 12

Virus Assay

Hemagglutination:
–Highly concentrated viral particles causes RBC to form an extensive network with the viral particles binding to the RBCs. (Appears Red Throughout)
(b/c prevents the RBCs from sinking to the bottom)
–Dilution of patient serum (dilution of virus particles) causes there to be no hemagglutination and so the RBCs form a “Dot” b/c sedimentation by gravity.

Hemagglutination Inhibition:
–Tests for Antibody from the infected patient that inhibits hemagglutination.
–High concentration of specific Antibodies neutralizes the Virus, inhibiting Hemagglutination.
(Appears as Dot)
–Dilution of patient serum (dilution of Anti-Virus Antibodies) allows Virus to Hemagglutinate the RBCs
(Appears Red throughout)

Question 13

Virus Neutralization Assay

Answer 13

Control Tube: Cytopathic effect occurs (Host Cell Lysis by the Virus)

Antibody that is effective will prevent CPE: tube will show living host cells

Antibody that is ineffective will not inhibit CPE: tube will show CPE, like the Control.

Question 14

Complement Fixation Test

Answer 14

Reactive:

1) Serum with Antibodies
2) Add the Specific Antigen, which then binds Antibodies
3) Complement is added to bind Antigen-Antibody Complexes
4) Sensitized RBCs added, but there is no surplus complement
5) Result: Intact RBCs settle in pellet

Reactive prevents RBC lysis because there is no extra complement around to lyse the RBCs.
–This test is used to detect and quantitate antibody.

Nonreactive:

1) Serum but no antibodies
2) Free antigen added
3) Added Complement remains Unbound
4) Free Complment binds Sensitized RBCs
5) Result: RBCs Lysis

Question 15

Enzyme-Linked Immunosorbent Assay (ELISA)

Answer 15

1) Bind Target Molecule sample to the support plate (usually plastic or a membrane)
2) Add Primary Antibody; wash
3) Add Secondary Antibody-Enzyme conjugate; wash
4) Add Substrate

Substrate is initially colorless. The enzyme converts it to a *Colored product, able to be visualized.

Question 16

Direct vs. Indirect ELISA

Answer 16

Difference = What is bound to the plate

Direct ELISA:

1) Antibody is bound to the plate.
2) Test Antigen is Added and binds to Antibody.
3) Enzyme-linked Antibody specific for test antigen then binds to Antigen, forming a Double Antibody Sandwich.
4) Enzyme’s Substrate is added; reaction produces a visible color change.

Indirect ELISA:

1) Antigen is bound to plate
2) Test Antibody is added and binds to Antigen.
3) Enzyme-linked Anti-Antibody binds to Bound Antibody.
4) Enzyme’s Substrate is added; reaction produces a visible color change.

Question 17

Immunofluorescence

Answer 17

Direct:

1) Antigen is fixed to plate.
2) Fluorescent-labeled Antibody is added and binds to Antigen.

Indirect:

1) Antigen is fixed to plate.
2) Primary Antibody is added and binds to Antigen.
3) Fluorescent-labeled Anti-Antibody is added and binds to Primary Antibody.

Examples: CMV, HSV

Question 18

Western Blot

Answer 18

Gold standard for HIV-1.

Seropositivity is diagnosed when Antibodies against both the **Env and the **Gag proteins are detected.

1) Add target protein Antigen.
2) Primary Antibody from patient’s serum binds to Target protein Antigen.
3) Enzyme-linked Secondary Antibody binds to Primary Antibody
4) Enzyme’s Substrate is added and color change to view.

Question 19

DNA Probe Hybridization

Answer 19

1) A single-stranded (denatured) target Nucleic acid is bound to a membrane plate.
2) A DNA probe with attached Enzyme that is complementary to the Target DNA binds and forms a Double-stranded Hybrid.
3) A colorless Substrate is added, which is converted by the enzyme to a colored substrate.

Question 20

Agarose Gel Electrophoresis

Answer 20

DNA can be visualized on gels.

Separated in an electrophoretic field in an agarose gel.

Migration depends on size.

With a known gene, you know how big the sequence is.

When sample DNA is seen on a gel, it can be determined whether the gene is present and whether it has the correct length segment and is the expected organism.

Question 21

DNA Fingerprinting

Answer 21

DNA Fingerprint is the pattern of DNA as it appears on a gel.

Fragments are created after Digestion with Restriction Endonucleases.

DNA Fingerprinting is done when a specific organism is suspected in order to determine which strain of the organism is present.

Can do this with chromosomal DNA or with Plasmid DNA.

Question 22

Polymerase Chain Reaction (PCR)

Answer 22

To identify which strain is present in a specimen.

Allows for the identification of microbes in tissues that cannot be cultured.

Makes multiple copies of a piece of DNA that is unique to the suspected microbe via Amplification.

Amplification makes it easier to detect an organism’s DNA.

PCR can start with very small amounts of DNA.

Start with a sample of DNA from a clinical specimen.

1) Primer is added to sample. (The Primer is what provides the Specificity for this test.)
2) Polymerase enzyme will read a sequence of DNA and will create only copies of the DNA that matches the primer
3) Building Blocks (nucleotides) of DNA bases to make copies.

Results:
–Amplification Occurs:
DNA in the specimen matched the Primer

–No Amplification Occurs:
Particular DNA that primer was designed to was not present.
(You probably ran a non-specific PCR rxn and just amplified whatever genetic material was present.)
(–Next step would be to **Sequence the DNA with the genetic material obtained from Amplification.
–This particular sequence can then be compared with known sequences of an organism or strain.)

Question 23

Ribotyping

Answer 23

Only used to identify **BACTERIA not viruses.

(Viruses don’t have ribosomes!)

It’s the same steps of digestion with specific restriction enzymes and putting the fragments in gel electrophoresis.
–This is specifically for the genes encoding the Bacterial rRNA (which is part of the bacterial ribosome).