Unit 10 - Diagnosis of Infections

Question 1

List some non-culture based methods

Answer 1

• Microscopy (staining techniques)
• Immunodetection
• Molecular diagnostics

Question 2

What are culture based methods

Answer 2

where we grow organisms in the lab

Question 3

What are the goals of a clinical microbiology laboratory?

Answer 3

• Identification of the microorganism in the patient specimen involved in the disease process
• Detection of microorganisms or their products in specimens collected from the patient
• Detection of the patient’s immune response to infection
• Provide antimicrobial susceptibility of the isolated microorganism

Question 4

Identification of microorganisms by _____ and ______

Answer 4

isolation and culture

*This determines presence/absence or number of microorganisms and susceptibility

Question 5

Identification of a specific microbial gene or product helps determine ?

Answer 5

Susceptibility? Presence of a resistance gene

Question 6

Detection of specific antibodies to a pathogen determines ?

Answer 6

• Pathogens that cannot be cultivated
• High risk group pathogens
• Retrospective diagnosis

Question 7

When processing specimens, we need to determine if the site is ..?

Answer 7

sterile or a site with commensal flora present

Question 8

Specimen processing can either be ______ or ______

Answer 8

culture based
or
non-culture based

Question 9

Describe culture based specimen processing

Answer 9

• > 18 hours
• slow growing organisms
• blood culture
• culturable

Question 10

Describe non-culture methods

Answer 10

Advantages:

• fast
• less labor intensive
• suitable for organisms that cannot be cultured in the lab
• Microscopy
• Immunodiagnostics
• Molecular diagnostics

Question 11

What are the two branches of microscopy?

Answer 11

• light microscopy

- electron microscopy

Question 12

Bright field microscopy uses a ___ ____

Answer 12

gram stain

Question 13

A gram stain shows you ______

Answer 13

morphology

as well as gram positive or gram negative

Question 14

Briefly describe the gram stain process

Answer 14

• Stain cells with crystal violet (purple)
• Add iodine which makes the stain stick
• Wash with ethanol
• Crystal violet will be lost only from cells that have thin peptidoglycan (gram negative)
• Stain with safranin (pink)

Purple = gram positive
Pink = gram negative

Question 15

Describe acid fast light microscopy

Answer 15

mycolic acid cells are easy to stain but very hard to remove the stain ?

you determine which ones are acid fast by green vs pink

Question 16

Light microscopy can also show ______

Answer 16

macromolecules

Question 17

______ staining is another form of light microscopy

Answer 17

Fluorescent

Question 18

Describe fluorescent staining

Answer 18

• naturally fluorescent

- stained with fluorescent dyes

Question 19

Describe electron microscopy

Answer 19

• Resolution = 0.1 - 1.0 nm
• Electron beam instead of light
• Magnets instead of lenses
• Thin specimen
• SEM vs TEM
• Viruses
• Not routinely used in clinical labs

Question 20

Describe immunodetection

Answer 20

• A lot more rapid than culturing
• Specific Ab coated onto latex bead -> visible clumping
• Can cause false-positives

Question 21

Common causes of bacterial meningitis?

Answer 21

• Streptococcus pneumoniae
• Haemophilus influenzae
• Neisseria meningiditis in CSF

Question 22

How do we quantify Ag/Ab?

Answer 22

ELISA

enzyme-linked immunosorbent assay

Question 23

Describe ELISA for an antigen

Answer 23

• Bind antibody to solid phase
• WASH
• Add unknown antigen
• WASH
• Detect bound antigen with labeled 2nd antibody
• WASH
• Measure label

Question 24

Describe ELISA for an antibody

Answer 24

• Bind antigen to solid phase
• WASH
• Add unknown antibody
• WASH
• Detect bound antibody with labeled anti-antibody
• WASH
• Measure label

Question 25

Describe how monoclonal antibodies are produced using tumor cells injected into a mouse

Answer 25

1) Tumor cells are injected into a mouse to stimulate production of B cells, which produce different types of antitumor antibodies 2) Immortalized myeloma cells are collected 3) The B cells are fused with the myeloma cells to produce immortalized, antibody-producing hybrid cells 4) The hybrid cell that produced the needed antibody is selected and cloned to produce unlimited quantities of a monoclonal antibody

Question 26

What 2 types of methods are available for detection of specific genes?

Answer 26

1) probe-based methods - labelled, single-stranded nucleic acid fragment to hybridize with the target DNA 2) amplification-based methods

Question 27

Describe probe-based methods

Answer 27

- Hydrogen bond formation between bases - Need a probe that is long enough to ensure specificity, but can't be too long because it will bind to itself - Probe is fluorescently labelled, if you see it, it is positive ex. HCV RNA

Question 28

Describe amplification-based methods

Answer 28

PCR can be used to amplify a specific DNA sequence to produce millions of copies within a few hours

Question 29

Describe culture based methods

Answer 29

- Solid and liquid media (isolation, solid media) ?? wtf kumar - Selective - Differential - Antimicrobial susceptibility

Question 30

Describe selective media

Answer 30

- selective against different pathogens - allows growth of certain bacteria ex. Mannitol salt agar - Allows growth of salt-loving bacteria (gram positive) - Inhibits growth of gram negative

Question 31

Describe differential media

Answer 31

-allows growth of both types of pathogens, but allows you to distinguish them (usually by color)

Question 32

What 3 types of hemolysis are possible on blood agar?

Answer 32

alpha hemolysis beta hemolysis gamma hemolysis

Question 33

alpha hemolysis

Answer 33

partial hemolysis of red blood cells

Question 34

beta hemolysis

Answer 34

complete hemolysis of red blood cells

Question 35

gamma hemolysis

Answer 35

no hemolysis of red blood cells

Question 36

Obligate intracellular organisms produce a _____ effect

Answer 36

cytopathic * labor intensive and slow * immunodetection, molecular methods

Question 37

see slide 21

Answer 37

alright

Question 38

Successful clinical diagnosis of infection depends on?

Answer 38

proper specimen collection, transport, and processing

Question 39

Diagnosis is based on?

Answer 39

detecting microorganisms, their products, or patient's immune response to the presence of infectious agent

Question 40

Interpretation of culture results depends upon the source of the specimen: ________ vs _________

Answer 40

sterile sites vs normal flora