Chapter 11

Question 1

What are the goals of a clinical microbiology laboratory?

Answer 1

Identify the microorganism in the patient involved in the disease
Provide antimicrobial susceptibility of the isolated microorganism

Question 2

What is the identification process of a microorganism?

Answer 2

Identification of microorganisms by isolations and culture (presence or number of microorganisms, susceptibility)
Identification of a specific microbial gene or product (presence of a resistance gene)
Detection of specific antibodies to a pathogen for pathogens that are high risk or cannot be cultivated, retrospective diagnosis

Question 3

Where are the two possible sites for specimen collection?

Answer 3

Sterile sites (blood, urine, CSF) and sites with commensal flora (skin, GI tract, urethra)
Need to make sure collection from the sterile site isn't contaminated.

Question 4

What are some of the downsides of culture-based methods for specimen growth?

Answer 4

Can take up to 2 days (>18 hours), organism must be culturable

Question 5

What must be done specially for blood culture?

Answer 5

Low numbers of cells thus rely on detection of an antibody response to a pathogen,

Question 6

What are the advantages of non-culture methods?

Answer 6

Fast, less labour intensive and suitable for organisms that cannot be cultured in the lab

Question 7

What are some non-culture methods?

Answer 7

Microscopy (light and electron), immunodiagnostics and molecular diagnostics

Question 8

What are the types of light microscopy? What are they used for?

Answer 8

Bright field used for stained preparations (gram stain, acid fast for mycobacteria) and wet preparations (for macromolecules like vesicles, granules).
Dark field for motility and thin cells
Fluorescent for naturally fluorescent or organisms and antibodies stained with fluorescent dyes

Question 9

What colours show up in a gram stain?

Answer 9

Gram positive are purple

Gram negative are pink

Question 10

How does electron microscopy work? What is it used for?

Answer 10

Uses a beam of electrons and magnets rather than light
Specimen must be cut into thin sections
Used for viruses (not usually used in a clinical lab)

Question 11

How does a gram stain work?

Answer 11

Crystal violet, iodine (forms complex with crystal violet), alcohol (removes complex not trapped in gram + peptidoglycan) then pink dye

Question 12

What could be the reason nothing show up under the microscope after a gram stain?

Answer 12

Mycobacterium or viral infection

Question 13

What is the difference between a scanning (SEM) and transmission (TEM) electron microscope?

Answer 13

Scanning simply scans the surface of the cell

Transmission is used to see cell structures as it passes through

Question 14

What organisms commonly cause bacterial meningitis?

Answer 14

Streptococcus pneumoniae, haemophilus influenzae, niesseria meningitis in CSF

Question 15

How can immunodetection be done?

Answer 15

A specific antibody is coated onto the latex bead and when it binds to the organism there is visible clumping for bacterial meningitis
Much faster than culturing

Question 16

What may cause a false positive in immunodetection?

Answer 16

If not enough of the patient’s specimen is tested. Cross reacting antigens

Question 17

What is ELISA? How does it work?

Answer 17

Enzyme linked immunosorbent assay
Direct: Coat plate with antibodies, add patient sample (antigens bind) and then add fluorescent or chemically labelled antibodies will bind to patient antigen
Indirect: Coat plate with antigens then add patient sample (antibodies bind) then add a labelled anti-immunoglobulin

Question 18

What is the difference between direct and indirect immunodetection?

Answer 18

Direct detects the antigen, indirect detects the antibody (more frequently used, can use same secondary antibody for multiple tests)

Question 19

What can ELISA be used for?

Answer 19

HIV, HBV and herpes

Question 20

What are monoclonal antibodies used for?

Answer 20

To distinguish between species and between strains of the same on the basis of antigenic differences
Diagnostic tests (really specific)

Question 21

How are monoclonal antibodies formed?

Answer 21

From hybridomas produced by fusion of immortal B cell tumours and normal antibody-producing cells

Question 22

What is the probe-based method for specific gene detection? What is it used for?

Answer 22

Label a single-stranded nucleic acid fragment to hybridize with the target DNA.
A with T, G with C hydrogen bonding
Hepatitis C virus RNA

Question 23

What are some problems with a probe-based method?

Answer 23

Must know the sequence of the target gene

Can’t make the strand too long or else it will bind to itself or if too small it will not be specific enough.

Question 24

What is an amplification based method for specific gene detection?

Answer 24

Polymerase chain reaction (PCR) is the most common
Amplifies a target sequence from any source and copies it.
Design specific primers for the gene sequence using taq polymerase from 5’ (works at high temperature when DNA strands separate)
Doubling of DNA content for 30-40 cycles
Tells if gene is absent or present

Question 25

How are culture-based methods done?

Answer 25

Use solid (isolation, differentiation) and liquid (turbidity shows presence) media
Selective, differential, antimicrobial susceptibility

Question 26

Why is agar used as a solid growth media?

Answer 26

Because it is not broken down by most bacteria

Question 27

What is a selective growth media?

Answer 27

A media that allows the growth of some organisms while inhibiting the growth of others. Know the natural conditions the organism grows in.
Add bile salts for GI tract bacteria

Question 28

What are some examples of differential growth media?

Answer 28

Lactose medium for salmonella and E. coli (produces acid, use pH indicator)
Blood medium to see which is capable of hemolysis (zone of clearing)

Question 29

What are the different types of hemolysis? How do they show up on a blood medium?

Answer 29

Beta gives a clear zone (complete hemolysis)
Alpha gives green zone (heme) (incomplete hemolysis)
Gamma is no hemolysis

Question 30

How can we culture viruses, chlamydia and rickettsia?

Answer 30

In cell or tissue culture (eukaryotic growth cultures)

Observe the presence by the cytopathic effect (changing of cell morphology due to cell killing)

Question 31

What are some problems with eukaryotic growth cultures?

Answer 31

Labour intensive and slow, contamination because culture is so rich.
Usually use immunodetection or molecular methods instead

Question 32

What are the steps taken when you want to identify a gram positive organism in culture?

Answer 32

Gram stain, shape and grouping, coagulase and hemolysis
(all for cocci)
Rods are aerobic or anaerobic

Question 33

What are the steps taken when you want to identify a gram negative organism in culture?

Answer 33

Gram stain, shape, lactose fermentation, oxidase
(all for rods, aerobic)
Also cocci

Question 34

What are fastidious organisms?

Answer 34

Hard to grow, must add additional growth factors. You have seen the cells but they aren’t growing on the plate

Question 35

How are fungi and protozoa identified?

Answer 35

Fungi can be identified by their colonial characteristics and cell morpholoy
Protozoa is identified by direct examination

Question 36

How are biochemical tests used to identify microbes in culture?

Answer 36

Phenotypic array

Can test acid production, using sugar as a food source, lower pH, degradation of vitamins, ability to resist antibiotics