Lecture 2 - Laboratory

Question 1

culture dependent

Answer 1

can grow organism in lab and study it

*Collect specimen > Isolate > Investigate / test > identify

Question 2

culture independent

Answer 2

don’t need to grow it in lab to be able to identify and study it
*collect specimen> Investigate (test) > identify

Question 3

what is important when it comes to specimens collection, why?

Answer 3

quality. Effects speed and accuracy of organism identification.
- contributes to better pt outcomes and avoids inappropriate treatment

Question 4

what types of specimens can you collect?

Answer 4

throat swab, sputum, blood samples (sterile), wound swabs, urine, genital swabs, stool

Question 5

what is an example of a sterile specimen, why?

Answer 5

blood sample.
arent in contact with bacteria, virus, fungus that is all over body – no chance for cross contamination.
– able to identify bacteria and such that isnt supposed to be in there

Question 6

what are important details to label on specimen collection

Answer 6

• sources (niches) –> can aid in identification (rash vs cat bite)
• patient status can effect work up (healthy vs diabetic)

Question 7

culture

Answer 7

a population of bacteria grown in defined medium and defined conditions in a laboratory

Question 8

pure culture

Answer 8

growth of a single known species - what we want

Question 9

mixed culture

Answer 9

holds two or more species (starting point)

-need to separate normal flora from causative agent

Question 10

contaminated culture

Answer 10

includes unwanted microbes, usually introduced inappropriately
-living on pt but not actually the cause of infection

Question 11

what is inoculation?

• how is it completed
• types?

Answer 11

Adding a small amount of sample (ex: urine, swab) to media to grow a culture
-only certain bacteria can grow on certain media

-Inoculated media is incubated to grow culture at 20 to 40 ºC, different atmospheres (aerobic, anaerobic)

Types of inoculation:
Streak plate (\/\/\)
Spread plate (L)

*goal is to isolate colonies

Question 12

isolated colony

Answer 12

A population of cells on an agar plate that arise from division of a single cell
-dif species form colonies with distinct characteristics (morphology) like size and colour

Question 13

what is colony morphology

Answer 13

• differs between species - an be used to identify causative agent
• media type also aids in identification (differential and selective or enriched)

Question 14

differential media

Answer 14

• Contain chemicals that allow distinction of different types of bacteria based on colony morphology (d/t specific trait like metabolism)
  ex) mackonkey agar - identify lactose fermentation or not

Question 15

Selective Media

Answer 15

• prevents growth of a specific group of microbes, while allowing growth of others
• target general groups (gram + or gram - or staphylococcus)

Question 16

enriched media

Answer 16

contains organic molecules to grow Fastidious Microbes

Question 17

what are fastidious microbes

Answer 17

microbes that have complex nutrient requirements Ex. vitamins, growth factors

• many pathogens are fastidious and rely on their host for nutrients
  ex) hemophelus influenza that causes meningitis needs heme (iron) and vitamins

Question 18

what are biochemical tests?

Answer 18

identify bacteria based on the enzymes they produce

-test for metabolic enzymes by growing in different nutrient sources

Question 19

what are immunological tests?

Answer 19

use antibodies to detect specific antigens that are found on microbes

• can be culture dependent or independent
• ex) O-antigen test would identify V. cholerae O139, E. coli O157

Question 20

what is agglutination

Answer 20

• Antibodies detect and bind specific surface antigens on microbes
• Antibodies can bind multiple microbes, cause visible clumping, indicating a positive reaction

Question 21

what is the reasoning behind staining

Answer 21

bacterial cells are clear and require staining to be visible

Question 22

what are the types of stains?

Answer 22

Positive stain: stains the bacteria
Negative stain: stains the background
differential staining - distinguished btw bacterial toes (gram +/- stain, endospore stain)

Question 23

how is investigation for independent cultures different than dependant cultures?

Answer 23

• important in obligate parasites (chlamydia, viruses)
• tests specific for organism
• immunological tests - detection of antigens
• molecular tests like PCR, DNA sequencing

Question 24

explain PCR

Answer 24

• amplifies small amount of dana with specific sequence
• allows detection of causative agent even in mixed samples at low concentration
• detection of TB, chlamydia

Question 25

How does PCR work in the presence of mixed samples

Answer 25

DNA primers bind to specific microbial DNA, an enzyme duplicated DNA by exponential application –> microbial DNA can now be detected

Question 26

what is DNA sequencing?

Answer 26

direct identification by sequencing all DNA in a sample and comparing to data base of known organism DNA