lecture 6) clinical microbiology 2

Question 1

why study the cause of infection?

Answer 1

prognosis
treatment
isolation
care

Question 2

name 5 ways the cause of infection can be determined

Answer 2

microscopy
molecular testing
biochemical tests
rapid test and immunoassays
culture

Question 3

what type of microscopy is used to identify infections of bacteria, fungi and protozoa?

Answer 3

light

Question 4

what type of microscopy is used to identify viral infections?

Answer 4

electron

Question 5

what molecular test can you do to identify infections of bacteria, fungi, protozoa and viruses?

Answer 5

nucleic acid amplification, sequencing, fingerprinting etc

Question 6

what biochemical tests can be used to identify bacterial and fungal infections?

Answer 6

identification and sensitivity

Question 7

what rapid tests can be used to identify bacterial and fungal infections?

Answer 7

biochemical identification

Question 8

what immunoassay tests can be used to identify bacterial, fungal, protozoa and viral infections?

Answer 8

ELISA
flow cytometry
complement fixation

Question 9

how would you culture bacterial and fungal infections?

Answer 9

purify and amplify

Question 10

how would you culture viral infections?

Answer 10

cytopathology

Question 11

what are the 4 steps in historical workflow of determining the cause of infection?

Answer 11

1) stain based methodologies
2) microbial cultures
3) biochemical/antigenic techniques
4) antimicrobial susceptibility testing

Question 12

what is the aim of stain based methodologies and what do they support?

Answer 12

classification of microscopic morphology

supports early diagnostic and therapeutic decisions

Question 13

what does microbial culture involve in historical workflow?

Answer 13

propagtion of organism on agar/liquid medium

Question 14

what do biochemical/antigenic techniques involve and what does this lead to?

Answer 14

metabolic and phenotypic analysis

leads to identification of organism

Question 15

what does antimicrobial susceptibility testing lead to?

Answer 15

confirming or tailoring therapeutic agents

Question 16

give 3 examples of stains for microscopy

Answer 16

gram stain
acid fast stain
periodic acid schiff (PAS)

Question 17

what is acid fast stain also known as?

Answer 17

zahl-neeson stain

Question 18

how is acid fast stain done and what does it test?

Answer 18

performed directly on sputum (saliva + mucus coughed up from resp tract), no need for culture, specific
performed on mycobacteria

Question 19

what is PAS used for?

Answer 19

stains glycoproteins in fungi

Question 20

what type of background is needed for PAS?

Answer 20

high background

Question 21

what is a disadvantage of PAS?

Answer 21

not very clear as it reacts with other cells that contain glycoproteins

Question 22

name a selective media used for microbial culture

Answer 22

mannitol salt agar that is used for isolation of staphylococci

Question 23

what does a selective media do?

Answer 23

supports the growth of one group of organisms out of a highly variable sample eg faeces

Question 24

name a differential media used for microbial culture

Answer 24

MacConkey agar for the recovery of enterobacteriaceae

Question 25

what property of mannitol salt agar plates prevents many bacterial strains from growing?

Answer 25

7.5% sodium chloride

Question 26

what species of bacteria thrive in mannitol salt agar plates?

Answer 26

staphylococcus | ferment mannitol and produce an acidic product

Question 27

what is the colour change for a positive result on mannitol salt agar?

Answer 27

red to yellow

Question 28

what dye is used on mannitol salt agar plates?

Answer 28

phenyl red

Question 29

what does differential media do?

Answer 29

supports the growth of many bacteria and then you can isolate certain ones

Question 30

what type property of bacteria would cause a positive result on MacConkey agar plates?

Answer 30

lactose fermenters

Question 31

what is the colour change for a positive result on MacConkey?

Answer 31

red to pink

Question 32

what dye is used for MacConkey plates?

Answer 32

neutral red

Question 33

why are tests that differentiate staphylococcus and streptococcus important?

Answer 33

because staph and strep are both gram positive bacteria that are found on the skin

Question 34

name 3 biochemical techniques that can be used after gram staining?

Answer 34

coagulase beta galactosidase catalase

Question 35

what does coagulase detect?

Answer 35

detects the presence of coagulase that causes plasma to clot

Question 36

what bacteria does coagulase differentiate?

Answer 36

staphylococcus aureus and epidermis

Question 37

what does beta galactosidase detect?

Answer 37

detects the presenc of an enzyme that cleaves lactose to form glucose and galactose

Question 38

what does beta galactosidase differentiate?

Answer 38

separates the enterics and identifies pseudonomads

Question 39

what does the biochemical test catalase detect?

Answer 39

catalse | converts hydrogen peroxide to water and oxygen

Question 40

what dose catalse differentiate between?

Answer 40

staphylococcus and streptococcus

Question 41

name 2 tests that differentiate between staphylococcus and streptococcus

Answer 41

catalase | mannitol salt agar plate

Question 42

what test is the first form of information?

Answer 42

gram staining

Question 43

what is used to carry out biochemical tests?

Answer 43

API strips (analytical profile index)

Question 44

how many biochemical tests does API test for?

Answer 44

20

Question 45

what does ELISA stand for?

Answer 45

enzyme linked immunoabsorbent assay

Question 46

why would indirect ELISA be used?

Answer 46

if you want to find out if an individual has been infected with a certain pathogen

Question 47

what are the steps involved in indirect ELISA?

Answer 47

take a serum sample from the individual clone the antigen and stick it to the plastic wells if they have antibodies for the antigens on the well they will stick to it and others wont wash everything that doesnt stick away indicator antibody linked to enzyme attached to any bound antibody rinse well to get rid of anything bound to them colourless substrate added enzymes that are linked to the indicator antibody hydrolyse the substrate releasing a dye

Question 48

what results are positive and negative from indirect ELISA?

Answer 48

coloured wells are positive for the antibody | colourless wells are negative for th antibody

Question 49

what viruses can be detected using antibody sandwich ELISA?

Answer 49

orthohantavirus | measles virus

Question 50

explain the steps in antibody sandwich ELISA

Answer 50

antibody absorbed to the well test antigen added enzyme linked antibody specific to test antigen binds to another antigen forming a sandwich enzyme substrate added if positive there will be a colour change

Question 51

what type of infections do agglutination assays commonly test for?

Answer 51

viral infections

Question 52

what do agglutination assays use?

Answer 52

uses the patients immune system to tell us what we have got

Question 53

what beads are used in agglutination of red blood cells and what do they do/tell us?

Answer 53

latex beads coated with an antigen latex beads coated with a protein belonging to the virus if you are infected with that virus you probably have antibodies for the virus therefore the antibodies with bind to the antigens on the latex beads agglutination of the antibodies to the antigens on the beads can be monitored in the blood sample

Question 54

what happens to red blood cells that arent agglutinated?

Answer 54

the will pellet at the bottom of the well

Question 55

explain how you would test for antimicrobial suscpetibility using an agar plate

Answer 55

put the antibiotic on the agar plate dot the microbe around the agar plate if there is mirobial growth = resistant to antibiotic no microbial growth = susceptible to antibiotic

Question 56

what is an Etest strip and what is it used for?

Answer 56

graded concentration strip | used for antibiotic susceptibiliy testing

Question 57

name 5 techniques used in modern clinical microbiology

Answer 57

next generation sequencing of whole bacterial genomes metagenomics and community profiling multiplex PCR mass spectrometry

Question 58

which is the best technique used in modern microbiology?

Answer 58

mass spectrometry

Question 59

what can also be measured in mass spectrometry?

Answer 59

PCR

Question 60

how is mass spectrometry used?

Answer 60

detects mass/charge ratio | fragment will be in a certain pattern

Question 61

describe NGS of bacterial genomes

Answer 61

currently culture dependent species identification becoming cheaper gives a wealth of information

Question 62

what are some of the advantages of using multiplex PCR?

Answer 62

differentiates strains based on the bands you have no need for culture cheap and sensitive no comparison standard

Question 63

what are some of the disadvantages of multiplex PCR?

Answer 63

need 1 set of primers specific for each organism too laborious prone to error in set up

Question 64

what is used as an alternative to multiplex PCR?

Answer 64

NAAT | PCR

Question 65

what is culturomics also known as?

Answer 65

metagenomics and community profiling

Question 66

give some examples of gram positive bacteria

Answer 66

``` streptococcus staphylococcus mycobacteria listeria bacillus clostridium ```

Question 67

give some examples of common negative bacteria

Answer 67

neisseria enteronacteriacae bacteriodes campylobacter

Question 68

mycobacteria are classed as being gram positives. what would make them qualify as being gram positive and gram negative?

Answer 68

1 phospholipid bilayer 1 peptiodglycan layer no asymmetric membrane (gram positive) mycolic acids serve as pseudophospholipid bilayer retain gram stain