PHA112 > Microbiology 7 > Flashcards

Microbiology 7 Flashcards

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1
Q

what is typing?

A

distinguishing between strains within species

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2
Q

what is diagnostic microbiology?

A

identification usually to species level

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3
Q

what is involved in diagnostic microbiology?

A
  • antimicrobial susceptibility testing
  • pathogenicity profiling
  • clinical applications but also vet micro, plant pathology, food micro and pharmaceutical micro
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4
Q

what are the culture based methods?

A
  • colony morphology
  • selective media e.g. mannitol sat agar
  • chromogenic media e.g. macConkey agar
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5
Q

give examples of microscopic examination?

A
  • grams staining

- electron microscopy

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6
Q

what are the 4 methods of microbial identification?

A
  • culture based methods
  • microscopic examination
  • biochemical profiling
  • molecular analysis
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7
Q

what are the different methods within molecular analysis?

A
  • immunologic
  • genomic
  • proteomic
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8
Q

using colony morphology to identify species is highly…

A

inaccurate

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9
Q

to increase accuracy, what must you do?

A

purify individual colonies

this is done by sub culturing them

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10
Q

give the agars involved in selective media?

A
  • macConkey agar
  • sabouraud dextrose agar
  • baird-parker agar
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11
Q

selective media?

A

allows for growth of particular species

  • suppresses other species
  • antibiotic/nutrient supplements
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12
Q

macConkey agar

A
  • inhibits G+ organisms
  • due to bile salts and crystal violet

NEUTRAL RED DYE —> PINK (in acid)

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13
Q

sabouraud dextrose agar

A
  • selective for certain fungi
  • due to low pH (5.6)
  • & high glucose conc (3-4%)
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14
Q

baird-parker agar

A
  • selective for staphylococci
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15
Q

what do pyruvate and glycine promote?

A

staph growth

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16
Q

what do lithium chloride and tellurite inhibit?

A

non-staph growth

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17
Q

what is tellurite reduced by?

A

s.aureus

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18
Q

what does s.aureus show?

A

shiny black colonies with clear zone around them

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19
Q

differential media?

A

enables bacterial species to = distinguished from one another on same medium

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20
Q

give the agars in differential media?

A
  • mannitol salt agar

- eosin methylene blue (EMB) agar

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21
Q

mannitol salt agar (selective)

A
  • inhibits G- organisms and some G+
  • due to high salt content (7.5-10%)
  • mannitol content and phenol red differentiate between staphylococci
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22
Q

eosin methylene blue (EMB) agar

A
  • selective for G- (e.coli) organisms
  • have 2 stains: eosin & methylene blue
  • toxic to G+
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23
Q

what does lactose fermentation cause?

A

dye uptake and stains G- purple

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24
Q

what does e.coli develop?

A

metallic green sheen

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25
light microscopy properties
- use visible light - medium = air - magnification: >1000x - resolution: 0.2 micrometres
26
electron microscopy properties
- uses electron beam - medium: high vacuum - magnification: >100000x - resolution: 0.5nm
27
give the different types of electron microscopy?
- cryo - transmission - scanning
28
what are the common stains used in bacteria?
- gram stain (gram pos vs gram neg) - ziehl-neelson stain (acid fast bacteria; mainly mycobacteria) - malachite green stain (endospore staining)
29
what are the common stains used in fungi?
- periodic acid-schiff stain (used detect polysaccharides, only work on living fungi) - india ink staining (used on bacteria capsules also) - calcofluor white binds chitin and cellulose
30
what are the common stains used in protozoa?
- giemsa stain direct microscopy (e.g. trypanosome brucei in blood)
31
what can most viruses not be visualised with?
light microscopy
32
what is the first step in identifying pure bacterial cultures?
differentiating between gram positive and gram negative
33
gram positive staining procedure?
- heat fix cells to slide cell walls have single: membrane enclosed by thick, cross linked peptidoglycan - saturate with crystal violet dye for 60 seconds: thick peptidoglycan takes up dye. appears PURPLE - add iodine (mordant) for 60 seconds: dye and mordant complex forms. adheres firmly to thick peptidoglycan layer - rinse slide with alcohol for 20 seconds: alcohol cannot wash out dye-mordant complex because = firmly secured in thick layer - stain slide with safranin (counter stain): saturated with crystal violet dye. cell counter stain has little to no effect - cell wall ranges in colour from mid to dark purple
34
gram negative staining procedure?
- heat fix cells to slide: walls have thin layer of peptidoglycan in periplasmic space in inner and outer lipid membrane - saturate with crystal violet dye for 60 seconds - add iodine (mordant) for 60 seconds: dye and mordant = complex doesn't adhere to thin layer - rinse slide with alcohol for 20 seconds: dye and mordant complex easily removed from peptidoglycan layer with alcohol - stain slide with safranin (counter stain): colourless wall can easily uptake counter stain - cell wall, counter stained with safranin ranges in colour from PINK TO RED
35
what can biochemical profiling identify?
organisms that have been isolated in pure culture needs gram state = known
36
what is the purpose of an oxidase test?
tests if bacteria = able to produce certain cytochrome oxidases/indophenol oxidases
37
what do TMPD and DMPD act as?
redox indicators
38
ox + indicator
pseudomonadacae
39
ox - indicator
enterobacteriacae
40
what are commercially available kits?
miniaturised biochem tests - API strips - BD BBL crystal produce numerical code which finds bacterium from database
41
biochemical profiling: ATP determination
- ATP quantified by amount of light released following breakdown by firefly luciferase - provides direct measure of bacteria no. - doesn't need info on another contaminant - susceptible to contamination
42
immunological identification example?
ELISA test
43
genomic identification examples?
- PCR - whole genome sequencing - 16s rRNA
44
proteomic identification example?
MALDI TOF
45
give examples of molecular identification?
- immunological - genomic - proteomic
46
what is ELISA used in?
medical diagnostics (HIV, TB)
47
what are the 2 main types of ELISA?
- direct ELISA - non specific coating of antigens | - sandwich ELISA - allows for capture of specific antigens
48
what does ELISA need?
needs specific monoclonal antibodies
49
what does PCR involve?
amplifies short sequence of DNA sequences amplified determined by primers
50
in PCR, what does the efficacy depend on?
how selective primers are | - provides pos/neg results
51
what is PCR used for?
identification of antibiotic resistance e.g. mecA
52
what does DNA sequencing involve?
can sequence whole genome/ targeted regions (e.g. 16s rRNA) - can find multiple changes at once (presence/absence of genes, SNPs etc)
53
what does MLST stand for?
multi locus sequence typing
54
what does MLST involve?
amplifies housekeeping genes, then sequences
55
how many genes are used in s.aureus?
7
56
what do you need to balance in MLST?
turnaround time and sensitivity
57
MLST is used less frequently as...
WGS (whole genome sequencing) cost decreases
58
why is MALDI TOF increasingly used?
due to speed of identification & lower cost compared to conventional methods
59
what does MALDI TOF involve?
- identifies sample based on ionised samples | - spectra peaks compared
60
what can MALDI TOF be used to identify?
bacterial species from selective media can also provide info about resistance in certain cases
61
what should the ideal diagnostic be?
- accurate - sensitive - cost effective - fast
62
what are the 4 different ways of identifying microbial contaminants?
- morphology - biochem - immunologic - genetic
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