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PHA112 > Microbiology 7 > Flashcards

Microbiology 7 Flashcards

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
Q

light microscopy properties

A
  • use visible light
  • medium = air
  • magnification: >1000x
  • resolution: 0.2 micrometres
26
Q

electron microscopy properties

A
  • uses electron beam
  • medium: high vacuum
  • magnification: >100000x
  • resolution: 0.5nm
27
Q

give the different types of electron microscopy?

A
  • cryo
  • transmission
  • scanning
28
Q

what are the common stains used in bacteria?

A
  • gram stain (gram pos vs gram neg)
  • ziehl-neelson stain (acid fast bacteria; mainly mycobacteria)
  • malachite green stain (endospore staining)
29
Q

what are the common stains used in fungi?

A
  • 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
Q

what are the common stains used in protozoa?

A
  • giemsa stain

direct microscopy (e.g. trypanosome brucei in blood)

31
Q

what can most viruses not be visualised with?

A

light microscopy

32
Q

what is the first step in identifying pure bacterial cultures?

A

differentiating between gram positive and gram negative

33
Q

gram positive staining procedure?

A
  • 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
Q

gram negative staining procedure?

A
  • 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
Q

what can biochemical profiling identify?

A

organisms that have been isolated in pure culture

needs gram state = known

36
Q

what is the purpose of an oxidase test?

A

tests if bacteria = able to produce certain cytochrome oxidases/indophenol oxidases

37
Q

what do TMPD and DMPD act as?

A

redox indicators

38
Q

ox + indicator

A

pseudomonadacae

39
Q

ox - indicator

A

enterobacteriacae

40
Q

what are commercially available kits?

A

miniaturised biochem tests

  • API strips
  • BD BBL crystal

produce numerical code which finds bacterium from database

41
Q

biochemical profiling: ATP determination

A
  • 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
Q

immunological identification example?

A

ELISA test

43
Q

genomic identification examples?

A
  • PCR
  • whole genome sequencing
  • 16s rRNA
44
Q

proteomic identification example?

A

MALDI TOF

45
Q

give examples of molecular identification?

A
  • immunological
  • genomic
  • proteomic
46
Q

what is ELISA used in?

A

medical diagnostics (HIV, TB)

47
Q

what are the 2 main types of ELISA?

A
  • direct ELISA - non specific coating of antigens

- sandwich ELISA - allows for capture of specific antigens

48
Q

what does ELISA need?

A

needs specific monoclonal antibodies

49
Q

what does PCR involve?

A

amplifies short sequence of DNA

sequences amplified determined by primers

50
Q

in PCR, what does the efficacy depend on?

A

how selective primers are

- provides pos/neg results

51
Q

what is PCR used for?

A

identification of antibiotic resistance e.g. mecA

52
Q

what does DNA sequencing involve?

A

can sequence whole genome/ targeted regions (e.g. 16s rRNA)

  • can find multiple changes at once (presence/absence of genes, SNPs etc)
53
Q

what does MLST stand for?

A

multi locus sequence typing

54
Q

what does MLST involve?

A

amplifies housekeeping genes, then sequences

55
Q

how many genes are used in s.aureus?

A

7

56
Q

what do you need to balance in MLST?

A

turnaround time and sensitivity

57
Q

MLST is used less frequently as…

A

WGS (whole genome sequencing) cost decreases

58
Q

why is MALDI TOF increasingly used?

A

due to speed of identification & lower cost compared to conventional methods

59
Q

what does MALDI TOF involve?

A
  • identifies sample based on ionised samples

- spectra peaks compared

60
Q

what can MALDI TOF be used to identify?

A

bacterial species from selective media

can also provide info about resistance in certain cases

61
Q

what should the ideal diagnostic be?

A
  • accurate
  • sensitive
  • cost effective
  • fast
62
Q

what are the 4 different ways of identifying microbial contaminants?

A
  • morphology
  • biochem
  • immunologic
  • genetic

PHA112 (37 decks)

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