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Clinical food and microbiology > 14. Methods for isolation and enumeration of microorganisms in food > Flashcards

14. Methods for isolation and enumeration of microorganisms in food Flashcards

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1
Q

Why is isolating microorganisms from food a challenge?

A
  • affects abundance and state of microorganisms
  • time frame: shelf life of food products
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2
Q

Why are current and emerging pathogens a challenge?

A
  • virulent strains
  • low infectious doses
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3
Q

Why do current consumer demands present a challenge?

A
  • fewer preservatives
  • minimally processed foods
  • longer shelf-life
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4
Q

What do we require for isolation and enumeration?

A
  • presence/ absence
  • type
  • numbers
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5
Q

What are method limitations for isolation and enumeration?

A
  • sensitivity
  • specificity
  • cost
  • ease of use
  • speed; culture based or rapid methods?
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6
Q

How are samples made suitable for analysis?

A
  • 25g required
  • must be representative
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7
Q

What different types of samples are there?

A
  • liquid and semi solid
  • solid foods- require homogenisation
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8
Q

How is a stomacher used?

A
  • food sample (25g) is diluted in a buffer (225ml)
  • there is a 1:10 ratio food sample: diluent
  • buffer minimises stress to microorganism
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9
Q

How are samples measured?

A

homogenised food sample

analysis:
- presence/ absence of particular organisms
- total number of organisms: direct methods
- viable number of organisms: culture methods

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10
Q

How can you know how many viable organisms are in the original sample?

A

Total Viable Count (TVC or ACC)
- use a non-selective, nutrient medium
- plate count agar PCA or nutrient agar NA

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11
Q

What are elective agents?

A

They encourage growth of a desired organism
- eg.. high salt for Staphylococci

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12
Q

What is a selective agent?

A

Makes the desired organisms resistant
- eg.. bile salts for coliforms

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13
Q

What are differential agents?

A

They discriminate desired organism from others
- eg.. pH indicators; acid production from CHO’s

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14
Q

What are incubation conditions for samples?

A
  • 37 degrees celsius
  • 44 degrees celsius
  • 30 degrees celsius for ACC
  • 24/ 48 hours
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15
Q

What are different atmospheres needed?

A
  • aerobic
  • anaerobic
  • micfroaerobic
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16
Q

How is bacteria isolated from food samples?

A
  1. pre-enrichment; non-selective media, resuscitation
  2. selective enrichment; media for preferential growth of organisms
  3. streak plating; sample onto selective agars to separate mixed cultures
17
Q

What is a cultures workflow?

A
  1. 25g food in diluted 225ml buffer
  2. TVC: eg.. nutrient agar, interpret results
  3. Selective count: eg.. MacConkey agar, interpret results
  4. Atmosphere?
  5. Duration?
18
Q

What are indicator organisms?

A

organisms easily cultured indicative of the potential presence of pathogens
- often in large numbers and easily identified; E.coli
- faecal contamination

19
Q

Why is an ELISA a rapid method?

A
  • reduces time between receiving a sample and confirming results
  • standardisation and automation
20
Q

Why is a lateral flow immunoassay test a rapid method?

A
  • results within 20 minutes
  • costs 8pounds per test
  • minimum of 48 hours
  • sensitive; detection limit
21
Q

What is immunocapture?

A
  • it isolates and concentrates pathogens
  • uses immuno-magnetic separation
  • antibodies are coated in magnetic beads
22
Q

What is a PCR-based method?

A

it is an in vitro amplification of DNA region specific for microorganism of interest
- requires primer; specific to microorganism
- dNTPs Taq polymerase buffer; Tris, MgCl2, Kcl
- template - DNA or pathogen
- thermal cycler; 20-30 cycles

23
Q

What is electrophoresis?

A
  • separation of DNA fragments based on size
  • amplicon of expected size= positive result
24
Q

What are benefits of PCR-based methods?

A
  • highly specific
  • highly sensitive
  • rapid; results within 6 hours
  • identifies variants
25
Q

What are drawbacks of PCR-based methods?

A

a positive result has no interpretation

Clinical food and microbiology (12 decks)

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