Trials and methods

Question 1

What is the purpose of culture based methods?

Answer 1

isolate the target microbes to study their metabolism,
morphology or strain typing

Question 2

How do non-culturing methods work?

Answer 2

Use genetic material to identify,
classify and quantify the target microbes

Question 3

What method do you use if you want to investigate what a probiotic drink from a supermarket contains?

Answer 3

Culturing

Question 4

What method do you use if you want to see how the microbiome has changed as a result of consuming a probiotic?

Answer 4

16S sequencing

Question 5

What method do you use if you want to test if a probiotic has live bacteria in it?

Answer 5

Culturing

Question 6

If the aim is related to live bacteria which method should come to mind?

Answer 6

If the aim is related to live microorganisms, culturing can be
the 1st option. There are non-culture based quantification
methods for live microbes as well.

Question 7

If the aim does not require live bacteria which method should come to mind?

Answer 7

If the aim does not require alive microbes, genetic material
based methods should come to mind in first place.

Question 8

How would you quantify Enterobacteriaceae in
the contaminated food?

Answer 8

Take a food sample, make a dilution serie and
incoluate VRBD (Violet Red Bile Dextrose) agar
plates, incubate at aerobic conditions at 37 °C for
24 h.

Calculate the amount using dilution factor. For
example at 10-8, 5 colonies are found. Then it
should be 5x10^8 in the un-diluted inoculation.

If you took 10 g of contaminated food from 200 g
of the food, then the total amount of
Enterobacteiraceae should be 1x10^10 CFU/g

Question 9

What is the purpose of End point PCR?

Answer 9

presence/absence of the target, verify the correct size for cloning

To see if it is there or not

Question 10

What is the purpose of nested PCR?

Answer 10

1st PCR amplicon is used as template for 2nd PCR, increased sensitivity, false positive risk

Question 11

What is the purpose of Multiplex PCR?

Answer 11

Use multiple sets of primers/probes to target multiple region in a single PCR reaction

Question 12

What is the purpose of real time PCR?

Answer 12

Quantification (absolute/relative, standard curve/housekeeping gene)

Question 13

What is the purpose of Reverse transcription PCR (RT-PCR)?

Answer 13

convert mRNA to cDNA then same as real time PCR

Question 14

What are the principles (3 steps) of PCR?

Answer 14

• Denaturation - Open up the double helix structure (heating 94-96 °C)
  – Annealing – primers/oligonucleotides bind on your separated DNA strands (50-60 °C)
  – Elongation – DNA polymerase help dNTPs (nucleotides added in the solution) find the
  complementary strand and extend (72 °C)

Question 15

What is SYBR green?

Answer 15

Non-specific flourescent DNA dye with high affinity for double stranded DNA

Number of cycles required to reach a flourescence threshold

Question 16

What is TAQMAN?

Answer 16

Fluorescently labeled oligonucleotide probes
A probe is designed with a fluorescent dye
reporter in one end (5 ́) and a quencher in the
other end (3 ́)
* During new DNA strand synthesis,the Taq DNA
polymerase comes into contact with the probe
and cleavesit
* The dye emitslight when not in contact with the
quencher
The light is measured and translated to amounts of
copied strands

Question 17

What are the pros of QPCR?

Answer 17

Can quantitatively detect both
bacteria (total), bacterial groups, and
specific genes based on primers
Small amount of template
Highly sensitive
Affordable cost

Question 18

What are the cons of QPCR?

Answer 18

SYBR green bind to any dsDNA
(e.g. primer timer, non-specific
reaction products)
Not possible to distinguish
between live and dead cells

Question 19

When you want to target bacteria you use 16S, what do you use for yeast?

Answer 19

18S

Question 20

What is first generation sequencing?

Answer 20

Sanger sequencing
4 PCR reactions
piece it together
time consuming and cannot scale up

Question 21

What is 2nd generation sequencing?

Answer 21

454 Pyrosequencing
Ion torrent
Illumina
Oxford Nanopore

Question 22

What are the pros and cons of culturing?

Answer 22

Pros: easy and cheap
Cons: Many microbes unculturable and it is time consuming

Question 23

What are the pros and cons of PCR/Sanger sequencing?

Answer 23

Pros: It is fast and cheap and can target unculturable microbes.
Cons: Contamination and false positives. Time consuming and expensive if the samples are large

Question 24

What are the pros and cons of NGS?

Answer 24

Pros: Can identify rare taxa, can handle large datasets
Cons: Expensive for small scale use, the data handling is not easy

Question 25

If you are looking for live microbes which method should you go for, culturing or gene based?

Answer 25

Culturing

Question 26

If you are looking to quantify your microbes which method should you go for, PCR or qPCR?

Answer 26

qPCR for quantification. PCR for just confirming presence/absence

Question 27

If you are looking for absolute quantification, should you go for flow cytometry or NGS?

Answer 27

Flow cytometry

Question 28

What type of mouse models exist?

Answer 28

Inbred strains (individuals are nearly identical to each other in
genotype due to long inbreeding).
* Outbred strains (wildtype in nature, as little inbreeding as
possible).
* Knockout (an existing gene has been inactivated by replacing
or disrupting it with DNA).
* Knock-in (one-for-one substitution of a DNA sequence in a
genetic locus or the insertion of DNA not found within the
locus).
* Transgenic mutants (a gene sequence has been isolated from
one organism and is randomly introduced into a another,
often into the germ line).
* Germ-free animals (microbiologically sterile).
* Gnotobiotic animals (only certain known strains of bacteria
and other microorganisms are present).

Question 29

What percentage of genes are similar between mice and humans?

Answer 29

99 %

Question 30

What does double blind mean?

Answer 30

neither the participants nor the researchers know who is receiving a
particular treatment.

Question 31

What does wash out mean?

Answer 31

a period before the trial starts e.g., participants are not allowed to consume
probiotics to get rid of effects from products that they normally consume.

Question 32

What does endpoint mean?

Answer 32

address the primary objective of the trial e.g., increased diversity, improved
disease activity index.

Question 33

What is a cohort study design?

Answer 33

observe a group of individuals suffering from celiac disease and records their intake of probiotics.

Question 34

Is this a placebo-controlled trial, single arm trial or crossover trial

patients with celiac disease are randomized to receive a probiotic- or a placebo
product. The participants are followed over time and responses are compared between groups.

Answer 34

Placebo-controlled

Question 35

Is this a placebo-controlled trial, single arm trial or crossover trial

participants are randomized to a sequence of treatments that either starts with
consumption of a probiotic- or a placebo product.

Answer 35

Crossover

Crossover trials still remains a comparison of treatment but
provide information of individual variation.

Question 36

What is a benefit of crossover trials?

Answer 36

Crossover trials still remains a comparison of treatment but
provide information of individual variation.

Question 37

Is this a placebo-controlled trial, single arm trial or crossover trial

a group of individuals is given the probiotic product and is followed over time to observe
the survivability of the bacteria.

Answer 37

Single arm