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Microbiome > MHD2 Methods to characterise the microbiome > Flashcards

MHD2 Methods to characterise the microbiome Flashcards

1
Q

What factors determine what microbiota will be present?

A

nutrient availability
pH
temperature
atmospheric conditions

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2
Q

What is the taxonomic hierarchy mnemonic?

A

Delicious KP Crisps Often Follow Good Sex

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3
Q

What is the taxonomic hierarchy

A 
Domain – Bacteria
Kingdom – Bacteria 
Phylum – Proteobacteria
Class – Gammaproteobacteria
Order – Enterobacteriales
Family – Enterobacteriaceae
Genus – Escherichia
Species – Coli
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4
Q

What are the major phyla present in a human?

Angry flies probably fart badly

A 
Actinobacteria
Firmicutes
Proteobacteria
Fusobacteria
Bacterioidetes
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5
Q

Where are Actinobacteria found?

A

Nares (nostrils), Skin retroaurical crease (inner elbow), Skin antecubital fossa (ear)

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6
Q

Give two examples of Actinobacteria

A

o Corynebacterium

o Propionibacterium

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7
Q

Where are Proteobacteria found?

A

Saliva, Nares, Tongue, Tonsils, Skin antecubital fossa

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8
Q

Where are Bacterioidetes found?

A

Gut stool, Saliva, Gingiva (gums), Tonsils, Tongue

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9
Q

Where are Fusobacteria found?

A

Saliva, Tongue, Tonsils

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10
Q

Where are Firmicutes found?

A

Vagina, Gut stool, Saliva, Gingiva, Nares, Tongue, Tonsils

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11
Q

Give 3 examples of Firmicutes

A

o Lactobacillus
o Staphylococcus
o Streptococcus

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12
Q

What methods can be used to characterise microbiota?

A

By isolating the individual prokaryotes, viruses, fungi and parasites and using phenotypic or genotypic methods for analysis

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13
Q

Which bacterial genus of the Neisseria family CAN grow on Thayer-Martin medium?

A

Within the Neisseria family. N. gonorrhoeae and N. meningitis

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14
Q

Which bacterial genus of the Neisseria family CANNOT grow on Thayer-Martin medium?

A

N. flavescens, N. sicca, and N. subflava

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15
Q

Which between N. gonorrhoeae and N. meningitidis cannot use maltose?

A

N. gonorrhoeae cannot

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16
Q

What genotypic tests can be used to classify new species of bacteria?

A

16S rRNA gene sequencing is used to determine whether the new species shares less than 97% nucleotide sequence similarity with the 16S rRNA sequences of all the known species of bacteria

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17
Q

What is phylogeny the study of?

A

The evolutionary history of an organism

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18
Q

How does phylogeny studying work?

A

Molecular sequence data is used in order to determine which organisms share a common ancestor by examining genetic properties

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19
Q

What is systematics?

A

The study of the diversity of organisms and their relationships

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20
Q

What is used to accurately name species in two terms

A

binomial nomenclature

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21
Q

How was microbial characterisation first performed?

A

Cultivation methods: to distinguish 6 types of bacteria

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22
Q

What conditions do obligate aerobic bacteria grow in?

A

same O2 level as the atmosphere

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23
Q

What conditions do obligate anaerobic bacteria grow in?

A

complete absence of O2

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24
Q

What conditions do facultative anaerobic bacteria grow in?

A

presence or absence of O2, but prefer O2 presence

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25
Q

What conditions do micro-aerophile bacteria grow in?

A

low levels of O2

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26
Q

What conditions do capnophile bacteria grow in?

A

increased levels of CO2

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27
Q

What conditions do aero-tolerant bacteria grow in?

A

Equally well in O2 presence of absence

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28
Q

What other phenotypical tests are there?

A
  • gram-staining
  • cell-shape
  • susceptibility to various antimicrobials
  • characterising cell-wall fatty acids
  • biochemical tests to observe the gases and short-chain fatty acids produced from sugar and glucose fermentation.
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29
Q

Rather than phenotypic methods, what is more accurate?

A

molecular-based methods should be used alongside phenotypic one

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30
Q

What is a combination of phenotypic and molecular based approaches called?

A

Polyphasic approach

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31
Q

Name two biochemical test kits

A
  • API strips

- Biolog systems

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32
Q

How can you control which type of bacteria grow?

A

Culture media
atmospheric conditions
incubation temperature

Media can be either agar or broth based and have varying nutrient compositions

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33
Q

Why are agar plates more useful than broth based?

A

Most bacteria can be isolated and each colony observed is potentially derived from a different single bacterium

34
Q

How are bacteria cultured?

A

Mixed cultures are recovered from samples of microbiota and individual colonies are purified before identification using phenotypic and genotypic tests

35
Q

What type of media is used to increase the probability of isolating specific bacterial groups?

A

Selective media

36
Q

Give 2 examples of selective media

A

MacConkey agar

Man-Rogosa-Sharpe agar

37
Q

What does MacConkey agar distinguish between?

A

Gram-negative and gram-positive as it inhibits gram-positive species

38
Q

How does MacConkey agar inhibit gram-positive species?

A

it contains bile salts and crystal violet

39
Q

What type of medium is MacConkey also known as? Why?

A

‘Differential medium’

It can distinguish species that ferment lactose as it contains the pH indicator neutral red.

40
Q

What can API 20E strips be used to identify?

A

Isolated colonies

41
Q

Name three lactose-positive colonies and what colour they turn MacConkey agar

A

Escherichia, Enterobacter, Klebsiella

pink

42
Q

Name three lactose-negative colonies, and what colour colonies do they have on MacConkey agar?

A

Salmonella, Proteus, Shigella

white

43
Q

When is chromogenic agar used? why?

A

in clinical and commercial laboratories to distinguish bacteria which are closely-related, as it allows differentiation.

44
Q

What type of medium is Columbia blood agar?

A

rich, non-selective medium

45
Q

What types of microbes does Columbia blood agar allow to grow?

A

Theoretically allows all types of microbes to grow

46
Q

What bacteria can you isolate with Columbia blood agar?

A

Allows the isolation of fastidious (fussy) aerobic and anaerobic bacteria with complex nutritional requires.

47
Q

What type of agar is nutrient agar and what types of bacteria can it isolate?

A

It is an example of a non-selective medium that can isolate non-fastidious aerobes and anaerobes

48
Q

How is chocolate agar produced?

A

By heating blood agar to cause the blood cells to burst and release NAD and heamin into the medium

49
Q

What does chocolate agar allow to grow?

A

very fastidious bacteria, such as Neisseria meningitidis (i.e. the bacterium lacks enzymes necessary for lysing red blood cells)

50
Q

The isolation and growth of anaerobic bacteria requires what specialist equipment?

A
  • Hungate tubes
  • Sealed cabinet
  • Gas pack
51
Q

How do hungate tubes aid the growing of anaerobic bacteria?

A

They are used to prepare anaerobic broth medium, anaerobic bacteria added using a needle inserted into the rubber stopper of the tube. The anaerobic environment in the tube is maintained by creating the rubber seal by heating.

52
Q

How does a sealed cabinet

aid the growing of anaerobic bacteria?

A

It is used to isolate and grow obligately anaerobic and microaerophilic bacteria in anaerobic conditions

53
Q

How does a gas pack aid the growing of anaerobic bacteria?

A

It is used to maintain atmospheric conditions in gas jars to isolate and grow facultative, oxygen-tolerant anaerobes, or capnophiles

54
Q

What is the problem with studying bacteria from the gastrointestinal tract by collecting faecal samples?

A

these are not representative of the microbiota in the GI tract as a whole, specifically with regard to more proximal regions of the tract.

55
Q

Explain why faecal samples are not representative of the gut microbiota as a whole?
Give an example

A

The same strain of bacteria can metabolise substances differently when found in different regions of the GI tract, which is seen in the way E.coli in the faeces and the caecum have varying concentrations of their metabolites.

56
Q

What bacteria dominates the vaginal microbiota?

A

Lactobacillus

57
Q

Why may thrush of bacterial vaginosis develop?

A

If the vaginal microbiota environment is disrupted by antibiotics of infection

58
Q

How can lactobacillus be isolated?

A

Using the selective medium Man-Rogosa-Sharpe agar

59
Q

What species does the mouth contain?

A

Streptococci (aerobic or anaerobic) and Bacteroidetes (fastidious obligatory anaerobic bacteria.)

60
Q

Who do Bacteroidetes survive in the mouth?

A

Bacteroidetes live in cavities and the gaps between teeth which are oxygen-free and contain food debris for the species to survive.

61
Q

What allows Bacteroidetes and Proteobacteria to be present in the faeces?

A

the colon is an anaerobic, nutrient-rich environment

62
Q

What type of anaerobic are proteobacteria?

A

facultative anaerobic

63
Q

Why is the isolation of novel bacteria important?

A

in microbiology as gaps in our knowledge of microbial diversity and function can be filled by isolating as many bacteria and archaea as possible.

64
Q

What do metagenomic studies do?

A

sequence and annotate all the genes in all the DNA of the microbiota

65
Q

No function has been found for how many of the microbial genes cultivated?

A

50%

66
Q

What % of the gut microbiome has been cultivated?

A

75%

67
Q

What advances have been made in culturing technology

A

Multiple culturing conditions, matrix-assisted laser desorption/ionisation, time of flight mass spectrometry, and sequence analysis to identify bacterial species.

68
Q

Why do housekeeping genes change far more slowly over time?

A

They are essential for the proper functioning of a cell

69
Q

What house keeping gene is used to distinguish individual prokaryote species?

A

16S ribosomal ribonucleic acid gene

70
Q

When were archaea distinguished from bacteria and how?

A

in 1977 through phylogenetic analysis of 16S rRNA sequences

71
Q

Why are rRNAs known as molecular clocks?

A

rRNAs are responsible for assembling the ribosome therefore their sequences change over time as translation is essential for life

72
Q

How can PCR primers be designed for use in diversity studies?

A

There are large databases of 16S rRNA gene sequences

73
Q

How do you ensure identification of bacteria up to genus level?

A

use PCR primers that flank the V3 and V4 variable regions as they amplify up to 450 nucleotides

74
Q

Name a machine that can be used to sequence PCR products

A

The Illumina Miseq machine

75
Q

What software can you use to process and analyse the PCR sequence products?

A

QIIME2, mothur, DADA2, phyloseq

76
Q

When doing 16S rRNA sequencing, do you need to isolate the bacteria into pure cultures?

A

No

77
Q

What are the pros of 16S rRNA sequencing?

A

inexpensiveness, small quantity of starting material required and availability of large public databases for analysis

78
Q

What are the cons of 16S rRNA sequencing?

A

Bias: as many bacteria have more than one copy of 16S rRNA genes.
Sample handling: atmospheric conditions and temperature must be maintained. Storage: of the samples by freezing can lead to the loss of Bacteroidetes-associated sequences and freeze-thaw cycles will affect the microbial composition present in the sample
DNA extraction methods: can affect the results as bead-beating must be involved in order to minimise the underestimation of gram-positive populations.

79
Q

Too vigorous an extraction method can cause what?

A

damage to genes

80
Q

How can PCR biases arise?

A 
differences in the specificity of the polymerases
interfering substances
contaminated reagents
artefacts
differential amplification
81
Q

Why are negative controls integral in microbiota profiling studies?

A

DNA extraction kits have also been found to not be sterile, so can be a source of contamination

82
Q

When are negative controls especially important during microbiota profiling studies?

A

In studies involving low-biomass samples from the nose, skin, lungs and vagina

Microbiome (10 decks)

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