Typed summary of the lecture content and articles. Includes sampling, detection and identification, metabolism of lactic acid bacteria, lactic acid bacteria in food, probiotics, bacteriocins and recombinant protein production.
Sampling, detection and identification
Sampling of microbes
Considerations when developing a sampling technique
What are we looking for? (influences the analysis and storage) — bacteria/yeast/viruses, pathogens, reasons to test:
testing for contamination, isolating new industrial strains
Where is the sample? (influences equipment needed and collection method) — environmental (eg. air/soil/water),
food (eg. meat/dairy), medical (eg. blood), process (eg. factory surfaces)
Sample consistency (solid, liquid, viscous)
Sampling method — destructive (alters sample) vs non-destructive sampling, sterile sampling, how many samples to
get an accurate representation?
Sample storage/transport — temperature sensitive samples (must be kept in their original state), hazardous samples
Type of analysis required — culturing or molecular
Sampling equipment required? (commercial sampling/testing kits are expensive but validated)
When and where should sampling take place?
For contamination: the areas where the contamination is most likely to occur
Bioprospecting (shotgun: sample whichever microbes are present) vs objective approaches (looking for a specific
organism in the area it is most likely to inhabit)
Basic workflow (sampling strategy)
1. looking for? bacteria, yeast, virus
2. where is the sample? environmental, food, medical, process
3. sample consistency? solid, liquid, viscous, other
4. analyses required? culturing, molecular, other
5. sampling method and how many? destructive, non-destructive, n=x
6. is the sample temperature-sensitive? yes / no
7. is the sample hazardous? yes / no
Importance of sampling
Quality control
Identification of potential industrial strains
Disease diagnosis
Answering fundamental questions (research)
Discussion questions
1. Why are standard operating procedures (SOPs) important when it comes to sampling?
Ensures samples are the same, such that results are valid and accurate, and can be compared.
2. What would be the difference between sampling SOPs in a food processing facility vs a medical diagnostic
facility?
These facilities would have different regulations/standards.
3. Is there an advantage to using commercial sampling kits compared to preparing these “in-house”?
Sampling, detection and identification 1
, These kits are validated for certain purposes/uses and would save time as nothing has to be prepared. However, they
are more expensive.
4. What should be considered when analysis requires samples to have intact nucleic acid?
Environmental conditions (extreme conditions could denature the nucleic acids)
No DNA/RNA contamination
No proteases (which could degrade the nucleic acids)
Organism grown on specific media. No culturing required.
Determines viable cells. Less time-consuming but may require special skills
and complex extraction methods.
Disadvantages: some microbes are unculturable,
labour intensive and time-consuming. Can be used to detect by-products.
Detection: selective or differential media is used to detect if an organism is present (qualitative).
Enumeration:
1. Dilution series and plating to determine colony forming units (CFUs) by counting.
2. Sample filtered through gridded membrane filter, leaving the microbes on the filer, which can then be counted to
determine the CFUs/L.
3. Ten-fold dilution series and determine the most probable number (MPN) using reference tables. Colour change /
turbidity is used to evaluate growth.
Factors to consider when selecting culturing or molecular methods
Time available
Cost
High- or low-throughput
Culture-dependent methods
Growth media contains the components that support microbial growth.
Complex media — unknown chemical composition; provides the nutritional growth requirements for cultivation of
most microorganisms.
Synthetic media — chemically-defined media; contains the minimal nutritional growth requirements for
cultivation of specific microorganisms.
Selective media selects for the growth of a specific organism, excluding all others.
Differential media supports the growth of various microorganisms but contains compounds that allow certain
genera/species to be identified visually.
Selective media:
Antibiotic media — antibiotic prevents the growth of non-resistant microbes (selection of antibiotic strains). Used to
selectively inhibit microorganisms, and is used in cloning.
Mannitol salt agar — high salt concentration inhibits most bacteria (selection of pathogenic/non-pathogenic
Staphylococci). Can also differentiate between organisms that ferment mannitol (which will alter pH).
Sampling, detection and identification 2
, Violet red bile agar — bile salts and crystal violet inhibit most gram-positive bacteria (selection of coliform gram-
negative bacteria).
Neutral red pH indicator
Differential media:
Columbia blood agar — 5% sheep blood detects hemolytic reactions (blood cell breakdown).
Bismuth sulfite glucose glycine yeast agar — bismuth sulfite inhibits bacteria, but Candida species reduce the
compound which results in colony pigmentation.
MacConkey sorbitol agar — E. coli serotype O157:H7 ferments sorbitol more slowly than other microbes and can
therefore be easily differentiated.
EC media with MUG — bile salts inhibit gram-positive bacteria, while E. coli produce glucoronidase which
hydrolyses MUG to a fluorogenic product.
MI agar — E. coli produce β-D-glucuronidase which cleaves IBDG to form a blue compound, while total coliforms
produce β-galactosidase which cleaves MUGal to produce a fluorescent compound.
Kligler iron agar — differentiates between members of Enterobacteriaceae on the basis of their ability to ferment
dextrose and lactose, releasing sulfides which cause a pH change that can be measured (phenol red responds to
acid production).
Black colour indicates hydrogen sulfide production
Culture-independent (molecular) methods
Primers are short DNA fragments that initiate the amplification reaction. Specific to the organism.
Basic polymerase chain reaction (PCR) — amplification of single DNA fragment (DNA denatures, primers anneal
and are then extended), requiring a template, primers, polymerase and dNTPs.
Multiplex PCR (mPCR) — amplification of multiple DNA fragments (additional primer sets added), eg. simultaneous
detection of multiple pathogens.
Real-time PCR (qPCR) — amplifies a specific DNA fragment and quantifies the amount of DNA, more accurate than
PCR. Adds a probe and fluorescence.
SYBR green — binds newly-synthesised double strand resulting in measurable fluorescence (used to quantify
accumulated PCR product).
Labelled probes — reporter in probe results in measurable fluorescence during amplification (used to quantify
specific target gene sequence); useful for multiplex qPCR, fewer false positives.
Reverse transcription PCR (RT-PCR) — additional first step: reverse transcription (RNA → cDNA), eg. the
detection of COVID-19.
ATP-based assays: firefly luciferase assay quantifies the amount of ATP in a sample (indicates overall
microbiological content).
Advantages: quick, gives total microbial load.
Sampling, detection and identification 3
, Disadvantages: ATP is unstable (short half-life) and therefore if the stabilisers used are insufficient, readings will
be misleading.
Custom antibodies can be generated for specific antigens.
Example: Sandwich/indirect/direct ELISA.
Lateral flow tests: sample flows through the strip, moving through several detection lines — detection Ab that
binds to sample (Ag) — test line with secondary Ab (generates line indicating the presence of absence of Ag),
and the control line (indicates whether test worked or not)
Biosensors: bioreceptor detects analyte (recognises target) while the transducer converts bio-recognition energy
(biological interactions) into measurable optical/electrical signals.
Examples of bioreceptors — antibodies, phages, GMO bacteria.
Discussion questions
1. Why is validation of methods and reagents important?
Accuracy and consistency.
2. What are the advantages and disadvantages of using traditional culture methods for detection?
Advantages: validated
Disadvantages: time-consuming, laborious, not all microbes can be cultured
3. What are the advantages and disadvantages of using molecular methods for detection?
Advantages: quick, many options available, able to detect a variety of different things
Disadvantages: expensive equipment, skills required to use equipment
4. How can molecular methods be used to detect viable cells?
Addition of a compound that binds free nucleic acids (which are released by damaged, inviable cells). Washing will
remove the DNA-binding molecules with the corresponding DNA. Viable DNA can then be extracted.
Alternatively: If the cell membrane is damaged but the nucleic acids are still in the cell — SDO denatures the membrane
proteins of injured cells. PMA penetrates the injured cells and binds to DNA. After washing, the viable DNA can then be
extracted.
Identification of microbes
Traditional identification methods
Gram stain — gram-positive bacteria retain crystal violet and turn purple, while gram-negative bacteria remain
pink
Colony morphology — size, shape, colour and consistency of cultured cells
Catalase test — catalase enzyme protects bacteria against oxidative damage by neutralising hydrogen peroxide
(effervesces), used to differentiate between catalase-positive and catalase-negative bacteria
Sporulation tests
Metabolite production
Presumptive identification: growth on differential/selective media
Usually based on the appearance of microbes on the specific media. Chromagenic substrates can be added
which release differently coloured compounds upon enzymatic degradation.
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