FMLec | M2 Non-Culture-Dependent Techniques

3 types of non-culture-dependent techniques

• Direct microscopic counts

• Rapid end-detection methods

• Nucleic acid- and protein-based methods

Non-culture-dependent technique

• Total count

• Commonly used for milk and other dairy products (also known as Breed count)

• A measured volume (0.01 mL) of bacterial suspension is placed within a defined area (1 cm²) on a microscope slide

• Steps:

  • Smear

  • Stain

  • Count

Direct microscopic counts

3 steps to direct microscopic counts

1. Smear

2. Stain

3. Count

Explain Petroff-Hausser Cell Counter procedure

1. Grid with 25 large squares, cover glass slide, bacterial suspension is then added and fills shallow volume over squares by capillary action

2. Depth under cover glass and area of squares are known, so volume of bacteria suspension over squares can be calculated (depth x area)

3. Microscopic count: All cells in several large squares are counted, and numbers are averaged. Large square has 14 bacterial cells.

4. Volume of fluid over large square = 1 / 1,250,000

   1. 14 cells x 1,250,000 mL = 17,500,000 cells/mL

Advantages and Disadvantages of Petroff-Hausser Cell Counter

• Advantages rscs

  • Rapid

  • Simple

  • Cell morphology can be assessed

  • Suited for samples with low numbers of bacteria

• Disadvantages enfihm

  • Exhausting to analyst

  • Nonviable cells are enumerated

  • Food particles are not always distinguishable from microorganisms

  • Invariably higher than SPC counts

  • High limit of detection (1.0×10⁷)

  • Motile bacteria are difficult to count

Explain Viability Staining Protocol

• This allows differentiation between viable and nonviable cells because

• Propidium iodide can only penetrate membrane of nonviable lysed cells, turning these into red

5 rapid end-detection methods

erfba

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Reversed passive latex agglutination

• Flow cytometer

• Biosensor

• ATP bioluminescence technique

ELISA limit of detection

• 10⁴ - 10⁶ CFU/mL

• May require pre-enrichment / selective enrichment

Petroff-Hausser Cell Counter limit of detection

1.0×10⁷ (high)

Rapid end-detection technique

• Antigen-antibody based detection system

• May require pre-enrichment or selective enrichment

• Commercially available kits: Campylobacter, Salmonella,

  L. monocytogenes, E. coli O157:H7

ELISA

Direct vs. Indirect vs. Sandwich ELISA

• Direct ELISA for rapid antigen detection

• Indirect if high sensitivity is needed (since this uses secondary antibodies that amplify signals)

• Sandwich if high specificity is required for complex samples, e.g., blood

T/F: Sandwich ELISA cannot be used if the antigen has only one epitope

TRUE

It requires two antibodies binding to different sites on the same antigen; if there’s only one epitope, this wouldn’t work

T/F: The signal intensity in an Indirect ELISA is lower than in a Direct ELISA

FALSE

Indirect ELISA has a higher signal because of the amplification effect from the secondary antibody

T/F: Sandwich ELISA is useful for detecting large, complex antigens with multiple epitopes

TRUE

Since two antibodies target different epitopes, it works well for large, multi-epitope molecules

Rapid end-detection technique

• Used for detection of microbial toxins, e.g., Shiga toxin of Shigella dysenteriae

Reversed passive latex agglutination

Explain principle behind reversed passive latex agglutination

• Latex particles coated with antibodies against target antigen (V-shaped well in microtiter plate)

• Absence of antigen = particles form a dense dot

• Presence of antigen = particles remain dispersed in solution

Flow cytometer limit of detection

10⁴ CFU/mL

Rapid end-detection technique

• Detects microorganisms based on light scattering by cells and fluorescent labels

• Sort cells based on size, shape, complexity, other properties

• Fluorescence‐labeled antibodies or nucleic acid

Flow cytometry

Rapid end-detection technique

• _ refers to analytical device that integrates biological material for target recognition with a physicochemical transducer

• Use either _ to give high specificity

• Biosensors

• DNA probes or specific antibodies

ATP bioluminescence technique limit of detection

1 pg (1000 bacterial cells)

Rapid end-detection technique

• _ has ATP as indicator of living bacterial cells

• Limit of detection = 1 pg = 1000 bacterial cells

• Primarily used as hygiene or cleaning regime monitoring method

ATP bioluminescence technique

T/F: Indirect ELISA is the preferred method when detecting small molecules due to its ability to amplify the signal through multiple secondary antibodies

TRUE

When performing cell sorting via flow cytometry, _ reflects cell size while side scatter (SSC) reflects shape; SSC and fluorescence can also be indicators of viability

forward scatter (FSC)

T/F: Flow cytometry is more sensitive than ATP bioluminescence in detecting bacterial contamination because it does not require cell enrichment

FALSE

ATP bioluminescence can detect as low as 1000 cells, whereas flow cytometry typically requires at least 10⁴ CFU/mL

T/F: ATP bioluminescence can be used to quantify both bacterial and fungal contamination, but it cannot distinguish between them

TRUE

ATP is a universal indicator of living cells, but it does not differentiate between bacteria and fungi

T/F: The ATP bioluminescence technique is limited to detecting only culturable bacterial cells

FALSE

It detects ATP from all living cells, regardless of culturability

T/F: ATP bioluminescence can be used to confirm the effectiveness of antibiotic treatment by measuring a decrease in ATP levels over time

TRUE

T/F: Reversed passive latex agglutination can be used to detect bacterial toxins but cannot differentiate between viable and non-viable bacterial cells

TRUE

10 nucleic acid- and protein-based methods

polpra rvrm

• Polymerase chain reaction (PCR)

• Oligonucleotide DNA microarrays

• Loop-mediated isothermal amplification (LAMP)

• Pulsed-field gel electrophoresis (PFGE)

• Restriction fragment length polymorphism (RFLP)

• Amplified fragment length polymorphism (AFLP)

• Random amplification of polymorphic DNA (RAPD)

• Multi-locus variable number tandem repeats (VNTR) analysis

• Ribotyping

• Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Nucleic-acid and protein-based method

• Amplifies DNA to levels that can be detected

• Repetitive cycle of heating and cooling to amplify the target DNA

• Heat-stable DNA Polymerase and specific primers

PCR

Advantages vs. Disadvantages of PCR

• Advantages hssq

  • High specificity

  • High sensitivity

  • Quick turnaround time to obtain results

• Disadvantages etcp

  • Expensive and high-maintenance equipment

  • Trained personnel

  • Need for extensive DNA clean-up to avoid cross-contamination

  • Need to pre-incubate

    • Limit of detection: ~100 cells

    • Allows to differentiate live and dead cells

PCR limit of detection

~100 cells

The presence of DNA amplification in LAMP can be detected by a _

color change

5 PCR-based methods

em 3r

• EMA/PMA-PCR

• Multiplex PCR

• Real-time or quantitative PCR (qPCR)

• Reverse transcriptase PCR (RT-PCR)

• Rep-PCR

Explain EMA/PMA-PCR procedure

• Sample pretreatment with Ethidium monoazide (EMA) and Propidium monoazide (PMA), which can only enter bacterial cells with damaged cell membranes and intercalate / cross-link their DNA after exposure to strong visible light, preventing DNA amplification

• Low amplicons = high amount of dead bacterial cells (whose DNA amplification has been inhibited)

PCR-based method

• Different primers are used to amplify different DNA

  regions simultaneously

Multiplex PCR

PCR-based method

• Monitors [DNA] as amplification progresses through fluorescence

Real‐time or quantitative PCR (qPCR)

PCR-based method

• Detection and quantification of RNA expression

Reverse transcriptase PCR (RT-PCR)

PCR-based method

• Amplifies noncoding (introns) intergenic repetitive sequences

• Number of amplicons differs between unrelated, non-clonal strains

Repetitive-element PCR

_ can simultaneously detect different sequences in mixed DNA samples

Multipathogen microarray

T/F: LAMP amplification results in linear DNA fragments, similar to PCR

FALSE

LAMP produces long, looped, and cauliflower-like DNA structures instead of linear fragments

LAMP can be performed in a _ instead of a PCR machine.

water bath or simple heat block

T/F: Loop primers in LAMP help accelerate amplification by facilitating strand displacement

TRUE

The loop primers create faster strand displacement and rapid DNA amplification

Nucleic acid-based method

• Consist of hundreds to thousands of specific oligonucleotides on a solid support

• Oligonucleotides (25–80bp)

  • PCR amplified

  • Positions (in a grid pattern) are recorded by spot location

Oligonucleotide DNA microarrays

_ allows autocycling amplification under constant temperature (60-65C)

Bst DNA polymerase (Geobacillus stearothermophilus)

• _ is an alternative to PCR

• Amplification at one fixed temperature

• Colorimetry, fluorescence, bioluminescence

Loop-mediated isothermal amplification (LAMP) technique

LAMP vs. PCR

ttpda	LAMP	PCR
Temperature	65 C only	Varies (cycling)
Time	20-40 mins (shorter)	1.5+ hr
Primers	4-6 primers	2 primers
Detection	Fluorescence (could be real-time or endpoint), color, turbidity	Fluorescence (could be real-time or endpoint)
Amplicon	Concatemeric	Discrete

Explain important role of Bst DNA polymerase in LAMP

Bst DNA polymerase has a strong strand displacement activity that eliminates the need for heat-induced denaturation of strands and thus allows continuous amplification at constant temperature

T/F: Loop primers in LAMP accelerate amplification by providing additional binding sites for DNA synthesis.

TRUE

Loop primers reduce reaction time by creating more initiation points for DNA extension, leading to faster amplification

Nonculture-dependent technique

• Used to separate or resolve very large DNA fragments

• RE digestion of gDNA

• Size-dependent net migration of DNA fragments

• Periodic reversal of polarity of electric field that reorients DNA to move in different direction or travel across the gel matrix

Pulsed-Field Gel Electrophoresis

Nonculture-dependent technique

• Detects variations in restriction sites using DNA probes

• Fragment size = determined by position of restriction sites on gel matrix (identifier)

• Number of bands = determined by number of restriction sites

Restriction Fragment Length Polymorphism (RFLP)

T/F: The pulsed electric field in PFGE is applied at random intervals to maximize DNA movement

FALSE

The pulses are carefully controlled and alternated at specific angles to optimize the separation of large DNA fragments

Nonculture-dependent technique

• Specific co-amplification of restriction fragments formed after RE digestion (50-100 bp)

Amplified fragment length polymorphism (AFLP)

Explain RFLP vs. AFLP

• RFLP = DNA is cut using restriction enzymes, then resolved via southern blot

• AFLP = Amplifies a subset of restriction fragments produced from RE digestion; gel electrophoresis

Nonculture-dependent technique

• Parallel amplification of a set of fragments using short arbitrary primers (10 bp long)

• Differentiates bacteria based on number and length of PCR amplicons

Random amplification of polymorphic DNA (RAPD)

Explain how RAPD vs. RFLP, AFLP

• RAPD

  • No RE digestion step

  • Random regions were amplified due to the use of short arbitrary primers

  • But it is a quick way to detect genetic variation without prior knowledge of genome

_ is unique for each strain and can be used to generate profile for genotyping

Number of variable number tandem repeats (VNTR) copies

Nonculture-dependent technique

• Amplifies VNTR regions

• Allows microorganism identification because the number of VNTR copies is unique for each strain and this can be used to generate profile for genotyping

Multiple-locus Variable Number Tandem Repeats (VNTRs) analysis

Nonculture-dependent technique

• Bacterial identification and characterization based on rRNA genes (DNA is being analyzed)

• Steps ggp

  • gDNA RE digestion

  • Gel electrophoresis

  • Probing with 16S or 23S rRNA sequences (southern blotting; fluorescence)

Ribotyping

Nonculture-dependent technique

• Measures masses of bacterial proteins and/or lipids

• Effective and rapid tool for genus- and species-level identification

• Requires single colony or pure culture

MALDI-TOF MS

T/F: Microarrays can be used to detect SNPs (single nucleotide polymorphisms), but they are less effective for detecting large structural variants in the genome

TRUE

T/F: Southern blotting is commonly used in RFLP analysis to detect specific DNA fragments after electrophoresis

TRUE

T/F: VNTR analysis is more discriminatory than SNP-based genotyping for closely related bacterial strains

TRUE