General Microbial Detection Methods and Drawbacks

Sampling Medium Method Type Details Drawbacks

Air Laser particle counter Total particulates (0.5 to 5.0 µm) Large volume is required
Active air samplers Air sampling units per cubic unit
Liquids and Membrane filtration <0.45 micron pore size filter Time-consuming
emulsions Spread-plate Neat or diluted sample
Pour-plate neat or diluted sample
solution/emulsion
Surfaces Contact-plate Useful for flat surfaces Sampling is always a challenge
Swab Useful for non-flat surfaces;
Surface rinse Useful for tanks, bioreactors, fill lines,
pipes
Tape/adhesive Useful for flat or curved surfaces
Cell suspension Flow cytometry Fluorescently stained microbes, Expensive and requires sample
generally for deal and live assays preparations
Cells or cell Electrical impedance Change in electrical impedance Requires optimization protocols
suspension
Fixed cells Fluorescence staining Imaging under microscopes Requires efficient fluorophores
Lysis of cells Nucleic acid amplification PCR techniques Very costly, time-consuming,
technologies high skill sets required
Cell suspension Light scatter Centrifuge to pellet down cells Purifications required 1
Process of detecting E. coli using colloidal gold

A sensitive colorimetric method for the detection of

Escherichia coli 0157:H7 (E. coli 0157:H7) in the
urine samples. Patients were established with urinary
tract infections (UTI). It was found that a red shift of
the plasmon peak with considerable broadening of the
spectra was achieved, and it correlated with the
concentration of the bacterial population.

‘Plasmons’ are collective oscillations of free

electrons in metals

Bigger size: Low SPR

Assembly: Low SPR
Au nanospheres with the size of 2–50 nm show
only one plasmon band centred at about
520nm, while two SPR bands appear when the
symmetry is reduced from spherical to
cylindrical, i.e. in Au NRs
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 Gold NPs with different shape

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Adopted from: https://iopscience.iop.org/article/10.1088/1361-648X/aa60f3/pdf
Process of detecting Bacteria using Enzyme activity

Cationic gold NPs featuring quaternary amine

headgroups electrostatically bound to an enzyme
(β-galactosidase [β-Gal]). As analyte bacteria
binds to the NPs, it releases the β-Gal and
restores its activity giving an enzyme-amplified
colorimetric readout. This method was
developed on a paper surface to achieve a visual
sensitivity of 104 bacteria/mL

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 Summary
• Bioactives: Materials of biological activity and can be of synthetic or
biological origin

• Bio-factories can be used to produce bioactives

• Novel biosensing techniques can help improving the product quantity

and quality

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 PART IV

Biocompatibility of Biomaterials: Protein structure,

interaction of proteins with synthetic material and
characterization of cell material interactions

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Proteins: Why It is Important to Study Surface Interaction with Biomaterials
• In as short a time as can be measured after implantation in a living system (<1 sec), proteins are already deposit on
biomaterial surfaces.

• In seconds to minutes, a monolayer of as a tightly bound adsorbate, adsorbs to most surfaces.

• The protein adsorption event deposits have on subsequent cellular interactions well before cells arrive at the surface.

• Therefore, cells see primarily a protein layer, rather than the actual surface of the biomaterial.

• Since cells respond specifically to proteins, this interfacial protein film may be the event that controls subsequent
bioreaction to implants.

• Protein adsorption is also of concern for biosensors, immunoassays, marine fouling, a host of other phenomena.

• After proteins adsorb, cells arrive at an implant surface propelled by diffusive, convective, or active (locomotion) mech-
layer on a solid substrate.

• After cells arrive and attach at surfaces, they may multiply to tissues. Synthetic materials can interact with or disrupt
living tissues. The organization of tissues must be understood to appreciate the response to synthetic materials implanted
in those tissues.
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Cell interaction with an adsorbed protein layer on a solid substrate
FIG. 1. The cell is shown as a circular space with a
bilayer membrane in which the adhesion receptor protein
molecules (the slingshot-shaped objects) are partly
embedded. The proteins in the extracellular fluid are
represented by squares and triangles. The receptor
proteins recognize and cause the cell to adhere to only the
surface-bound form of one protein, the one represented by
a solid circle. The bulk phase of this same adhesion
protein is represented by a triangle, indicating that the
solution and solid-phase forms of this same protein have a
different biological activity. The figure is schematic and
not to scale.

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 Amino Acids: Structures in Side Chain
In addition, phenomena at the air-water interface (e.g., interfacial coagulation and foaming), at the oil-water
interface (e.g., the "receptor" proteins located in cell mem branes that serve many important signalling functions), and at the
solid—liquid interface in nonbiomaterial settings (e.g., marine fouling, bacterial adhesion, and cell growth on surfaces in
culture), are all strongly influenced by the behavior of proteins at interfaces,
 Amino Acids: Nature of Side Chain

http://www.cryst.bbk.ac.uk/PPS2/course/section2/SideChains/primary1.html 10
pKa is the negative base-10 logarithm of
the acid dissociation constant (Ka) of
a solution.
pKa = -log10Ka
The lower the pKa value, the stronger
the acid. For example, the pKa of acetic acid
is 4.8, while the pKa of lactic acid is 3.8.
Using the pKa values, one can see lactic acid
is a stronger acid than acetic acid.

HA ⇆ A- + H+

Ka = [A-][H+]/[HA]

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