Cell Culture Techniques

What limits early detection?

For bloodstream infections:

< 10 bacteria among a billion RBCs, million WBCs, 100 million platelets per mL
of blood. For direct optical imaging at 90x magnification:

Cell concentra+on in sample No. of cells per field of view (FOV)

107 cfu/mL 20 – 30
106 cfu/mL 2 – 3
105 cfu/mL 2 – 3 cells per 10 frames
104 cfu/mL 2 – 3 cells per 100 frames
103 cfu/mL 2 – 3 cells per 1000 frames
102 cfu/mL 2 – 3 cells per 10,000 frames

CFU: Colony forming units

This is a mammoth task! One can either slow scan and collect a large
number of images or apply principles of pre-concentration. In other words,
target cell amplification.
 Bacterial cell culture
Solid media are useful for observations of characteristic colonies, for isolation
of pure cultures and for short-term maintenance of cultures.

Streaking Spreading

For single colony isolation For colony forming units analysis in a sample

** Nutrient agar medium, semisolid

http://www.microbiologyinfo.com/nutrent-agar-composition-preparation-and-uses/
Liquid media are used for growth of large numbers of organisms
or for physiological or biochemical studies and assays.

Broth Slant

For growth in suspension To maintain isolated cultures for long term

** Nutrient broth medium

http://www.microbiologyinfo.com/nutrient-agar-composition-preparation-and-uses/
Types of media
Nutrient media – General cultivation (all nutrients glucose, salt, beef)

Selective media – For isolation of selected bacteria

Antibiotics for antibiotic resistant,
Methylene blue for Gram positive
MacConkey for Gram negative

Minimal media – for wild type strain isolation from recombinants

Differential media – Distinguish between types of bacteria

Blood agar for hemolytic bacteria
Mannitol salt for mannitol fermentation
MacConkey for lactose fermentation

Transport media - For temporary storage (buffer and salt)

Enriched media - For growth of wide variety (blood agar, chocolate agar)
 Selective growth on blood agar
for Gram status

Differential growth based

on bacterial metabolism

Selective charcoal based

detection

Centers for Disease Control and Prevention

 Mammalian cell culture
Culture Flasks for adhesion culture Semi solid medium plates

Culture tubes for suspension culture

DMEM–Dulbecco's Modified Eagle Medium, RPMI Media

Fun with trypsinized cells
(movie speeded 40x)
Planar gold electrode Electrode
Top view

30 - 50 V
30 - 200 Hz
Electrode

Electrode

Maximum field intensity

Electrode
EF-driven assembly: Reversible process

Before electric field During AC field After AC field off

Cell

Permanent binding using Lectin (Concanavalin A)

functionalized particles Particle

Polysaccharides (e.g.
D-mannose)
Cell
Permanent assemblies
In optical microscopy Complementary fluorescence image

Optically responsive

Magnetically responsive
Examples of cell viability of cell chains
formed in AC fields

Yeast cells Mouse fibroblast cells

Using FUN-1 staining method Using Trypan Blue staining method

Electric field does not affect the viability of cells in any significant way.
Magnetically responsive membranes
Point needle electrode

Cell suspension
75 - 100 V
30 - 200 Hz Two-directional alignment &
annealing

2D & 3D Translation & Stretching 3D Rotation & Folding