**4.2 Automated mechanical counting**

The most commonly used automatic cell counting methods are direct electrical impedance, flow cytometry, computer-aided image analysis, and serological counting. Through changes in electrical properties, the direct electrical impedance method quantifies the number and the volume of cells in the blood. Using a photomultiplier to filter and detect the signal, flow cytometry records both the density and height of fluorescent pulses and then converts them to the number of bacteria; the method is fast and sensitive and can simultaneously analyze the cell morphology and protein biomarkers. Computer-aided image analysis [47] and serology [48] counting methods analyze the image or 2D picture to obtain accurate quantification and morphological structure. So far, both methods have been used successfully in biology, materials science, mineralogy, and neurological science.

**45**

**Table 2.**

*Cell Growth Measurement*

culture solution [49].

*4.3.2 BACTEC MGIT method*

*4.3.3 Fluorescent dye method*

**Fluorescent dye Stain** 

SYTO nucleic acid stains

SYTOX green nucleic acid stain

Propidium iodide (PI)

Sulforhodamine B (SRB)

Calcofluor white

*Summary of fluorescent dye.*

M2R

PHK26, 67 Membrane

DiO Membrane

DiD Membrane

DiBAC4(3) Membrane

suitable for high-throughput plate-based AST assays.

**subject**

Nucleic acid [52]

Nucleic acid [53]

Nucleic acid [54]

Protein [55]

[56]

[57]

[58]

Cytoderm [59]

[60]

*DOI: http://dx.doi.org/10.5772/intechopen.86835*

*4.3.1 Turbidity assay by spectrophotometry*

Turbidity can be observed when the cell density reaches certain level; within a certain range, the number of cells is proportional to the turbidity of the bacterial culture. The cell turbidity is measured by a spectrophotometer or a colorimeter, and a standard curve is generated by plotting the absorbance at OD600nm and the actual cell numbers in the sample. Photoelectric turbidimetric counting is a simple, rapid, and continuous measurement suitable for high-throughput screening. However, its optical density is less sensitive, cannot differentiate between dead or live bacteria, and is greatly affected by cell size, morphology, and the color of the

BACTEC MGIT [50, 51] measures microbial growth by oxygen depletion which requires anaerobic conditions, so the bacteria must be grown in a sealed tube or compartment. This method has been widely applied in medical diagnosis but is not

Live or dead cells that cannot be differentiated by the light microscope can be counted after fluorescent labeling. **Table 2** showed the commonly used dyes that

> **Excitation/ emission wavelength**

Yes 420~657/441~678 Live or

~496/~520

Yes ~551/~567,

No ~504/~523 Dead cell Fungus,

No ~530/~635 Dead cell Fungus,

Yes ~565/~586 Live cell Mammals

Yes 482~487/ 501~504 Dead cell Mammals

Yes ~646/~665 Dead cell Mammals

Yes 385~405/ 437~445 Live cell Fungus

Yes 506/ 526 Dead cell Fungus,

**Function Detection**

Live cell Mammals

Fungus, bacteria

bacteria

bacteria, mammals

bacteria, mammals

dead cell

**Membrane permeability**

**4.3 Indirect cell counting methods**

**Figure 4.** *The main methods for cell growth measurement.*
