Implementing Best Practices and Validation of Cryopreservation Techniques for Microorganisms

Table 2

Some commonly used methods used to assess strain stability following cryopreservation.

Method	Test
Anatomical	Microscopical observation of anatomical structures. For example, spores, conidia, flagella, plastids, and hyphal form.
Culture characters	Analysis of culture morphology in plate culture. For example, pigmentation, abundance of sporulation, presence or absence of sectors, or abnormal growth
Growth rate	Measurement of radial growth of fungi and other mycelial organisms in plate culture [9]
Cell density	Cell counts at set time points using microscopical counting methods, flow cytometry of spectrophotometric approaches
Molecular integrity	PCR fingerprinting approaches (ISSR, AFLP) which assess the whole genome [10, 11]
Viability of cells	The use of chromatogenic or fluorogenic viability indicators. Many available, commonly used ones for fungi and bacteria include fluorescein diacetate (FDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [12] fluorescein isothiocyanate (FITC), FUN-1 viability staining
Enzymic capacity	APIZYM utilisation of naphthyl-bound substrates that yield a chromatogenic change [13, 14]
	4-methylumbelliferone [15–17]
Metabolic stability	High performance liquid chromatography (HPLC) of secondary metabolites [18]
	Thin layer chromatography (TLC) of secondary metabolites [18, 19]
Pathogenicity	The target organisms are inoculated onto test media with the potential control strain (or metabolite/protein extract from the control strain)/or directly onto a plant or animal, and the extent of pathogenicity and mortality are recorded.