CELL VIABILITY ASSAY

Presented By

K.PARAMESWARI

CONTENTSIntroduction

Definition

Methods for viable cell counting

Indirect –direct methods

conclusion

References

Determination of viability haveplayed a central role in bacteriology (Postgate, 1967,1969; Barer and Harwood, 1999). In medicalmicrobiology one is concerned primarily withmeasuring the effects of antimicrobial agents,sterilization and disinfection regimens, and withimmune effector mechanism. In addition, only viablebacteria are considered capable of spreadinginfection. For many years it has been the acceptedpractice to equate viability with culture-basedestimations such as colony or most probable number(MPN) counts.

INTRODUCTION

CONCEPTUAL DEFFINITION:-

Retaining the capacity for replicationovera statedor generallyacceptedtime frame.

OPERATIONAL DEFINITION:-

Explicit demonstration of replication in a validation laboratory system.

DEFINITION

Methods for viable counting Indirect viable-cell counting.

1. Dilution methods ( solid, liquid media).

2. Surface viable count (miles & mizra ).

3. Roll tube technique.

Direct viable-cell counting

1. Nalidixic acid method

2. Vital fluorogenic dyes

3. Trypan blue assay

4. Microradioautography

INDIRECT VIABLE-CELL COUNTING

If we want to count only livingcells in a sample, then we will need to use aprocedure that does not count dead cells. Sincewe know that one living cell isolated from anyother living cells will produce an isolated colony,we can culture a well-distributed group ofindividual cells for 24-48 hours on or in an agarplate medium. we would then simply count thetotal number of colonies and assume that eachcolony represents one viable cell from theoriginal sample.

1. Dilution method

In pour plate method the culture is diluted serially and inoculated in to respective test tubes or petri plates. Then the required dilution is taken and used.

In the same way the dilutions can also be used in spread plate and for streaking.

DILUTION METHOD

2 . Surface viable countThis is probably most

accurate of viable count techniques. Here alsoserial dilution is done and culture is placed onagar plates. The plates are incubated and plateswith approximately 20 colonies should becounted.

for e.g: plate 10 has 21 colonies

there fore 21 x 10 = no of colonies in one dropof original suspension.

21 x 10 x 50= 10500000 viable organisms /ml

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3. Roll tube technique

Here, prepare ten-fold dilution and add 0.1 ml of diluted culture to molten agar cooled to 50°c poured in test tube.

Now tilt the tube and roll so that medium is formed as a thin film around the wall of tube

Incubate and count the no of colonies on next day

DIRECT VIABLE-CELL COUNTING

Nalidixic acid method It helps the grow bigger and more stainable but not

divide. Here we compares a fixed (2% formaldehyde) aliquot

of the sample with an unfixed aliquot to which 0.025% yeast extract is added and 0.002% nalidixic acid as an inhibitor of DNA synthesis

Incubate the amended aliquot for a period of 6hr Filtrate the samples, and stain with 0.01% acridine

orange or fluroescein isothiocyanate for epifluroscence microscopy.

Viable cells elongate when exposed to yeast extract and nalidixic acid, inducing non septate filaments in bacillary forms elongated cells are enumerated as viable cells

NALIDIXIC ACID

VITAL FLUOROGENIC DYES

Vital fluorogenic dyes such as 0.02 to 0.05% fluorescein diacetate, when added to the sample are non-fluorscent when taken up by cells but are hydrolyzed by the nonspecific esterases in live cells and remain unchanged in dead cells.

The released fluorescein is easily detected by fluorescence microscopy (with either transmitted illumination or epi-illumination),and the method may be applied to a sample on filtrates by epifluorescence microscopy.

FLUROGENIC DYES

Epi-fluorescence

Trypan Blue Cell Viability Assay

Trypan Blue is a stain commonlyused to differentiate viable and nonviable cells. Thisapplication brief discusses the importance of TrypanBlue and cell viability assays in increasing ourunderstanding of normal and defective apoptoticprocesses.

Here uptake and incroption of H-labelled glucose, acetate aminoacids, or thymidine by specific activities of the active cells.

Treat the samples for 2.5hrs and then fix in 2% formaldehyde and filter by 0.2µm porosity filter

Remove the filter which is developed as silver grains around labelled cells. Add acridine orange and destain it with citrate buffer, covered by spraying thin layer of gelatin. Place a cover slip and observed under epi-fluorescence microscope

Microradioautography

3

FUNCTIONASSAYED

GENERAL METHOD EXAMPLES

Cell integrity Quantitation of cells Microscopy

Optical density, nephelometry, flow cytometry coulter counting,Morphology of cell by light and electron microscopy. Change in phase-contrast appreance.

Cellpermeability

Dye exclusionRetention of intracelluar components.

Propidium iodide exclusion Assay of released DNA or enzyme (eg: lactate dehydrogenase )

Cell nucleic acid content

Cell association DNA and RNA

DAPI staining of DNA . rRNA in-situ hybridisation

Cell enzyme content

Modification of chromogenic or fluorogenic substrate

Fluoresecein in diacetate hrdrolysis, tretrazolium reduction

Energy status Presence of property that requires constant energy input

ATP content, ATP/ADP ratio. Rhodamine 123 accumulation (membrane energisation)

Evidence of integratedfunction

Assay of property that requires function of multiple cell system

Reporter gene expression (e.g. luxAB). Inducible reporter gene expression (e.g. lacz). Labelled precursor incorporation into specific cell production via multiple processing steps (amino acid incorporation,Co2 production, thymidine incorporation)

Transcription Detection of mRNA Uracil incorporation. Specific transcript detection by hybridisation or RT PCR

Evidence of growth potential

Dection of properties that always occur in growing cells

Presence of septa or multiple nucleoids. Cell elongation (kogure test)

CONCLUSION

Analysis of cell viability in cell culture is an important means of evaluating in vitro drug or environmental effects in cell-mediated cytotoxicity assays or for monitoring cell proliferation and health in cell culture.

Gerhardt.P, Murray.R.G.E, Wood.W.A, 1994, Cell Viability assay, In: spublications.

Baker .F.J, Breach .M.R, Leighton.I, Taylor.P, 1980, Cell Viability assay In: Medical microbiological techniques, edn:1st Pp-(422-425), Buther Worth & Co.

Sassmam.M, 2002, Cell Viability assay, In: Molecular medical microbiology, edn: 1st Pp-(221-227), Academic press.

http://www.accademicpress.com. Studzinski.G.P,1999,Viability of cells In: Cell

growth , differentiation and senenscence,edn:1st Page:4,34,66,246.Oxford university press.

http://www.oup.co.uk/PAS.

REFERENCES