Chapter 4 SILVER NANOPARTICLES AND THE YEAST...

Chapter 4

SILVER NANOPARTICLES AND THE YEAST CELL:

INHIBITORY CONCENTRATIONS, EFFECT ON GROWTH,

CELLULAR LOCALIZATION, PERMEABILITY AND

METABOLIC ACTIVITIES

4.1 Introduction

The procedure for the synthesis of nanoparticles in growth media was

standardized and the nanoparticles were characterized by UV-Vis, XRD and

TEM analysis. These aspects are described in the previous chapter. Following the

successful synthesis protocol, the next phase of experiments was designed to

evaluate the effect of nanoparticles on the test organism. The current chapter

describes the experimental findings on the effect of silver nanoparticles (Ag NPs)

on growth, morphology and some physical and biochemical parameters of

Saccharomyces cerevisiae. A discussion on the experimental findings concludes

the chapter.

In assessing the toxicity of an agent the first step, inevitably, is to determine the

minimum dosage limits of treatment. There are reports available on a variety of

protocols for determination of minimum inhibitory and killing concentrations of

nanoparticles including agar disk diffusion (Kim et al., 2007), conductimetric

assay (Sawai, 2003) and culture turbidity and plate count assays. We have

followed the NCCLS recommended protocol as described by Taso et al. (2002).

The yeast growth phases were studied by the turbidity method while the cell

cycle was analyzed in a flow cytometer. Williams et al. (2004), in a pioneering

work, had evaluated the growth responses of microbes to inorganic nanoparticles

and described the importance of such studies. The analysis of yeast cell cycle

under silver nanoparticle stress described in the present study is, to the best of our

knowledge, a novel approach and complements the findings in mammalian cell

lines described by others.

Cell morphology as also the localization of nanoparticles was studied by SEM

and High-resolution TEM analysis of nanoparticle stressed cells. Electron

Silver nanoparticles and the yeast cell 77

micrographs representing the ultra-structure of cells are a potent method for

understanding the effect of nanoparticles and have been widely described in

literature. In one of the most cite works, Sondi and Salopek-Sondi (2004) has

reported the formation of “pits” in the cell wall of bacteria, while the silver

nanoparticles were found to accumulate in the bacterial membrane. In the present

study we have included a cell permeability assay to confirm the observations

from electron micrographs pertaining to membrane/cell wall damage.

The concentration of key cellular metabolites viz. RNA, amino acids and protein

as also the stress protectant – trehalose was evaluated in nanoparticle stressed

cells. While there are no specific literatures pertaining to the impact of

nanoparticles on the metabolic activities of microorganisms; it is well

documented that yeast cells may change their level of metabolic activity, such as

their carbon metabolism and nitrogen metabolism, as well as their stress response

to environmental conditions (Higgins et al., 2001).

4.2 Materials and Methods

A list of all buffers and reagents used in the following protocols are described in

Appendix I.

4.2.1 Saccharomyces cerevisiae strain and growth conditions

The yeast, Saccharomyces cerevisiae, used throughout this study was procured

from the Microbial Type Culture Collection & Gene Bank, Institute of Microbial

Technology, Chandigarh, India as an active culture on slant.

The strain MTCC 36 (equivalent to NRRLY–11857, ATCC26602, and

NCYC975) was originally isolated from sugar refinery.

Cells were grown on yeast malt (YM) (HiMedia Labs) agar slants at 30ºC with

an incubation time of 48 h. Cultures were then transferred and maintained at

refrigerated temperature and routinely sub-cultured at 30 days intervals.


4.2.2 Determination of MIC and MKC

Silver nanoparticles (Ag NPs) of different concentrations ranging from 5.39

µg/ml to 70.07 µg/ml were synthesized in YM media in 100 ml flasks as

described in Chapter 3. One flask containing 40 ml YM media without Ag NPs

was kept as control.

Procedure

(i) The media were then sterilized at 15 psi for 15 m and cooled to ambient

inside a laminar hood.

(ii) Each of the flasks were inoculated aseptically with 100 µl of a 0.1 OD*

culture of MTCC 36.

(iii) Flasks were incubated at 30ºC/120 rpm in a shaker incubator (Orbitek) for

48h.

(iv) At the end of the growth period, turbidity was checked visually as well as

by a spectrophotometer (Spectronic-20) set at 625 nm.

(v) 100 µl culture from each flask was diluted 5-6 times (105 to 10

6) in 0.85%

NaCl and spread on YM agar plates in triplicate.

(vi) The plates were then incubated (NSW) at 30ºC for 48 h and counted to

determine the cfu/ml.

Interpretation

(i) The flask with lowest concentration of Ag NPs that shows no visual

turbidity or gives a transmission of 100% is the minimum inhibitory

concentration (MIC).

(ii) The minimum concentration of Ag NP treated cultures that when plated

on YM agar gives a cfu/ml of < 0.1% of the initial concentration of yeast

was taken to represent the MKC.

*Note: 0.1 OD culture – 40 ml of YM media was inoculated with a loop of MTCC

36 taken from a slant stored at 4°C. Incubated at ambient at 150 rpm for 24 hr.

100 µl of this culture was used to inoculate a fresh YM media of 40 ml. OD at

625 nm checked after 24 h. For MTCC 36 OD after 24 h was found to be 0.125 at

625 nm. 100 µl of this culture was spread on YM agar plates after 6-7 dilutions

to determine the cfu/ml. For MTCC 36, OD of 0.125 corresponds to ~ 1.5 x 108

cfu/ml.


4.2.3 Growth Studies

Silver nanoparticles (Ag NPs) of concentrations 26.95, 48.51 and 70.07 µg/ml

were synthesized in YM media in 100 ml flasks as described in Chapter 3. One

flask containing 40 ml YM media without Ag NPs was kept as a control.

Procedure

(i) Growth was initiated in each flask with 106 cells and flasks were then

incubated in a shaker incubator set at 30ºC/120 rpm.

(ii) Aliquots of 200 µl were collected aseptically at intervals of 0, 6.42, 9.33,

12.25, 26.25, 30, 33.08 and 36.25 h from each of the flasks and stored at

4ºC after adding sodium azide (NaN3) to a final concentration of 10 mM.

(iii) OD of all aliquots were recorded together in a microplate reader (iMark,

Bio-Rad) at 595 nm after an initial mixing for 10 s.

(iv) Data were analyzed with GraphPad Prism®

Version 5.03.

4.2.4 Cell Cycle Analysis

Nanoparticles of concentration 5.39, 26.95, 48.51 and 70.07 µg/ml were

synthesized in YM media as described in Chapter 3. One flask containing 40 ml

YM media without Ag NPs was kept as control. 100 µl of a log phase culture (as

described in Section 4.2.7) was used to inoculate the flasks. The flasks were then

incubated overnight at 30ºC/120 rpm. These cultures were used for the study of

cell cycle by flow cytometry.

Reagents

§ Cold absolute ethanol

§ 0.5 M sodium citrate stock (filtered), 50 mM diluted stock

§ 10 mg/ml RNase A

§ 10 mg/ml propidium iodide (filtered)


Procedure

(i) Cell number in the cultures was determined with a haemocytometer and

107 cells were pelleted by spinning down cultures at 2000 rpm at 4ºC for

5 min.

(ii) 1.0 ml of cold 70% ethanol (Amresco) was added under constant vortex.

Cells thus fixed were stored at 4ºC till further process.

(iii) 0.3 ml of EtOH fixed cells (~ 2-3 x 106 cells) were taken in a 5 ml falcon

tube and 3 ml of 50 mM Na (Hi-Media) citrate was added.

(iv) Cells were mixed by pipetting, centrifuged at 2000 rpm for 5 min and the

supernatant discarded.

(v) Pellets were then re-suspended in 0.5 ml of 50 mM Na citrate containing

0.1 mg/ml RNase A (Sigma-Aldrich) and the tubes were left at 37ºC for

2 h for RNA digestion.

(vi) For staining, 0.5 ml 50 mM Na citrate containing 50 µg/ml propidium

iodide (Sigma-Aldrich) was added so that the final concentration in the

samples was 25 µg/ml.

(vii) Stained cells were processed immediately in a FACSCalibur (BD

Biosciences) flow cytometer and data analyzed with BD CellQuest Pro

software.

Flow cytometer settings

Parameter Detector Voltage Amp Gain

P1 FSC E00 5.57

P2 SSC 427 1.00

P3 FL1 600 1.00

P4 FL2 580 2.00

P5 FL3 650 1.00

P6 FL2-A 2.00

P7 FL2-W 1.00


4.2.5 Cell Viability and Cell Volume

The percentage of viable cells in yeast populations grown under Ag NP stress

was determined by the methylene blue staining method. Nanoparticles of

concentration 26.95, 48.51 and 70.07 µg/ml were synthesized in YM media in

100 ml flasks as described in Chapter 3. Flasks were sterilized, inoculated with

MTCC 36 cells and then incubated for 48 h at 30ºC/120 rpm. At the end of the

growth period, aliquots were taken out for microscopic examination.

Preparation of methylene blue solution

0.01 g of methylene blue (Blue Star) was dissolved in 10 ml distilled water. Then,

2 g sodium citrate dihydrate (HiMedia) was added and dissolved under constant

stirring. The solution was filtered through Whatman No.1 and volume was made

upto 100 ml with distilled water.

Procedure

(i) Cell viability

Equal volumes of the yeast cultures and methylene blue solution were mixed on a

haemocytometer using a wire loop. The cells were examined for staining using 40

x objective in a bright field microscope (Olympus BX51).

Approximately 350 cells were examined in each culture and the percentage of

unstained cells was recorded. This figure was taken to represent the percentage

viability of the cultures.

(ii) Cell volume

For cell volume measurements, the length and width of approximately 100 cells

was measured using a micrometer. Volume was calculated according to the

equation of an ellipse

where, a = length/2; b = width/2


4.2.6 Electron Microscopy of Yeast Cells

Nanoparticles of concentration 26.95, 48.51 and 70.07 µg/ml were synthesized in

YM media in 100 ml flasks as described in Chapter 3. One flask without

nanoparticles was kept as a control. Flasks were sterilized, inoculated with

MTCC 36 cells and then incubated for 24 h at 30ºC/120 rpm. At the end of the

growth period aliquots of 1 ml cultures were collected in 1.5 ml tubes for TEM

and SEM analysis.

4.2.6.1 Analysis by Transmission Electron Microscope (TEM)

Cultures were centrifuged at 3500 rpm for 5 min at 4ºC and supernatant was

discarded. The following standard protocol was then followed for sample

processing:

Fixation

(i) Primary fixation was done in Karnovsky’s fixative for 1 h at 4ºC.

(ii) Tubes were centrifuged and fixative was decanted. Pellets were then

washed thrice with 0.1 M sodium cacodylate buffer for 15 min at 4ºC.

(iii) Post-fixation was done in 1% osmium tetroxide in 0.1 M sodium

cacodylate buffer at 4ºC for 1 h.

(iv) Pellets were again washed thrice with 0.1 M sodium cacodylate buffer

for 15 min at 4ºC.

Dehydration

(v) Dehydration was carried out successively in 30, 50, 70, 80, 90, 95 and

100% acetone at 4ºC. At each concentration the pellets were kept for 15

min with two changes. A final dehydration step was done in 100%

acetone for 30 min at room temperature.

Clearing

(vi) Clearing was carried out twice using propylene oxide at room

temperature for 15 min.


Infiltration

(vii) Infiltration of samples was done according to the scheme below:

Propylene oxide : Embedding medium – 3:1 (overnight)

Propylene oxide : Embedding medium – 1:1 (1 h)

Propylene oxide : Embedding medium – 1:3 (1 h in vaccum)

Pure embedding medium – 1 h at 50ºC

Embedding

(viii) Infiltrated samples were transferred into BEEM capsules and oriented.

Pure embedding medium was then poured into the capsules and

incubated overnight in oven at 50ºC.

Polymerization

(ix) Oven temperature was then raised to 60ºC and kept for 24 h for

polymerization.

Sectioning and Staining

(x) The samples were cut into 60-90 nm sections with an ultramicrotome,

placed on copper grid (200 mesh) and then stained by double staining

method with uranyl acetate and lead citrate.

Imaging

(xi) Viewing of samples and capture of electron micrographs was done in a

JEM-2100 (JEOL, Japan) high resolution transmission electron

microscope (HRTEM) operating at a maximum of 200 kV.

4.2.6.2 Analysis by Scanning Electron Microscope (SEM)

Cultures were centrifuged at 3500 rpm for 5 min at 4ºC and supernatant was

discarded. The following standard protocol was then followed for sample

processing:


(i) Primary fixation was done in 2.5% glutaraldehyde for 1 h at 4ºC.

(ii) Tubes were centrifuged and fixative was decanted. Pellets were then

washed thrice with 1X PBS pH 7.2 for 15 min at 4ºC.

(iii) Dehydration was carried out successively in 30, 50, 70, 80, 90, 95 and

100% acetone at 4ºC. At each concentration the pellets were kept for 15

min with two changes.

(iv) Dehydrated samples were spread on a cover slip and dried at 30ºC for

1 h.

(v) The samples were then mounted on brass stubs with double-sided tape

(vii) Finally samples were sputter coated with gold particles to a thickness of

~ 35 nm and stubs loaded into the instrument for viewing.

(viii) Viewing of samples and capture of electron micrographs was done in

JSM-6360 scanning electron microscope operating at 20 kV.

4.2.7 Cell Permeability Assay

Nanoparticles of concentration 5.39, 16.17, 26.95, 37.73, 48.51 and 70.07 µg/ml

were synthesized in de-ionized water in 100 ml flasks as described in Chapter 3.

A loopful of yeast cells from slants were inoculated into 50 ml YM media and

grown overnight at 30ºC/120 rpm. 100 µl of this culture was then inoculated into

100 ml YM media and grown at 30ºC/120 rpm to log phase (24 h). Cells for the

assay were harvested from this culture by spinning down at 3000 rpm for 10 min

in centrifuge (Remi).

Procedure

Cell membrane damage was studied by adopting the procedure described by

Yphantis et al. (1967).


(i) 2.0 mg cells/ml (wet weight) was mixed in 5 ml Ag NP solutions taken

in falcon tubes. Tubes were incubated in a water bath (GeNei) at 30ºC

for 1 h.

(ii) At the end of the treatment period, cells were spinned down at 3000 rpm

for 10 min and the supernatant was collected.

(iii) Absorbance of the supernatant fluid was recorded at 260/280 nm in a

spectrophotometer (Chemito UV Scan 2600) to account for the UV-

absorbing compounds released from the cells due to Ag NP treatment.

Notes: The data was adjusted to account for the spontaneous release of UV-

absorbing compounds by cells in de-ionized water. Total extractable compounds

was estimated by incubating 2.0 mg cells/ml in 1.0 N HClO4 (perchloric acid) at

100ºC for 1 h.

4.2.8 Metabolite Concentrations

Nanoparticles of concentration 5.39, 16.17, 26.95, 37.73, 48.51 and 70.07 µg/ml

were synthesized in YM media as described in Chapter 3. One flask containing

40 ml YM media without Ag NPs was kept as control. 100 µl of a log phase

culture (as described in Section 4.2.7) was used to inoculate the flasks. The flasks

were then incubated overnight at 30ºC/120 rpm. These cultures were used for the

determination of cellular metabolites viz. – RNA, protein, amino acid and

trehalose

4.2.8.1 Cellular RNA

The total cellular RNA content was determined according to the method

described by Benthin et al. (1991).


Procedure

(i) 1 ml culture was collected from the flasks and OD was adjusted to 0.1

with sterile nuclease free water (Amresco).

(ii) Cells were spinned down at 5000 rpm at 4ºC for 10 m. Supernatant was

aspirated and the pellet was frozen in liquid N2 and stored at -20ºC till

analysis.

(iii) Thawed samples were washed thrice with 600 µl of cold 0.7 M HClO4

and re-suspended in 300 µl of 0.3 M KOH.

(iv) The tubes were incubated at 37ºC for 90 min to hydrolyze RNA.

(v) Samples were then cooled in an ice bath and 100 µl of 3 M HClO4 was

added and volume adjusted to 1 ml with 0.5 M HClO4.

(vi) Supernatant was collected by spinning the samples at 10000 rpm for 10

min at 4 ºC.

(vii) RNA was quantified by measuring the absorbance at 260 nm according

to the Schmidt-Tannhauser method:

where,

A = absorbance at 260 nm

ε = Molar extinction co-efficient = 10800 M-1

cm-1

l = path length

Molecular weight of average nucleotide = 340 g mol-1

4.2.8.2 Total Cellular Protein

Estimation of cellular protein content was done using a GeNei™

Protein

Estimation kit by Lowry’s Method.


Kit Components

(i) Protein standard: BSA

(ii) Solution I: Copper sulphate solution

(iii) Solution II: Alkaline tartarate

(iv) Solution III: Folin reagent

Preparation of Protein Standard and Reagents

5 mg BSA was reconstituted with 1 ml distilled water to get 5mg/ml solution

which is stable at 4ºC for two weeks. 0.1 ml of this solution was diluted with 0.9

ml distilled water just prior to use to get 0.5 mg/ml BSA standard.

To one volume of Solution I, 100 volume of Solution II was added just before use

to make the complex forming reagent.

Procedure

(i) Protein standards and samples were pipetted according to the table below

and the final volume was made up to 200 µl with distilled water.

S. No. Std/Sample

Concentration

(µg)

Amount

(µl)

Water

(µl)

1. Blank 0 200

2. 10 20 180

3. 20 40 160

4. 40 80 120

5. 60 120 180

6. 80 160 40

7. Sample 120 80


(ii) 2 ml of the complex forming reagent was added and the solutions kept at

ambient for 10 min.

(iii) 200 µl of Solution III was added under constant vortex and the solutions

incubated at ambient for 30 min.

(iv) The OD of the solutions was then recorded at 660 nm with a

spectrophotometer (Chemito UV Scan 2600).

(v) A calibration curve was constructed by plotting the OD readings against

protein standards and the sample concentrations were interpolated.

4.2.8.3 Total Free Amino Acid

Measurement of free amino acids was done according to the procedure described

by Holmes et al. (1991) with slight modifications.

Procedure

Sample Preparation

(i) 10 ml cultures were spinned down at 3000 rpm for 10 min to collect the

pellets.

(ii) Pellets were re-suspended in 1 ml of 600 mM cold HClO4 and vortexed

for 2 min.

(iii) The tubes were then centrifuged at 3000 rpm for 5 min.

(iv) The supernatant was collected and neutralized by adding 200 µl of 2 M

K2CO3.

(v) Crystals were removed by spinning at 10000 rpm for 5 min and the

samples were then frozen in liquid N2 and stored at -20ºC till analysis.

(vi) Thawed samples were analyzed by staining with ninhydrin using glycine

as the standard.


Standards

50 mg glycine was dissolved in 50 ml distilled water and 10 ml of this stock

solution was diluted to 100 ml in a volumetric flask to get the working standard

solution. A series of volume from 0.1 to 1 ml of this standard solution gives a

concentration range of 10 µg to 100 µg.

Ninhydrin Assay

(i) To 100 µl of the sample/standards 1 ml of ninhydrin solution was added

and the volume was made up to 2 ml with distilled water.

(ii) The tubes were then heated in a boiling water bath for 20 min.

(iii) 5 ml of the diluents (water:n-propanol) was added and contents were

mixed by pipetting.

(iv) After 15 min, the intensity of the solutions was read in a

spectrophotometer at 570 nm. The reagent blank was prepared as above

by taking 100 µl of 80% ethanol instead of the sample.

(v) A calibration curve was constructed by plotting the OD readings against

glycine standards and the sample concentrations were interpolated.

4.2.8.4 Trehalose

Intracellular and released trehalose from nanoparticle stressed cells was

determined according to Parrou and Francois (1997) with slight modifications.

Yeast cultures, as described in Section 4.2.8, grown at nanoparticle concentration

of 26.95, 48.51 and 70.07 µg/ml were used for the measurements.

(i) 1 ml cultures (1.2 x 107 cells) were spinned down at 12000 rpm for 20

min.

(ii) Supernatant (released trehalose) was transferred to a new microfuge tube

while pellets (intracellular trehalose) were re-suspended in 1 ml ice-cold

water.


(iii) Pellets were then centrifuged at 12000 rpm for 15 s, liquid drained out

completely and stored at – 20 ºC till analysis.

(iv) To the supernatant of step (ii) 0.05 U/ml of trehalase (Sigma-Aldrich)

was added and the tubes were incubated at 37 ºC for 1 h.

(v) 500 µl of the reaction suspension was then added to 500 µl of 16% DNS

reagent.

(vi) The mixture was boiled in a water bath for 5 min and then cooled before

recording the colour intensity in a spectrophotometer (Chemito UVScan)

at 525 nm.

(vii) The pellets from step (iii) were dried and weighed and then 0.25 ml of

0.25M Na2CO3 was added and tubes were incubated in a water bath set at

95-98 °C for 4 h.

(viii) Then 0.15 ml of 1 M acetic acid and 0.6 ml of 0.2 M sodium acetate

were added to each sample.

(ix) Samples were then incubated overnight with 0.05 U/ml of trehalase at

37ºC.

(x) At the end of incubation, samples were centrifuged at 12000 rpm for 3

min.

(xi) Reaction suspensions were separated and steps (v) – (vi) were followed.

(xii) Trehalose content were determined from interpolating the absorbance

values obtained in step (vi) and (xi) in a standard graph. The standard

curve was prepared from the absorbance reading obtained from the

reaction of a series of glucose (0 to 50 µg) with 16% DNS reagent.


4.3 Experimental Findings

4.3.1 Minimum Inhibitory and Killing Concentrations

The procedure followed to determine the minimum inhibitory concentration

(MIC) and minimum killing concentration (MKC) of Ag NPs against yeast was

based on culture turbidity and plate count.The experimental findings are

presented below:

Figure 4.1 Growth of cells in different concentrations of nanoparticles. (a) YM

media (w/o Ag NPs) and YM media with 5.39, 26.95, 43.12, 48.51, 53.90 µg/ml

Ag NPs (left – right) and (b) YM media (w/o Ag NPs) and YM media with 59.29,

64.68 and 70.07 µg/ml Ag NPs (left – right).


Turbidity of the cultures was clearly visible in the tubes containing up to 43.12

µg/ml nanoparticle concentration (Figure 4.1). This observation was confirmed

by recording the percent (%) transmission at 625 nm of each culture (Table 4.1).

For cultures with higher nanoparticle concentrations the % transmission

decreased progressively indicating that the growth of cells was inhibited.

Table 4.1: Evaluation of turbid cultures by spectrophotometer.

Concentration Turbidity % Transmission

Yeast Malt Media + 49.5

5.39 µg/ml + 34.5

26.95 µg/ml + 88.0

43.12 µg/ml + 95.2

48.51 µg/ml - 100.0

The lowest concentration of Ag NPs at which no visible turbidity was observed

and which recorded a transmission of 100% was the MIC value. We found that

48.51 µg/ml was the minimum inhibitory concentration of silver nanoparticles for

S. cerevisiae.

Each of the non-turbid cultures (of concentrations 48.45 µg/ml and higher) was

then plated onto YM agar and the number of colonies were counted (Figure 4.2

and Table 4.2).


Figure 4.2 Growth of cells from non-turbid cultures of different Ag NP

concentrations after re-inoculation in YM agar. Figures represent cultures from

(a) 48.51 9, (b) 53.90, (c) 59.29, (d) 64.68 and (e) 70.07 µg/ml Ag NPs

respectively. A progressive decrease in the number of CFUs/ml at increasing

nanoparticle concentrations is evident.

Table 4.2: Colony forming units per ml in non-turbid cultures.

CONCENTRATION CFU/ML % DECREASE#

48.51 µg/ml 2.1 x107 86.00%

53.90 µg/ml 1.6 x 107 89.33%

59.29 µg/ml 3.1 x 106 97.93%

64.68 µg/ml 2.3 x 106 98.46%

70.07 µg/ml 1.0 x 105 99.93%

# initial CFU/ml was 1.5 x 10

8


It was observed that growth of cells in presence of 70.07 µg/ml of nanoparticles

led to decrease of > 99% of the initial cell concentration. According to the criteria

that the MKC is minimum concentration of Ag NPs where CFU/ml (Colony

Forming Unit/ml) was < 0.1% of the initial concentration, we concluded that

70.07 µg/ml was the minimum killing concentration (MKC) of Ag NPs for S.

cerevisiae.

4.3.1.1 Comparison of antimicrobial effect of Ag NPs and Silver ions

To compare the antimicrobial activity of silver nanoparticles and silver ions, the

susceptibility constants or Z values of silver materials were calculated according

to the following equation:

Where, N is the number of CFUs after exposure to silver material; N0 is the

number of CFUs in the absence of silver materials and C is the concentration of

silver material (ppm or µg/ml).

Using the Z value, a given C value, and the exposure time, the survival fraction

can be predicted. A higher Z value implies that the microbes are more sensitive to

the material, indicating that the materials are more toxic to the microbes (Lee et

al, 2009).

In the present study, the yeast cells were exposed to equivalent concentrations of

Ag NPs and silver ions (Ag NO3) prepared in the growth media (YM media) for a

period of 48 h. At the end of the incubation period, cells were plated onto YM

agar after appropriate dilutions in normal saline and the CFUs were counted.

The experimental findings are presented below in Table 4.3 and Figure 4.3.


Table 4.3: Susceptibility constants of silver nanoparticles and ions.

Ag NPs Silver ions

Concentration

(µg/ml)

Z value

(ml/µg)

Z value

(ml/µg)

5.39 0.0128 0.0023

26.95 0.0145 0.0017

48.51 0.0413 0.0080

70.07 0.1045 0.0522

Figure 4.3 Antimicrobial effects of silver nanoparticles and silver ions. After

growth of 48 h, the CFUs (Colony Forming Unit) were measured to count the

surviving cells.

From the Z values depicted in Table 4.3 it is clear that yeast cells are more

susceptible to silver nanoparticles than to silver ions of equivalent concentrations.

Susceptibility constants for all concentrations of nanoparticles were higher than

those of silver ions. For Ag NPs, Z values ranges from 0.0128 to 0.1045 while for

silver ions it ranges from 0.0023 to 0.0522. At a given concentration the


susceptibility constants were higher for Ag NPs than those of silver ions. The

corresponding values obtained at the MIC (48.51 µg/ml) and MKC (70.07 µg/ml)

doses are significantly higher in case of nanoparticles being 0.0413 (Ag NPs)

against 0.0080 (ions) and 0.1045 (Ag NPs) against 0.0522 (ions) respectively.

This indicates that S. cerevisiae are more sensitive to silver nanoparticles.

The antimicrobial effects of nanoparticles and ions depicted in Figure 4.3

demonstrate that the effects of Ag NPs are more potent than those of silver ions

of an equivalent concentration. Starting with an initial number of 1.5 x 108

CFUs/ml, the decrease in the number of CFUs/ml is significantly higher in case

of cells exposed to nanoparticles than those exposed to silver ions of the same

concentration. This is particularly evident at the MIC and MKC doses where

surviving population was 2.1 x 107 (Ag NPs) against 1.0 x 10

8 (ions) and 1.0 x

105

(Ag NPs) against 3.4 x 106 (ions) respectively. This comparison of the

antimicrobial efficacy of Ag NPs and silver ions was done to preclude the

possibility that the effect observed on yeast cells as a result of exposure to the

nanoparticle solutions might be influenced by any residual silver ions present

therein.

As has been discussed in the previous chapter, a large excess of the reducing

agent NaBH4 (up to 2 x 102 times) than that of the silver salt (Ag NO3) was used

during the preparation of nanoparticles. Under the synthesis conditions employed,

all of the silver salt is reduced and any proportion of silver ions left is negligible.

The effect on the yeast cells arising due to exposure to Ag NPs in YM media is

likely due to nanoparticles alone. As is evident from the comparison of

antimicrobial effects of silver nanoparticles and ions of equivalent

concentrations, the presence of any residual ions left-over from the synthesis

process would not influence the effect of Ag NPs on yeast cells.

In all subsequent experiments of this study, the effect of silver nanoparticles

alone was considered as it was evident that the test solutions was composed

almost entirely of nanoparticles and also, as discussed in the findings above, Ag

NPs have a more potent effect on yeast cells than that of silver ions of equivalent

concentrations.


4.3.2 Cell Viability and Cell Volume

The percentages of viable cells in cultures with different concentration of Ag NPs

were assessed by the methylene blue staining method. Cells in the same cultures

were also measured to determine any changes in volume. The results are

presented below in Tables 4.4 and 4.5.

Table 4.4: Percentage cell viability under silver nanoparticle stress

S. No Nanoparticle

concentration

(µg/ml)

Blue cells Un-stained

cells

% viable

cells

1 Control 17 338 95.2

2 26.95 25 321 92.7

3 48.51 319 32 9.1

4 70.07 343 4 1.2

Table 4.5: Effect of nanoparticles on cell dimensions

S. No Nanoparticle

concentration

(µg/ml)

Mean width

(µm)

Mean length

(µm)

Volume

(Mean ± SD)

(µm3)

1 Control 5.67 7.30 123.32 ±

61.10

2 26.95 5.85 7.44 133.63 ±

101.7

3 48.51 6.11 7.83 153.61 ±

149.8

4 70.07 6.43 8.11 186.28 ±

118.4


The methylene blue assay involves mixing the dye with a yeast sample, with live

cells either excluding or reducing the dye, such that only dead cells stain blue

(Sami et al, 1994). Methylene blue in a sample containing microorganisms gets

reduced to ‘leuko’ or colorless form of the dye at the cell surface via reductase

enzymes present in the cell membrane. The sample is then analysed by light

microscopy. The assay confirmed the earlier observation that yeast cultures lose

viability upon growth in presence of nanoparticles (Table 4.2). There was a

progressive decrease in the number of viable (colourless) cells at higher

concentrations of nanoparticles (Table 4.4). While >95% of cells were found to

be viable in the control culture the percentage of viable cells decreased to 92.7%

at 26.95 µg/ml, 9.1% at 48.51 µg/ml and 1.2% at 70.07 µg/ml Ag NP

concentration. Thus at the MKC less than 98% of the cells were found to be

viable.

The change in volume of yeast cells grown at different concentrations of

nanoparticles is depicted in Table 4.5. The cell volume was observed to increase

at higher nanoparticle concentrations. While the mean volume of cells grown in

pure YM media (control) was found to be 123.32 µm3, it increased to 133.63 µm

3

at 26.95 µg/ml, 153.61 µm3 at 48.51 µg/ml and 186.28 µm

3 at 70.07 µg/ml Ag

NP concentrations.

Table 4.6 t-Test of mean volumes in control and nanoparticle stressed cell

Nanoparticle

Concentration

SD N P value Significance*

26.95 µg/ml 101.7 100 0.3859 No

48.51 µg/ml 149.8 100 0.0626 No

70.07 µg/ml 118.4 100 0.0001 Yes

* Significance at 95% CI

To determine the statistical significance of volume changes due to nanoparticle

exposure, a t-Test was performed between control and stressed cells in the

GraphPad QuickCalcs online software (Table 4.5). It was found that the volume


of cells grown at the sub-lethal concentration of 26.95 µg/ml was not

significantly different (P = 0.3859) from those of the control while the volume

changes of cells grown at MIC of 48.51 µg/ml was close, but not quite

significantly different (P = 0.0626). However, the change in volume of cells

grown at the MKC of 70.07 µg/ml was found to be extremely significant (P =

0.0001).

4.3.3 Growth Kinetics and Cell Cycle

Having determined the MIC and MKC values, we followed the growth curves of

cells under sub-lethal and lethal doses of Ag NPs; a control set of cells were

grown in plain YM media for the sake of comparison. The observations are

presented below.

Table 4.7 Data of growth curves: Mean OD at different time intervals

Time (h)

Nanoparticle Concentrations

Control 26.95 µg/ml 48.51 µg/ml 70.07 µg/ml

Mean (OD) ± SD Mean (OD)± SD Mean (OD)± SD Mean(OD)± SD

0.00 0.017 ± 0.020 0.120 ± 0.031 0.109 ± 0.026 0.004 ± 0.007

6.42 0.019 ± 0.020 0.125 ± 0.026 0.022 ± 0.023 0.012 ± 0.009

9.33 0.153 ± 0.035 0.132 ± 0.029 0.035 ± 0.036 0.011 ± 0.006

12.25 0.410 ± 0.041 0.323 ± 0.035 0.041 ± 0.033 0.014 ± 0.008

26.25 0.828 ± 0.052 0.627 ± 0.041 0.033 ± 0.040 0.004 ± 0.008

30.00 0.577 ± 0.047 0.493 ± 0.038 0.034 ± 0.043 0.001 ± 0.005

33.08 0.415 ± 0.031 0.517 ± 0.034 0.021 ± 0.029 0.001 ± 0.006

36.25 0.410 ± 0.027 0.506 ± 0.036 0.020 ± 0.037 0.003 ± 0.005

OD values are mean of three independent experiments.

Table 4.7 presents the optical density measurements of yeast cultures grown in

different concentrations of silver nanoparticles. These data were used to plot the

growth curves of cultures, represented in Figure 4.4.


Figure 4.4 The effect of various concentrations of Ag NPs on the growth of S.

cerevisiae. (a) Control; (b) 26.95 µg/ml; (c) 48.51 µg/ml and (d) 70.07 µg/ml Ag

NP concentrations.

As is clear from Figure 4.4, Ag NPs at the concentrations of 48.51 µg/ml and

70.07 µg/ml completely abolished yeast growth, whereas it was marginal at a

concentration of 26.95 µg/ml. The control sample (grown in the absence of Ag

NPs) showed no growth inhibition.

The growth data were analyzed with a nonlinear regression model (curve fit)

using the exponential growth equation:

Where,

Y0 is the Y value when X (time) is zero. It is expressed in the same units as Y,

k is the rate constant, expressed in reciprocal of the X axis time units.

Tau is the time constant, expressed in the same units as the X axis. It is computed

as the reciprocal of k.

Doubling-time is in the time units of the X axis. It is computed as ln(2)/K.


The results are presented below in Table 4.8.

Table 4.8 Growth parameters of S. cerevisiae under Ag NP stress

Figure Nanoparticle

Concentration

(µg/ml)

Y0 K

(h-1

)

Tau

(h)

Doubling

Time

(h)

4.4 a 0 0.1721 0.03407

29.35

20.35

4.4 b 26.95 0.1620 0.03617

27.65

19.17

4.4 c 48.51 0.08147 -0.05196

-19.25

-13.34

4.4 d 70.07 0.01057 -0.03098

-32.28

-22.37

It is evident that cells grown in the presence of silver nanoparticles witness

significant changes in growth kinetics. While the growth rate (k) and doubling

time were observed to be similar for the control and 26.95 µg/ml concentrations;

the cultures reported negative growth rates at the MIC and MKC concentrations.

It can be concluded that silver nanoparticles have little effect on the growth of

cells at concentrations well below the lethal doses; however they are able to

significantly alter the growth parameters at higher doses.

We studied the cell cycle stages of S. cerevisiae under nanoparticle stress in an

effort to determine whether the cells get arrested at a certain stage of growth and

also the percentage of apoptotic cells, if any. The results of flow cytometry

analysis pertaining to Ag NP stressed cells are presented below.


Figure 4.5a Analysis of yeast cells by flow cytometry. The scatter plots indicate

the granularity and staining (by PI). The purple plots (below) show the effect of

Ag NPs on the cell cycle. The x-axis indicates the fluorescent intensity of PI,

while y-axis indicates cell numbers (events). Cells in this analysis were treated

with (bottom, left to right) control, 5.39 µg/ml and 26.95 µg/ml Ag NPs.

Figure 4.5b Analysis of yeast cells by flow cytometry. The scatter plots indicate

the granularity and staining (by PI). The purple plots (below) show the effect of

Ag NPs on the cell cycle. The x-axis indicates the fluorescent intensity of PI,

while y-axis indicates cell numbers (events). Cells in this analysis were treated

with (bottom, left to right) 48.51 µg/ml and 70.07 µg/ml Ag NPs.


Table 4.9 Percentage events (Gated) and Markers in cultures grown at different

Ag NP concentrations.

Nanoparticle

Concentration

Markers

G0/G1 S G2/M

%

Gated

CV %

Gated

CV %

Gated

CV

0 26.10 9.36 51.39 12.11 14.50 7.43

5.39 µg/ml 14.68 9.85 43.26 11.75 31.77 7.63

26.95 µg/ml 13.50 8.70 45.30 10.98 33.58 7.73

48.51 µg/ml 4.20 9.15 40.18 12.10 45.65 7.16

70.07 µg/ml 5.21 9.78 32.12 11.48 52.17 7.55

Figure 4.6 Histogram indicating cell cycle progressions under nanoparticle

stress. Blue bars (1) are for control cells (without Ag NPs) while the green are for

cells grown under different nanoparticle concentrations viz. (2 – 5) 5.39, 26.95,

48.51 and 70.07 µg/ml respectively. Error bars represent % CV.


As shown in Figure 4.6, silver nanoparticles clearly has an effect on the cell cycle

progression of yeast cells. A total of 10000 events were analyzed for each of the

different nanoparticle concentrations. It was observed that the percentage of cells

in the G2/M phase increased by over 37% (from 14.5 in control cultures to 52.17

in cultures with 70.07 µg/ml Ag NPs). While on the other hand, the percentage of

cells in the G1 phase significantly decreased by about 22% (from 26.1 in control

cultures to 5.21 in cultures with 70.07 µg/ml Ag NPs) in the presence of Ag NPs

(Table 4.9). These variations are positively correlated with increasing Ag NP

concentrations. The percentage of apoptotic cells in each of the cultures was

variable ranging from about 0.6% (at 26.95 µg/ml Ag NPs) to ~ 8% (at 48.51

µg/ml Ag NPs); the control set had ~ 6% apoptotic cells. There was no strong

evidence to suggest that apoptosis was induced by silver nanoparticles. The flow

cytometry data are presented in Appendix III.

4.3.4 Electron Microscopy and Cellular Localization of Nanoparticles

Yeast cells were studied by transmission and scanning electron microscope to

observe changes in the surface and intracellular morphology induced by growth

under silver nanoparticles stress. The micrographs were compared with those of

cells grown without Ag NP stress. The observations are presented in the

photographs below.

Figure 4.7 Scanning electron micrograph (SEM) of yeast cells grown in YM

media (Magnifactions: 3500X, 15000X & 25000X).


Figure 4.8 SEM micrograph of yeast cells grown in YM media with 5.39 µg/ml

Ag NPs (Magnification 3500 X & 25000X).

Figure 4.9 SEM micrograph of yeast cells grown in YM media with 26.95 µg/ml

Ag NPs (Magnification 3500X & 25000X).

Figure 4.10 SEM micrograph of yeast cells grown in YM media with 48.51

µg/ml Ag NPs (Magnification 3500X & 25000X).


Figure 4.11 SEM micrographs of yeast cells grown in YM media with

70.07µg/ml Ag NPs (Magnification 3500X, 15000X & 25000X)

Yeast cells were grown for 48 h in YM media and in YM media having different

concentrations of nanoparticles before fixation in 2.5% glutaraldehyde for SEM

analysis. The filamentous morphology of cells may be attributed to prolonged

growth and subsequent limitation of nitrogen in the growth media.

When compared to the control, those cells taken from cultures having Ag NPs

show a distorted surface morphology i.e. the cell wall/membrane is not smooth as

is observed in the case of cells grown in YM media without nanoparticles. The

outer layer may be said to be ruptured or segmented. This observation was found

to be more pronounced in the case of cultures with higher concentrations of

nanoparticles particularly at the lethal concentrations of 48.51 and 70.07 µg/ml. It

is obvious from the SEM micrographs that yeast cells also show significant

changes in their outer layers, which might be crucial in explaining the mechanism

of silver nanoparticle toxicity.

Further changes in cellular morphology were studied by transmission electron

microscope, the observations are presented in the following micrographs.


Figure 4.12 TEM micrographs of yeast cells grown in YM media (left) and 48.51

µg/ml Ag NP concentration (right). Arrows in the right image indicate deposition

of nanoparticles in the cell wall and breakage of outer layer.

Figure 4.13 High-resolution TEM micrographs of yeast cells grown in YM

media without nanoparticle stress. (a) Different growth forms (Scale bar = 1µm)

(b) budding cell (Scale bar = 100 nm) (c) & (d) A typical ellipsoidal and

filamentous cell at high magnification (Scale bar = 100 nm)


Figure 4.14 HRTEM micrograph of a yeast cell grown in YM media without

nanoparticles. Image on right shows a magnified view of the intact cell wall.

Figure 4.15 HRTEM micrograph of yeast cells grown in YM media with 70.07

µg/ml Ag NPs. Arrows indicate deposition of nanoparticles on the cell wall (b)

and in cytoplasm (c). Chromatin distribution is irregular (d).


Figure 4.16 HRTEM micrograph of yeast cell grown in YM media with 70.07

µg/ml Ag NPs showing deposition of nanoparticles on the outer layers and

destruction of cell wall.

The electron micrographs reveal that yeast cells in the cultures are dimorphic, i.e.

exhibiting both the typical ellipsoidal as well as filamentous forms (Figure 4.13).

Cells grown in YM media only have a well-defined and intact cell wall (Figure

4.14). The distribution of chromatin is uniform and in general the cultures exhibit

‘good health’ such as budding (Figure 4.13 b) and a well defined shape.

In cultures that were grown in the presence of nanoparticles, the striking feature

is the lack of a defined cell wall Figures 4.12 & 4.16). These cells appear to be

‘diffuse’ with part of the cytoplasm exposed to the elements (Figure 4.15 c & d).

The chromatin is either unrecognisable (Figure 4.16) or sequestered towards the

outer envelope (Figure 4.15 d). It was observed that a majority of the

nanoparticles were localized in the cell wall/membrane (Figures 4.15 b & 4.16)

seemingly embedded in the outer matrix. Some particles were also detected

inside the cytoplasm (Figure 4.15 c) in areas where the cell wall was completely

destroyed. Possibly the particles are tightly bound to the cell surface groups and

diffuse inside once the cell wall has dissolved. The disruption of outer layers has

been reported in previous studies as an important mechanism of nanoparticle

mediated inhibition of microorganisms (see discussion below). This aspect was

validated by an in vitro cell permeability assay, the results of which are presented

below.


4.3.5 Cell Permeability Assay

The assay was performed to investigate any change in membrane permeability of

yeast cells exposed to silver nanoparticles. As cells may spontaneously release

constituents into the suspending medium, an appropriate control (water) was set.

Values in Figure 4.17 represent readings after adjustment with the control.

Table 4.10 Data of cell permeability assay: Absorbance readings of culture

supernatants

Sample

(incubated in)

Absorbance Adjusted Values (Mean ± SD)

260 nm 280 nm 260 nm 280 nm

Water 0.404 0.248 - -

Perchloric Acid 0.974 0.784 0.570 ± 0.035 0.536 ± 0.042

5.39 µg/ml Ag NP 0.552 0.349 0.148 ± 0.041 0.101 ± 0.028

16.17 µg/ml Ag NP 0.581 0.347 0.177 ± 0.049 0.099 ± 0.028

26.95 µg/ml Ag NP 0.443 0.304 0.215 ± 0.025 0.125 ± 0.042

37.73 µg/ml Ag NP 0.908 0.468 0.504 ± 0.032 0.220 ± 0.028

48.51 µg/ml Ag NP 1.203 0.645 0.799 ± 0.042 0.397 ± 0.056

70.07 µg/ml Ag NP 2.164 1.327 1.760 ± 0.038 1.079 ± 0.046

Figure 4.17 Release of UV-absorbing compounds from cells after treatment with

perchloric acid (PCA) and nanoparticles. Values are mean of three readings, error

bars represent SD.


The in vitro membrane permeability assay confirmed the damage caused by Ag

NPs to cell membrane. The release of UV-absorbing compounds from cells

increased in proportion to the concentration of nanoparticles (Figure 4.17) present

in the growth media. At the MIC of 48.51 µg/ml nanoparticles mediated the

release of metabolites which was similar to that of perchloric acid (~ 1.08 times

that of PCA). There was a dramatic increase in the permeability of cells at the

MKC of 70.07μg/ml nanoparticle when the release of metabolites were more

than 2.5 times than that released after digestion in 1N HClO4 at 100°C for 1 hour.

4.3.6 Cellular Metabolites

Changes in intracellular concentrations of metabolites viz. RNA, amino acid,

protein, and trehalose in yeast cells grown under nanoparticle stress are presented

below.

Table 4.11: Total RNA content in nanoparticle stressed cells

Nanoparticle Concentration

(µg/ml)

RNA Content

(Mean ± SD)#

(µg/ml)

0.00 27.30 ± 4.31

5.39 25.70 ± 7.09

16.17 26.65 ± 7.11

26.95 26.65 ± 12.73

37.73 42.30 ± 6.65

48.51 3.64 ± 2.83

70.07 2.31 ± 1.70 # Mean of three separate experiments

Figure 4.18 Histogram representing variations in RNA content in response to

nanoparticle concentrations in growth media. Error bars indicate SD.


Table 4.12: Free amino acid content in nanoparticle stressed cells


(µg/ml)

Amino acid

(Mean ± SD) #

(µM/ml)

0.00 0.320 ± 0.040

5.39 0.156 ± 0.007

16.17 0.148 ± 0.016

26.95 0.141 ± 0.006

37.73 0.129 ± 0.011

48.51 0.008 ± 0.004

70.07 0.005 ± 0.002

# Mean of three separate experiments

Figure 4.19 Histogram representing variations in free amino acid content in

response to nanoparticle concentrations in growth media. Error bars indicate SD.


Table 4.13: Total cellular protein content in nanoparticle stressed cells


(µg/ml)

Protein

(Mean ± SD) #

(mg/ml)

0.00 0.084600 ± 0.00410

5.39 0.037800 ± 0.00085

16.17 0.034400 ± 0.00212

26.95 0..030900 ± 0.00085

37.73 0.020500 ± 0.00113

48.51 0.008950 ± 0.00035

70.07 0.003800 ± 0.00010


Figure 4.20 Histogram representing variations in protein levels in response to



Table 4.14: Trehalose content in nanoparticle stressed cells

Nanoparticle

Concentration

(µg/ml)

Trehalose

(Mean ± SD) #

(µg/mg)

Intracellular Released

0.00 11.2 ± 2.97 9.8 ± 2.54

26.95 17.8 ± 3.25 16.6 ± 1.98

48.51 25.8 ± 1.41 47.3 ± 1.55

70.07 26.2 ± 1.55 49.6 ± 2.05


Figure 4.21 Histogram representing variations in trehalose levels in response to



Cellular metabolites levels in yeast cells were clearly influenced by presence of

nanoparticles in the growth medium. Free amino acid and total cellular protein

content showed a gradual decrease when cells were grown in increasingly higher

concentrations of Ag NPs (Tables 4.12, 4.13 and Figures 4.19, 4.20). At the MIC

concentration of 48.51µg/ml and higher there was very low level of nitrogenous

metabolites. Interestingly RNA content (Table 4.11 and Figure 4.18) was almost

stable and then decreased significantly at the MIC and MKC concentrations.

It might be possible that nanoparticles adversely affect protein synthesis which

leads to lowered expression of genes while RNA tends to accumulate at normal

levels in stressed cells. This aspect was further investigated by expression

profiling of genes involved in protein synthesis, the results of which are described

in Chapter 5.

Trehalose levels, both intracellular and released, increased in nanoparticle

stressed cells (Table 4.14 and Figure 4.21). Extracellular trehalose was more than

five times higher at the MIC and MKC compared to control. This can be

explained by the fact that nanoparticles damage cell membrane and increases

permeability which leads to extrusion of intracellular compounds. This was

demonstrated in the cell permeability assay described in section 4.3.5.

Nanoparticle stressed cells also accumulated more trehalose (intracellular) than

the control set.

4.4 Discussion

The minimum inhibitory and killing concentrations, MIC and MKC values were

found to be 48.51 µg/ml and 70.07 µg/ml respectively. There have been

numerous reports on the anti-bactericidal effects of nanoparticles (see Rai et al.,

2011 for review), however data on eukaryotic cells are limited. Kim et al. (2007)

reported a MIC value of >6.6 nM against yeast (ATCC19636) while Panacek et

al. (2009) report that against Candida albicans, non-stabilized Ag NPs have an

MIC of 0.42 mg/L. These values are lower than those found in the current study.

In a recent study, Nasrollahi et al. (2011), investigated the antifungal effects of


silver nanoparticles on Candida albicans (ATCC 5027) and Saccharomyces

cerevisiae (ATCC 5027). They reported MIC50 of 0.5 mg/ml and MIC90 of 2

mg/ml against C. ablicans and against S. Cerevisiae the MIC50 was 4 mg/ml

while MIC90 was reportedly 32 mg/ml. When compared to our study these

values are significantly higher.

It is pertinent to note that inhibitory concentrations are dependent on the species

tested and the method employed. It is also reported (Ruparelia et al., 2008) that

strain specific variation in MIC/MBC (minimum bactericidal concentration) is

present in case of silver and copper nanoparticles against E. coli. The use of

stabilizing agents in particle synthesis also contribute towards inhibition,

however in the present study this was avoided by direct synthesis of nanoparticles

in growth media without the use of stabilizers. The MKC value of 70.07 µg/ml

provided an upper limit of treatment in experiments concerning metabolic and

gene expression studies.

Viability is an essential analytical measurement for quantifying cell culture. It is

most frequently determined by microscopic examination of stained cells. The dye

most widely used to measure yeast viability in is methylene blue. Although

relatively inexpensive and easy to perform, the assay produces inconsistent

results, with many observations of both overestimation and underestimation of

viability. Furthermore, analysis is subjective, and the relatively low number of

cells that can be analysed is not representative of the entire yeast population

(Byod et al., 2003). We found that the methylene blue assay of cell viability

largely confirmed the experimental results obtained for determining the MIC and

MKC of Ag NPs by plate count. At 70.07 µg/ml i.e. the minimum killing

concentration, only 1.2% of the cells were found to be viable or in other words a

>98% reduction in cell viability. It might be recalled that at this concentration

there was a decrease of 99.93% in the number of CFUs/ml when assessed by

plate count (Table 4.2).

The mean cell volume was observed to increase in cultures grown at higher

concentrations of Ag NPs, this change was found to be extremely significant (P =


0.0001), when cells were grown at 70.07 µg/ml nanoparticle concentration.

Under suboptimal conditions, cells developed higher specific volumes

(surface/volume ratio), and in the case of inefficient oxygenation, this could be

explained by an increase of the cell surface in response to a lack of dissolved

oxygen in the growth medium (Furukawa et al., 1983). In a recent study,

Reisetter et al. (2011) determined whether reduced plasma membrane integrity

affected cell size/volume of human macrophage cell line after exposure to carbon

black (CB) nanoparticles. The authors report an increase in both the size and

cellular volume with CB nanoparticle exposure.

To the best of our knowledge there has been no previous study on the growth

cycle of S. cerevisiae under silver nanoparticle stress. The absorbance readings

were recorded in a microplate reader, which was a novel approach. As all

samples from the entire growth period were analyzed together, variations in

absorbance readings were minimized and the process was convenient for high-

throughput. Toussaint and Conconi (2006) had described a method to determine

the sensitivity of growing yeast to DNA-damaging agents that is based on

automatic measurements of the optical density of very small (100 µl) liquid

cultures. Starting with the treatment of cells with different doses of damaging

agents, pre-prepared growing media containing 96-well plates were inoculated

and cell population automatically monitored every 10 min for 48 hours.

Reportedly the sensitivity of a number of yeast strains to several concentrations

of drug can be tested in triplicate in less than 4 hours. Microplate based assays for

cell growth may be an effective alternative to the traditional spectrometer and cell

counts.

Representative growth profiles of the yeast cultures in presence of varying

concentration of silver nanoparticles are depicted in Figure 4.4. At concentrations

below the MIC, the doubling time was not significantly different from the control

cells. The lag phase however was longer being 10 h for cells grown in 26.95

µg/ml Ag NPs compared to 6 h for the control. A greater lag phase and lower

maximum absorbance (at 600 nm) in eight different strains of bacteria were

reported by Ruparelia et al. (2008) as the concentration of silver and copper


nanoparticles increased. Similar observation was reported by Sondi and Salopek-

Sondi (2004) in their studies on effect of silver nanoparticles on a single strain of

E. coli. In the current study we had found a negative growth rate (k) for cultures

grown at the MIC and MKC concentrations, the trace of the curves being similar

to those reported by Gogoi et al. (2006) for E. coli grown under silver

nanoparticle stress. However, at the sub-lethal concentration of 26.95 µg/ml the

growth rate was similar to that of the control. A study by Williams et al. (2006)

evaluated the growth of E. coli exposed to silica, silica/iron oxide, and gold

nanoparticles. Growth curves were generated for E. coli growing in 100 ml of LB

media containing silica/iron oxide nanoparticles at a concentration of 2.2 × 10-3

g/ml of solution. Under these growth conditions, there was no evidence that the

nanoparticles prevented the microbial cells from growing. The authors confirm

that the growth curves for E. coli growing with and without silica/iron oxide

nanoparticles have little difference and that it is possible that there may be more

subtle changes in cell function and behaviour detectable at the gene or protein

level. It must however be noted that in their study the growth was evaluated at

only one concentration of nanoparticles and the inhibitory and bactericidal

concentrations of the nanoparticles were not assessed. The important similarity is

that as in the present study, the growth of cells exposed to non-lethal doses of

nanoparticles closely resembles to that of control cells not exposed to

nanoparticles.

The use of flow cytometry to monitor the cell cycle position of individual cells in

a population has been a standard tool for cell cycle studies in both mammalian

cells and yeast. Fluorescence can be measured for cells by a flow cytometer

following fixation and staining with a fluorescent dye that binds DNA

quantitatively. Individual cells in a population can then be assigned to a cell cycle

position based on DNA content, as defined by fluorescence intensity (Haase and

Reed, 2002). In order to elucidate the physiological changes of the yeast cells

induced by silver nanoparticles, a flow cytometric analysis of the cell cycle was

performed. The cells were cultured in the presence or absence of nanoparticles

and their DNA content was determined via flow cytometry by staining with


propidium iodide (PI). PI is a DNA-staining dye that intercalates between the

bases of DNA or RNA molecules (Tas and Westerneng, 1981). The flow

cytometric profiles of control cultures showed that they had about 26% of cells

with the DNA content of the G1 phase and only 14.5% of cells with the DNA

content of the G2 phase, during the 24 h of incubation (Figures. 4.5a,b & 4.6). In

cultures with Ag NPs, the proportion of cells with the DNA content of the G2

phase more than doubled, accounting for upto 52% of all cells at the MKC of

70.07 µg/ml. The increase was seen to be positively correlated with the

nanoparticle concentration. The fact that cells tend to accumulate in the G2/M

phase seems to indicate that nanoparticles do not affect DNA replication but

inhibited normal bud growth. Endo et al. (1997) reported similar results with 80-

85% of cells in G2 upon treatment with Aureobasidin A, a fungicide. The authors

conclude that inhibition of bud growth correlates with maintenance of the

integrity of the cell membrane.

The G2/M phase arrest seems to be a recurring phenomena in Ag NP mediated

toxicity. It was earlier reported that starch-coated silver nanoparticles caused cell

cycle arrest in G2/M phase in human lung fibroblast cells (IMR-90) and

glioblastoma cells (U251) (Asha Rani et al., 2009). The authors speculate that the

accumulation of cells at the G2/M interface is associated with DNA repair which

could lead to cell death at a later stage if repair pathways were unsuccessful.

In a study on the cellular uptake of AS-COOH nanoparticles by A549 cell lines

(Kim et al., 2011) it was reported that uptake of nanoparticles by cells is also

influenced by their cell cycle phase. Although cells in different phases of the cell

cycle were found to internalize nanoparticles at similar rates, after 24 h the

concentration of nanoparticles in the cells could be ranked according to the

different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells

are not exported from cells but are split between daughter cells when the parent

cell divides. As from our study, we found a G2/M phase arrest; a higher

concentration of nanoparticles in a large proportion of the cell population could

lead to increased toxicity as particle toxicity is dose dependent.


The study of yeast cells by electron microscope revealed changes induced by

nanoparticle stress (Section 4.3.4). A large proportion of cells (both control and

stressed) were elongated. During vegetative growth, the ascomycete

Saccharomyces cerevisiae is able to adopt one of two distinct morphologic forms,

designated the yeast form and the filamentous form (Edgington et al., 1999). In

the phenomenon known as pseudohyphal growth, diploid cells become

filamentous when grown on solid agar medium containing a rich carbon source

but limited for nitrogen (Gimeno et al., 1992). In the present study cells were

cultured for 24 h in 100 ml flasks, before fixation and image capture. Such

conditions can be attributed for the filamentous growth of cells.

Scanning electron micrographs (Figures 4.8 – 4.11) show damage to cell

wall/membranes in cultures exposed to nanoparticles. This aspect is further

confirmed in the TEM images (Figures 4.12 – 4.16) where the disruption of

membrane integrity and uneven distribution of chromatin is evident. Cells grown

in absence of Ag NPs (Figure 4.14) show an intact outer layer and homogenous

distribution of chromatin, whereas deformities in the cell wall are observed for

those cells grown in presence of Ag NPs (Figure 4.16). Dark spots, construed to

be nanoparticles, were deposited on the outer layers as well as in the cytoplasm

indicating deposition and penetration of the particles inside cells. It is likely that

the interaction between the particles and the membrane is non-specific rather than

specific between the nanoparticles and a particular component of the membrane

such as a surface expressed protein (Williams et al., 2006). There are reports in

the literature that show that electrostatic attraction between negatively charged

bacterial cells and positively charged nanoparticles is crucial for the activity of

nanoparticles as bactericidal materials (Stoimenov et al., 2002). In contrast,

Sondi and Salopek-Sondi, (2004) used negatively charged silver nanoparticles

which interacted with the constituents of the outer membrane of E. coli and

despite their negative surface charge, they somehow interacted with “building

elements” of the bacterial membrane, causing structural changes and degradation

and finally, cell death. The authors showed by TEM analysis the existence of

elementary silver in the membranes of treated bacteria, detected by EDAX,


thereby confirming the incorporation of silver nanoparticles into the membrane

structure. This observation is crucial for explaining the antibacterial mode of

these particles. It is clear that treated bacteria also show significant changes in

and damage to membranes, which are recognized by the formation of “pits” on

their surfaces (Sondi and Salopek-Sondi, 2004). A similar effect was described

by Stoimenov and co-workers (2002) when E. coli bacteria were treated with

highly reactive metal oxide nanoparticles. A recent study (Despax et al., 2011)

reported sulphur-containing silver clusters preferentially located at the cell wall

periphery together with nodules composed of silver, sulphur and phosphorus all

over the cell. Electron-dense regions and apparent destruction of chromatin are

visible inside cells treated with Ag NPs.

A yeast membrane with this aberrant morphology exhibits a significant increase

in permeability, leaving the cells incapable of properly regulating transport

through the plasma membrane and, finally, causing cell death. Endo et al. (1997)

had measured amino acids released by S. Cerevisiae treated with Aureobasidin A

to assess disruption of the cell membrane. In the present study, an in vitro

membrane permeability assay confirmed the damage caused by Ag NPs to cell

membrane. The release of UV-absorbing compounds from cells increased in

proportion to the concentration of nanoparticles (Figure 4.17) present in the

growth media. Nanoparticles of 70.07μg/ml mediated the release of metabolites

which were >2.5 times than that released after digestion in 1N perchloric at

100°C for 1 hour. These observations on the yeast cell suggest that silver

nanoparticles are potent cytotoxic agents and have a profound impact on their cell

wall leading to altered diffusion across membranes and cell death.

The intracellular metabolite concentrations have been investigated to assess the

impact of nanoparticles on the metabolic activities of yeast cells. Baker’s-yeast

cells may change their level of metabolic activity, such as their carbon

metabolism and nitrogen metabolism, as well as their stress response to

environmental conditions (Higgins et al., 2001). As is described in Section 4.3.6,

the amino acid and cellular protein concentrations of stressed cells showed a

steady decrease when cells were exposed to increasingly higher doses of Ag NPs.


The total RNA levels however, were relatively consistent at nanoparticle

concentrations below the lethal dose and then showed a substantial reduction at

the MIC concentration and beyond. This observation seems to predict that in cells

stressed with nanoparticles, but still viable, some aspect of metabolism related to

protein synthesis is inhibited leading to a state where RNA levels are normal but

protein content has decreased. Bond (2006) has discussed that exposure of yeast

cells to environmental stresses can disrupt essential intracellular processes,

especially those carried out by large macromolecular complexes. The production

of mature, translatable mRNAs is most sensitive to stress owing to the inhibition

of messenger RNA splicing and alterations in the export of mRNA from the

nucleus.

In the yeast Saccharomyces cerevisiae, a strong correlation between trehalose

content and stress resistance has been demonstrated in different strains, in a

variety of growth conditions, during sub-lethal heat treatment and other stress

conditions, and in mutants affected in the Ras-adenylate cyclase pathway (Dijck

et al., 1995). In the present study we found that stressed cells accumulated more

trehalose as the nanoparticle concentration increased and at the MKC of 70.07

µg/ml, trehalose content more than doubled from that of control cells. Most of the

trehalose content however was extruded from cells and extracellular trehalose

was found to be more than five times higher in cultures exposed to MIC and

MKC of nanoparticles compared to the control. Higher levels of extracellular

trehalose can be explained by increased membrane permeability as a consequence

of nanoparticle stress. In S. cerevisiae, it is known that glycerol and trehalose

function as major stress protectants and that the synthesis of glycerol or trehalose

is induced by many stress conditions at the transcriptional level (Shima and

Takagi, 2009).

To summarize, we have assessed the inhibitory and killing concentrations of

silver nanoparticles for the yeast strain. This has provided us with the dosage

limit for treatment of cells for metabolic and gene studies. The growth and cell

cycle for stressed cells was investigated as also the concentrations of cellular

metabolites. The observations on the levels of nitrogenous metabolites presented


some clues about deficiencies in protein synthesis process; this aspect was further

investigated and is described in the next chapter. Finally, high-resolution electron

microscopic analysis of cells confirmed the damage incurred during nanoparticle

exposure which was further confirmed by a cell permeability assay.

Chapter 4 SILVER NANOPARTICLES AND THE YEAST...

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