4.3 Culturing and Counting BacteriaMicrobes in nature exist in.docx

4.3 Culturing and Counting Bacteria
Microbes in nature exist in complex, multispecies communities,
but for detailed studies they must be grown separately in pure
culture.
After 120 years of trying, we have succeeded in culturing less
than 1% of the microorganisms around us.
The vast majority has yet to be tamed.
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Bacteria Are Grown in Culture Media – 1
Bacteria are grown in culture media, which are of two main
types:
Liquid or broth
Useful for studying the growth characteristics of a pure culture
Solid (usually gelled with agar)
Useful for trying to separate mixed cultures from clinical
specimens or natural environments
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Bacteria Are Grown in Culture Media – 2

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FIGURE 4.12 ■ Separation and growth of microbes on an agar
surface. A. Colonies (diameter 1–5 mm) of Acidovorax citrulli
separated on an agar plate. A. citrulli is a plant pathogen that
causes watermelon fruit blotch. B. A mixture of yellow-
pigmented bacterial colonies, wrinkled bacterial colonies, and
fungus separated by dilution on an agar plate. As time passed,
the fungal colony overgrew adjacent bacterial colonies.
Dilution Streaking and Spread Plates – 1
Pure colonies are isolated via two main techniques:
Dilution streaking
A loop is dragged across the surface of an agar plate.
Spread plate
Tenfold serial dilutions are performed on a liquid culture.
A small amount of each dilution is then plated.
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FIGURE 4.13 ■ Dilution streaking technique. A. A liquid
culture is sampled with a sterile inoculating loop and streaked
across the plate in three or four areas, with the loop flamed
between areas to kill bacteria still clinging to it. Dragging the

loop across the agar diminishes the number of organisms
clinging to the loop until only single cells are deposited at a
given location. B. Salmonella enterica culture obtained by
dilution streaking.
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FIGURE 4.15 ■ Tenfold dilutions, plating, and viable counts. A.
A culture containing an unknown concentration of cells is
serially diluted. One milliliter (ml) of culture is added to 9.0 ml
of diluent broth and mixed, and then 1 ml of this 1/10 dilution
is added to another 9.0 ml of diluent (10–2 dilution). These
steps are repeated for further dilution, each of which lowers the
cell number tenfold. After dilution, 0.1 ml of each dilution is
spread onto an agar plate. B. Plates prepared as in (A) are
incubated at 37°C to yield colonies. By multiplying the number
of countable colonies (107 colonies on the 10–5 plate) by 10,
you get the number of cells in 1.0 ml of the 10–5 dilution.
Multiplying that number by the reciprocal of the dilution factor,
you can calculate the number of cells (colony-forming units, or
CFUs) per milliliter in the original broth tube (107 x 101 x 105
= 1.1 x 108 CFUs/ml). TNTC = too numerous to count.
Types of Media – 1
Complex media are nutrient rich but poorly defined.
Minimal defined media contains only those nutrients that are
essential for growth of a given microbe.
Enriched media are complex media to which specific blood
components are added.
Selective media favor the growth of one organism over another.
Differential media exploit differences between two species that

grow equally well.
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Types of Media – 2 MediumIngredientsAmount per
literOrganisms culturedLuria Bertani (complex)Bacto tryptone,
a pancreatic digest of casein (bovine milk protein)10
gramsMany Gram-negative and Gram-positive organisms (such
as Escherichia coli and Staphylococcus aureus,
respectively)Luria Bertani (complex)Bacto yeast extract5
gramsEmpty cellLuria Bertani (complex)Upper N a upper C l
Adjust to pH 710 gramsEmpty cellM 9 medium
(defined)Glucose2.0 gramsGram-negative organisms such as E.
coliM 9 medium (defined)Upper N a 2 upper H upper P upper O
46.0 grams (42 millimolar)Gram-negative organisms such as E.
coliM 9 medium (defined)Upper K upper H 2 upper P upper O
43.0 grams (22 millimolar)Gram-negative organisms such as E.
coliM 9 medium (defined)Upper N upper H 4 upper C l1.0
grams (19 millimolar)Gram-negative organisms such as E.
coliM 9 medium (defined)Upper N a upper C l0.5 grams (9
millimolar)Gram-negative organisms such as E. coli
M 9 medium (defined)Upper M g upper S upper O 42.0
millimolarGram-negative organisms such as E. coliM 9 medium
(defined)Upper C a upper C l 2 Adjust to pH 70.1
millimolarGram-negative organisms such as E. coliSulfur
oxidizers (defined)Upper N upper H 4 upper C l0.52
gramsAcidithiobacillus thiooxidansSulfur oxidizers
(defined)Upper K upper H 2 upper P upper O 40.28
(defined)Upper M g upper S upper O 4 times 7 upper H 2 upper
O0.25 gramsAcidithiobacillus thiooxidansSulfur oxidizers
(defined)Upper C a upper C l 20.07 gramsAcidithiobacillus

thiooxidansSulfur oxidizers (defined)Elemental sulfur1.56
(defined)Upper C upper O 2 Adjust to pH 35
percentAcidithiobacillus thiooxidans
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Types of Media – 3
Several media used in clinical microbiology are both selective
and differential.
e.g., MacConkey medium
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FIGURE 4.16 ■ MacConkey medium, a culture medium both
selective and differential. Only Gram-negative bacteria grow on
lactose MacConkey (selective). Only a species capable of
fermenting lactose produces pink colonies (differential),
because only fermenters can take up the neutral red and
peptones that are also in the medium. Gram-negative
nonfermenters appear as uncolored colonies.
Growth Factors, Unculturable Microbes, and Obligate
Intracellular Bacteria – 1
Microbes can evolve to require specific growth factors
depending on the nutrient richness of their natural ecological
niche.
Growth factors are specific nutrients not required by other

species.
A microbe needs them in order to be able to grow in laboratory
media.
e.g.: Streptococcus pyogenes requires glutamic acid and alanine
because it can no longer synthesize them.
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Intracellular Bacteria – 2 OrganismDiseasesNatural
habitatsGrowth factorsShigellaBloody
diarrheaHumansNicotinamide, which is derived from upper N
upper A upper D, nicotinamide adenine
dinucleotideHaemophilusMeningitis, chancroidHumans and
other animal species, upper respiratory tractHemin, upper N
upper A upper DStaphylococcusBoils,
osteomyelitisWidespreadComplex
requirementAbiotrophiaOsteomyelitisHumans and other animal
speciesVitamin K, cysteineLegionellaLegionnaires’ diseaseSoil,
refrigeration cooling towersCysteineBordetellaWhooping
coughHumans and other animal speciesGlutamate, proline,
cysteineFrancisellaTularemiaWild deer, rabbitsComplex,
cysteineMycobacteriumTuberculosis, leprosyHumansNicotinic
acid, which is derived from upper N upper A upper D,
nicotinamide adenine dinucleotide, and alanine (M. leprae is
unculturable)Streptococcus pyogenesPharyngitis, rheumatic
feverHumansGlutamate, alanine
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Some species have adapted so well to their natural habitats that
we still do not know how to grow them in the lab.
Some of these “unculturable” organisms depend on factors
provided by other species that cohabit their niche.
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FIGURE 4.17 ■ “Unculturable” marine organism MSC33. To
grow in natural environments, many bacterial species rely on
factors produced by other species within their niche. The
microbe shown, MSC33, will not grow in laboratory media
unless a peptide growth factor from another species is included.
Obligate intracellular bacteria are also unculturable.
e.g.: Rickettsia prowazekii, the cause of epidemic typhus fever,
has adapted to grow within the cytoplasm of eukaryotic cells
and nowhere else.
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FIGURE 4.18 ■ Rickettsia prowazekii growing within
eukaryotic cells. A. R. prowazekii growing within the cytoplasm
of a chicken embryo fibroblast (SEM). B. Fluorescent stain of

R. prowazekii (approx. 0.5 μm long), growing within a cultured
human cell (outline marked by dotted line). The rickettsias are
green (FITClabeled antibody, arrow), the host cell nucleus is
blue (Hoechst stain), and the mitochondria are red (Texas Red
MitoTracker). The bacterium grows only in the cytoplasm, not
in the nucleus.
Techniques for Counting Bacteria
There are many reasons why it is important to know the number
of organisms in a sample.
Counting or quantifying organisms invisible to the naked eye is
surprisingly difficult.
Each of the available techniques measures a different physical
or biochemical aspect of growth.
Thus, cell density values derived from these techniques may not
necessarily agree with one another.
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Direct Counting of Living and Dead Cells – 1
Microorganisms can be counted directly by placing dilutions on
a special microscope slide called a Petroff-Hausser counting
chamber.
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FIGURE 4.19 ■ The Petroff-Hausser chamber for direct
microscopic counts. A precision grid is etched on the surface of
the slide. The organisms in several squares are counted, and

their numbers are averaged. Knowing the dimensions of the grid
and the height of the coverslip over the slide makes it possible
to calculate the number of organisms in a milliliter.
Living cells may be distinguished from dead cells by
fluorescence microscopy using fluorescent chemical dyes.
Dead bacterial cells fluoresce orange or yellow because
propidium (red) can enter the cells and intercalate the base pairs
of DNA.
Live cells fluoresce green because Syto-9 (green) enters the
cell.
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FIGURE 4.20 ■ Live/dead stain. Live and dead bacteria
visualized on freshly isolated human cheek epithelial cells using
the LIVE/DEAD BacLight Bacterial Viability Kit. Dead
bacterial cells fluoresce orange or yellow because propidium
(red) can enter the cells and intercalate the base pairs of DNA.
Live cells fluoresce green because Syto-9 (green) enters the
cell. The faint green smears are the outlines of cheek cells.
Direct counting without microscopy can be done using an
electronic technique that not only counts but also separates
populations of bacterial cells according to their distinguishing
properties.
The instrument is called a fluorescence-activated cell sorter
(FACS) or flow cytometer.
Fluorescent cells are passed through a small orifice and then
past a laser.
Detectors measure light scatter in the forward direction

(measure of particle size) and to the side (particle shape or
granularity).
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FIGURE 4.21 ■ Fluorescence-activated cell sorter (FACS). A.
Schematic of a FACS apparatus (bidirectional sorting). B.
Separation of GFP-producing E. coli from non-GFP-producing
E. coli. The low-level fluorescence in the cells on the left is
baseline fluorescence (autofluorescence). The scatterplot
displays the same FACS data, showing the size distribution of
cells (x-axis) with respect to the level of fluorescence (y-axis).
The larger cells may be cells that are about to divide.
Other Techniques
A viable bacterium is defined as being capable of replicating
and forming a colony on a solid medium.
Viable cells can be counted via the pour plate method.
Microorganisms can be counted indirectly via biochemical
assays of cell mass, protein content, or metabolic rate.
Also by measuring optical density
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4.4 The Growth Cycle
Most bacteria divide by binary fission, where one parent cell
splits into two equal daughter cells.
However, some divide asymmetrically.
Hyphomicrobium divides by budding.
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FIGURE 4.22 ■ Symmetrical and asymmetrical cell division. A.
Symmetrical cell division, or binary fission, in Lactobacillus sp.
(SEM). B. Asymmetrical cell division via budding in the marine
bacterium Hyphomicrobium (approx. 4 μm long).
Exponential Growth
The growth rate, or rate of increase in cell numbers or biomass,
is proportional to the population size at a given time.
Such a growth rate is called “exponential” because it generates
an exponential curve, a curve whose slope increases
continually.
If a cell divides by binary fission, the number of cells is
proportional to 2n.
Where n = number of generations
Note: Some cyanobacteria divide by multiple fission.
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Generation Time
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Stages of Growth in a Batch Culture
Exponential growth never lasts indefinitely.
The simplest way to model the effects of a changing
environment is to culture bacteria in a batch culture.
A liquid medium within a closed system
The changing conditions in this system greatly affect bacterial
physiology and growth.
This illustrates the remarkable ability of bacteria to adapt to
their environment.
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Bacterial Growth Curves
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FIGURE 4.23 ■ Bacterial growth curves. A. Theoretical growth
curve of a bacterial suspension measured by optical density
(OD) at a wavelength of 600 nm. B. Phases of bacterial growth
in a typical batch culture.

Continuous Culture – 1
In a continuous culture, all cells in a population achieve a
steady state, which allows detailed study of bacterial
physiology.
The chemostat ensures logarithmic growth by constantly adding
and removing equal amounts of culture media.
Note that the human gastrointestinal tract is engineered much
like a chemostat.
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FIGURE 4.25 ■ Chemostats and continuous culture. A. The
basic chemostat ensures logarithmic growth by constantly
adding and removing equal amounts of culture media. B. The
human gastrointestinal tract is engineered much like a
chemostat, in that new nutrients are always arriving from the
throat while equal amounts of bacterial culture exit in fecal
waste. C. A modern chemostat.
The complex relationships among dilution rate, cell mass, and
generation time in a chemostat are illustrated here.

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FIGURE 4.26 ■ Relationships among chemostat dilution rate,
cell mass, and generation time. As the dilution rate (x-axis)
increases, the generation time decreases and the mass of the
culture increases. When the rate of dilution exceeds the division
rate, cells are washed from the vessel faster than they can be
replaced by division, and the cell mass decreases. The y-axis
varies depending on the curve, as labeled.
4.5 Biofilms – 1
In nature, many bacteria form specialized, surface-attached
communities called biofilms.
These can be constructed by one or multiple species and can
form on a range of organic or inorganic surfaces.
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FIGURE 4.27 ■ Biofilms. A. A greenish-brown slime biofilm
found on cobbles of the streambed in High Ore Creek, Montana.
B. The biofilm that forms on teeth is called plaque.
4.5 Biofilms – 2
Bacterial biofilms form when nutrients are plentiful.
Once nutrients become scarce, individuals detach from the
community to forage for new sources of nutrients.
Biofilms in nature can take many different forms and serve
different functions for different species.
The formation of biofilms can be cued by different
environmental signals in different species.

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4.5 Biofilms – 3
Chemical signals enable bacteria to communicate (quorum
sensing) and in some cases to form biofilms.
Biofilm development involves:
The adherence of cells to a substrate
The formation of microcolonies
Ultimately, the formation of complex channeled communities
that generate new planktonic cells
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Biofilm Development – 1
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FIGURE 4.28 ■ Biofilm development. The stages of biofilm
development in Pseudomonas, which generally apply to the
formation of many kinds of biofilms. Inset: A mucoid
environmental strain of P. aeruginosa produces uneven, lumpy
biofilms in an experimental flow cell (see Fig. 2.18). Cells in
the biofilm were stained green with the fluorescent DNAbinding
dye Syto-9 (3D confocal laser scanning microscopy). Source: H.

C. Flemming and J. Wingender. 2010. Nat. Rev. Microbiol.
8:623–633.
Biofilm Development – 2
For many bacteria, sessile (nonmoving) cells in a biofilm
chemically “talk” to each other in order to build microcolonies
and keep water channels open.
Bacillus subtilis also spins out a fibril-like amyloid protein
called TasA, which tethers cells and strengthens biofilms.
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FIGURE 4.29 ■ Floating biofilm (pellicle) formation of
Bacillus subtilis. Cells were grown in a broth for 48 hours
without agitation at 30°C. The pellicles formed by wild-type
and tasA mutant B. subtilis are strikingly different. Wild-type
pellicles are extremely wrinkly (A), whereas tasA mutant
pellicles are flat and fragile (B). Insets: Electron micrographs of
wild-type (A) and tasA mutant (B) cells.
Background
Is a non-pathogenic bacterium, which is the model organism for
studying the formation and growth of bacterial biofilms.
B. subtilus is non-pathogenic, which means does not cause
disease.
Several bacterial species, prefer to live under B. subtilus
biofilms, as they are robust, and provide a barrier of protection
from environmental stressors (chemicals, or protection from
other microorganisms).
Pathogenic (disease causing) bacteria in the wild primarily
prefer B. subtilus biofilms.
An example of a pathogen frequently found in B. subtilus
biofilms is Bacillus cereus.

In the same genus as Bacillus
A well known food poisoning bacterium
Forms its own biofilm, but when grown together with B.
subtilus, will send signals to have B. subtilus create the biofilm.
Bacillus subtilus
Bacillus cereus
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B. subtilus biofilm formation is possible by adding 1%
glycerol/0.1mM MnSO4 to LB media
LB only
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Biofilm composition in B. subtilus and B. cereus
B. subtilus and B. cereus share homology between genes of the
epsA-epsO and tapA operons that are involved in biofim
formation.
The epsA-epsO is an operon composed of 15 genes, which
encode proteins responsible for the formation of a
exopolysaccharide (sugars) in the extracellular matrix that hold
bacteria (not only B. subtilus and B. cereus) in these biofilms
together.
The tap A operon contains a gene known as tasA, which encodes
a protein that produces amyloid fibers.
The epsA-epsO and tapA are inducible operons, meaning the
genes are not expressed until needed.

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Triggers of biofilm formation in B. subtilus
Biofilm formation requires specific nutrients in order to create
the exopolysaccharide (EPS), and amyloid fibers.
The two nutrients needed by B. subtilus and B. cereus to create
biofilms are glycerol (C3H8O3), and manganese (Mn++).
The picture on the right is a depiction of a signal transduction
pathway in B. subtilus and B. cereus, which ultimately leads to
the induction of the epsA-epsO and tapA operons.
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1% Glycerol/0.1mM MnSO4 is necessary and sufficient for
complete biofilm formation
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Escherichia coli: a Gram-negative bacterium of the gut
microbiome, Part 1
FIGURE 3.1 ■ Escherichia coli: a Gram-negative bacterium of

the gut microbiome. The envelope includes the outer membrane;
the cell wall and periplasm; and the inner (cell) membrane.
Embedded in the membranes is the motor of a flagellum. The
cytoplasm includes enzymes, messenger RNA extending out of
the nucleoid, and ribosomes. Ribosomes translate the mRNA to
make proteins, which are folded by chaperones. The nucleoid
contains the chromosomal DNA wrapped around binding
proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding
protein, 1P78; RNA polymerase, 1MSW)
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Bacterial Cell Structure: What is seen in Gram-negative &
Gram-positive bacteriaBacteria can be placed in 2 groups based
on the thickness and placement of the cell wallGram-
negativeGram-positivePlasma membraneAbsence of a
nucleusDNA is located in the nucleiod regionNo histone
proteins, but DNA-binding proteins present to keep genomic
DNA compactPlasmids: DNA that is independent of the genome.
Flagellum
Biochemical composition of bacteriaWaterEssential IonsNeeded
for enzymatic reactionsSmall organic molecules: lipids and
sugarsLipids are almost as abundant as RNA moleculesFound in
the cell wall peptidoglycanMacromolecules: nucleic acids,
proteins, fats, & sugars

Goal: Isolate proteinsPurpose of cell fractionation is to isolate
components of choice from a bacterial cellThe first step is cell
lysisEDTASucroseLyzozymesUltracentrifugation
FIGURE 3.2 ■ Fractionation of Gram-negative cells.
Cell periplasm fills with sucrose, and lysozyme breaks down the
cell wall. Dilution in water causes osmotic shock to the outer
membrane, and periplasmic proteins leak out. Subsequent
centrifugation steps separate the proteins of the periplasm,
cytoplasm, and inner and outer membranes. Photo
Source: Lars D. Rennera and Douglas B. Weibel. PNAS
108(15):6264.
*
Goal 2:Protein Analysis
FIGURE 3.3 ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer
membrane proteins from cell fractionation. B. Outer membrane
proteins are identified by tryptic digest and mass spectrum
analysis. The resulting peptide sequence is compared with those
predicted from genome data.
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FIGURE 3.3a ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer
membrane proteins from cell fractionation.

*
FIGURE 3.3b ■ Protein analysis.
B. Outer membrane proteins are identified by tryptic digest and
mass spectrum analysis. The resulting peptide sequence is
compared with those predicted from genome data.
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Understanding the role of a protein
FIGURE 3.4 ■ Genetic analysis of FtsZ.
A. E. coli with aspartate (D) at position 45 replaced by alanine
(A) (D45A) elongate abnormally, forming blebs from the side,
with no Z-rings. Cells with aspartate replaced by alanine at
position 212 (D212A) elongate to form extended nondividing
cells that contain spiral FtsZ complexes. FtsZ was visualized by
immunofluorescence. B. Model of FtsZ protein monomer based
on X-ray crystallography shows the position of the mutant
residues, D212A and D45A.
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FIGURE 3.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with
hydrophobic fatty acid chains directed inward, away from water.
The bilayer contains stiffening agents such as hopanoids. Half

the membrane volume consists of proteins.
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LeuT sodium/leucine cotransporterHomology to human
neurotransmitter sodium sympotersHas been used as a blueprint
to understand structure and function, and pharmacology of NSS
human transporters.
FIGURE 3.7 (part 1) ■ A cell membrane–embedded transport
protein: the LeuT sodium/leucine cotransporter of Aquifex
bacteria.
The protein complex carries leucine across the cell membrane
into the cytoplasm, coupled to sodium ion influx. (PDB code:
3F3E)
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Transport across bacterial membranesPassive
diffusionMembrane proteinsAquaporinsPermease (lac
operon)OsmosisGreater osmotic pressure can lead to bacterial
cell lysis (seen with certain antibiotics)Membrane-permeant
weak acids and bases: can cross the plasma
membraneTransmembrane ion gradients
FIGURE 3.8 ■ Common drugs are membrane-permeant weak

acids and bases.
In its charged form (A– or BH+), each drug is soluble in the
bloodstream. The uncharged form (HA or B) is hydrophobic and
penetrates the cell membrane.
*
FIGURE 3.8a ■ Common drugs are membrane-permeant weak
acids and bases.
*
FIGURE 3.8b ■ Common drugs are membrane-permeant weak
acids and bases.
*
NAM and NAG are linked together by a β-(1,4)-glycosidic
bondLysozymes target this bondThe peptidoglycan monomer
will have 5 peptidesOnce this monomer becomes incorporated
into the existing polymer, 4 peptides are seen.

FIGURE 3.14b ■ The peptidoglycan sacculus and peptidoglycan
cross-bridge formation.
B. A disaccharide unit of glycan has an attached peptide of four
to six amino acids.
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FIGURE 3.16 ■ Cell envelope: Gram-positive (Firmicutes) and
Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with
multiple layers of peptidoglycan, threaded by teichoic acids. A
inset: Gram-positive envelope of Bacillus subtilis (TEM). B.
Proteobacteria (Gram-negative) cells have a single layer of
peptidoglycan covered by an outer membrane; the cell
membrane is called the inner membrane. B inset: Gram-negative
envelope of Pseudomonas aeruginosa (TEM).
*
Gram +
FIGURE 3.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride

inserted in the inward-facing leaflet of the outer membrane. The
C-terminal lysine forms a peptide bond with the m-
diaminopimelic acid of the peptidoglycan (murein) cell wall.
*
FIGURE 3.20 ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide
and O antigen linked to a lipid A. Lipid A consists of a dimer of
phosphoglucosamine esterified or amidated to six fatty acids. B.
Repeating polysaccharide units of O antigen extend from lipid
A.
*
FIGURE 3.20a ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide
and O antigen linked to a lipid A. Lipid A consists of a dimer of
phosphoglucosamine esterified or amidated to six fatty acids.
*
FIGURE 3.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide units of O antigen extend from
lipid A.
*

Name: ______________________
Bio 351 Homework 2 (10 POINTS): DUE MONDAY AT 11PM
ON BLACKBOARD.
Background Information: The delivery of antibiotics, or
antimicrobials has been extensively studied. Nanoparticles
provide a promising alternative to treating infections due to
their small size, and variety of applications in the study of
microbial medicine. Your job is apply what you have learned in
lecture to something you have not seen before. It is necessary to
use the knowledge you have to begin or continue to interpret
scientific research articles, particularly in the field of
microbiology.
Nanoparticles are transport vehicles that deliver ions (at least in
the research article I provided). The article provided used two
types of CuO (copper oxide nanoparticles) as an antimicrobial:
CuO nanosheets (flat structures that contain copper oxide), vs.
CuO nanospheres (spherical structures containing copper
oxide). Scientists wanted to determine if there was one
structure, or if both were effective as a vehicle in delivering
copper ions, and if it is an effective antimicrobial towards E.
coli, B. subtilus, P. vulgaris, and M. luteous. Word limit of
maximum 40 words per answer. Be concise with your answers.
Do not copy the wording from the article, or your textbook.
1. Before you can begin to interpret figures from the research
article attached, you must gather information first by answering
questions a-c below. Once you have done this, then questions d
& e can be answered.
a. Please indicate which of the bacterial species listed are gram-
positive and gram-negative. (1 point)
b. What is the difference between antimicrobials and
antibiotics? You can perform a Google search to find the
answer. (1 point)
c. What are differences seen in cell wall composition between
gram-positive and gram-negative bacteria? Use your textbook
and lecture notes. (1 point)

d. In Figure 2, what is the optical density from each of the
bacterial species? Over a period of time what does this tell you
about bacterial growth from each species when exposed to CuO
nanosheet (Panel A), and CuO nanospherical (Panel B)? Are
gram-positive and gram-negative bacteria used in this study
affected similarly or different? If so, how do you know based on
Figure 2? (2 points)
e. One method scientists use to determine if a drug-delivery
system is effective is by measuring the amount of reactive
oxygen species (ROS) generated. ROS are considered free
radicals that can harm cells, and their likelihood for survival.
Please interpret Figure 3, Panel A & B. (2 points)
f. What were the findings from Figure 5? What could be
occurring at the cell wall for gram-positive and gram-negative
bacteria? (2 points)
g. Based on the evidence from Figures 2, 3, and 5., which
nanoparticle delivery system was most effective as an
antimicrobial? (1 point)
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Shape-dependent bactericidal activity of copper oxide
nanoparticle
mediated by DNA and membrane damage
Dipranjan Laha a, Arindam Pramanik a, Aparna Laskar c,
Madhurya Jana a,
Panchanan Pramanik b, Parimal Karmakar a,*
a Department of Life Science and Biotechnology, Jadavpur
University, 188, Raja S C Mallick Road, Kolkata 700032, India
b Department of Chemistry, Indian Institute of Technology,
Kharagpur 721302, India
c CSIR-Indian Institute of Chemical Biology, Kolkata 700032,
India
A R T I C L E I N F O
Article history:
Received 7 May 2014
Received in revised form 14 June 2014
Accepted 22 June 2014
Available online 10 July 2014
Keywords:
B. Chemical synthesis
A. Metals
C. Atomic force microscopy
A B S T R A C T
In this work, we synthesized spherical and sheet shaped copper
oxide nanoparticles and their physical
characterizations were done by the X-ray diffraction, fourier
transform infrared spectroscopy,
transmission electron microscopy and dynamic light scattering.

The antibacterial activity of these
nanoparticles was determined on both gram positive and gram
negative bacterial. Spherical shaped
copper oxide nanoparticles showed more antibacterial property
on gram positive bacteria where as sheet
shaped copper oxide nanoparticles are more active on gram
negative bacteria. We also demonstrated that
copper oxide nanoparticles produced reactive oxygen species in
both gram negative and gram positive
bacteria. Furthermore, they induced membrane damage as
determined by atomic force microscopy and
scanning electron microscopy. Thus production of and
membrane damage are major mechanisms of the
bactericidal activity of these copper oxide nanoparticles. Finally
it was concluded that antibacterial
activity of nanoparticles depend on physicochemical properties
of copper oxide nanoparticles and
bacterial strain.
ã 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Nanostructured materials offer promising opportunities for
improved applications in different area of modern life due to
their
unique physicochemical properties, caused by their nanosized
dimensions and large surface/volume ratios [1]. More recently,
several natural and engineered nanomaterials have been shown
to
possess strong antimicrobial properties including silver nano-
particles [2], TiO2 [3], ZnO [4] and SiO2 [5]. Some
nanocomposite
consisting of different materials are possess bactericidal
activity.
For example, microfibril bundles of cellulose substance with

titania/chitosan/silver-nanoparticle composite films and hierar-
chical nanofibrous titania–carbon composite material deposited
with silver nanoparticles are lethal to various bacterial strains
[6,7].
Application of antibacterial agents in the textile industry, water
disinfection, medicine, food packaging etc. are well known.
Unlike
conventional chemical disinfectants, the antimicrobial
nanomate-
rials are not expected to produce harmful disinfection by
products
(DBPs). Among these several metal based nanoparticles (e.g.,
copper
based nanoparticle) are increasingly recognized as a suitable
alternative due to its high redox potential property and
relatively
lowercostofproduction[8]. Previously, ithas
beenreportedthatCuO
NPs exibit strong antimicrobial activity against broad spectrum
of
gram positive and gram negative bacteria [9]. Though, the
constit-
uents of cell wall in gram-positive and gram-negative bacteria
are
mainly responsible for their sensitivity to CuO NPs but other
factors
can also influence the sensitivity. For instance, gram negative E
(!) is
highlysensitive,but
S.aureus(+)andB.subtilis(+)arelesssensitiveto
CuO NPs [8]. On the other hand bactericidal activity of such
nanoparticles in part depends on size, stability, shape and
concentration in the growth medium [10,11].

The mechanisms by which such metal oxide nanoparticles
induce bactericidal activities is not fully known but amount of
ion
release and subsequent production of ROS is supposed to be the
main cause [12]. The rate of dissolution of such nanoparticles
depends on their morphology as well as their nature [13].
Additionally, by electrostatic interaction nanoparticles are able
to attach to the membrane of bacteria and interfere with
bacterial
membrane [14]. Depending on these two factors many metal
oxide
nanoparticles act differentially on different strain. As the way
by
which bacteria is killed by such nanoparticles is different from
the
* Corresponding author. Tel.: +91 3324146710; fax: +91
3324137121.
E-mail address: [email protected] (P. Karmakar).
http://dx.doi.org/10.1016/j.materresbull.2014.06.024
0025-5408/ã 2014 Elsevier Ltd. All rights reserved.
Materials Research Bulletin 59 (2014) 185–191
Contents lists available at ScienceDirect
Materials Research Bulletin
journal homepage: www.else vie r.com/locat e/mat resbu
antibiotic their proper evaluation is necessary. Thus a
comprehen-
sive knowledge about their size and morphology depended anti

bacterial activity must be evaluated. In light of these, we
undertook
the effort to assess the morphology dependent activity of CuO
NPs
on different bacterial strain. We have synthesized two different
shapes of CuO NPs and characterized them for their
antimicrobial
activity. The antibacterial activity was examined on a broad
range
of bacterial species including E.scherichia coli wild type,
Micrococ-
cus luteus, Bacillus subtilis and Proteus vulgaris. While sheet
shape
CuO NPs are potentially active against gram positive bacteria
and
spherical shaped CuO NPs are more effective on gram negative
bacteria. Both membrane damage and ROS mediated DNA
damage
are responsible for their antimicrobial activity.
2. Materials and methods
2.1. Materials
In this study all chemicals of analytical grade were used.
Copper
acetate [Cu(CH3COO)2], glacial acetic acid [CH3COOH],
sodium
hydroxide [NaOH], copper nitrate trihydrate [Cu(NO3)2"3H2O]
was
obtained from SRL, India, ethanol (99%), sodium acetate
[CH3COONa]
from Qualigen, India. Alizarin red S (ARS), Hanks balanced salt
solution (HBSS), nitroblue tetrazolium (NBT) were obtained
from
Sigma–Aldrich, USA. Hydrochloric acid (35%), dimethyl

sulfoxide
(DMSO), Muller–Hinton agar (MHA) medium and Muller–
Hinton
broth (MHB) medium were obtained from Hi-media, India.
2.2. Synthesis of CuO NPs (nanospherical, nanosheet)
Different shaped CuO NPs were prepared using co-precipitation
method where either copper acetate or copper nitrate is used to
form CuO NPs and NaOH acts as stabilizing compound [15,16].
2.2.1. Synthesis of CuO nanospherical
300 ml of 0.02 M copper acetate was taken in a conical flask.
1 ml of glacial acetic acid was added to it. The solution is
heated at
80–90 #C on a hot plate with vigorous stirring for 10 min by a
magnetic stirrer. 0.8 g NaOH was added rapidly to maintain the
pH
6–7. The mixer was kept for 1 h in stirring condition. The
resultant
solution was centrifuged at 8000 rpm for 10 min. Pellet was
dried
at 37 #C for 3 days. After that it was homogenized by pestle–
mortar
and stored.
2.2.2. Synthesis of CuO nanosheet
80 ml of 0.02 M copper nitrate was slowly added to 5 M NaOH
solution in a conical flask at 82 #C. Additional 80 ml of same
copper
nitrate solution was added to above solution, a total of 32 g of
NaOH
pellet was added to the flask reactor to maintain the constant
concentration of NaOH. The resultant solution was centrifuged

at
8000 rpm for 10 min. The pellet was collected and washed with
water. Pellet was dried at 37 #C for 3 days. After that it was
homogenized by pestle–mortar and stored.
2.3. Particle characterization
Thephaseformationandcrystallographicstateofdifferentshaped
CuO NPs were determined by XRD with an Expert Pro
(Phillips) X-ray
diffractometer using CoKa radiation (a = 0.178897 nm).
Samples
were scanned from 20# to 80# of 2u increment of 0.04# with 2 s
counting time. Presence of surface functional groups was
investi-
gated by FTIR spectroscopy (Thermo 132 Nicolet Nexus FTIR,
model
870). The particle size and nanostructure were studied by high-
resolution transmission electron microscopy in a JEOL 3010
(HRTEM), Japan operating at 200 KeV. Dry powder of particles
was
suspended in de-ionized water at a concentration of 1 mg/mL
and
then sonicated at room temperature for 10 min at 40 W to form
a
homogeneous suspension. After sonication and stabilization, the
samples were prepared by coating on carbon-coated copper
grids
and air dried before TEM analysis. The hydrodynamic size of
dispersed CuO NPs in aqueous phase was measured in a
Brookhaven
90 Plus particle size analyzer. Copper based nanoparticles were
dispersed in water to form diluted suspension of 0.5 mg/ml
using
sonicator for 30 min. The particles were analyzed by DLS after

they
were completely dispersed in water.
2.4. Bacterial strains and culture conditions
Well characterized cells of B. subtilis (ATCC 6633), M. luteous
(ATCC 9341), E. coli (ATCC 10,536), P. vulgaris (ATCC
13,387), DH5a
(k12) were maintained on MHA. Prior to incubation with NPs,
the
bacteria were cultured overnight in 4 ml of MHB in shaker at 37
#C
until the optical density (OD) of the culture reached 1.0 at 600
nm,
which indicates 109 CFU ml!1. The overnight cultures were
diluted
to 107 CFU ml!1 with sterile broth.
2.5. Antibacterial assay
Antibacterial activity of different shaped CuO NPs was affirmed
through determination of minimum inhibitory concentration
(MIC)
and minimum bactericidal concentrations (MBC) [17,18]. MIC
is
defined as the lowest concentration of antimicrobial agent at
which
no growth is observedin broth medium. Test tubescontaining 4
ml of
broth was inoculated with overnight cultures of the bacteria and
then various concentrations of different shaped CuO NPs (0
mg/ml–
0.4 mg/ml) were added in each tube. The tubes were left for
shaking
at 37 #C for 24 h. Then optical density of each tube was
measured at

600 nm for the determination of bacterial growth. To
substantiate
antibacterial activity further, MBC was determined by
inoculating a
loop of NPs treated bacterial culture on MHA plates and left at
37 #C
for 24 h. MBC is defined as the lowest concentration of NPs
where no
growth of bacteria is noted on agar plates. Growth curve was
studies
for both gram positive and gram negative bacteria with and
without
LD50 dose of these nanoparticles for 8 h.
2.6. Reactive oxygen species (ROS) assay
The production of intracellular reactive oxygen species (ROS)
was measured using the same protocol mentioned in our earlier
publication [19].
2.7. In vitro copper ion release study
Release of copper ion from the adsorbed NPs in nutrient broth
was studied by the metallochromic dye ARS. To each test tube,
4 mg
of different shape CuO NPs (nanospherical, nanosheet) were
added
in 1 ml of MHB. Then the test tubes were kept under shaking
condition at 37 #C. Supernatant from each test tube was
collected
after 2, 4, 6, 12 and 24 h by centrifugation at 10,000 rpm for 10
min.
Next, to each collected supernatant a 100 ml of ARS was added
from
stock (10!5 M) along with sodium acetate buffer to maintain
acidic

pH. The solution was kept for 10 min and then optical density
(OD)
was measured at 510 nm by UV–vis spectrophotometer. The
intensity of absorption depends on the amount of Cu–ARS
complex
which in turn depends on the concentration of Cu2+. The
experiment was carried out three times and reproducible data
were obtained [20].
2.8. DNA damage assay
The effect of different shaped CuO NPs on DNA was observed
inside bacterial cell. Reporter (b-galactosidase) gene expression
186 D. Laha et al. / Materials Research Bulletin 59 (2014) 185–
191
assay was performed. They were inoculated on agar plates
containing X-gal and IPTG in the medium and incubated for 12
h
at 37 #C for blue color forming colonies [20].
2.9. Cell morphology study by AFM
The effect of different shaped CuO NPs on bacterial cell
morphology was studies using atomic force microscopy (AFM,
Vecco, USA). Fresh E. coli bacterial culture (OD 0.2) were
treated
with LD50 dose of NPs for 3 h and then washed with phosphate
buffered saline (pH 7) for three times and the cells were fixed
with
2.5% glutaraldehyde. A drop of diluted cell suspension was
placed
on a cover slip and allowed to dry before AFM study [21].

2.10. Cell morphology study by SEM
The effect of different shaped CuO NPs on bacterial cell
morphology was studies using scanning electron microscopy
(SEM,
Vecco, USA). Fresh bacterial culture (OD 0.2) were treated with
LD50 dose of different shaped CuO NPs for 3 h and then
washed
with phosphate buffered saline (pH 7) for three times and the
cells
were fixed with 2.5% glutaraldehyde. A drop of diluted cell
suspension was placed on a cover slip and allowed to vacuum
dry
before SEM study [22].
2.11. Data analysis
A Student’s t-test was used to calculate the statistical
significance of changes. In all cases, differences are significant
for p < 0.05. Data analysis was performed using the Origin Pro
v.8 software(Origin Lab).
3. Results and discussions
3.1. DLS and TEM analysis
The hydrodynamic size of different shaped CuO NPs was
measured by DLS. Table 1 summarize their physical
characteriza-
tion. The TEM micrograph of different shaped copper oxide is
shown Fig. 1A. From the Fig. 1A, the size of spherical and
sheet
shaped CuO NPs were 35 $ 5.6, 257.12 $ 13.6 % 42 $ 5.10,
respec-

tively. As seen in the table, the hydrodynamic sizes of the
Table 1
Characterization of the different shaped CuO NPs used in this
study morphology primary size hydrodynamic diameter zeta
potential pDia (TEM) TEM (nm) DLS (nm).
Morphology Primary size Hydrodynamic diameter Zeta potential
pDia
(TEM) TEM (nm) DLS (nm)
CuO spherical 33.20 $ 6.18 235 !27.6 0.305
CuO sheet 257.12 $ 13.6 % 42 $ 5.10 372 !23.1 0.346
a Polydispersity index.
Fig. 1. Physical characterization of different shaped CuO NPs
(A) X-ray diffraction patterns of CuO nanosheet and CuO
nanospherical; (B) FTIR spectra of of CuO nanosheet and
CuO nanospherical; (C) transmission electron microscopic
(TEM) image and dynamic light scattering CuO nanosheet and
CuO nanospherical.
D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191
187
synthesized NPs were significantly larger than those indicated
by
their TEM images. This is possibly due to the fact that TEM
measures size in the dried state of the sample, where as the DLS
measures the size of the hydrated state of particle.
3.2. X-ray diffraction pattern

We first characterized the purity of CuO NPs by XRD. The
XRD
pattern of CuO NPs was compared and interpreted with standard
data of the JCPDS file (JCPDS International Center for
Diffraction
Data, 1991). Fig. 1B shows the XRD pattern of two different
shaped
CuO NPs, the characteristic peaks at 2u = 32.25#, 33.12#,
35.28#,
48.62#, 53.42#, 58.09#, 65.95#,67.90# and 72.24# which are in
agreement with JCPDS card no. 44-0706.
3.3. Compositional and optical analysis of synthesized different
shaped
copper oxide nanoparticles (CuO NPs)
The functional or composition quality of the synthesized
product was analyzed by the FTIR spectroscopy. Fig. 1C shows
the FTIR spectrum in the range of 500–4,000 cm!1. The pure
CuO
NPs exhibited strong band at 1640 cm!1, characteristic of the
CO
stretch and the broad band around 3440 cm!1, indicates the
presence of !!OH groups (Fig. 1C) for both CuO NPs. Table 1
summarized the physical characteristic of CuO NPs.
3.4. Evaluation of antibacterial properties
The antibacterial activities of these two different shaped CuO
NPs against gram positive and negative bacteria were investi-
gated using E. coli, P vulgaris, B.subtilis and M. luteus as
model
organisms. Shape dependent activity of CuO NPs was measured
by
determining minimum inhibitory concentration (MIC) and mini-
mal bactericidal concentration (MBC) as shown in Tables 2 and

3,
respectively. The growth of gram negative bacteria P. vulgaris
and
E.coli was completely inhibited by spherical CuO NPs at a
concentration of 0.16 mg/ml and 0.20 mg/ml, respectively
where
as CuO nanosheet was more active on gram positive bacteria B.
subtilis and M. luteous (0.22 mg/ml and 0.20 mg/ml,
respectively).
Significance of each MIC value is also determined. Difference
in
dose required for both types of nanoparticles to inhibit the
growth
of same bacterial strain is also shown on the last column of
Table 2. From the Table 2, it is seen that for nanosheet the MIC
value is 120–140 ug/ml less than nanoshperical for gram
positive
bacteria where as for gram negative bacteria, spherical CuO NPs
is
120–80 ug/ml less than nanosheet indicating nanosheet CuO NP
are more effective in gram positive bacteria and spherical CuO
NP
is effective in gram negative bacteria. We also determined the
MBC of all bacterial strains after treating them with different
shaped CuO NPs. A comprehensive table, showing MIC and
MBC of
different bacterial strain and the ratios of MIC and MBC are
Table 2
MIC value of different shaped CuO NPs on different strain.
Bacterial strain
(106 CFU/ml)
Nanospherical (mg/ml) Nanaospherical (mg/ml) p value
Difference doses between nanospherical and nanosheet

B. subtilis (+) 0.22 $ 0.028 0.36 $ 0 Nanosheet > nanospherical
(p < 0.05)
140 mg/ml
M. luteous (+) 0.20 $ 0.010 0.32 $ 0 Nanosheet > nanospherical
(p < 0.01)
120 mg/ml
E. coli (!) 0.28 $ 0.024 0.20 $ 0.05 Nanospherical > nanosheet
(p < 0.01)
80 mg/ml
P. vulgaris (!) 0.28 $ 0.0 0.16 $ 0 Nanospherical > nanosheet
(p < 0.05)
120 mg/ml
Table 3
MBC and MBC/MIC value of different shaped CuO NPs on
different strain.
B. subtilis (+ve) M. luteus (+ve) P. vulgaris (!ve) E. coli (!ve)
Sph Sheet Sph Sheet Sph Sheet Sph Sheet
MBC(mg/ml) 0.36 0.24 0.32 0.24 0.36 0.36 0.36 0.32
MBC/MIC 1 1.12 1 1.5 1.28 1.28 1.5 1
Fig. 2. (A,B) Growth curve (optical density) of E. coli, P.
vulgaris, B. subtilis, M. luteous treated with respective LD50
dose of CuO nanosheet and CuO nanospherical respectively.

191
presented in Table 3, For all the cases the ratio of MBC to MIC
is 1
or greater than 1 indicating the potential bactericidal activity.
LD50 value of different shaped CuO nanoparticles on different
strain was also determine (data not shown). Fig. 2 represents
growth kinetics of different strain bacteria in the presence of
sheet (Fig 2A) and spherical (Fig. 2B). As seen in the Fig 2 the
growth of CuO nanosheet treated bacteria was inhibited after 10
h
whereas CuO NPs spherical treated bacteria reached a stationary
phase after 10 h of growth. In case of all the four microbial
strains,
it was observed that with the increase in time of incubation
beyond 10 h, with different shaped CuO NPs, OD value was
decreased.
To determine the possible mechanism of different shaped
CuO NPs on bacterial strains, we assayed in vitro copper ion
release by ARS. As shown in Fig. 3A, copper ion release from
spherical shaped CuO NPs was less than sheet shaped CuO NPs
at early time point but with increasing time the ion release
became same for both the nanoparticles. One step further, we
assayed ROS for bacterial strain E.coli and B. subtilis after the
treatment of CuO NPs at LD50 dose. In E. coli spherical NPs
produced more ROS compared to sheet but for B. subtillis ROS
production was almost same for both the NPs. To check the
DNA
damage induced by NPs we used plasmid based reporter gene
assay. In Fig. 3C, reporter gene b-galactosidase was assayed by
transforming DH5a with the plasmid and followed by NPs
treatment. The amount of blue colonies (due to the hydrolysis of

X-gal by b-galactosidase enzyme) reduced significantly for the
bacterial cells treated with NPs. We also used atomic force
microscope to determine the effect of CuO NPs on E. coli. As
seen
in Fig 4, both spherical and sheet CuO NPs attached to bacterial
cell membrane. Finally, we used SEM to visualize any
membrane
damage of bacteria. From the SEM image it was observed that
Fig. 3. (A) In vitro copper ion release of these two different
shaped CuO NPs. (B) Determination of reactive oxygen species
(ROS) of E. coli and B. subtilis in presence of these of
different shaped CuO NPs. (C) Reporter gene (b-galactosidase)
assay on nanoparticle treated and mock treated pUC 19
transformed DH5a.
Fig. 4. Atomic force microscopy (AFM) images of different
shaped copper oxide nanoparticles treated or mock-treated gram
negative E. coli bacterial cells.
189
spherical shaped produced more membrane damage on E. coli
compared to sheet and sheet shaped induced more membrane
damage on B. subtilis (Fig. 5).
4. Discussion
In this study, we have reported the antibacterial activity of
spherical and sheet shaped CuO NPs. Our results showed that
the
antibacterial effect of CuO NPs not only depends on size, but
also on

specific morphology and nature of the bacterial strain. Being
transition metal, copper plays an important role in cellular
redox
cycling and antibacterial activity of copper based NPs are
reported
earlier [23,24]. Here we showed that apart from its size, CuO
NPs
morphology is also important for antibacterial activity.
Previously
Marsili et al. reported morphology dependent antibacterial
activity
of zinc oxide nanoparticles [25]. In our case we observed
differential antibacterial activity of rod and spherical shaped
CuO NPs. However, the mechanism of bactericidal actions of
these
nanoparticles are still not well understood, but it was proposed
that surface charge of free metal surface is responsible for the
interaction with the bacterial membrane [26]. As a matter of
fact
nanoparticles may associate with bacteria through several types
of
interaction such as hydrophobic, electrostatic or van der Waals
interaction which may help to damage the cell membrane [27].
In a
previous report it was shown that the interaction between silver
nanoparticles and constituents of the bacterial membrane caused
structural changes in membranes and finally leading to cell
death
[28]. Similarly surface modification of gold nanoparticles with
BSA
has been shown to determine its biological effects [29].
We observed that gram positive bacteria are more sensitive to
nanosheet CuO NPs where as gram negative are more sensitive
to
spherical CuO NPs. It may be due to the fact that large sheet

shaped
CuO NPs can not penetrate the outer membrane of gram
negative
bacteria, where as small spherical shaped CuO NPs easily
penetrate
inside the bacterial cell. On the other hand having more surface
charge, sheet shaped CuO NPs induced more damage to gram
positive bacteria. Such large surface area of diethylaminoethyl
dextran chloride (DEAE-D) functionalized gold nanoparticles
also
shown to induce hemolysis in RBC [29].
Previously, several studies reported that two possible mecha-
nisms are involved in the toxicity of nanoparticles on bacterial
cell
(1) production of increased level of ROS mostly hydroxyl
radicals
and singlet oxygen (2) deposition of nanoparticle on the surface
of
bacteria, resulting accumulation of nanoparticles either in the
cytoplasm or in the periplasmic region causing disruption of
cellular function. We have also seen the accumulation of CuO
NPs
on bacterial cell surface by AFM. The differential activity of
these
two shaped nanoparticles may be due to their difference in ROS
generation inside the cells. In vitro Cu ion release is almost
same at
the higher time for both shaped CuO NPs and the amount of
ROS
generation is also same by two CuO NPs in B. subtilis strain. It
is
likely that sheet shaped NPs have less access inside the cells
but
their accumulation in the membrane or periplasmic region

perturb
the structure of membrane of such bacteria. This is also
observed in
our SEM studies where more membrane damage are observed in
B.
subtilis by nanosheet CuONPs. The thick shield of
peptidoglycan
layer or its constituents may thus be the target of sheet shaped
CuONPs where as small size spherical CuO NPs easily
permeable to
thin peptidoglycan layer of gram negative bacteria and produce
more ROS inside the cell. As a matter of fact these NPs can
locally
change microenvironments near the bacteria and produce ROS
or
increase the NPs solubility, which can induce bacterial damage.
Thus both spherical and sheet shaped CuO NPs produce
membrane
damage to gram negative or gram positive bacteria, as observed
by
SEM. The exact mechanisms of action is not known but it seems
likely that constituent of bacterial cell surface may contribute
largely by interacting with specific nanoparticles. Additionally
we
found both the nanoparticles produce DNA damage. Large
amounts
of ROS could be generated even when only small amounts of
CuO
NPs are incorporated into cells. Nanoparticles can induce ROS
directly, once they are exposed to the acidic environment of
lysosomes or interact with oxidative organelles, such as mito-
chondria. Thus, antibacterial activity of these two CuO NPs may
depend on several factors including physiochemical properties
of
Fig. 5. Scanning electronic microscopic image (SEM) of

different shaped copper oxide nanoparticles treated or mock-
treated gram negative and gram positive E. coli bacterial
cells.
191
nanoparticles and nature of bacterial surface. Thus the nature of
bacterial strain and the surface properties of CuO NPs (e.g.,
size,
shape, zeta potential etc.) are responsible for the antibacterial
activity.
5. Conclusion
In this study, we presented the antibacterial activity of two
different shaped CuO NPs on different strain. The particles size
and
morphology were characterized by DLS and TEM. Chemical
characterization was done by XRD, FTIR. The studies of
antibacte-
rial activity of different shaped CuO NPs showed that the NPs
were
effective on variety of gram positive and gram negative bacteria
as
well as sheet shaped CuO NPs is more active on gram positive
where as spherical shaped CuO NPs is more active gram
negative
bacteria. ROS induced DNA damage and membrane ruptures are
the possible mechanisms of antibacterial activity of both shaped
CuO NPs.
Acknowledgements

The authors would like to acknowledge for financial support for
this research work the Department of Biotechnology,
Government
of India (No. BT/PR14661/NNT/28/494/2010). We also express
sincere thanks to Indian Institute of Chemical Biology (IICB),
Kolkata, India for providing the facilities to transmission
electron
microscopy and atomic force microscopy.
References
[1] M.J. Sweet, A. Chessher, I. Singleton, Review: metal-based
nanoparticles; size,
function, and areas for advancement in applied microbiology,
Adv. Appl.
Microbiol. 80 (2012) 113–142.
[2] F. Okafor, A. Janen, T. Kukhtareva, V. Edwards, M. Curley,
Green synthesis of
silver nanoparticles, their characterization, application and
antibacterial
activity, Int. J. Environ. Res. 10 (2013) 5221–5238.
[3] L. Sun, Y. Qin, Q. Cao, B. Hu, Z. Huang, L. Ye, X. Tang,
Novel photocatalytic
antibacterial activity of TiO2 microspheres exposing 100%
reactive {111} facets,
Chem. Commun. 47 (2011) 12628–12630.
[4] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, G.
Manivannan,
Selective toxicity of ZnO nanoparticles toward Gram-positive
bacteria and
cancer cells by apoptosis through lipid peroxidation, Nanomed.
Nanotech. Biol.
Med. 7 (2011) 184–1921.

[5] S. Wang, W. Hou, L. Wei, H. Jia, X. Liu, B. Xu,
Antibacterial activity of nano-SiO2
antibacterial agent grafted on wool surface, Surf. Coat. Tech.
202 (2007) 460–
465.
[6] W. Xiao, J. Xu, X. Liu, Q. Hu, J. Huang, Antibacterial
hybrid materials fabricated
by nanocoating of microfibril bundles of cellulose substance
withtitania/
chitosan/silver-nanoparticlecomposite films, J. Mater. Chem. B
1 (2013) 3477–
3485.
[7] X. Liu, Y. Luo, T. Wu, J. Huang, Antibacterial hybrid
materials fabricated by
nanocoating of microfibril bundles of cellulose substance
withtitania/
chitosan/silver-nanoparticlecomposite films, New J. Chem. 36
(2012) 2568–
2573.
[8] B. Fahmy, S.A. Cormier, Copper oxide nanoparticles induce
oxidative stress and
cytotoxicity in airway epithelial cells, Toxicol. In Vitro 7
(2009) 1365–1371.
[9] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, S.S. Habib, A.
Memic, Antimicrobial
activity of metal oxide nanoparticles against Gram-positive and
Gram-
negative bacteria: a comparative study, Int. J. Nanomed. 7
(2012) 6003–6009.
[10] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S.

Mukherji, Strain specificity in
antimicrobial activity of silver and copper nanoparticles, Acta
Biomater. 3
(2008) 707–716.
[11] S.K. Misra, S. Nuseibeh, A. Dybowska, D. Berhanu, T.D.
Tetley, E. Valsami-Jones,
Comparative study using spheres, rods and spindle-shaped
nanoplatelets on
dispersion stability, dissolution and toxicity of CuO
nanomaterials, Nano-
toxicol. 4 (2014) 422–432.
[12] J. Beranová, G. Seydlová, H. Kozak, O. Benada, R. Fišer,
A. Artemenko, I.
Konopásek, A. Kromka, Sensitivity of bacteria to diamond
nanoparticles of
various size differs in gram-positive and gram-negative cells,
FEMS Microbiol.
Lett. (2014), doi:http://dx.doi.org/10.1111/1574-1111/6968.
[13] M. Shoeb, B.R. Singh, J.A. Khan, W. Khan, B.N. Singh,
H.B. Singh, A.H. Naqvi,
ROS-dependent anticandidal activity of zinc oxide nanoparticles
synthesized
by using egg albumen as a biotemplate, Adv. Nat. Sci.: Nanosci.
Nanotechnol. 4
(2013) 35015.
[14] Y. Li, W. Zhang, J. Niu, Y. Chen, Surface-coating-
dependent dissolution,
aggregation, and reactive oxygen species (ROS) generation of
silver nano-
particles under different irradiation conditions, Environ. Sci.
Technol. 47
(2013) 10293–10301.

[15] P.B. Santhosha, A. Velikonjab, C. Š. Perutkovad, E.
Gongadzed, M. Kulkarnid, J.
Genovaf, K. Elerši9cg, A. Igli9cd, V. Kralj-Igli9ch, N.P. Ulrih,
Influence of
nanoparticle-membrane electrostatic interactions on membrane
fluidity
and bending elasticity, Chem. Phys. Lipids 178 (2014) 52–62.
[16] Y. Chang, H. Chun Zeng, Controlled synthesis and self-
assembly of single-
crystalline CuO nanorods and nanoribbons, Cryst. Growth Des.
2 (2004) 397–
402.
[17] D. Bhattacharya, S. Samanta, A. Mukharjee, C.R. Santra,
A.N. Ghosh, S.K. Niyogi,
P. Karmakar, Antibacterial activities of polyethylene glycol,
tween 80 and
sodium dodecyl sulphate coated silver nanoparticles in normal
and multi-drug
resistant bacteria, J. Nanosci. Nanotechnol. 12 (2012) 2513–
2521.
[18] J.M. Andrews, Determination of minimum inhibitory
concentrations, J.
Antimicrob. Chemother. 48 (2001) 5–16.
[19] A. Pramanik, D. Laha, D. Bhattacharya, P. Pramanik, P.
Karmakar, A novel study
of antibacterial activity of copper iodide nanoparticle mediated
by DNA and
membrane damage, Colloids Surf. B: Biointerfaces 96 (2012)
50–55.
[20] B. Bagchi, S. Kar, S. Kr Dey, S. Bhandary, D. Roya, T. Kr

Mukhopadhyay, S. Das, P.
Nandy, In situ synthesis and antibacterial activity of copper
nanoparticle
loaded natural montmorillonite clay based on contact inhibition
and ion
release, Colloids Surf. B: Biointerfaces 108 (2013) 358–365.
[21] M. Gad, A. Itoh, A. Ikai, Mapping cell wall
polysaccharides of living microbial
cells using atomic force microscopy, Cell Biol. Int. 21 (1997)
697–706.
[22] M. Ramani, S. Ponnusamy, C. Muthamizhchelvan, J.
Cullen, S. Krishnamurthy, E.
Marsili, Morphology-directed synthesis of ZnO nanostructures
and their
antibacterialActivity, Colloids. Surf. B: Biointerfaces 1 (2013)
24–30.
[23] B. Fahmy, S.A. Cormier, Copper oxide nanoparticles
induce oxidative stress and
cytotoxicity in airway epithelial cells, Toxicol. In Vitro 23
(2009)
1365–1371.
[24] J. Xu, Z. Li, P. Xu, L. Xiao, Z. Yang, Nanosized copper
oxide induces apoptosis
through oxidative stress in podocytes, Arch. Toxicol. 87 (2013)
1067–1073.
[25] N. Talebian, S. Matin, M. Doudi, Controllable synthesis of
ZnO nanoparticles
and their morphology-dependent antibacterial and optical
properties, J.
Photochem. Photobiol. B. 120 (2013) 66–73.

[26] M.A. Ansari, H.M. Khan, A.A. Khan, S.S. Cameotra, Q.
Saquib, J. Musarrat,
Interaction of Al2O3 nanoparticles with Escherichia coli and
their cell envelope
biomolecules, J. Appl. Microbiol. 116 (2014) 772–783.
[27] P. Khullar, V. Singh, A. Mahal, P.N. Dave, S. Thakur, G.
Kaur, J. Singh, S.S. Kamboj,
M.S. Bakshi, Bovine serum albumin bioconjugated gold
nanoparticles:
synthesis, hemolysis, and cytotoxicity toward cancer cell lines,
J. Phys. Chem.
C 116 (2012) 8834.
[28] J. Li, K. Rong, H. Zhao, F. Li, Z. Lu, R. Chen, Highly
selective antibacterial activities
of silver nanoparticles against Bacillus subtilis, J. Nanosci.
Nanotechnol. 10
(2013) 6806–6813.
[29] V. Singh, P. Khullar, P.N. Dave, G. Kaur, M.S. Bakshi,
Ecofriendly route to
synthesize nanomaterials for biomedical applications: bioactive
polymers on
shape-controlled effects of nanomaterials under different
reaction conditions,
ACS Sustain. Chem. Eng. 1 (2013) 1417–1431.
191
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