METHODS - Shodhganga : a reservoir of Indian theses @...

Fabrication Of Coatings That Prevent Microbial Adhesion: Antimicrobial Effect Of Biomedical Products

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Materials and methods

METHODS

The present study was carried out in Microbiology laboratory, CMS College

of Science and Commerce, Coimbatore, India. FTIR analysis of the test samples

were carried out in Central Research Laboratory, PSG College of Arts and Science,

Coimbatore, India. Scanning electron microscopic (SEM) analysis was carried out

at SASTRA University, Tanjore, India. Physical parameters of the drug treated and

untreated textile materials were tested in SITRA, Coimbatore and NFIT-TEA,

Tirupur, India.

3.1. Determining the surface colonizing capability of test bacteria on

biomedical materials

The surface colonizing capability of test bacteria on biomedical implantable

materials (Silicone, Polyurethane, Teflon, Polyester and Silk Suture) and non-

implantable materials (Cotton, Cotton-Polyester, Polyamide and Viscose) was

determined using standard preliminary (Exit-Site challenge test) and confirmatory

tests (Congo red agar biofilm assay and microtitre plate biofilm assay).

3.1.1. Preliminary Exit-site challenge test (Bayston et al., 2009)

Exit-site challenge test was performed as preliminary test. This test was

used to identify the ability of specific test organism to grow on a type of biomedical

materials used in the study. In this method, three-quarter strength Iso-sensitest

semi solid Agar (Annexure-1) was poured into a sterile boiling tube and allowed to

solidify. The surface of the agar was then inoculated with 18h cultures of test

organisms [Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus

(ATCC 29213), Escherichia coli (ATCC 43894), Proteus mirabilis (ATCC 49565),

Pseudomonas aeruginosa (ATCC 700829), Bacteroides fragilis (ATCC 25285),

Fusobacterium nucleatum (ATCC 10953) and Prevotella intermedia (ATCC 2564)].

Different implantable materials and non-implantable materials were partially

inserted into the semi-solid medium through the inoculated area and incubated at

37°C. In the case of anaerobic test bacteria, Wright’s tube method was followed.

Migrating ability of the test bacteria from the ‘‘exit site’’ down the material track

i.e., outside of the materials were assessed visually up to 24-48 hours.


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3.2. Assessing the biofilm forming capability of test bacteria on biomedical

materials using two standard confirmatory test methods

The efficiency of test organisms to form biofilm on the biomedical materials

was confirmed after the preliminary exit-site screening test using two standard

methods, Congo red agar biofilm assay (Freeman et al., 1989) and Microtitre plate

biofilm assay (Christensen et al., 1985).

3.2.1. Biofilm forming ability of test bacteria by Congo red agar biofilm

assay (Freeman et al., 1989)

The biofilm forming ability of test organisms were confirmed by Congo red

agar biofilm assay method. In the method, specially prepared Brain heart infusion

agar (Annexure-2) was used to isolate the colonies of biofilm producing organisms.

Congo red was prepared as concentrated aqueous solution and autoclaved at

121°C for 15 minutes, separately from other medium constituents and was then

added when the agar had cooled to 55°C. Plates were inoculated and incubated

aerobically for 24 to 48 hours at 37°C. In the case of anaerobes, the seeded media

was incubated anaerobically in McIntosh field jar at 37°C for 24 to 48 hours.

Positive result was indicated by black colonies with a dry crystalline consistency.

Weak slime producers usually remain pink, though occasional darkening at the

centres of colonies was observed. A darkening of the colonies with the absence of a

dry crystalline colonial morphology indicated an indeterminate result. The

experiment was carried out in triplicates to confirm the biofilm production.

3.2.2. Confirmatory test on biofilm formation using Microtitre plate biofilm

assay (Christensen et al., 1985)

Bacterial attachment to an abiotic surface is assessed by measuring the

stain taken up by adherent biomass in a 96-well plate format by means of

microtitre biofilm assay. The test organisms were grown in 96-well microtitre plate

for 48 hours. The anaerobes were grown inside McIntosh Field jar simultaneously.

The wells were washed to remove any unbound test bacteria. Cells remaining

adhered to the wells were subsequently stained with a dye that allowed

visualization of the attachment pattern. Each of the test organisms was inoculated


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in a 5 ml culture broth and grown to stationary phase. Cultures were diluted at

1:100. Following this, 100 μl of each diluted cultures was pipetted into eight wells

in a fresh microtitre plate. The plate was covered and incubated at optimal growth

temperature for 24-48 hours. Four small trays were set up in a series and 1 to 2

inches of tap water was added to the last three. The first tray was used to collect

waste, while the others were used to wash the assay plates. Unbound bacteria if

any were removed from each microtitre dish by briskly shaking the dish out over

the waste tray.

About 125 μl of 0.1% crystal violet solution was added to each well. Staining

was done for 10 min at room temperature. The crystal violet solution was removed

by shaking each microtitre dish out over the waste tray. The dishes were washed

successively in each of the next two water trays and as much liquid as possible

was shaken out after each wash. To remove any excess liquid, each microtitre dish

was inverted and vigorously tapped on paper towels. The plates were allowed to

air-dry. Added 200 μl of 95% ethanol to each stained well. The plates were covered

to allow solubilisation by incubating for 10 to 15 min at room temperature. The

contents of each well were briefly mixed by pipetting. Following this, 125 μl of the

crystal violet-ethanol solution was transferred from each well to a separate well in

an optically clear flat-bottom 96-well plate. The optical density (OD) of each of

these 125μl samples was measured at a wavelength of 500 to 600 nm. Optical

density (OD) of stained adherent bacteria was determined with a micro ELISA auto

reader. The OD values were considered as an index of bacteria adhering to surface

and forming biofilms. Based on the OD value the adherence of organism in the

plate can be classified as below (Table-5).

Table-5: Classification of biofilm formation

Mean OD values Biofilm formation Biofilm index

<0.120 Nil Non / weak

0.120-0.240 Moderately Moderate

>0.240 Strong High

Table adapted from Mathur et al., (2006)


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3.3. Determining the synergistic activity of fluoroquinolone and

nitroimidazole drugs against test bacteria (Eliopoulos and Moellering, 1991)

The synergistic activity of a fluoroquinolone drug and a nitroimidazole drug

on all the test bacteria was determined by the standard checker board titration

method. To determine the inhibitory concentrations of each drug separately and in

combinations, the minimal inhibitory concentrations (MIC) was simultaneously

identified in this method. The fractional inhibitory concentrations (FIC) of the drugs

were calculated from MIC values to determine the synergism between the

fluoroquinolone drugs (norfloxacin, ofloxacin and ciprofloxacin) and nitroimidazole

drugs (metronidazole, ornidazole and tinidazole).

3.3.1. Assessing the antimicrobial combinations against test bacteria using

standard checker board titration method (Qasiasgar and Kermanshahi, 2009)

To assess antimicrobial combinations in vitro the checkerboard method was

selected. In this technique by using agar dilution method, the concentrations

tested for each antimicrobial agent were typically ranged from four or five

below the expected MIC to twice the anticipated MIC as in the 45 degree line

in Figure- 2 (each square represents one plate).

Figure-2: Checkerboard model to determine synergism of two drugs

(The picture was adapted from Qaziasgar and Kermanshahi, 2008)

Bottom row: To determine MIC of Drug-A, Left column: to determine MIC of drug-B, Centre: MIC of Drugs A + B


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The predetermined concentrations (µg/ml) used for this method were 0.015,

0.03, 0.06, 0.12, 0.25, 0.5, 1.0 and 2.0. According to figure-2, the plates in the left

hand column was used for the predetermined concentrations of first drug

(fluoroquinolone), the plates in bottom row was used for second drug

(nitroimidazole) and the plates in the 45 degree line was used for mixed drug

combinations. In all the arranged plates, 1ml of predetermined dilutions of the

antimicrobial agents were added with sterile and molten Muller-Hinton agar.

Then the surface of each plate was inoculated with 1 X 104 CFU/spot of bacteria.

After 16-20 hours incubation at 37 ºC, the plates were examined for evidence of

visible growth. Experimental set up was made for all the drug combinations

(norfloxacin-metronidazole, ofloxacin-ornidazole and ciprofloxacin-tinidazole) in

triplicate.

3.3.2. Evaluating the synergism between fluoroquinolone and nitroimidazole

drugs by fractional inhibitory concentration index (Bharadwaj et al., 2003)

Fractional inhibitory concentration index (FICI) was calculated by using the

following equation.

Formula to determine synergy

FIC index = FICA + FICB

MICA in combination FICA= ................................ MICA

MICB in combination FICB= ............................... MICB

where A was the minimal inhibitory concentration (MIC) of drug A in a plate that

was the lowest inhibitory concentration in its row, and B was the MIC of drug

B in a plate that was the lowest inhibitory concentration in its column. MICAB

was the lowest inhibitory concentration of drug A and B in combination in the 45

degree line. With this method, synergism has traditionally been defined as an FIC

index of 0.5 or less and partial synergy as a FIC index of >0.5 - ≤1.0; antagonism

has been defined as a FIC index of ≥2.0.


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Interpretation:

Mean FICI ≤ 0.5 → Synergy, (p< 0.5),

Mean FICI >0.5 - ≤1.0→ Partial synergy, (p> 0.5)

Mean FICI ≥ 2.0→ Antagonism

Synergy: Synergistic action of a combination of antibiotics is present if the effect of

the combination exceeds the additive effects of the individual components.

In simple terms, synergism is defined as the ability of a pair of drugs to produce a

more rapid rate of bactericidal action within the first 24 hours of exposure than

either member of the pair alone, and killing of great numbers of bacteria that could

be expected from simple summation of single drug effects.

Partial synergy: The additive effect of combination is one in which the effect of

combination is equal to that of the sum of the effects of the individual components.

3.4. Determining the mode of action of fluoroquinolone and nitroimidazole

drugs on bacterial DNA (Rajendran et al., 2011)

Fluoroquinolone drug and nitroimidazole drug inhibits the DNA replicative

enzymes of anaerobic and facultative anaerobic bacteria. To confirm this mode of

action, the test organisms were exposed to the synergistic drugs under optimum

conditions. Two groups of similar test organisms were selected. One group of the

representative test organisms [Staphylococcus epidermidis (ATCC 35984) and

Escherichia coli (ATCC 43894)] were exposed to the synergistic drugs. 100 ml of 18

hours test cultures were treated with 1 % each of the synergistic drug for 3 hours.

Another group of similar organisms was cultured separately without exposing to

the drugs. Drug exposed test organisms and unexposed test organisms were then

processed to extract DNA. DNA samples of both the samples were electrophoresed

to observe the difference in width of DNA bands under UV light to determine the

mode of action of synergistic drugs. Following were the steps involved in DNA

extraction and electrophoresis.

a) Culturing the test bacteria for DNA extraction

About 50 ml of LB broth was prepared and inoculated each of the

representative test organisms separately (S. epidermidis (ATCC 35984) as gram-


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positive representative and E. coli (ATCC 43894) as gram-negative representative

organisms). Cultures were incubated in the appropriate condition (37 °C for 12

hours). Dispensed 5 ml of the culture separately and centrifuged at 8,000 rpm for

15 minutes. The pellets were washed in normal saline twice and then suspended

in 2ml normal saline buffer.

b) Exposing the test bacteria with synergistic drugs

Norfloxacin-metronidazole (D1) and ofloxacin-ornidazole (D2) drug

combinations prepared at 1mg/ml were added to the bacterial pellet suspension

and incubated overnight at 37°C. Another set of bacterial pellet suspension was

handled in parallel without the addition of drugs (test organisms unexposed to

drugs)

c) Extraction of DNA from drug exposed and unexposed test bacteria

About 2ml of drug-exposed test bacterial culture was taken after overnight

incubation. Both aerobic and anaerobic culture suspensions were centrifuged at

10,000 rpm for 15 minutes to pelletize the cell mass. The supernatant was

removed and the cell pellet was incubated on ice till next use. To the obtained

pellet 500µl of solution-A (Annexure-3) was added and gently mixed to make a

uniform cell suspension. The cell suspension was incubated on ice for 30 minutes.

To the mixture 100µl of freshly prepared solution-B (Annexure-4) was added and

vortexed well. The suspension was incubated in ice for 5 minutes (cells get lysed

and the solution becomes clear). About 750µl of solution-C (Annexure-5) was

added and vortexed for 2 min. The suspension was kept in ice for 60 minutes

(chromosomal DNA and cell material will precipitate into whole viscous clump).

The tubes were centrifuged at 6000 rpm for 10 minute at 4°C. Transferred the

supernatant to fresh eppendorf tube and centrifuged at 10,000 rpm for 10 minute

at 4°C. The supernatant was discarded and to the pellet, 50 µl of ice cold ethanol

was added along the sides of the tube. The solvent used was removed immediately

without disturbing the pellet. The tubes were blot dried using blotting paper by

inverting the tube over it. The pellets of DNA were stored by adding 100µl of 1X

TAE buffer and placed in a deep freezer. Similar procedure was carried out for

drug-unexposed DNA samples of test organism.


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d) Agarose gel electrophoretic analysis of DNA

The extracted DNA from the drug exposed test organism was subjected to

agarose gel electrophoresis to observe the DNA band width by comparing with the

DNA of drug unexposed bacteria. Agarose (1 %) was suspended in 100 ml of 1X

TAE buffer and placed in boiling water bath. The entire agarose was allowed to

dissolve and the solution was left in the water bath until it becomes transparent.

The gel solution was kept at room temperature and allowed to reach 55-60°C.

About 100µl of ethidium bromide solution (Annexure-6) was added and mixed

gently for even distribution using glass rod without making bubbles. Gel casting

tray with the comb was kept ready for agarose gel preparation. The gel solution

was slowly poured into the tray and allowed to solidify. This assembly was left

undisturbed for 35-45 minutes and after gel casting the comb was carefully

removed. The gel was placed inside the electrophoretic tank and the tank buffer

(1X TAE) was poured to cover the gel. About 10µl of DNA sample was mixed with

5µl of gel loading dye (Annexure-7) and allowed to settle. With a power pack,

constant voltage of 50V was supplied to the tank buffer and the well poured with

DNA sample was placed near to negative terminal so that this facilitates DNA

movement towards positive terminal. The gel was supplied with power until

bromophenol blue in the tracking dye reaches 0.5cm close to the end of the gel.

After disconnecting the terminals from the power pack, the gel was placed over a

transilluminator to observe the DNA bands.

IMPLANTABLE MATERIALS

After analysing the synergistic activity of different fluoroquinolone drugs

and nitroimidazole drugs and its mode of action on bacterial DNA, the drug

combination mixtures were added with different drug-carriers to fabricate the

selected implantable materials (silicone, PTFE-teflon, polyurethane, polyester and

silk suture). The implantable materials were fabricated by imparting antibacterial

drugs and carriers using a standard two-dip-coating method. Following were the

protocols of antibacterial drug preparations, dip-coating the materials,


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antibacterial activity of the drug coated materials and its durability

determinations.

3.5. Preparation of antibacterial drugs for coating the implantable

materials (Gollwitzer et al., 2003)

The antibacterial drugs for coating the implantable materials were prepared

using a solvent-casting technique. Each carrier (beta-cyclodextrin, DL-lactic acid,

tocopherol acetate) was mixed with each of the synergistic drugs (a fluoroquinolone

drug and a nitroimidazole drug) by the following procedure. All the carriers were

selected based on their biological properties like; enhancement of antibacterial

activity; provides sustained and constant release of drugs, food and medical grade

polymers.

a) Beta-cyclodextrin + synergistic drugs

Carrier, 650 mg of β-Cyclodextrin was added in 5 ml of 0.1N NaOH at a

concentration of 130 mg/ml. To the solvent carrier mixture, Norfloxacin and

Metronidazole or Ofloxacin and Ornidazole or Ciprofloxacin and Tinidazole were

added to attain a final concentration of 1%.

b) DL-Lactic acid + synergistic drugs

Carrier, 0.78 ml (650mg) (1.21 ml = 1g) of DLLA was added in 4.22 ml of

0.1N sodium hydroxide (NaOH) at a concentration of 130 mg/ml. To the solvent

carrier mixture, Norfloxacin and Metronidazole or Ofloxacin and Ornidazole or

Ciprofloxacin and Tinidazole were added to attain a final concentration of 1%.

c) Tocopherol acetate + synergistic drugs

Carrier, 0.84 ml (650mg) (1.4 ml = 1g) of Tocopherol acetate was added in

4.16 ml of 0.1N NaOH at a concentration of 130 mg/ml. To the solvent carrier

mixture, Norfloxacin and Metronidazole or Ofloxacin and Ornidazole or

Ciprofloxacin and Tinidazole were added to attain a final concentration of 1%.

The test was carried out in such a way that, all the three combinations of

synergistic drugs were mixed individually with all the three types of carriers. Thus

drug and carrier combinations were D1C1, D1C2, D1C3, D2C1, D2C2, D2C3 and D3C1,

D3C2, D3C3. In these combinations, norfloxacin-metronidazole was indicated as


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(D1), ofloxacin-ornidazole as (D2), ciprofloxacin-tinidazole as (D3) and beta-

cyclodextrin as (C1), DL-Lactic acid as (C2) and tocopherol acetate as (C3).

3.6. Coating the implantable materials with prepared antibacterial drugs

using the standard two-dip-coating method (Matl et al., 2008)

All the implantable materials (Silicone, Polyurethane, PTFE, Polyester and

Silk Suture) were subjected for coatings in 3 different groups (Group-A, B and C).

The materials in Group-A was coated with synergistic drugs and carriers. Materials

in Group-B were coated using synergistic drugs without carriers and the materials

in Group-C were coated using carriers without drugs. The surface

functionalization of the implantable materials was carried out using a standard

two-dip-coating method. The coated materials in Group-A, B and C were referred

below as drug-carrier coated (dcc), drug-coated (dc) and carrier-coated (cc). Uncoated

(uc) implantable materials were also used in parallel to differentiate from other 3

groups of materials.

All the materials were cut according to the type of experimental needs and

also based on the standard references. Silicone was cut in two different sizes (5

mm disc for antibacterial assay and 1 cm disc pieces for FTIR analysis).

Polyurethane of 1 cm in length was selected for antibacterial assay. PTFE and

polyester of 1 cm2 pieces was selected for antibacterial assay and Silk suture with

length of 2.5 cm was cut to determine the antibacterial activity.

All the materials were autoclaved at 121°C for 15 minutes before coating.

Sterile materials were coated separately with drugs, carriers and drug-carrier

conjugates. The dip-coating procedure was carried out in sterile glass beakers on a

shaker (120 rpm) for 3 hours, with a drying period of about 15 minutes between

the two coating procedures. The coated materials were rinsed in phosphate

buffered saline (PBS) to remove surface accumulation of particles, followed by

drying at room temperature. All coating steps were carried out under aseptic

conditions in a laminar airflow hood. The weight of each coating i.e., the amount of

the drug incorporated, was assessed by the difference in the weight of the test

material before and after the coating procedure.


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3.7. Determining the drug-add on percentage on the implantable materials.

Weight determination of coated materials (Shanmugasundaram et al., 2011)

Add on concentration of antibacterial drugs on each implantable materials

were calculated using a standard formula of drug add on percentage. After coating

with antibacterial drugs all the implantable materials were subjected to determine

the weight. To measure the drug concentration, the materials were weighed using

Schimadzu weighing balance before and after coating. To ensure the coatings on

the implantable surfaces each material was weighed separately. The average

weight of the samples was assessed by weighing each sample three times. The

increase in weight after coating with drugs was determined in percentage.

Drug add on (%) = [(W1 – W2)/W2] x 100

Where,

W1 = weight of the material after coating

W2 = weight of the material before coating

3.8. Assessing the qualitative antibacterial activity of dip-coated implantable

materials (El-rehewy et al., 2009)

The method was performed for analysing the antibacterial activity of

different implant materials after dip-coating with synergistic antimicrobial drugs

and carriers. In this qualitative method the pre-measured size (Section 3.6) of all

sterilized implant materials were tested from each preparation [drug-carrier coated

(dcc), carrier-coated (cc) and uncoated (uc) implantable materials]. The materials

were all rinsed twice in phosphate buffered saline (Annexure-9) before testing to

remove any surface accumulation of drug. All implant materials were placed on the

surface of Mueller-Hinton agar plate which had previously been seeded with an

overnight broth culture of the test organisms and incubated at 37°C for 24 to 48

hours. In the case of anaerobes, the seeded media were incubated anaerobically in

McIntosh field jar at 37°C for 24 to 48 hours. The experiment was carried out in

triplicate. Antibacterial activity was expressed as the diameter of the zone of


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inhibition. From the qualitative analysis, for each implantable material, atleast

three drug carrier combinations were selected for the in vitro drug release analysis.

3.9. Analysing the drug release profiles from coated implantable materials

using the standard in vitro dissolution method (Mashru and Saikumar, 2010)

The release profile of synergistic drugs from the drug-carrier coated (dcc) and

drug coated (dc) implantable materials was studied by in vitro dissolution method.

This test was performed in triplicate to determine the sustained release of

synergistic antimicrobial drugs from the drug-carrier coated implantable materials.

The concentration of each drug released from the implant materials were

measured using the calibration curves constructed from standards of each drug.

3.9.1. Development of standard calibration curve of antibacterial drugs

A stock solution of Norfloxacin, Metronidazole, Ofloxacin, Ornidazole,

Ciprofloxacin and Tinidazole (100 µg/ml) was prepared by adding 10mg of each

drug in 100ml of phosphate buffer (pH - 6.8). From the stock, series of standards

(10µg to 200µg) were prepared and the absorbance was measured to evaluate the

concentration of drug. The absorbance was plotted against the respective

concentration to obtain the calibration curves. The absorbance of known drug

concentration was measured spectrophotometrically using UV-VIS

spectrophotometer at their respective wavelengths (Norfloxacin - 300 nm,

Metronidazole - 320 nm, Ofloxacin - 293.4 nm, Ornidazole - 319.6 nm,

Ciprofloxacin - 277 nm, Tinidazole - 240 nm).

3.9.2. Determining the drug release concentrations from (drug coated and

drug-carrier coated materials using in vitro dissolution method

Drug release from drug-carrier coated (dcc) and drug coated (dc) implantable

materials was studied in 50 ml phosphate buffer (pH - 6.8) kept at 300 rpm in a

thermomixer. The release profiles of each of the drugs were evaluated

spectrophotometrically using UV-VIS spectrophotometer. Absorbance was

measured for 4 ml of the sample solution at their respective wave lengths against

Phosphate buffered saline as a blank. Fresh medium of the same volume was


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replaced each time after measuring absorbance. The release profile of the drugs

was determined from 30 minutes to 120 hours (30 min, 1, 2, 4, 8, 12, 24, 48, 72,

96 120 hours) using UV-Vis spectrophotometer. Specific drug-carrier combinations

for each type of implantable materials showing sustained release of the drugs were

alone selected for further parameters like bacterial adherence test and in vitro

challenge test.

3.10. Quantitative antibacterial activity of coated materials using the

standard bacterial Adherence test (El-Rehewy et al., 2009)

Antibacterial activity for each implantable material coated with the selected

drug-carrier combinations were quantitatively analysed using bacterial adherence

test (specific drug-carrier combination for each material was selected after in vitro

dissolution method).

The effect of dip-coated implantable materials (drug-carrier coated - dcc) was

tested against the test organism using a standard bacterial adherence test. The

materials were placed separately in a tube with 5 ml of each of the test bacteria

and incubated at 37 °C for 18 h. During the incubation period the bacterial cells

adhere on the surface of the implantable materials. After incubation, the numbers

of viable adherent cells were determined as follow: Implantable materials were

collected aseptically and washed in a sterile test tube of 10 ml normal saline to

remove the non-adherent cells. The washed pieces were transferred each in 10 ml

fresh sterile saline, and sonicated for 30 seconds to dislodge the sessile adherent

cells using an ultra-sonicator. After sonication, serial dilution of the sonicated

saline was made and the number of sessile bacteria which indicates degree of

adherence was determined by viable count technique. Similar experimental set up

was run in parallel for uncoated (uc) and carrier coated (cc) materials. The

difference in number of adhered cells on dcc, and cc were determined statistically

using chi square analysis.

The percentage reduction of adhered organisms on the drug-carrier coated

materials was determined using a standard percentage reduction formula.


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Bacterial reduction (%) = A – B/A X 100

Where,

A = number of adhered organisms (in CFU) obtained from the uc materials

B = number of adhered organisms (in CFU) obtained from the dcc or cc materials

3.10.1 Statistical analysis of total viable bacteria on coated materials

Chi-square non parametric test using SPSS-9 for Windows 7 was used as a

statistical tool to determine the effect of antimicrobial drug on bacterial adherence.

The hypothesis selected (H0) was that “There is significant effect of antimicrobial

drug on the test organisms”. The difference in the bacterial reduction percentage

between the drug-carrier coated (dcc) implantable materials and the carrier-coated

(cc) implantable materials were statistically calculated with P<0.05 considered

significant.

3.11. Determining the persistence of drugs on coated materials using the

standard in vitro challenge test (Bayston et al., 2009)

The In vitro challenge (IVC) test was designed to determine the ability of dip-

coated implantable materials to resist bacterial colonization with multiple

challenges in flow conditions. In vitro challenge test was performed in triplicate to

determine the persistence of the most effective antimicrobial drug during the

implantation period. Briefly, the pre-measured size (Section 3.6) of each material

coated with the selected antimicrobial drug combination was used for the study.

The drug-carrier coated (dcc) implantable materials were inserted into a controlled-

environment chamber containing pre-sterilized nutrient broth. The broth

containing dcc implantable material was inoculated with the test organism and

incubated in a shaker incubator (80 rpm) at 37°C. A challenge dose (1 ml

overnight culture) of each test organism was inoculated into the broth. Similar set

up was made for the uncoated implantable materials as control. All the materials

were examined visually each day and samples of the medium were collected

periodically for culture. If no colonisation was detected after 7 days incubation, a


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second challenge dose was administered. In the case of absence of turbidity after

the second challenge dose, further challenge dose was administered at 7 days

interval until turbidity was observed.

NON-IMPLANTABLE MATERIALS

Plain weave cotton and cotton blend polyester (C/P) woven fabrics (mostly

used in woven surgical gowns, patients, physicians and nurse uniforms);

polyamide and viscose (routinely used in several applications like medical gauze,

diapers, wet wipes, facial mask and incontinence products) were selected as non-

implantable materials in the present study. The specifications of cotton and C/P

fabrics were given in table-6. Polyamide and Viscose with the following

specifications, weight of 50 g/m2 (GSM), white coloured and width of 100-2100

mm was selected.

Table-6: Specification of cotton and C/P fabrics

S. No Particulars Cotton C/P

1 Count – Warp Weft

40s

40s 2/40s

2/40s

2 Ends/Inch (EPI) 60 60

3 Picks/Inch (PPI) 56 56

4 Weave Plain Plain

3.12. Different antibacterial finishing methods on non-implantable textile

materials

Two standard methods used in textile applications were selected in this

research to impart antibacterial drugs on non-implantable materials. In the first

method, standard reactive dye method was used to impart the reactive drugs onto

textile materials. The method was a standard protocol handled in textile industries

to impart reactive dye colours onto textiles. Secondly, microencapsulation method

was used to increase the wash durability properties in finished textile materials.

Following were the protocols of two standard methods used to fabricate the non-

implantable test materials for antibacterial finish and its durability

determinations.


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3.12.1. Direct attachment of synergistic drugs onto textile materials using

the standard reactive dye method (Chun and Gamble, 2007)

The approach in this study was to modify two well described antibacterial

drugs (fluoroquinolone and nitroimidazole compounds) for direct attachment to

textile fabrics.

a) Synthesis of reactive synergistic drugs

To convert the drug reactive and imparting to non-implantable materials,

the method was performed. Synthesis of reactive fluoroquinolone drug was

accomplished by suspending 2 % each of the drugs (norfloxacin, ofloxacin,

ciprofloxacin) in 20 ml deionized water in an ice bath at 5 °C. To this suspension,

0.04 M cyanuric chloride was added to convert the drug reactive. The suspension

was maintained at 5 °C during the drop-wise addition of 0.04 M NaOH. Synthesis

of reactive nitroimidazole drug was similarly prepared suspending 2 % each of the

drugs (metronidazole, ornidazole, tinidazole) in 20 ml deionized water in an ice

bath at 5 °C. To this suspension, 0.03 M cyanuric chloride was added to convert

nitroimidazole drug reactive. The suspension was maintained at 5 °C during the

drop-wise addition of 0.03 M NaOH.

b) Exhaust dyeing method to bind reactive antibacterial agents to textile

materials

An exhaust dyeing method was used to bind the synthesized reactive

antibiotic to the non-implantable textile fabric. The dye bath was prepared by

adding 0.5 ml of Triton-X-100, 75 g of sodium sulfate, and 6.5 g of the reactive

antibacterial drug, or 3.25 g of each of the two reactive antibacterial drugs (a

fluoroquinolone and a nitroimidazole drug) to 1.2 L of deionized water. To the

suspension each of the carriers (beta-cyclodextrin, DL-lactic acid and tocopherol

acetate) was added at a concentration of 2 %. Three, 20 g squares of the test fabric

(cotton) were submerged in the dye bath heated to 60 °C. After 30 min of

incubation, 12 g NaOH that had been dissolved in 100 ml of deionized water was

added. The temperature was then raised to 80 °C, and the fabrics heated for

another 30 min. The fabric was then rinsed in deionized water and heated for 10


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min at 80 °C in deionized water, then rinsed and kept in a convection oven at 105

°C until dried. Similar procedure was carried out for all the test fabrics (C/P,

polyamide and viscose).

3.12.2. Determining the durability of drugs using the standard

microencapsulation method (Sathianarayanan et al., 2010)

Fabrics treated with microencapsulated antibacterial agents were reported

to be durable for few number of wash cycles compared to other methods. Hence, in

the present study the test materials were treated with encapsulated drugs by

adding it as core material and acacia powder as wall material.

a) Synthesis of microencapsulated synergistic drugs

About 10 grams of acacia powder was allowed to swell for 15 min in 100ml

of hot water. To this mixture, 50 ml of hot water was added and stirred for 15 min

maintaining the temperature between 40 °C and 50 °C. Around 2% of core-

material (a fluoroquinolone drug and a nitroimidazole drug) was slowly added

under stirring condition. Stirring was continued for another 15 min and then 10

ml of 20% sodium sulphate was added. To this suspension, 2 % of a carrier (beta-

cyclodextrin, DL-lactic acid, or tocopherol acetate) and 2 % of citric acid

(crosslinker) was added. The stirrer was stopped and the mixture was freeze-dried

overnight to develop microcapsules.

b) Characterization of microcapsules

The developed microcapsules were observed under conventional light

microscope to determine its shape and outer structure. The method was also used

to identify the particle size and its homogenous distribution.

c) Binding microencapsulated drugs with textile materials

Each of the fabric samples (cotton, cotton-polyester, polyamide and viscose)

was immersed in the microcapsule solution and padded through a pneumatic

padding mangle at a pressure of 3 psi to get a wet pick up of 100% on weight of

fabric. The treated fabric was dried at 80°C for 5 min and cured at 150°C for 5


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min. Similar experiment was carried out for each material separately, treated with

synergistic drugs (D1, D2 or D3) coupled with each carriers (C1, C2 or C3).

3.13. Evaluating the antibacterial activity of treated fabrics using the

standard AATCC Test Method 100 – 2004.

The antibacterial properties of materials can be studied by quantitative

(AATCC-100) test methods. Quantitative test is the proper indicator of degree of

antibacterial activity when the antibacterial agents are fixed on to the textile

material or are unable to leach out. The different tests carried out in this study

were based on such consideration.

Assay for antibacterial properties of textile materials (AATCC Method 100-2004)

All the treated fabrics and untreated fabrics by both the methods (reactive

dye and microencapsulation method) were subjected to antibacterial assay. The

assay used for measuring antibacterial properties was based on the AATCC Test

Method 100-2004. Briefly, 1.0 ml of 12 hours challenge bacterial inoculum was

dispersed as droplets over the 3 swatches (test fabrics) using a micropipette. The

swatches were inoculated in pre-sterilized 250 ml Erlenmeyer flasks. After all the

samples were inoculated, the flasks were incubated at 37 ± 2 °C for 18 h before

being assayed for bacterial population density. The bacterial population density

was determined by extracting the bacteria from the fabric by adding 100 ml of

distilled water to each flask and shaken using an orbital shaker for 1 min. Then

aliquots were serially diluted and pour plated to determine the bacterial density.

The difference in number of viable bacteria was evaluated on the basis of the

percentage reduction. Percentage reduction was calculated using the following

formula.

R = (A-B) / A X 100

Where, R is percentage reduction; A is the number of bacteria in the broth

inoculated with treated test fabric sample immediately after inoculation i.e., at zero

contact time and B is the number of bacteria recovered from the broth inoculated

with treated test fabric sample after the desired contact period of 18 hours.


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3.14. Determining the wash durable properties of treated textile materials

using the standard wash fastness test (AATCC Test Method 124-2010)

Assay for antibacterial properties of after wash materials (AATCC Method 100-2004)

All the treated fabrics and untreated fabrics by both the methods (reactive

dye and microencapsulation method) were subjected to wash fastness test. The

fabric was washed based on the AATCC Test Method 124-1996 laundering

procedure to see if the drug would persist through multiple washings. The treated

samples were washed 2, 5, 10 and 15 times. After each wash, antibacterial activity

of each fabric were quantitatively assayed (AATCC Method 100-2004) using the

method described in Section – 3.13.

3.14.1. Statistical analysis of wash fastness

Statistical analysis of viable bacteria on encapsulated drug treated textiles after 15th

wash

Chi-square non parametric test using SPSS-19 for Windows-7 was selected

as a statistical tool to determine the effect of antimicrobial drug on the number of

viable bacteria at 18th hour after 15th wash. The hypothesis selected (H0) was that

“There is significant effect of antibacterial drug on the test organisms”. The

difference in the number of viable bacteria after ‘0’ contact time and 18 hours

contact time for the encapsulated drug treated fabrics (after 15th wash) were

statistically calculated with P<0.05 considered significant.

3.15. Analysing the physical properties of textile materials after antibacterial

finishing

Different physical properties of reactive drug treated and encapsulated drug

treated textile materials were analysed and compared with the normal untreated

materials. The methods were performed to determine whether the normal physical

properties of the textile materials were unaffected after the treatment of

antibacterial drugs.

3.15.1. Tensile strength test (ASTM D 5035-2006 test method)

Tensile strength is the measure of the resistance of the fabric tensile load or

stress in either warp or weft direction. It is the strength shown by a specimen


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subjected to tension as distinct from torsion, compression, or shear. Elongation

defines the length to which a fibre may stretch before breaking. A sample of 12” X

2” was taken for the test. The tensile strength of the fabric was determined by

cloth tensile strength tester. Tensile strength is performed using cut strip method.

This test is used for treated or heavily sized fabrics. Three readings for every

sample were taken and the average was calculated.

3.15.2. Abrasion test (AATCC 119-2004 test method)

Abrasion test determined the ability of a fabric to withstand damage by

friction. This is dependent on the fineness of the fiber, the amount of twist of the

yarn and the weave structure of the fabric. Yarns that have a firmer and tighter

twist were generally more resistant to abrasion. The fabric specimen was mounted

over a foam rubber cushion and rubbed multi-directionally against a wire screen

mounted on a weighted head. Abrasion was carried out for 75 revolutions for the

fabrics. The initial and final weight of the fabric was noted. Four readings for every

sample were taken and the average was calculated.

Abrasion resistance % = weight before abrasion – weight after abrasion X 100

Weight before abrasion

3.15.3. Resistance to Pilling

A piece of fabric measuring 5 inch x 5 inch is stitched so as to be a firm fit

when placed round a rubber tube 6 inch long, 1¼ inch outside diameter and 1.8

inch thick. The cut ends of the fabric (9 inch x 9 inch x 9 inches) lined with cork

1.8 inch thick. The instrument, I.C.I. Pill box tester was used for this method. The

pre-measured size of the test fabric was then rotated at 60 rpm for 5 hours. For

fabrics that are normally subjected to repeat washing as well as to wear, washing

may be done prior to sample preparation. After the test was over, the extent of

pilling was assessed visually by comparison with the arbitrary standards 1, 2, 3, 4

and 5. Under the test conditions, fabrics of standard 1 become hairy but do not

pill, fabrics of standard 2 become hairy and pills lightly, while fabrics of standard 3

become hairy and pill more severely, fabrics of standard 4 refers to good and

fabrics of standard 5 refers to excellent.


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3.15.4. Dimensional stability

Length and width bench marks were made on a woven fabric specimen. The

materials were then washed and dried followed by re-measuring the bench marks

distance. The laundering process was repeated for the indicator cycle and the

dimensional change was calculated from the measurement.

Materials used to determine the dimensional stability of the textile materials

Cylindrical wash wheel of the reversing type, flat-bed press (at a

temperature not less than 135 °C), dryer of the rotary type, hand iron, measuring

device and ATCC detergent.

Method

The samples measuring the size of 60cm X 60cm for each textile materials

were placed in the washing machine with specified level of water. 66 ± 1 gm of

standard reference detergent was added with preselected washing temperature.

The specimen was dried by flat-bed dry method. After washing and drying,

materials were pressed and hand ironed at temperature of 120 0C - 180 0C. For

dimensional stability, the distance between each part of benchmarks to the

nearest millimetre were measured using the following formula

(B –A) Dimensional change = -------------- X 100 A Where,

A - Average of three original measurements for the length wise (or) width wise

direction in the specimen.

B - Average of three measurements after cycle completed for the lengthwise (or)

width wise direction of the specimen.

3.15.5. Air- permeability test (ASTM D 737-96 test method)

Air permeability of a fabric is the volume of air measured in cubic cm

passed per second through 1 sq. cm for the fabric at a pressure of one cm. head of

water. Air permeability can be measured using an instrument called Shirley Air

Permeability Tester. Air permeability was determined in accordance with Test

Method ASTM D-737-96. The conditioned specimens in the standard atmosphere

for testing textiles, 21 ± 1°C and 65 ± 2 % relative humidity was tested unless


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otherwise specified in a material specification or contract order. The test

specimens were carefully handled to avoid altering the natural state of the

material. Placed each test specimen onto the test head of the test instrument, and

performed the test as specified in the manufacturer’s operating instructions. Read

and recorded the individual test results in SI units as cm3/s/cm2.

3.16. Surface characterization of coated biomedical materials

The surface properties of biomedical materials before and after coating with

antibacterial drugs were characterized using two standard parameters, Fourier

transform infra-red spectroscopy (FTIR) and scanning electron microscopy (SEM).

Silicone and cotton alone were selected as a representative material for

implantable and non-implantable material. Both the material was subjected for

experimentation physically and chemically. Chemical analysis was made using

FTIR and physical characterization was analysed by SEM.

3.16.1. Determining the presence of drugs and its chemical interventions on

coated biomaterials using FTIR analysis (Coates, 2000).

The alteration in the functional groups of test materials (silicone and cotton)

due to the addition of drugs and carriers was determined chemically using FTIR

spectroscopy. The FTIR absorption spectra of the implantable material (silicone),

non-implantable material (cotton), pure drugs (ofloxacin, ornidazole), carriers (DL-

lactic acid and beta-cyclodextrin) and drug-carrier coated silicone and cotton were

recorded in the range of 400-4000 cm-1 by KBr disc method. FTIR spectra of the

samples were determined using Shimadzu FTIR spectrophotometer. All the

samples were prepared in KBr discs with a hydrostatic press at a force of

5.2 τ cm-2 for 3 minutes to reduce the moisture content on the disc surface. Each

disc was dried under radiation to remove excess moisture content.

3.16.2. Examining the homogenous coatings on biomaterials using

topographic analysis – SEM (Matl et al., 2008; Rajendran et al., 2012)

The surface coatings of the drugs and carriers on implantable (silicone) and

non-implantable materials (cotton) were observed using Scanning electron


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microscopy (SEM). SEM evaluation was also used to know the uniformity of

coating of finishing chemicals over the specimen. The topographic analysis of

coated and uncoated test materials was prepared for SEM using a suitable

accelerating voltage (10 KV), vacuum (below 5 Pa) and magnification (X 3500).

Metal coating was used as the conducting material to analyse the sample.

3.17. Determining the tissue reactions of coated biomedical materials using

the standard HET-CAM test (Luepke, 1985 and Valdes et al., 2002)

Hen´s Egg Test on the Chorioallantoic Membrane (HET-CAM) of chick eggs

To understand the inflammatory tissue reactions of coated biomedical

materials on the live tissues, the materials were placed on the surface of chorio-

allantoic membrane (CAM) of embryonated chick eggs. The inflammatory response

on CAM was evaluated by direct evaluation method and histological method.

a) Fertile eggs used in the study

Freshly laid fertile eggs were collected from the chicken farm and incubated

at 36-37° C for 8 days before implanting the sample materials (drug-carrier coated

and uncoated implantable and non-implantable). During the incubation time, the

eggs were turned twice daily.

b) Egg windowing

On the day of implantation, the eggs were candled to determine the position

of the air sac and the embryo. A square, with sides approximately 18-20 mm, was

marked on the shell where the chorio-allantoic membrane was best developed.

Areas with large blood vessels were avoided to obviate possible haemorrhage.

Using a dental drill fitted with a straight hand-piece the sides of the marked

square were drilled, taking care not to pierce the underlying shell membrane. In

one corner of this large triangle a second smaller square was drilled, with sides of

approximately 5 mm. A small slit was drilled in the shell over the air sac. A

mixture of molten paraffin wax and vaseline was painted over the drilled surfaces

to prevent fragments of shell from falling on to the membrane when it was later

exposed.


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c) Application of test material, positive and negative control on CAM

Aseptic technique was used for the implantation of the test material. For

dropping the material onto chorio-allantoic membrane the egg was mounted on a

stand, with the drilled area of shell uppermost; a straight Hagedorn's needle was

gently inserted under one corner of the smaller square of shell and this square was

raised and removed. The shell and shell membrane circumscribed by the larger

square were then removed, and the sterile pre-measured size of sample

implantable materials and non-implantable materials (drug-carrier coated

materials placed on CAM of separate eggs) were inserted and carefully lowered on

to the exposed membrane. Inorder to compare the inflammatory and non-

inflammatory patterns of the implanted test material, 0.3 ml of the substance

(positive and negative control) was applied to the surface of the CAM. 0.1 N NaOH

was added on the CAM of separate egg as a positive control and 0.9 % NaCl was

used as an appropriate negative control. After a 20-second exposure period, the

CAM is rinsed with 5 ml of water.

3.17.1 Direct evaluation of CAM (Luepke, 1985)

The CAM was evaluated for development of irritant endpoints like

hyperemia (increased blood flow), hemorrhage (blood from a ruptured vessel), and

coagulation (presence of blood clots)

a) Time to Development of Observed Endpoints after Exposure to the Test

Substance

A procedure used to evaluate the time to development of endpoints after

exposure to the test substance was to continually observe the CAM during the 5-

minute observation period and record (typically in seconds) the time at which each

of the endpoints developed. Therefore, three separate time values were obtained

and recorded for each egg (one time value for each endpoint). Individual values for

the observed endpoints were then used to determine the irritation potential of the

test substance.


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b) Irritation Scores (IS)

A majority of the test method protocols calculated a score (referred to as

irritation score, irritation index, or irritation potential) that represented the

irritation potential of the test substance based on endpoint development. This

score could be determined by a mathematical model which was discussed below.

c) IS calculation – IS [B] analysis method

The time taken for the development of each endpoints, hyperemia,

hemorrhage and coagulation was substituted in the standard formula as per IS [B]

analysis method. The time values assigned/obtained to each endpoint were

totalled to give an overall IS value for the test substance. An IS score could be

calculated using the following general formula,

Where, Hemorrhage time = time (in seconds) of the first appearance of blood

hemorrhages, Lysis time = time (in seconds) of the first appearance of vessel lysis

Coagulation time = time (in seconds) of first appearance of protein coagulation

d) Relationship of scores with category of irritation

After calculating the IS scores for the developed endpoints, the totalled

score was compared to identify the category of irritation caused by the test

materials on CAM. In Table-6 the final IS value ranged from 0 (for test substances

that do not induce development of any of the observed endpoints) to 21 (for test

substances that induce development of all three endpoints within 30 seconds of

application of the test substance) was presented. The relationship between scores

and category of irritation was tabulated below.

Table-7: Relationship of scores with category of irritation

Scores on HET-CAM Category of irritation

0 – 0.9 No irritation

1 – 4.9 Weak or slight irritation

5 – 8.9 Moderate irritation

9 - 21 Strong or severe irritation

From Cazedey et al., (2009)


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3.17.2 Histological evaluation of CAM (Valdes et al., 2002)

a) Incubation of eggs with test materials for microscopic analysis

After calculating the initial irritation scores of the test materials by

comparing with the scores of positive and negative controls, another set of similar

test materials were reimplanted on CAM of fresh eggs. The opening in the shell was

sealed with heated adhesive tape and the hole over the air sac region sealed with

molten paraffin wax. The eggs were then incubated at 37°C for 9 days, without

turning.

At the end of this period, the chorio-allantoic membrane was exposed by

cutting around the long circumference of the egg with a pair of curved scissors; the

chorio-allantoic membrane remained in the top half of the shell, together with the

implantable sample material. The sample material and the underlying portion of

thickened membrane were particularly dissected out. The membrane was gently

eased out of the shell using forceps and placed in a dish containing 10 % formal-

saline solution. After 24 hours fixation, implants were teased away from

underlying CAM which were then trimmed, paraffin embedded and prepared for

staining by using haematoxylin and eosin staining method. Prepared specimen

was observed for the inflammatory response on the CAM at the implanted site

under bright field microscopy.

b) Haematoxylin and eosin staining

The paraffin section was cut (4 μm to 8 μm thick) and placed on a labelled

slide. The slides were placed in a manual staining rack and dried in a 60 °C oven

for 30 to 40 minutes. Then the slides were stained manually using the following

reagent bath. Xylene was poured on the specimen and allowed to withstand for 4

min. The step is repeated twice using fresh xylene. 100% alcohol was added and

kept for 3 min (repeated thrice using fresh alcohol). The slide was then rinsed

using tap water. Haematoxylin stain was added and kept for 5 min. Again the slide

was rinsed using tap water. Acid water (0.5% conc HCl aqueous solution) was

added as haematoxylin differentiators and allowed to stand for 10 seconds

(repeated for 5 to 10 times). The slides were then rinsed with tap water and added

5% aqueous eosin for 3 min. Then slides were dipped in 95% and 100% ethanol


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solution for 2 times (repeated 4 times with fresh ethanol). Finally xylene was added

on to the slides for 10 seconds (repeated thrice with fresh xylene). Stained

specimens were covered with a glass coverslip before obtaining the microscopic

image.

c) Interpretation of the stains on tissue cells (Gamble and Wilson, 2002)

Haematoxylin

Haematoxylin is a very versatile stain and can be used to demonstrate many

different tissue components in a highly selective way. The type of mordant used

alters the specificity and colour of the stain. It stains the nucleus of the cell,

specifically, the chromatin within the nucleus and the nuclear membrane. The

active ingredient in haematoxylin solution is hematein complexed with mordant,

aluminium potassium sulphate (potash alum). In acid solutions, the alum dye

lakes are quite soluble and have a strong red colour. In alkaline conditions, the

dye lakes are less soluble and have a strong blue colour. The dyeing bath is

usually acidified and once staining is complete the section is rinsed in an alkaline

solution. In hard water areas, the tap water is alkaline and simply rinsing in tap

water will ‘blue’ the section.

Eosin

It stains nearly everything that haematoxylin will not stain. Eosin is a very

good cytoplasmic stain as it gives several shades to the tissue. The range of shades

can be extended even further if more than one dye is used in the solution. It

produces three different hues which can be used to differentiate various tissue

elements; like, red blood cells stain dark reddish orange, collagen stains a lighter

pastel pink and smooth muscle stains bright pink.

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