MATERIALS AND METHODS

Standard bacterial strain of Enterobacter sakazakii was procured from MTCC - 2958

(Microbial Type Culture Collection) Chandigarh, India and the isolate of Enterobacter

sakazakii was procured from the cell biotechnology lab, DEI, Agra.

1. Creating Aseptic Conditions for the Bacteria

Aseptic condition was created for the cultures. Glassware were washed with lab

detergent solution, and packed with the help of cotton, paper, aluminum foil and

thread. Subsequently they were autoclaved at a pressure of 15 atm and a

temperature of 1210C for 15-20 minutes. All the transfers were done inside the

laminar hood chamber which was properly sterilized with spirit.

2. Reviving the Standard Culture of Enterobacter sakazakii (MTCC- 2958)

Standard strain of Enterobacter sakazakii was first enriched in EE (Enterobacter

enrichment) broth and incubated for 24 hrs.

Glasswares were properly sterilized by autoclaving under 15 atm pressure at 1210C

for 15-20 minutes. EE broth prepared in a sterilized conical flask was poured into

the sterilized tubes and was steamed for 20-25 minutes and sterility was checked

before using it for enriching the culture, by incubating at 370C for 24 hrs.

EE Broth

CHEMICALS QUANTITY

EE broth dehydrated 43.5 gm

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Distilled water 1000ml

3. Reviving the isolated and confirmed Enterobacter sakazakii, procured

from lab

Enterobacter sakazakii, isolated from goat milk samples and confirmed by PCR

(Sharma and Prakash, 2013), was procured from the lab and was enriched in EE

(Enterobacter enrichment) broth and incubated for 24 hrs.

4. Biochemical characterization

The revived cultures of Enterobacter sakazakii (MTCC- 2958) and the isolate were

biochemically characterized at the beginning of the experiment.

4.1 Methyl Red Test

I. PRINCIPLE: Bacteria are able to ferment glucose to produce sufficient amount

of acidity which gives red color with the help of methyl red indicator.

II.

II. REAGENTS USED:

Glucose Phosphate Broth

CHEMICALS QUANTITY

MR-VP medium 17 gm

Distilled water 1000 ml

This was autoclaved at 15 atm, 1210C for 15- 20 minutes. The sterility of broth was checked by 24 hr incubation at 370C.

Methyl Red Indicator

CHEMICALS QUANTITY

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Methyl Red 0.1 gm

95% Alcohol 300 ml

Distilled Water 200ml

III. PROCEDURE:

In 1ml of sterile glucose phosphate broth, loopful of colonies were inoculated.

This was incubated overnight at 370C

A few drops of methyl red indicator were added and change in color was

observed.

IV. INTERPRETATION:

Red color - Positive test

Yellow or orange color- Negative test

4.2 Voges-Proskauer Test

I. PRINCIPLE: Some bacteria are able to produce acetone when cultured in

glucose phosphate broth. Acetone produced by the fermentation of glucose is

converted to diacetyl under alkaline condition and on exposure to air it forms a

red-pink compound with α-naphthol.

II. REAGENTS USED:

Glucose Phosphate Broth

CHEMICALS QUANTITY

MR-VP 17 gm

Distilled Water 1000 ml

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This was autoclaved at 15 atm, 1210C for 15- 20 minutesThe sterility of broth was

checked by incubating it at 370C for 24hrs.

Naphthol Solution

CHEMICALS QUANTITY

α-Naphthol 5 gm

Absolute Alcohol 100 ml

Potassium Hydroxide (40%)

CHEMICALS QUANTITY

KOH 40gm

Distilled Water 100ml

III. PROCEDURE:

In 1ml of sterile broth loopful of colonies was inoculated and was incubated

overnight at 370C.

0.6 ml α-naphthol solution and 0.2 ml potassium hydroxide (40%) was added.

After the addition of reagents the tube was vortexed without the cotton

plugs.

The test tubes were left at room temperature for an hour.

Development of red-pink color was awaited.

IV. INTERPRETATION

Red-pink color – Positive test

No pink color- Negative test

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4.3 Indole Test

I. PRINCIPLE: Certain bacteria have the ability to split amino acid tryptophan into

indole and pyruvic acid. Indole can be easily detected with kovac’s reagent,

which reacts with indole and produces a dark pink colored compound.

II. REAGENTS USED :

Tryptone Broth

CHEMICALS QUANTITY

Tryptone 10gm

Distilled Water 1000 ml

This was autoclaved at 15 atm, 1210C for 15- 20 minutes.

The sterility of broth was checked by 24hr incubation at 370C.

Kovac’s Reagent

*HCl- till pale yellow color develops

III. PROCEDURE:

In 1 ml of sterile tryptone broth, loopful of test organisms was inoculated and

was incubated overnight at 370C.

A few drops of kovac’s reagent were added.

Development of pink color on the surface layer was awaited for 10 minutes.

CHEMICALS QUANTITY

Para-dimethyl amino bezaldehyde 5 gm

Isoamyl alcohol 75 ml

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IV. INTERPRETATION:

Pink surface layer- Positive test

No pink surface layer – Negative test

4.4 Nitrate Test

I. PRINCIPLE: Certain bacteria have the ability to reduce nitrate to nitrite.

Sulphanilic acid reagent is used to detect the presence of nitrite. If nitrite is

present then reagent is diazotized and forms a dark red compound with α-

naphthalamine.

II. Reagents used:

Nitrate Broth

CHEMICALS QUANTITY

Potassium nitrate 1 gm

Beef extract 10 gm

Peptone 10 gm

NaCl 5 gm

Distilled Water 1000 ml

This was autoclaved at 15 atm, 1210C for 15- 20 minutes.

Sulphanilic Acid Reagent

CHEMICALS QUANTITY

0.8% sulphanilic acid 0.04 gm

5N acetic acid 5 ml

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α-naphthylamine reagent

CHEMICALS QUANTITY

0.5% α-naphthylamine 0.025 gm

5N acetic acid 5 ml

III. PROCEDURE:

In 1 ml of sterile nitrate broth loopful of test organism was inoculated and was

incubated for 24 hrs at 370C.

Both the reagents (sulphanilic acid and α-naphthylamine reagent) were mixed in a

ratio of 1:1.

1-2 drops of the reagent were added and color change was observed.

IV. INTERPRETATION:

Red color- Positive test

No red color- Negative test

4.5 Catalase Test

I. PRINCIPLE: Catalase is an enzyme, which converts hydrogen peroxide to water

and oxygen. Catalase producing organisms when brought into contact with

hydrogen peroxide produce oxygen bubbles.

II. Reagents used:

Hydrogen peroxide

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III. PROCEDURE:

Using a sterile loop the test organism cells were immersed in 1-2 ml of hydrogen

peroxide.

Immediately bubble formation was observed.

IV. INTERPRETATION:

Active bubbling- Positive test

No release of bubbles- Negative test

4.6 Oxidase Test

I. PRINCIPLE: Organism producing enzyme oxidase, oxidises phenylenediamine to

produce a blue-purple color.

II. Reagents used:

Oxidase Reagent

CHEMICALS QUANTITY

Tetra methyl p-phenylenediamine

dihydro-chloride

0.1 gm

Distilled water 10 ml

III. PROCEDURE:

On a piece of whatman no. 2 filter paper, placed in a petridish, several drops

of oxidase reagent were added.

With the help of tooth pick or glass rod a colony of test organism was picked

smeared was made over a small area of filter paper.

Purple-blue color within 10-15 seconds was awaited.

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IV. INTERPRETATION :

Blue-purple color- Positive test

No blue-purple color- Negative test

4.7 Triple Sugar Iron (TSI) Agar Test

I. PRINCIPLE: This test is used to differentiate Enterobacteria by producing multiple

tests on a single medium. Acid from sugars (glucose, sucrose and lactose) and S

production was observed.

II. REAGENTS USED:

TSI Agar

CHEMICALS QUANTITY

Peptone 20.0 gm

Yeast extract 3.0 gm

Beef extract 3.0 gm

Lactose 10.0 gm

Sucrose 10.0 gm

Dextrose 1.0 gm

NaCl 5.0 gm

Ferric citrate 0.3 gm

Sodium thiosulphate 0.3 gm

Phenol red 0.024 gm

Agar 22 gm

Distilled water 1000 ml

This was autoclaved at 15 atm pressure and 121 0C for 15-20 minutes.

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*Sugars were added after autoclaving the broth and when the temperature drops

near to room temperature. After addition of sugars steaming was done for 15-20

minutes.

Slants were prepared of the TSI agar and there after solified and sterility was

checked by incubating for 24hrs at 370C.

III. PROCEDURE:

With the help of sterile loop, the slants were streaked with the test organism

(revived in EE broth).

Next, the slants were stabbed with a sterile loop.

This was incubated at 370C for 24- 48 hrs.

IV. INTERPRETATION:

REACTION SLANT BUTT INTERPRETATION

Yellow color + + Acid from

lactose/sucrose/glucose

Red color/no color

change

+ + No acid from any sugar

Blackening In either

slant or butt

or both

In either

slant or butt

or both

S production

Gas

bubbles/cracking/lifting

of medium

At any place At any place Gas from sugars

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4.8 Citrate Utilization Test

I. PRINCIPLE: Test organism is cultured in a medium containing sodium citrate,

ammonium salt and bromothymol blue indicator which changes from green to

blue when the growth of organism causes alkalinity.

II. REAGENTS USED:

Simmon’s Citrate Agar

CHEMICALS QUANTITY

Simmon’s Citrate Agar dehydrated 22.28 gm

Agar 22 gm

Distilled Water 1000 ml

C for 15- 20 minutes and was later

poured into the test tubes

C.

III. PROCEDURE:

With the help of a sterile loop the slope was streaked with the test organism.

This was incubated at 370C for 24 hrs and color change was observed in the

medium.

IV. INTERPRETATION:

Bright blue color- Positive test

No color change- Negative test

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4.9 Acid from Sugar

I. PRINCIPLE: The test organism is cultured in the medium which contains

specific sugars that is fermented by the test organism and lactic acid id

produced. Lactic acid production is detected by the change of color due to the

phenol red indicator used.

II. REAGENTS USED:

Sugar Broth

CHEMICALS QUANTITY

Peptone 10 gm

Beef extract 3 gm

NaCl 5 gm

Distilled Water 1000ml

Phenol Red 12ml

The broth was autoclaved at 15 atm, 1210C for 15- 20 minutes

1% of broth, appropriate sugar was added to the broth (when temperature drops near to room temperature)

1 ml of broth was poured in test tubes and steaming was done for 25 minutes.

Sterility of broth was checked by overnight incubation at 370C.

III. PROCEDURE:

Loopful of test organism was inoculated in the broth and incubated overnight

at 370C.

Color change was observed.

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IV. INTERPRETATION:

Color change from red to yellow- Positive test

No color change or orange color- Negative test

5. Growing and Fixation of Biofilms for SEM

5.1. Growing of the biofilms of standard culture of Enterobacter sakazakii

(MTCC- 2958) and the isolate.

The revived cultures of standard Enterobacter sakazakii (MTCC- 2958) and

the isolate were streaked on TSA (Trptone Soya Agar) plates.

TSA (Trptone Soya Agar)

CHEMICALS QUANTITY

Caesin Peptone/Tryptone 15 gm

Soya Peptone 15 gm

Sodium Chloride 5 gm

Agar 22 gm

Distilled Water 1000 ml

This was autoclaved under 15 atm pressure at 1210C for 15-20 minutes.

Sterility of the plates was checked by incubating at 370C for 24hours

A colony, from the colonies so appeared on TSA was then picked up and

inoculated in 10ml TSB (tryptic soya broth) with 1% glucose for the growth

hike of biofilms (Bose et al., 2009) along with the three different substrates

such as glass, steel coupons and aluminium to compare the adhering of

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biofilms with the various type of substrata which are used in our day to day

life.

The substrates were cleaned properly with soap solution followed by acid

wash and were then autoclaved under 15 atm pressure at 1210C for 15-20

minutes.

Sterilized substrates were then introduced in different tubes containing

TSB+1%glucose and the colonies of standard Enterobacter sakazakii (MTCC-

2958) and the isolate. Such as :-

S.No. Substrates Revived Culture TSB

+1%glucose

Test tube 1 Aluminium Standard

(MTCC- 2958)

Test tube 2 Aluminium Isolate of

c.sakazakii

Test tube 3 Steel Standard

(MTCC- 2958)

Test tube 4 Steel Isolate of

c.sakazakii

Test tube 5 Glass Standard

(MTCC- 2958)

Test tube 6 Glass Isolate of

c.sakazakii

They were incubated for 24 hours at 370C.

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TSB (Tryptic Soya Broth)

CHEMICALS QUANTITY

TSB 30ml

Distilled water 1000ml

The broth was autoclaved at 15 atm, 1210C for 15- 20 minutes.

1% of broth, appropriate sugar was added to the broth (when temperature

dropped close to room temperature).

10 ml of broth was poured in tubes and steaming was done for 15-20minutes.

Sterility of broth was checked by overnight incubation at 370C.

5.2 Fixation of Biofilms on Substrates

REAGENTS FOR FIXING SOLUTION

PBS – phosphate buffer saline (pH=7.3)

CHEMICALS QUANTITY

0.2M Disodium hydrogen phosphate 1.4195 gm

0.2M Sodium dihydrogen phosphate 1.56 gm

0.8% (w/v) NaCl 0.4 gm

Distilled Water 100 ml

0.05M Sodium cacodylate buffer (pH=7.2)

CHEMICALS QUANTITY

Glutharaldehyde 2.5%

Formaldehyde 2.5%

Sodium cacodylate 0.05M in distilled water

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Calculation for 100ml 0.05M Sodium cacodylate buffer (pH=7.2)

Mass = concentration × volume × molecular weight

Mass = 0.05M × 100/1000 × 159.98

Mass = 0.7999gm sodium cacodylate in 100ml distilled water

Gluthaldehyde = 2.5% of 100ml

= 2.5ml

Formaldehyde = 2.5% of 100ml

= 2.5ml

0.001M Calcium chloride in distilled water

Calculation for 10ml 0.001M Calcium chloride

Mass = concentration × volume × molecular weight

Mass= 0.001 × 10/1000 × 110.984

Mass = 0.00110984gm

Osmium tetraoxide*

1% in distilled water (required in very small quantity)

*handle with care

Acetone gradients

25% acetone (For 20ml)

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CHEMICALS QUANTITY

Acetone 5ml

Distilled water 15ml

50% acetone (For 20ml)

CHEMICALS QUANTITY

Acetone 10ml

Distilled water 10ml

75% acetone (For 20ml)

CHEMICALS QUANTITY

Acetone 15ml

Distilled water 05ml

90%acetone (For 20ml)

CHEMICALS QUANTITY

Acetone 18ml

Distilled water 02ml

100% acetone (For 20ml)

CHEMICALS QUANTITY

Acetone 20ml

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After 24 hours of incubation the biofilms stick to the various substrates. The

broth was decanted and the substrata with biofilms was washed with PBS buffer

(pH=7.3)

After washing, the substrates were dried and were fixed in fixing solution by

modified Karnovsky’s method. Various steps followed up for the procedure were:

1. Substrates were incubated for 24hours in 0.05M sodium cacodylate

buffer of pH 7.2 and 0.001M calcium chloride.

2. After incubation, the substrates were washed with 0.05M sodium

cacodylate of pH 7.2 buffer three times for about 10 minutes.

3. Fixation was done in osmium tetraoxide (1% in distilled water) for 1

hour at ambient temperature.

4. Washed thrice with distilled water.

5. The biofilms on substrates were dehydrated in acetone gradients of

25%, 50%, 75%, 90% and 100% for 2-3 minutes in each gradient. The

respective gradient was added to the tube in which the substratum

was contained, thrice consecutively.

6. The substrates were transferred to critical point apparatus for

complete drying.

7. Every substratum was sputter coated with gold.

8. The samples were subjected to scanning electron microscopy (SEM)

for comparison of the biofilms.

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6. Preparation of planktonic cells for SEM

The enriched cultures of standard Enterobacter sakazakii (MTCC- 2958) and

the isolate were centrifuged at 6000rpm for 10 minutes.

The supernatant was discarded and pellet obtained was washed with PBS

buffer. This was again centrifuged for 5minutes at 6000 rpm. Supernatant was

discarded and pellet was obtained.

The pellet was used to make the smear on slide and was allowed to dry for

fixation process.

Fixation was carried out in the same manner as that for biofilms.

These arrested cells were then subjected for SEM.

7. Control of Biofilms

The biofilms on the substrates were washed with PBS buffer and were incubated

in EE broth overnight so that the biofilms adhering to the substrates get into the

broth.

7.1. Antibiotic susceptibility

Antibiotic susceptibility was checked by agar well diffusion method on MHA

(Muller-Hinton agar) plates. MHA was prepared and was inoculated with 150µl of

the culture of biofilms in EE broth. Wells of 6mm diameter were made in which

40µl of antibiotics of different concentrations was inoculated (Stock and

Wiedemann, 2002). This was incubated at 370C for 24hours. Cronobacter

sakazakii is found to be susceptible to gentamycin and tetracycline. Thereby

these antibiotics were selected for this experiment. Inhibition zones were

measured and recorded.

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MHA (Muller-Hinton Agar)

CHEMICALS QUANTITY

MHA 38gm

Agar 6gm

Distilled water 1000ml

Calculations for the drug Tetracycline amount for different concentrations

Powdered drug was diluted with distilled water as per the concentration stated by Stock and Weidemann, 2002. MIC of drug is 40mg/L = 40µg/ml

... 1ml = 40 µg of drug

And 1 µl= .04 µg of drug

As per serial dilution procedure the drug was diluted in the ratio of 9:1 (water: drug)

0.4 µl dilution 0.04µl dilution 0.004 µl dilution 0.0004µl dilution =1.6×10-2µg drug =1.6×10-3µg drug =1.6×10-4µg drug =1.6×10-5µg drug

100µl in

900µl H2O

40 µg in

1ml

100µl in

900µl H2O

100µl in

900µl H2O

100µl in

900µl H2O

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Calculations for the drug Gentamycin amount for different concentrations

7.2. MIC of natural products against the biofilms

Minimum inhibitory concentration was checked for plant extracts (obtained

from the lab) on the biofilms of standard and the isolate by agar well diffusion

method. Alcoholic crude extracts of Triphala, Har (Terminalia chebula),

Amla(Phyllanthus emblica) and Laung (Syngium aromaticum) were checked

against the cultures. 150 µl of the culture was inoculated in MHA which was

poured in plates and 6mm diameter wells were made in which 40 µl of extracts

were added and incubated for 24hours. Inhibition zones were measured and

recorded.

Powdered drug was diluted with distilled water as per the concentration stated by Stock and Weidemann, 2002. MIC of drug is 2.5mg/L =2.5µg/ml

... 1ml = 2.5µg of drug

And 1 µl= 2.5×10-3

µg of drug

As per serial dilution procedure the drug was diluted in the ratio of 9:1 (water: drug)

2.5×10-2

µl dilution 2.5×10-3

µl dilution 2.5×10-4

µl dilution 2.5×10-5

µl dilution =1×10-3 µg drug =1×10-4 µg drug =1×10-5 µg drug =1×10-6 µg drug

2.5 µg in

1ml

100µl in

900µl H2O

100µl in

900µl H2O

100µl in

900µl H2O

100µl in

900µl H2O

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7.3. Control of biofilms by LAB

The biofilms of the standard and the isolate cultured on different substrata were

subjected to LAB (lactic acid bacteria) to check the MIC for the biofilms. This was

done by agar well diffusion method. Antimicrobial effect of Lactobacillus

fermentum (MTCC- 903), Lactobacillus lactis (MTCC- 1423) and

Pediococcus acidilactici (MTCC-7742) was checked by inoculating 150 µl culture

in MHA and then 40 µl of LAB in the wells. This was incubated for 24 hours and

the inhibition zones were measured and recorded.

8. Control of Planktonic cells of the isolate and the standard.

The enriched planktonic cells were subjected to antimicrobial effect of plant

extracts, antibiotics and LAB by agar well diffusion method. This was done by

pouring 150 µl of the culture of these planktonic cells in MHA and the wells were

made of 6mm diameter after solidification of the plates. In the wells 40 µl of

extracts, antibiotics of various dilutions and LAB of the three strains were added.

Zone of inhibition was measured and recorded.

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