Internshipreport

Biotech-412

Food and Biotechnology Research Centre

In
Pakistan Council of Scientific and Industrial
Research, Lahore

Submitted By
Tahmina Sattar
2015-Uam-168
B.Sc (Hons.)Agriculture

MNS-University of Agriculture Multan

Institute of Plant Breeding & Biotechnology
<<2019>>
 CERTIFICATE

It is certified that Tahmina Sattar Registration No. 2015-uam-168, B.Sc. (Hons.)

Agriculture Biotechnology, Muhammad Nawaz Shareef University of Agriculture, Multan
has successfully completed his internship at PCSIR. Her work and behaviour was found to be
extremely satisfactory. We appreciate her sincere dedication towards her work.

Approved by:

Internship Supervisor:

_______________________

Dr.Yasir Saleem
(Senior Scientific officer
PCSIR, FBRC Lahore)

Internal Supervisor:

_______________________

Dr. Zulqurnain khan

Assistant professor
IPPB, MNSUA,Multan

2|Page
 Acknowledgement
At the beginning I would like to convey my sincere appreciation to almighty Allah for giving
me a strength and ability to finish the task within the planned time. Then I would like to
express my gratitude to my parents they contributed towards preparing and making this study
successfully.
I convey my sincere gratitude to my academic supervisor Dr.Zulkurnain khan, Associate
professor of institute of Plant Breeding and Biotechnology, university of Multan. Without his
kind direction and proper guidance this study would have been a little success. In every phase
of the project his guidance and supervision shaped this report to be completed perfectly.
I would also like to express my sincere gratitude to my internship supervisor Dr.Yasir
Saleem, Senior scientific officer, FBRC, PCSIR, Lahore and Director FBRC Dr.Muhamad
Ijaz With their guidance and appreciation I have learned most of the laboratory techniques.
I am humbly thankful to my seniors out there in the molecular biotechnology lab especially
Miss Maryam, Miss Rafia and Mr. Qasim javaid. They were really extremely helpful to
me throughout my internship.
At last but not least I am extremely grateful to our energetic and honourable chairman
Dr.Muhammad Hammad Nadeem Tahir and our hard working coordinator Mr. Ali Sher.
I perceive this opportunity as big milestone in my career development. I will strive to use
gained skills and knowledge in the best possible way, I will continue to work on their
improvement, In order to attain desired career objectives. Hope to continue cooperation with
all of you in future.

TAHMINA SATTAR

3|Page
 Table of Contents

Sr. Topic Page

No. No.
1 Executive Summary 5
2 Introduction Of PCSIR 6
3 Training Program 9
4 Tests Performed Besides Main Work 20

5 Learning Experience 27
6 SWOT Analysis 29
7 References 32

4|Page
 1. Executive Summary
I joined internship at Molecular Biotechnology Lab FBRC, PCSIRLahore. Basically, the

project was on testing antibiotic susceptibility pattern of aerobic bacteria isolated from

diabetic foot ulcers. The sample size was 100, collected from diabetic foot ulcer unit from

various hospitals of Lahore. The aim of the project was to identify and isolate diabetic foot

infection and to check antibiotic susceptibility pattern against bacteriocins. Besides this

project two milk testing were also performed. One sample from a branded milk company was

tested for the presence of antibiotics, Beta lactam and Tetracycline while the other sample

from another branded company was evaluated for the presence of lactose. I also witnessed

projects on antimicrobial susceptibility pattern testing through NPs, isolation of internal and

external micro flora of vegetables and Ozone application and Evaluation and identification of

biofilm resistance gene in Pseudomonas areuginosa.

5|Page
 2. Introduction to PCSIR

In 1953 Pakistan Council of Scientific and Industrial Research (PCSIR) was established with

an objective of utilizing available resources for the development of industrial sector in

Pakistan. It has its laboratories situated in all across the Pakistan in various cities including

Hyderabad, Karachi, Skardu, Quetta, Peshawar and Lahore. Its head office is situated in

Islamabad.

PCSIR, Lahore started functioning in 1953 in Punjab University and then got shifted to

current place, occupying 68.5 acres of land, in 1955.

PCSIR Lahore has a number of functional research centres under various domains including a

 Applied chemistry

 Applied physics

 Environmental protection

 Engineering

 Food and Biotechnology

 Glass and ceramics

 Mineral processing

PCSIR, Lahore has a number of international and national recognitions including ISO

(Pakistan), Saudi Arabian Standard Organization and Ministry of health and welfare japan.

In 1977 by emerging biological evaluation and food technology department FBRC came into

existence.FBRC is promoting food and biotechnology industry in country and aimed at

improving quality and utilization of food products. It is equipped with all kind of required

6|Page
instrumentation including Spectrophometer, High Performance Liquid Chromatography, Gas

Chromatography, Freeze dryers, ultra-centrifuges, sonicator etc.

Basic hierarchy:

Back in 2016 FBRC brought PCSIR in the international eyes, as a student of it won Queen

Elizabeth II young leaders award for her research on genomic characterization of different

members of brassicaseae family for their potential of making biofuels.

All the scientific officers there are not only

highly competent but also much cooperative.

They boost up their interns to think out of the

box and ensure the providence of any kind of

assistance or facilities to them.

7|Page
In the labs of PCSIR, a healthy diverse environment is found. There is a lot of diversity is

found among research fields, research ideas and intellect of researchers making it an ideal

place to do research.

PCSIR has many important achievements on its credit most importantly, establishment of

Asia’s first ever dioxin testing laboratory which is assisting persons related to fisheries and

sea foods to enhance its export to European market. Another big and critical achievement of

PCSIR is the establishment of Halal authentication laboratory to regulate the import and

export of Halal items. PCSIR has a number of products and processes ready for being

commercialize including photovoltaic cells, micro feeder, superabsorbent polymer, Nano

fertilizer, phytase enzyme for poultry feed, bio fertilizer, food grade pink dye, Nano filter

material and many more.

8|Page
 3. Training Program

“Antibiotic susceptibility pattern testing of aerobic bacteria

isolated from diabetic foot ulcer”

3.1. Introduction(Importance of study and review of literature):

Foot ulcers are usually hardly irreparable(Singh, Armstrong et al. 2005) erosions on the foot

skin that may get extend deep into tissues(Reiber, Lipsky et al. 1998). Occurrence of foot

ulcers in diabetic patients is a common phenomenon, in about 15 % of diabetic

patients(Reiber, Lipsky et al. 1998), termed as diabetic foot ulcer. It worsens the already

worse condition of diabetic patient .Diabetic patient having ulcer too has a more chance of

bone infection(Harris 1995). It badly damages patient socially, economically and

psychologically(Reiber, Lipsky et al. 1998; Boulton, Vileikyte et al. 2005). It most of the

times lead towards amputation of leg It most of the times lead towards amputation of leg

.Only the cost of amputation is US$993 to US$17 519(Boulton, Vileikyte et al. 2005). It

increases mortality rate in diabetic patients up to 2.39%(Boyko, Ahroni et al. 1996). Even

after amputation there is a chance of mortality from it, whose chance varies from country to

country(Reiber, Lipsky et al. 1998; Faglia, Favales et al. 2001) according to availability of

medical facilities in that country(Lee, Lu et al. 1993). Women and old men are more

susceptible to death after amputation(Faglia, Favales et al. 2001). There is a poor

understanding of these phenomena(Boyko, Ahroni et al. 1996).

9|Page
A lot of negligence towards identification and treatment of these ulcers has been shown in

health care centres(Jeffcoate and Harding 2003).

Swabbing from wound is done and it is immediately checked for the identification of bacteria

in both aerobic and anaerobic conditions. Diabetic foot ulcer is a complex phenomenon

having involvement of many aerobic/ anaerobic bacteria and gram positive/gram negative

bacteria(Lipsky and Berendt 2000) including Bacteroides species, peptococci , Proteus

specie, enterococci, Staphylococcus spp. and Escherichia coli(Louie, Bartlett et al. 1976).

However,Staphylococcus spp. is the single most common isolated bacteria,about79%(Dang,

Prasad et al. 2003)) from these ulcers.

The complexity of microbes is kept in mind while selecting antimicrobial therapy from it and

the design or prescription of antibiotic is based on the knowledge of infection causing

microbes obtained from the results of this identification test(Lipsky 1999; Lipsky and

Berendt 2000).

A special focus on elimination of Staphylococcus spp. has been given. In early times

methicillin was used to cure foot ulcers however bacteria developed resistance against

them(Dang, Prasad et al. 2003) than came the most prolong era of vancomycin usage as

antibiotic for treating ulcers associated with Staphylococcus spp. However Staphylococcus

spp. developed resistance against it too through vanA gene. First case of vancomycin

resistivity was reported in 1997(Control and Prevention 1997) and then a series of reports

start to come out(Rotun, McMath et al. 1999; Smith, Pearson et al. 1999; Hageman, Pegues et

al. 2001; Weinstein and Fridkin 2001). Many modifications were used in this antibiotic to

control Staphylococcus spp. but none of them proved to be effective over a long period of

time. These drug resistance cases were increasing the complications associated with diabetic

foot ulcers(Jeffcoate and Harding 2003).

10 | P a g e
So in these all conditions a new solution to the old problem has been suggested, named as

bacteriocin. Bacteriocins are “ribosomally synthesized antimicrobial peptides”(Okuda, Zendo

et al. 2013) secreted by different bacteria and have antimicrobial activity with broad

inhibition spectrum, high specificity, high stability and low toxicity, giving them edge over

antibiotics(Tagg, Dajani et al. 1976; Gálvez, Abriouel et al. 2007; Cotter, Ross et al. 2013). It

is found to be highly effective against Methicillin‐resistant Staphylococcus spp. (MRSA).

Isolation of anti-Staphylococcus spp. bacteriocins from soil inhibiting bacteria is in initial

stages now and looked to be much promising(Lee, Zainal et al. 2014).

3.2. Objective:

To isolate staphylococcus spp. from diabetic foot ulcer and to evaluate potential of

bacteriocins extracted from soil inhibiting bacteria against staphylococcus spp.

3.3. Hypothesis:

If bacteriocins may have potential against staphylococcus spp. than it will inhibit the growth

of staphylococcus spp. “on agar well plates”.

3.4. Material and Methods:

3.4.1. Isolation and identification of diabetic foot ulcer bacterial pathogens

Wound swabs of diabetic foot ulcer patients were collected from hospitals of Lahore and

were processed by standard microbiological analysis. After the collection of samples swabs

were placed in a plastic tube.These samples were swabbed on nutrient agar plates and these

plates were incubated at 37C for 24 hours. After the incubation different bacterial strains

were observed on the plates. After the identification,colonies were re-streaked on media for

isolation of strains.

11 | P a g e
3.4.2. Identification and confirmation of bacterial strain:

Fig.1, 2 (Isolated strains after swabbing)

3.4.2.1. Microscopic examination:

The relative number and type of microorganism and host cells were identified by gram-

staining after the formation of smear of all the samples. In the first step of gram-staining one

drop of distilled water was placed on glass slide. Red hot wire loop was used to pick single

colony from plate and smear was formed. After this the bacterial strain was fixed with the

help of burner. The bacterial strain was stained by placing one drop of crystal violet on smear

for one minute and then washing it with distilled water. Then we placed one drop of iodine

on it and again washed it with distilled water. Than we placed one drop of alcohol for 30 sec

and washed it with distilled water. At the end one drop of safranin was placed on it and it was

again washed with distilled water. Then dried glass slideswere observed under 100X power.

Fig.3,4,5,6 (Slide preparation and Microscopy)

12 | P a g e
3.4.2.2. Biochemical test:

A. Catalyse test:

For the confirmation of staphylococcus aureus biochemical test was performed. In catalyse

test, with the help of sterilized wire loop, single colony was picked and was placed on the

clean glass slide and 1-2 drops of hydrogen peroxide(H2O2) were added, bubbling was

checked if bubbling was formed than it was termed as catalyse positive otherwise negative.

Figure.7 (Catalase Test Results)

B. Oxidase test:

In oxidase test N,N, N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-

phenylenediamine (DMPD) reagent was used that determined certain cytochrome oxidases

produced by bacterium.We took piece of filter paper and wetted it with distilled paper and

transferred single colony of bacterium on it with the help of sterilized platinum loop. Then we

placed 1-2 drops of oxidase reagent on it and incubatedit for some minutes at room

temperature. Positive test result: Dark blue-purple colour change within 10-30 sec. Negative

test result: No colour change or colour change after more than 30 sec.

13 | P a g e
 Figure.8 (Oxidase Test)
C. Methyl red test:

In methyl red test, the test bacteria were grown in a broth medium that contained glucose.

Incubate the broth media at 37C for 24 hours. Thenwe took 1 ml of broth medium in an

aliquot and added 3-4 drops of methyl red reagent into the aliquot and observed it, colour

changed from yellow to red. If the inoculated bacteria have the ability to utilizeglucose, it

produced stable acid, changing colour from yellow to red. Positive result: colour changed

from yellow to red (bacteria have the ability to utilized glucose and produced stable acid)

negative result: colour not changed from yellow to red (bacteria don’t have the ability to

utilized glucose and produced stable acid).

Figure.9, 10(Methyl red test)

3.4.2.3.Selective media:

For the differentiation and isolation of particular organisms selective media were used, they

allow certain type of microorganism to grow and inhibit the growth of other. For the isolation

of staphylococcus spp. MSA media was used as selective medium, that contain high salt

concentration (7.5%), staphylococcus spp. was able to tolerate this salt concentration but

14 | P a g e
other gram positive or gram negative bacteria not. That’s way MSA used as a selective

medium for the isolation and differentiation of staphylococcus spp. species.

Figure.11 (differentiation ofstaphylococcus spp. on MSA

3.4.3. Antimicrobial susceptibility testing

Antimicrobial susceptibility test was done to analyse the antibiogram of the isolates by using

disc diffusion method. The discs of antibiotics used in diabetic foot ulcer treatment were

used, such as, ceftriaxone 30 µg, ceftazidime 30 µg, amoxyclave 30 µg, ampicillin 25 µg, co-

trimoxazole 25 µg, ciprofloxacin 30 µg, chloramphenicol 30 µg, gentamycin 30 µg,

Tetracycline30 µg, Cephalexin 30 µg,Cefuroxime25 µg ,Amikacin 30µg Fig.(12,13,14,15).

In this step after swabbing of isolated staphylococcus spp. strains on MHA(Mullen-Hilton

Agar) plates discs of antibiotics were placed by using sterilized forceps.

Some of the pictures clicked during antimicrobial susceptibility testing using disc diffusion

method can be seen below,

15 | P a g e
 Fig.12, 13 (Different antibiotic susceptibility testing plates having aforementioned
antibiotics in them)

3.4.4. Isolation of Bacillus from soil:

For the isolation of bacillus from soil, 1g soil was measured and then dissolved in 50ml

distilled water and vortexed vigorously to dissolve particles. Then the soil sample was boiled

for 10-15 minutes by using water bath and was allowed to cool. Then serial dilution of

samples were made by dissolving 1ml sample in 9ml distilled water in first dilution and then

ten-fold, hundred-fold and thousand-fold dilution of the re-suspended soil samples were

made. Then we took 0.1ml from each dilution was plated on nutrient agar plates by using

micropipette. The undiluted soil solution was also placed on the nutrient agar plates. Then

incubate these plates at 37C for 24 hours. The colonies were identified by colony morphology

and biochemical characterization after incubation period..

Fig.16 (soil solution) Fig.17 (serial dilution of Fig.18 (bacteriocin growth on

sample) agar plates

16 | P a g e
3.4.5. Bacteriocins extraction and purification

After the identification and characterization of bacteriocins, single colony of Bacillus spp.

was placed in a nutrient broth and incubated it at 37°C at 150rpm/minutes for about 24 hours.

Then the sample was centrifuged at 10,000 rpm/minutes for 20 minutes, and the supernatant

was collected. Then took 100ml supernatant and chloroform and was stirred vigorously after

this transferred it into a separate funnel. Then the bacteriocins was harvested from the

interface layer between the aqueous and organic phase, by using speed vacuum residual

chloroform was eliminated.

3.4.6. Antimicrobial activity of partially purified bacteriocins on DFU bacterial isolates

By using Agar well diffusion method inhibitory activities of partially purified bacteriocins of

all the DFU (Diabetic Foot Ulcer) isolates were done. In this step MHA plates were made, by

using blue tips, wells were formed on plates and dilution of bacteriocins were filled in each

well of the plate. These plates were incubated at 37°C for 24 hours, next day plates were

observed and zone of inhibition were measured by using measuring ruler.

Fig.19 (Antimicrobial activity of bacteriocin against staphylococcus

3.5. Results and discussion:

17 | P a g e
Antimicrobial susceptibility testing of staphylococcus spp. using disc diffusion method

showed resistance to following mentioned antibiotics.

Table.1 (Different Antibiotics pattern against staphylococcus spp.)

Sr. Antimicrobial agent Staphylococcus aureus %age of

no. No=26 effectiveness
1 Ceftriaxone 21 80.76
2 Ceftazidime 08 30.76
3 Amoxyclave 06 23.07
4 Ampicillin 26 100
5 Co-trimoxazole 25 96.15
6 Ciprofloxacin 12 46.15
7 Chloramphenicol 09 34.61
8 Gentamycin 08 30.76
9 Amikacin 03 11.53
10 Tetracycline 22 84.61
11 Cephalexin 20 76.92
12 Cefuroxime 11 42.30

Antimicrobial sensitivity test was performed against staphylococcus spp. using bacteriocins

from soil. Bacteriocins showed no activity to resist the growth of staphylococcus spp.

Different dilution of bacteriocins were used, not a single dilution of bacteriocins was able to

resist the growth of staphylococcus spp.

Fig.20 (Antimicrobial susceptibility pattern of staphylococcus spp. against

bacteriocins)

18 | P a g e
In diabetic foot ulceration multiple factors are involved, like neuropathy, abnormal foot

biomechanics and peripheral arterial diseases. In hospitals patients with diabetic foot

infections had multidrug resistant organisms that showed high level of antibiotic resistance.

So due to the polymicrobial nature of diabetic foot ulceration, multidrug resistance in

organism was increased. To reduce the risk of complications continuous surveillance should

be provided.

The most important bacteria are Bacillus spp. whichproduces various kinds of antimicrobial

proteins that inhibit the growth of gram positive bacteria. So, by the purification, SDS page

analysis, HPLC testing and genotypic identification by 16 S rRNA Bacillus spp. will be used

effectively to kill the staphylococcus spp. strains. Since, the bacteriocins and the strain of

Bacillus spp. are found to be safe for use,so, in humans they can be easily used to treat

diabetic foot ulcer related bacterial pathogens.

19 | P a g e
 4. Tests Performed Besides Main Work
4.1.Milk antibiotic residues identification kit:

On 21 of March molecular biotechnology lab received two milk samples from a branded

company to test presence of two antibiotics named beta lactam and tetracycline in it. To

evaluate presence of antibiotic we used milk antibiotic residues identification kit.

4.1.1.Review of literature and importance of work:

Antibiotics are like a gift to the entire mankind. From therapeutic to sub-therapeutic level

they have been beneficial for human beings. But their excess of use could result in the

formation of drug residues(Khaskheli, Malik et al. 2008).The antibiotics present in the milk

are usually the residues of antibiotics being injected in it for curing a particular disease. Beta

lactam is usually used for the treatment of Mastitis in cattle(Bengtsson, Unnerstad et al.

2009).

Antibiotics residues present in the milk not also deteriorates quality and processing of

milk(Ruegg 2013)but also pose a serious threat to human health and can cause severe health

complications(Samaržija and Antunac 2002).European Union’s several regulations are

focused on defining the maximum limit of antibiotic residues in milk in order to be

20 | P a g e
consumable(Commission 2010)After identification of possible hazards by scientists and EU

regulations, Now The practice of testing milk for the presence of any antibiotic has become a

common practice(Fejzić, Begagić et al. 2014) and in USA its even mandatory now(Moats and

Harik-Khan 1995) while in many other countries it is illegal to have amount of antibiotics

residues more than a limit as discussed by(Kempe, Cederfur et al. 1999) In Pakistan too

studies have been made to detect antibiotic residues in the milk(Khaskheli, Malik et al. 2008)

As most of the antibiotics being used are of Beta lactam origin and to somehow of

tetracycline origin so most of the tests are designed to detect these antibiotics only(Fejzić,

Begagić et al. 2014)

Various techniques have been used throughout the world to detect antibiotic residues in milk

like liquid chromatography (Straub, Linder et al. 1994; Moats and Harik-Khan 1995;

Andersen, Roybal et al. 2005), FT-MIR spectroscopy(Sivakesava and Irudayaraj 2002)and

MSPD(Long, Hsieh et al. 1990). A trend of using prepared kits is also getting popular(Harik-

Khan and Moats 1995)

4.1.3. Material and method:

We took two nestle samples and one control sample (having confirmed presence of both

antibiotics) to run in the wells of kit. Corresponding antigens were already present in these

wells. We poured down 200ul of all three samples in three separate wells and incubated them

for 5min for antigen-antibody interaction. After 5 minutes of incubation antibiotic detection

strips were placed in the wells for 5 minutes.

After 5 minutes strips were observed, control strip should have c two visible lines first of b-

lectin and second of tetracycline. We compared the pattern of lines on the control strip with

strips of other two wells to evaluate the presence of suspected antibiotics.

21 | P a g e
4.1.4. Results and discussion:

Sample 1 had b-lectin negative but tetracycline positive line. Sample 2 showed negative

results for both antibiotics (b-lectin and tetracycline).

Table.2 (Results of antibiotic detection in milk sample)

ILO#
B-lectin Tetracycline
608 Negative Positive
609 Negative Negative

Fig.20 (Milk antibiotic identification kit results)

Basically the presence of any antibiotic in milk sample can be easily tested using antibiotic
identification kit. It is one pot experiment, by using this type of identification technique we
can easily develop different antibiotic identification kit based on antigen-antibody interaction.

22 | P a g e
4.2. Lactose free milk identification through HPLC

4.2.1. Introduction:

On April 5th molecular biotechnology lab received lactose free claimed milk sample from

Day fresh. A test was performed to evaluate the credibility of the claim.

4.2.2. Review of literature/Importance of work:

Milk is complete nutrition having many important nutrients like proteins, fats and lactose.

Lactose is the major component of mammalian milk. It is found about 7.2g/100mL in mature

human milk and 4.7 g/100mL in cow’s milk(Solomons 2003). In the body, in intestine,

Lactase enzyme (B-galactosidase, E.C. 3.2.I.23) digests it into simpler sugars(Gilat, Russo et

al. 1972). However its production is vulnerable to intestinal cell damage as it is not found

much deeply into the intestine. Maintenance of lactase in the body is found to be genetically

regulated(Sahi 1974; Lisker, Gonzalez et al. 1975). In almost all mammals except humans,

the production of lactase enzyme is inhibited upon weaning. However in humans too, to an

extent, production of lactase is disturbed and termed as lactose intolerance. Its symptoms may

include Stomach pain, constipation, diarrhoea and bloating after consuming milk or milk

products(Swagerty, Walling et al. 2002). 50 g of lactose in a litter of milk can produce the

23 | P a g e
symptoms associated with Lactose intolerance. However contradiction is found regarding

exact amount producing symptoms(Suarez, Savaiano et al. 1995).

Many studies have been performed throughout the world to check the prevalence of lactose

intolerance (Cook and Al-Torki 1975; Rahimi, Delbrück et al. 1976; Ahmad and Flatz 1984).

There is no definite pattern about its manifestation in humans. It varies not only on

demographic basis but also on geographic one(Simoons 1978; Swagerty, Walling et al.

2002). In Asians and Africans it appears in the early childhood while in America and Europe

it appears after adolescence(Scrimshaw and Murray 1988). Asians especially east Indians are

found to be mostly lactose intolerant(Murthy and Haworth 1970). However the condition in

Pakistan is found to be much satisfactory as compared to its neighbour including Afghanistan

and India(Rab and Baseer 1976). This phenomenon is also found more commonly among old

people(Vesa, Marteau et al. 2000). Women are found to be more vulnerable to it(Jussila

1969).

So a trend of producing lactose free milk has been started throughout the world(Dahlqvist,

Mattiasson et al. 1973). Modifications producing lactose free milk can be proved much

promising for milk market throughout the world(Jelen and Tossavainen 2003). As only in

USA, according to estimation, there are 50 million consumers for lactose free milk(Sloan

1999)

4.2.3. Material and method:

In order to check the presence of lactose in milk, we performed HPLC after sample

preparation. First of all 0.1% lactose standard was prepared by mixing 0.01g powdered milk,

having lactose, in 10ml dH2O. After this TCA treated milk was prepared by dissolving 1g

TCA in 5ml day fresh milk sample. Then TCA treated milk was centrifuged at

24 | P a g e
5000rpm/20min and then we shifted the supernatant into a labelled falcon. In the next step we

washed the cuvette three times each with methanol and H2O. Lactose standard and

supernatant was syringe filtered and 1.5ml from each was put into separate cuvettes. HPLC

apparatus was equilibrated and standardized before running our samples.

Fig.21 (Sample & standard preparation) Fig.22 (HPLC running after sample
placement)

4.2.4.Results and Discussion:

For the calculation of lactose concentration in milk sample standard and sample was run in

HPLC. Then P.A of the standard and average P.A of the sample was noted and by using

above mentioned formula final concentration of the lactose present in the milk sample was

calculated by comparing the peaks of standard and sample in the retention time.

Table.3 (Results of lactose % in milk sample using HPLC):

Sr. Sample Peak Area (P.A) Retention Time (R.T)

no.
1 Standard (0.1%) 9066740+2257169 3.066+4.00
2 ILO# 331 10071419+3411505 3.531+3.861
3 ILO# 331 11101750+4037963 3.76+4.09
4 ILO# 331 6802544.51+198973355 3.76+4.063

25 | P a g e
Calculations:

Average P.A of standard (0.1%):

9066740+2257169= 11,323,909

Average P.A of the sample (ILO#331):

10071419+3411505 = 13,482,924

11101750+4037963 = 15,139,713

6802544.51+198973355 = 205,775,899.51

13,482,924 +15,139,713 +205,775,899.51= 234,398,536.51

=78,132,845.5033

Lactose % in sample= P.A of the sample× conc. of standard

P.A of standard

= 78, 132, 845, 5033× 0.1%

11,323,909

= 0.112%

26 | P a g e
 5. Learning Experience

5.1. Knowledge acquired:

We have learnt slide preparation and microscopy in our Cytogenetics course that helped us to

prepare slides during our internship experiments. In the course of principles of biotechnology

we have learnt basic techniques liked DNA extraction, PCR running and gel electrophoresis

so these techniques helped us. In molecular virology course we have learnt serial dilution,

microbial isolation and identification techniques that helped us a lot during our internship

work. During our course of Nano biotechnology we have learnt about the synthesis and

effectiveness of nanoparticles. We applied this study of nanoparticles here in order to check

the antimicrobial susceptibility pattern.

5.2. Skills learned:

We learnt equipment handling and basic biosafety rules. Hence, background knowledge

helped us in understanding experimental work during our internship. We have learnt report

writing skills, time management skills, how to work in a group as well as communication

skills there.

5.3. Observed attitude and gained values:

27 | P a g e
The coordinative and friendly environment helped us to work effectively in the lab. The

honest and helpful attitude of the seniors played an important role in our learning. There was

no communication barrier between seniors and juniors. So, it was quite easy for us to

understand and perform our practical work.

5.4. The most challenging task performed:

Though my entire internship work at PCSIR was enough challenging but not more

challenging than that day on which I had already 50 MSA plates to be streaked and then my

senior requested me to streak her 70 MSA plates too. It was quite a hectic task but I accepted

the challenge and it took me 6 consecutive hours of work to accomplish this task.

28 | P a g e
 6. SWOT Analysis

6.1. Strengths:

 Well-equipped labs:

The laboratories are well equipped having latest and up-to-date equipment

like HPLC, FPLC, Ozone generator, sonicator, ion exchange chromatography,

spectrophotometer, assorted fermenters, temperature control incubator, freeze dryer

and all required kits and equipment for any kind of food testing.

 Diversified laboratories:

In the labs of PCSIR, a healthy diverse environment is found. There is a lot of

diversity is found among research fields, research ideas and intellect of

researchers making it an ideal place to do research.

 Cooperative environment:

All the scientific officers there are not only highly competent but also much

cooperative. They boost up their interns to think out of the box and ensure the

providence of any kind of assistance or facilities to them.

6.2. Weaknesses:

29 | P a g e
  Unclean Labs:

Labs here are not kept cleaned. They are not swept on daily basis. One could easily

find a lot of dust on furniture and other equipment of labs. One has to make his/her

required stuff clean himself/herself.

 poorly furnished labs:

The labs here are not well furnished. They are seriously deficient in essentially

required furniture like nonporous stools, chairs tables and wood cabinets

 Poor management:

Lab assistant showed non cooperative behaviour with researcher and students. They

don’t provide required things on time. In labs there is no proper placement of

chemicals and related stuff.

 Absence of log register in labs:

There is no log in/log out register present in labs. There is no proper record of

chemicals. The students or interns from other labs took away things or chemicals

easily without any check and balance.

6.3. Opportunities:

 Diversified ideas:

As discussed above, it has a very diverse environment. So students can interchange

their ideas with others and they also have an access to diverse fields of research.

 Expert Faculty:

It has a competent and world-class faculty. Intellectually saturated mature minds are

available to guide the mentally scattered naïve minds to a straight path. It has

specially a lot of opportunities for students, coming from any background, to research

under the umbrella of research giants.

30 | P a g e
  Well renowned institute:

PCSIR is a well renowned institute. About 3 years back, A student of it won” Queen

Elizabeth II young leaders award” for her MS research, Making this institute to be

globally recognized.

6.4. Threats:
 Biosafety Breaches:
There is a threat of biosafety related issues in the labs. There is no proper arrangement
of waste disposal. Interns use to throw their waste material openly on the roof.
Although there is no such high biosafety level work is going on here but still who
knows when things got out of control.
 Careless attitude:
A lot of careless attitude has been seen in the labs. Interns most of the times ignore the
sterilization precautions. Unawareness about threats associated with a chemical, like
Ethidium Bromide, has been also seen. These practices can cost much in the future.

31 | P a g e
 7. References
Ahmad, M. and G. Flatz (1984). "Prevalence of primary adult lactose malabsorption in Pakistan."
Human heredity34(2): 69-75.

Andersen, W. C., J. E. Roybal, et al. (2005). "Determination of tetracycline residues in shrimp and
whole milk using liquid chromatography with ultraviolet detection and residue confirmation by mass
spectrometry." Analytica Chimica Acta529(1-2): 145-150.

Bengtsson, B., H. E. Unnerstad, et al. (2009). "Antimicrobial susceptibility of udder pathogens from
cases of acute clinical mastitis in dairy cows." Veterinary microbiology136(1-2): 142-149.

Boulton, A. J., L. Vileikyte, et al. (2005). "The global burden of diabetic foot disease." The
lancet366(9498): 1719-1724.

Boyko, E., J. Ahroni, et al. (1996). "Increased mortality associated with diabetic foot ulcer." Diabetic
medicine13(11): 967-972.

Commission, E.-E. (2010). "Commission regulation (EU) No 37/2010 of 22 December 2009 on

pharmacologically active substances and their classification regarding maximum residue limits in
foodstuffs of animal origin." Off. J. Eur. Union L15: 1-72.

Control, C. f. D. and Prevention (1997). "Reduced susceptibility of Staphylococcus aureus to

vancomycin--Japan, 1996." MMWR. Morbidity and mortality weekly report46(27): 624.

Cook, G. and M. Al-Torki (1975). "High intestinal lactase concentrations in adult Arbs in Saudi
Arabia." Br Med J3(5976): 135-136.

Cotter, P. D., R. P. Ross, et al. (2013). "Bacteriocins—a viable alternative to antibiotics?" Nature
Reviews Microbiology11(2): 95.

32 | P a g e
Dahlqvist, A., B. Mattiasson, et al. (1973). "Hydrolysis of β‐galactosides using polymer‐entrapped
lactase. A study towards producing lactose‐free milk." Biotechnology and Bioengineering15(2): 395-
402.

Dang, C., Y. Prasad, et al. (2003). "Methicillin‐resistant Staphylococcus aureus in the diabetic foot
clinic: a worsening problem." Diabetic medicine20(2): 159-161.

Faglia, E., F. Favales, et al. (2001). "New ulceration, new major amputation, and survival rates in
diabetic subjects hospitalized for foot ulceration from 1990 to 1993: a 6.5-year follow-up." Diabetes
care24(1): 78-83.

Fejzić, N., M. Begagić, et al. (2014). "Beta lactam antibiotics residues in cow’s milk: comparison of
efficacy of three screening tests used in Bosnia and Herzegovina." Bosnian journal of basic medical
sciences14(3): 155.

Gálvez, A., H. Abriouel, et al. (2007). "Bacteriocin-based strategies for food biopreservation."
International journal of food microbiology120(1-2): 51-70.

Gilat, T., S. Russo, et al. (1972). "Lactase in man: a nonadaptable enzyme." Gastroenterology62(6):
1125-1127.

Hageman, J. C., D. A. Pegues, et al. (2001). "Vancomycin-intermediate Staphylococcus aureus in a

home health-care patient." Emerging infectious diseases7(6): 1023.

Harik-Khan, R. and W. A. Moats (1995). "Identification and measurement of beta-lactam antibiotic

residues in milk: integration of screening kits with liquid chromatography." Journal of AOAC
International78(4): 978-986.

Harris, M. I. (1995). "Diabetes in America." Diabetes Research and Clinical Practice30(1): 75.

Jeffcoate, W. J. and K. G. Harding (2003). "Diabetic foot ulcers." The lancet361(9368): 1545-1551.

Jelen, P. and O. Tossavainen (2003). "Low lactose and lactose-free milk and dairy products-prospects,
technologies and applications." Australian Journal of Dairy Technology58(2): 161.

Jussila, J. (1969). "Milk intolerance and lactose malabsorption in hospital patients and young
servicemen in Finland." Ann. Clin. Res.1: 199-207.

Kempe, M., J. Cederfur, et al. (1999). "CREAM—Cartridges with Molecularly Imprinted Recognition
Elements for Antibiotic residues Monitoring in Milk." Lunds Universitet, Centre for Chemistry and
Chemical Engineering.

Khaskheli, M., R. Malik, et al. (2008). "Detection of ß-lactam antibiotic residues in market milk." Pak
J Nutr7(5): 682-685.

33 | P a g e
Lee, J. S., M. Lu, et al. (1993). "Lower-extremity amputation: incidence, risk factors, and mortality in
the Oklahoma Indian Diabetes Study." Diabetes42(6): 876-882.

Lee, L.-H., N. Zainal, et al. (2014). "Streptomyces pluripotens sp. nov., a bacteriocin-producing
streptomycete that inhibits meticillin-resistant Staphylococcus aureus." International journal of
systematic and evolutionary microbiology64(9): 3297-3306.

Lipsky, B. A. (1999). "A current approach to diabetic foot infections." Current infectious disease
reports1(3): 253-260.

Lipsky, B. A. and A. R. Berendt (2000). "Principles and practice of antibiotic therapy of diabetic foot
infections." Diabetes/metabolism research and reviews16(S1): S42-S46.

Lisker, R., B. Gonzalez, et al. (1975). "Recessive inheritance of the adult type of intestinal lactase
deficiency." American journal of human genetics27(5): 662.

Long, A. R., L. C. Hsieh, et al. (1990). "Matrix solid-phase dispersion (MSPD) isolation and liquid
chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk."
Journal-Association of Official Analytical Chemists73(3): 379-384.

Louie, T. J., J. G. Bartlett, et al. (1976). "Aerobic and anaerobic bacteria in diabetic foot ulcers." Ann
Intern Med85(4): 461-463.

Moats, W. A. and R. Harik-Khan (1995). "Liquid chromatographic determination of beta-lactam

antibiotics in milk: a multiresidue approach." Journal of AOAC International78(1): 49-54.

Murthy, M. and J. Haworth (1970). "Intestinal lactase deficiency among East Indians: an adaptive
rather than a genetically inherited phenomenon?" Amer. J. Gastroent.53(3): 246-251.

Okuda, K.-i., T. Zendo, et al. (2013). "Effects of bacteriocins on methicillin-resistant Staphylococcus

aureus biofilm." Antimicrobial agents and chemotherapy57(11): 5572-5579.

Rab, S. and A. Baseer (1976). "High intestinal lactase concentration in adult Pakistanis." British
medical journal1(6007): 436.

Rahimi, A., H. Delbrück, et al. (1976). "Persistence of high intestinal lactase activity (lactose
tolerance) in Afghanistan." Human Genetics34(1): 57-62.

Reiber, G., B. Lipsky, et al. (1998). "The burden of diabetic foot ulcers." The American journal of
surgery176(2): 5S-10S.

Rotun, S. S., V. McMath, et al. (1999). "Staphylococcus aureus with reduced susceptibility to
vancomycin isolated from a patient with fatal bacteremia." Emerging infectious diseases5(1): 147.

34 | P a g e
Ruegg, P. (2013). "Antimicrobial residues and resistance: Understanding and managing drug usage on
dairy farms." University of Winsconsin, Dept. of Dairy Scinece, Madison, Wisconsin.

Sahi, T. (1974). "The inheritance of selective adult-type lactose malabsorption." Scandinavian journal
of gastroenterology. Supplement30: 1.

Samaržija, D. and N. Antunac (2002). "Važnost dokazivanja prisutnosti antibiotičkih ostataka u

mlijeku." Mljekarstvo: časopis za unaprjeđenje proizvodnje i prerade mlijeka52(1): 61-70.

Scrimshaw, N. and E. Murray (1988). "Prevalence of lactose maldigestion." Am J Clin Nutr48(suppl):

1086-1098.

Simoons, F. J. (1978). "The geographic hypothesis and lactose malabsorption." The American journal
of digestive diseases23(11): 963-980.

Singh, N., D. G. Armstrong, et al. (2005). "Preventing foot ulcers in patients with diabetes."
Jama293(2): 217-228.

Sivakesava, S. and J. Irudayaraj (2002). "Rapid determination of tetracycline in milk by FT-MIR and
FT-NIR spectroscopy." Journal of dairy science85(3): 487-493.

Sloan, A. E. (1999). "The new market: foods for the not-so-healthy." Food technology (USA).

Smith, T. L., M. L. Pearson, et al. (1999). "Emergence of vancomycin resistance in Staphylococcus

aureus." New England Journal of Medicine340(7): 493-501.

Solomons, N. (2003). "Fermentation, fermented foods and lactose intolerance." European Journal of
Clinical Nutrition56(S4): S50.

Straub, R., M. Linder, et al. (1994). "Determination of. beta.-lactam residues in milk using perfusive-
particle liquid chromatography combined with ultrasonic nebulization electrospray mass
spectrometry." Analytical chemistry66(21): 3651-3658.

Suarez, F. L., D. A. Savaiano, et al. (1995). "A comparison of symptoms after the consumption of
milk or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance." New
England Journal of Medicine333(1): 1-4.

Swagerty, D. L., A. D. Walling, et al. (2002). "Lactose intolerance." American family physician65(9):
1845-1860.

Tagg, J. R., A. S. Dajani, et al. (1976). "Bacteriocins of gram-positive bacteria." Bacteriological

reviews40(3): 722.

35 | P a g e
Vesa, T. H., P. Marteau, et al. (2000). "Lactose intolerance." Journal of the American College of
Nutrition19(sup2): 165S-175S.

Weinstein, R. A. and S. K. Fridkin (2001). "Vancomycin-intermediate and-resistant Staphylococcus

aureus: what the infectious disease specialist needs to know." Clinical Infectious Diseases32(1): 108-
115.

Andersen, W. C., J. E. Roybal, et al. (2005). "Determination of tetracycline residues in shrimp and
whole milk using liquid chromatography with ultraviolet detection and residue confirmation by mass
spectrometry." Analytica Chimica Acta529(1-2): 145-150.

Bengtsson, B., H. E. Unnerstad, et al. (2009). "Antimicrobial susceptibility of udder pathogens from
cases of acute clinical mastitis in dairy cows." Veterinary microbiology136(1-2): 142-149.

Commission, E.-E. (2010). "Commission regulation (EU) No 37/2010 of 22 December 2009 on

pharmacologically active substances and their classification regarding maximum residue limits in
foodstuffs of animal origin." Off. J. Eur. Union L15: 1-72.

Fejzić, N., M. Begagić, et al. (2014). "Beta lactam antibiotics residues in cow’s milk: comparison of
efficacy of three screening tests used in Bosnia and Herzegovina." Bosnian journal of basic medical
sciences14(3): 155.

Harik-Khan, R. and W. A. Moats (1995). "Identification and measurement of beta-lactam antibiotic

residues in milk: integration of screening kits with liquid chromatography." Journal of AOAC
International78(4): 978-986.

Kempe, M., J. Cederfur, et al. (1999). "CREAM—Cartridges with Molecularly Imprinted Recognition
Elements for Antibiotic residues Monitoring in Milk." Lunds Universitet, Centre for Chemistry and
Chemical Engineering.

Khaskheli, M., R. Malik, et al. (2008). "Detection of ß-lactam antibiotic residues in market milk." Pak
J Nutr7(5): 682-685.

Long, A. R., L. C. Hsieh, et al. (1990). "Matrix solid-phase dispersion (MSPD) isolation and liquid
chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk."
Journal-Association of Official Analytical Chemists73(3): 379-384.

Moats, W. A. and R. Harik-Khan (1995). "Liquid chromatographic determination of beta-lactam

antibiotics in milk: a multiresidue approach." Journal of AOAC International78(1): 49-54.

Ruegg, P. (2013). "Antimicrobial residues and resistance: Understanding and managing drug usage on
dairy farms." University of Winsconsin, Dept. of Dairy Scinece, Madison, Wisconsin.

36 | P a g e
Samaržija, D. and N. Antunac (2002). "Važnost dokazivanja prisutnosti antibiotičkih ostataka u
mlijeku." Mljekarstvo: časopis za unaprjeđenje proizvodnje i prerade mlijeka52(1): 61-70.

Sivakesava, S. and J. Irudayaraj (2002). "Rapid determination of tetracycline in milk by FT-MIR and
FT-NIR spectroscopy." Journal of dairy science85(3): 487-493.

Straub, R., M. Linder, et al. (1994). "Determination of. beta.-lactam residues in milk using perfusive-
particle liquid chromatography combined with ultrasonic nebulization electrospray mass
spectrometry." Analytical chemistry66(21): 3651-3658.

Ahmad, M. and G. Flatz (1984). "Prevalence of primary adult lactose malabsorption in Pakistan."
Human heredity34(2): 69-75.

Andersen, W. C., J. E. Roybal, et al. (2005). "Determination of tetracycline residues in shrimp and
whole milk using liquid chromatography with ultraviolet detection and residue confirmation by mass
spectrometry." Analytica Chimica Acta529(1-2): 145-150.

Bengtsson, B., H. E. Unnerstad, et al. (2009). "Antimicrobial susceptibility of udder pathogens from
cases of acute clinical mastitis in dairy cows." Veterinary microbiology136(1-2): 142-149.

Boulton, A. J., L. Vileikyte, et al. (2005). "The global burden of diabetic foot disease." The
lancet366(9498): 1719-1724.

Boyko, E., J. Ahroni, et al. (1996). "Increased mortality associated with diabetic foot ulcer." Diabetic
medicine13(11): 967-972.

Commission, E.-E. (2010). "Commission regulation (EU) No 37/2010 of 22 December 2009 on

pharmacologically active substances and their classification regarding maximum residue limits in
foodstuffs of animal origin." Off. J. Eur. Union L15: 1-72.

Control, C. f. D. and Prevention (1997). "Reduced susceptibility of Staphylococcus aureus to

vancomycin--Japan, 1996." MMWR. Morbidity and mortality weekly report46(27): 624.

Cook, G. and M. Al-Torki (1975). "High intestinal lactase concentrations in adult Arbs in Saudi
Arabia." Br Med J3(5976): 135-136.

Cotter, P. D., R. P. Ross, et al. (2013). "Bacteriocins—a viable alternative to antibiotics?" Nature
Reviews Microbiology11(2): 95.

Dahlqvist, A., B. Mattiasson, et al. (1973). "Hydrolysis of β‐galactosides using polymer‐entrapped

lactase. A study towards producing lactose‐free milk." Biotechnology and Bioengineering15(2): 395-
402.

37 | P a g e
Dang, C., Y. Prasad, et al. (2003). "Methicillin‐resistant Staphylococcus aureus in the diabetic foot
clinic: a worsening problem." Diabetic medicine20(2): 159-161.

Faglia, E., F. Favales, et al. (2001). "New ulceration, new major amputation, and survival rates in
diabetic subjects hospitalized for foot ulceration from 1990 to 1993: a 6.5-year follow-up." Diabetes
care24(1): 78-83.

Fejzić, N., M. Begagić, et al. (2014). "Beta lactam antibiotics residues in cow’s milk: comparison of
efficacy of three screening tests used in Bosnia and Herzegovina." Bosnian journal of basic medical
sciences14(3): 155.

Gálvez, A., H. Abriouel, et al. (2007). "Bacteriocin-based strategies for food biopreservation."
International journal of food microbiology120(1-2): 51-70.

Gilat, T., S. Russo, et al. (1972). "Lactase in man: a nonadaptable enzyme." Gastroenterology62(6):
1125-1127.

Hageman, J. C., D. A. Pegues, et al. (2001). "Vancomycin-intermediate Staphylococcus aureus in a

home health-care patient." Emerging infectious diseases7(6): 1023.

Harik-Khan, R. and W. A. Moats (1995). "Identification and measurement of beta-lactam antibiotic

residues in milk: integration of screening kits with liquid chromatography." Journal of AOAC
International78(4): 978-986.

Harris, M. I. (1995). "Diabetes in America." Diabetes Research and Clinical Practice30(1): 75.

Jeffcoate, W. J. and K. G. Harding (2003). "Diabetic foot ulcers." The lancet361(9368): 1545-1551.

Jelen, P. and O. Tossavainen (2003). "Low lactose and lactose-free milk and dairy products-
prospects, technologies and applications." Australian Journal of Dairy Technology58(2): 161.

Jussila, J. (1969). "Milk intolerance and lactose malabsorption in hospital patients and young
servicemen in Finland." Ann. Clin. Res.1: 199-207.

Kempe, M., J. Cederfur, et al. (1999). "CREAM—Cartridges with Molecularly Imprinted Recognition
Elements for Antibiotic residues Monitoring in Milk." Lunds Universitet, Centre for Chemistry and
Chemical Engineering.

Khaskheli, M., R. Malik, et al. (2008). "Detection of ß-lactam antibiotic residues in market milk." Pak J
Nutr7(5): 682-685.

38 | P a g e
Lee, J. S., M. Lu, et al. (1993). "Lower-extremity amputation: incidence, risk factors, and mortality in
the Oklahoma Indian Diabetes Study." Diabetes42(6): 876-882.

Lee, L.-H., N. Zainal, et al. (2014). "Streptomyces pluripotens sp. nov., a bacteriocin-producing
streptomycete that inhibits meticillin-resistant Staphylococcus aureus." International journal of
systematic and evolutionary microbiology64(9): 3297-3306.

Lipsky, B. A. (1999). "A current approach to diabetic foot infections." Current infectious disease
reports1(3): 253-260.

Lipsky, B. A. and A. R. Berendt (2000). "Principles and practice of antibiotic therapy of diabetic foot
infections." Diabetes/metabolism research and reviews16(S1): S42-S46.

Lisker, R., B. Gonzalez, et al. (1975). "Recessive inheritance of the adult type of intestinal lactase
deficiency." American journal of human genetics27(5): 662.

Long, A. R., L. C. Hsieh, et al. (1990). "Matrix solid-phase dispersion (MSPD) isolation and liquid
chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk."
Journal-Association of Official Analytical Chemists73(3): 379-384.

Louie, T. J., J. G. Bartlett, et al. (1976). "Aerobic and anaerobic bacteria in diabetic foot ulcers." Ann
Intern Med85(4): 461-463.

Moats, W. A. and R. Harik-Khan (1995). "Liquid chromatographic determination of beta-lactam

antibiotics in milk: a multiresidue approach." Journal of AOAC International78(1): 49-54.

Murthy, M. and J. Haworth (1970). "Intestinal lactase deficiency among East Indians: an adaptive
rather than a genetically inherited phenomenon?" Amer. J. Gastroent.53(3): 246-251.

Okuda, K.-i., T. Zendo, et al. (2013). "Effects of bacteriocins on methicillin-resistant Staphylococcus

aureus biofilm." Antimicrobial agents and chemotherapy57(11): 5572-5579.

Rab, S. and A. Baseer (1976). "High intestinal lactase concentration in adult Pakistanis." British
medical journal1(6007): 436.

Rahimi, A., H. Delbrück, et al. (1976). "Persistence of high intestinal lactase activity (lactose
tolerance) in Afghanistan." Human Genetics34(1): 57-62.

Reiber, G., B. Lipsky, et al. (1998). "The burden of diabetic foot ulcers." The American journal of
surgery176(2): 5S-10S.

39 | P a g e
Rotun, S. S., V. McMath, et al. (1999). "Staphylococcus aureus with reduced susceptibility to
vancomycin isolated from a patient with fatal bacteremia." Emerging infectious diseases5(1): 147.

Ruegg, P. (2013). "Antimicrobial residues and resistance: Understanding and managing drug usage
on dairy farms." University of Winsconsin, Dept. of Dairy Scinece, Madison, Wisconsin.

Sahi, T. (1974). "The inheritance of selective adult-type lactose malabsorption." Scandinavian journal
of gastroenterology. Supplement30: 1.

Samaržija, D. and N. Antunac (2002). "Važnost dokazivanja prisutnosti antibiotičkih ostataka u

mlijeku." Mljekarstvo: časopis za unaprjeđenje proizvodnje i prerade mlijeka52(1): 61-70.

Scrimshaw, N. and E. Murray (1988). "Prevalence of lactose maldigestion." Am J Clin Nutr48(suppl):

1086-1098.

Simoons, F. J. (1978). "The geographic hypothesis and lactose malabsorption." The American journal
of digestive diseases23(11): 963-980.

Singh, N., D. G. Armstrong, et al. (2005). "Preventing foot ulcers in patients with diabetes."
Jama293(2): 217-228.

Sivakesava, S. and J. Irudayaraj (2002). "Rapid determination of tetracycline in milk by FT-MIR and
FT-NIR spectroscopy." Journal of dairy science85(3): 487-493.

Sloan, A. E. (1999). "The new market: foods for the not-so-healthy." Food technology (USA).

Smith, T. L., M. L. Pearson, et al. (1999). "Emergence of vancomycin resistance in Staphylococcus

aureus." New England Journal of Medicine340(7): 493-501.

Solomons, N. (2003). "Fermentation, fermented foods and lactose intolerance." European Journal of
Clinical Nutrition56(S4): S50.

Straub, R., M. Linder, et al. (1994). "Determination of. beta.-lactam residues in milk using perfusive-
particle liquid chromatography combined with ultrasonic nebulization electrospray mass
spectrometry." Analytical chemistry66(21): 3651-3658.

Suarez, F. L., D. A. Savaiano, et al. (1995). "A comparison of symptoms after the consumption of milk
or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance." New England
Journal of Medicine333(1): 1-4.

Swagerty, D. L., A. D. Walling, et al. (2002). "Lactose intolerance." American family physician65(9):
1845-1860.

40 | P a g e
Tagg, J. R., A. S. Dajani, et al. (1976). "Bacteriocins of gram-positive bacteria." Bacteriological
reviews40(3): 722.

Vesa, T. H., P. Marteau, et al. (2000). "Lactose intolerance." Journal of the American College of
Nutrition19(sup2): 165S-175S.

Weinstein, R. A. and S. K. Fridkin (2001). "Vancomycin-intermediate and-resistant Staphylococcus

aureus: what the infectious disease specialist needs to know." Clinical Infectious Diseases32(1): 108-
115.

41 | P a g e