Acknowledgments

Acknowledgments
All praises, hymns and countless thanks to ALMIGHTY ALLAH, the most gracious, the most
merciful, and millions of Darood-O-Salam for HAZRAT MUHAMMAD (S.A.W.W.) who is
forever a model of guidance and knowledge for humanity as a whole.
I deem it profound honor to express the depth of my gratitude to Dr.Waseem Shahzad, Director
of Institute of Biochemistry and Biotechnology for accommodating me in Biochemistry lab
under the guidance of Tahir and Umer.
I would like to express my gratitude to my supervisor, Miss Faiza Masood whose expertise,
understanding, and patience added considerably to my graduate experience. I appreciate their
vast knowledge and skill in many areas (e.g., vision, ethics, interaction with group members),
and their assistance in writing reports.
I would like to extend my deep appreciation and thanks to the members of Lab, Sir Tahir and
Umer for the assistance, exceptional guidance, inspiring attitude and creative suggestions they
provided at all levels of the internship work. They impressed and influenced me in my learning
experience, during my association with them.
It is a pleasure to express my gratitude whole heartedly to my dearest friend Jahanzaib Azhar
and Faisal Sheraz Shah for his kind assistance during my internship duration. I would like to
express my gratitude towards my teachers & professors of University of Veterinary & Animal
Sciences for their kind co-operation and encouragement which helped me in completion of this
internship.
Finally but profoundly, I pay my heartily thanks to my beloved mother for love, support and
countless prayers for my success during the course of study.
Mohsin Shad

Sr.no Content Page
no
1. Acknowledgement 1
2. Summary 2
3. Introduction 3
4. Buffer 5
5. Spectrophotometer 8
6. Bacteria Classification 10
7. Bacteria Culturing 12
8. Gram Staining 15
9.
Amylase Production on Submerged Fermentation by Bacillus spp
18

Introduction:
The Institute of Biochemistry and Biotechnology was instituted in September 2009 after the up
gradation and union of department of Molecular Biology and Biotechnology and department of
Biochemistry. Currently the undergraduate degree B.S.(Hons) Biotechnology and Bioinformatics
and postgraduate degrees (MPhil/PhD) in Molecular Biology and Biotechnology, Biochemistry,
Bioinformatics, and Forensic Sciences (only M.Phil.) are going on with an overall strength of
more than 250 students.
This success story will be incomplete if it will not be mentioned and recognized the contributions
of highly qualified and dedicated team of scientists of our institute, working flat out 24 hours a
day.
Objectives:
 Establishment of a Centre that will provide the detailed information about the field of
study both at basic and clinical level with continuous curriculum update.
 To provide facilities for advanced studies and research leading to MPhil/Ph.D. in the
areas of Biochemistry.
 To develop trained manpower able to make diagnosis based upon the basic knowledge of
biochemical diseases.
 To develop skilled human resources for biochemical disorders caused due to
consanguinity in our population.
Books and computing facilities:
The department has a pretty good collection of books and user manuals in the field of
Biochemistry and molecular biology. To upgrade their knowledge the students are encouraged to
consult latest research papers. Facility of online journals is accessible to them not only in the
main library but in the department as well. To increase the understanding of biological processes
the biological data must be combined to form a comprehensive picture of these activities. For
this purpose online freely available bioinformatics sites for databases and data analysis soft
wares need to be used. Computers having full access to these sites are available in the
department to facilitate the students.
Future Plans
 To improve the number of M. Phil. And PhD scholars in the department.

 To upgrade the department by providing state of the art lab facilities for advance research
and practical training in Molecular Genetics including cancer.
 To establish a laboratory to provide rapid genetic testing in different hereditary diseases.

Buffer
A buffer is a solution containing either a weak acid and its salt or a weak base and its salt, which
is resistant to changes in pH.
OR
Buffers are the solutions which resist changes in pH when small amounts of acid or alkali is
added to them.
A buffer is a pair of weak acid and its salt.
Buffers are of main importance in regulating the pH of the body fluids and tissues
Many biochemical reactions including those catalyzed by enzymes require pH control which is
provided by buffers
Examples: Blood, TRIS buffer, phosphate buffer.
• Mammalian tissues in the resting state have a pH of about 7.4
• In order to maintain the required pH in an in vitro biochemical experiment a buffer is
always used
• The pH of a buffer is given by Handerson-Hasselbalch equation
• pH= pKa + log [A-]
[HA]
• pKa= -logKa
• Ka is the dissociation constant of the acid
• [A-] is the concentration of the base
• [HA] is the concentration of the acid
• Let we make a buffer which is described below,
• Prepare a Phosphate Buffer whose concentration is 0.1M in 250ml and it pH is 7.
As we know that Handerson-Hasselbalch equation is,
[HA]
Calculation;

7 = 6.8 + log [salt]/ [acid]
7_6.8 = log [salt]/ [acid]
0.2 = log [salt]/ [acid]
Taking antilog on both side of the equation
Antilog [0.2] = [salt]/ [acid]
_1.609= [salt]/ [acid]
_1.609 /1= [salt]/ [acid]
_1.609 + 1= [salt] + [acid]
_0.609 = [salt] + [acid]
For Salt:
Salt = _1.609/ _0.609
Salt = 2.462
For acid:
Acid = 1 /_0.609
Acid =_1.642
Molecular weight of NaH2PO4=120
Molecular weight of Na2HPO4=142
For salt;
1M=142/1000
1M = 0.142g
For 0.1M
0.1M = 0.142/0.1
0.1M=1.42g
1000ml contain salt=1.42
1ml contain salt = 1.42/1000

250ml contain salt= 1.42/1000 x250
= 0.355g
For acid;
1M=120
1M=0.12
For 0.1 M
0.1M=0.12/0.1
0.1M=1.2g
1000ml contain salt=1.2g
1ml contain salt = 1.2/1000
250ml contain salt= 1.2/1000 x250
= 0.3g
Putting the values in Handerson equation
[HA
7=6.8 +log 0.355/0.3
7=7
Hence prove that the pH of buffer is 7

Spectrophotometer
Spectrophotometry is a method to measure how much a chemical substance absorbs light
by measuring the intensity of light as a beam of light passes through sample solution. The basic
principle is that each compound absorbs or transmits light over a certain range of wavelength.
Methods to detect molecules:
There are two methods to detect molecules
1=Indicator
2=Spectrophotometer
Two different types of Spectrophotometer:
 Ultraviolet (UV) Spectrophotometers. Uses ultraviolet light of wave lengths from 200 nm
to 350 nm.
 Visible (VIS) Light Spectrum Spectrophotometers. Uses visible light (white light) of
wave lengths from 350 nm to 700 nm.
Standard curve;
 The concentration of an unknown sample can be determined by comparing the
absorbance data to standards of known concentration.
 The data generated with the set of known standards is called a standard curve.
How a spectrophotometer works:
 Visible Spectrophotometer
 White light hits the prism or grating, it is split into the colors of the rainbow (Visible
Spectrum).
 The wavelength knob rotates the prism/grating, directing different color of light toward the sample.
 The wavelength of light produced by the tungsten lamp range from about 350 nm (Violet light) to 700 nm
(red light).
 The detector measures the amount of light being transmitted by the sample and reports that value
directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using
Beers Law.

Measurement of standard wavelength of Cobalt Chloride:
I prepare different concentration of cobalt chloride solution like 1%, 2%, 3%, 4%, 5%. Then
I note absorbance of standard wavelength. After standard wavelength I measured the absorbance
of different present solution and plot a graph and note results.
Calculated Concentrations Dilutions and Corresponding Absorbance
Test Tube Number Concentration g/L Absorbance
1 0.01 0.164
2 0.02 0.191
3 0.03 0.222
4 0.04 0.234
5 0.05 0.262
Standard wavelength of cobalt chloride = 500 nm
Concentration= X axis
Absorbation = Yaxis
`
absorbance= 2.39 concentration
0
0.05
0.1
0.15
0.2
0.25
0.3
0 0.01 0.02 0.03 0.04 0.05 0.06
Absorbance
Concentration
absorbance vs concentration graph of CoCl2
absorbance
Linear (absorbance)

Bacteria Classification
There are two major categories of bacteria: Gram positive and Gram negative.
Gram Positive Cell Wall:
Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-
90% of cell wall), which stains purple. Peptidoglycan is mainly a polysaccharide composed of
two subunits called N-acetyl glucosamine and N-acetyl muramic acid. As adjacent layers of
peptidoglycan are formed, they are cross linked by short chains of peptides by means of a
transpeptidase enzyme, resulting in the shape and rigidity of the cell wall. The thick
peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal
violet-iodine complex and stains the cells as purple.
Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria
which is embedded in the peptidoglycan layer. It consists of teichoic acids which are long
chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator of
autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell walls that cause
disintegration of the cell following injury or death.)
Gram Negative Cell Wall:
Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the
crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter
stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane
which contains lipids, which is separated from the cell wall by means of periplasmic space.
Fig: Gram positive bacteria Fig: Gram negative bacteria

Typical Gram-negative bacteria:
1. Bordetella pertusis, the causative agent of whooping cough
2. Salmonella typhi, the causative agent of typhoid
3. Vibrio cholera, the causative agent of cholera
4. Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria
Typical Gram-positive bacteria:
1. Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a
common cause of boils.
2. Streptococci such as the many species of oral streptococci, Streptococcus pyogenes which
causes many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar
pneumonia.
3. Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).
4. Actinomyces such as Actinomyces odontolyticus which is found in mouth.
5. Species of the genus Bacillus such as Bacillus subtilis which are common microbes living in
soil.
Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical
point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-
negative diplococcus looking very much like a pair of kidney bean.

Bacteria Culturing
Materials Required:
1. Mixed culture of bacteria.
2. Sterile petri dish with appropriate bacterial media(such as trypticase soy agar, nutrient agar).
3. Inoculating loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be a
single-use disposable plastic loop, which would be discarded between sectors rather than
resterilized).
4. Bunsen burner.
5. Marking pen
Procedure:
All the process is done in a laminar air flow cabinet aseptically.
Label a Petri dish:
Petri dishes are labelled on the bottom rather than on the lid. Write close to the edge of the
bottom of the plate to preserve area to observe the plate after it has incubated. Labels usually
include the organism name, type of agar, date, and the plater's name or initials. Using sterile
cotton swabs, remove any visible water on the agar in the plate or around the inner rim of the
petri plate. Observe the plate and mentally divide it into three sectors. The plate will then be
turned clockwise (if you are right handed) with the agar side up. The second sector will then be
at the top for streaking and then the plate is turned again so that the third sector can be streaked.
Sterilize the Transfer Loop before Obtaining a Specimen:
To streak a specimen from a culture tube, metal transfer loops are first sterilized by flaming the
wire loop held in the light blue area of a Bunsen burner just above the tip of inner flame of the
flame until it is red-hot. If a hot incinerator is available, the loop may be sterilized by holding it
inside the incinerator for 5 to 7 seconds. Once sterile, the loop is allowed to cool by holding it
still. Do not wave it around to cool it or blow on it. When manipulating bacteria, transfer loops
are usually held like a pencil. If plastic disposable loops are being utilized, they are removed
from the packaging to avoid contamination and after being used, are discarded into an
appropriate container. A new loop is recommended for each sector of an isolation streak plate.

Open the culture tube and collect a sample of specimen using the sterile loop:
Isolation can be obtained from any of a variety of specimens. This protocol describes the use of a
mixed broth culture, where the culture contains several different bacterial species or strains. The
specimen streaked on a plate could come in a variety of forms, such as solid samples, liquid
samples, and cotton or foam swabs. Material containing possibly infectious agents should be
handled appropriately in the lab using bio safety procedure.
Remove the test tube cap. It is recommended that the cap be kept in your right hand (the hand
holding the sterile loop). Curl the little finger of your right hand around the cap to hold it or hold
it between the little finger and third finger from the back. Modern test tube caps extend over the
top of the test tube, keeping the rim of the test tube sterile while the rim of the cap has not been
exposed to the bacteria. The cap can also be placed on the disinfected table, if the test tube is
held at an angle so that air contamination does not fall down into the tube. Insert the loop into the
culture tube and remove a loopful of broth. Replace the cap of the test tube and put it back into
the test tube rack.
Streak the Plate:
The lid of the agar plate has to be opened just sufficiently enough to streak the plate with the
inoculation loop. Minimize the amount of agar and the length of time the agar is exposed to the
environment during the streak process.
Three Sector Streak (t streak):
1. Sterilize the wire loop.
2. Cool the loop by touching it on the edge of the sterile agar plate.
3. Dip the loop into the broth culture containing the mixture of bacteria.
4. Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top
one-third of the plate back and forth in a "zig-zag" formation.
5. The loop has picked up thousands of bacteria which are spread out over the surface of the agar.
6. Sterilize the loop in the flame.

7. Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three
times and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate
without touching that area again.
8. Sterilize the loop in the flame.
9. Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the
area you just streaked, and fill in the remaining area of the plate (zig-zag formation), being very
careful not to touch any of the areas you previously streaked.
10. Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies in the
third sector. The heaviest growth will be in the first sector. There will be less growth and some
isolated colonies in the second sector. The third area should have the least growth with isolated
colonies.
Results:
Staphylococcus aureus colony on nutrient agar is observed in pattri dishs.

Gram Staining
Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of
the microscopic image. Stains and dyes are widely used in the scientific field to highlight the
structure of the biological specimens, cells, tissues etc.
Materials Required:
1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Lens paper and lens cleaner
7. Immersion oil
8. Distilled water
9. 18 to 24 hour cultures of organisms
Reagents:
1. Primary Stain - Crystal Violet
2. Mordant - Grams Iodine
3. Decolourizer - Ethyl Alcohol
4. Secondary Stain - Safranin
Procedure:
Part 1: Preparation of the glass microscopic slide
Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from
the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides
with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.
Part 2: Labeling of the slides:
Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to
clearly designate the area in which you will prepare the smear. You may also label the slide with

the initials of the name of the organism on the edge of the slide. Care should be taken that the
label should not be in contact with the staining reagents.
Part 3: Preparation of the smear:
 Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture
on the slide. Spread by means of circular motion of the inoculating loop to about one centimeter
in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory
smear will allow examination of the typical cellular arrangement and isolated cells.
 Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline
solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a
bacterial colony and gently stir into the drop of water/saline on the slide to create an emulsion.
 Swab Samples: Roll the swab over the cleaned surface of a glass slide.
Please note: It is very important to prevent preparing thick, dense smears which contain an
excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass
through, thus making it difficult to visualize the morphology of single cells. Smears typically
require only a small amount of bacterial culture. An effective smear appears as a thin whitish
layer or film after heat-fixing.
Part 4: Heat Fixing:
Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the
sample to more readily take up stains.
Allow the smear to air dry.
After the smear has air-dried, hold the slide at one end and pass the entire slide through the flame
of a Bunsen burner two to three times with the smear-side up.
Now the smear is ready to be stained.
Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.
Part 5: Gram Stain Procedure :
1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with crystal violet and let stand for 1 minute.
3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The
smear will appear as a purple circle on the slide.
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop
by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with safranin to counter-stain and let stand for 45 seconds.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
10. Blot dries the slide with bibulous paper.
11. View the smear using a light-microscope under oil-immersion.
Fig: Colour changes that occur at each step in the staining process
Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet
in the cell wall. Example: Staphylococcus aureus
Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called
Safranin. Example: Escherichia coli

Amylase Production on Submerged Fermentation by Bacillus spp
Abstract:
The production of extracellular amylase by Bacillus spp was optimized in a submerged
fermentation. The production of the enzyme was maximum at 10 h after inoculation. The effect
of incubation period, pH of the medium and incubation temperature was optimized. The
maximum production of enzyme was obtained at 35°C and pH 7.
Introduction:
Amylases are enzymes that break down starch or glycogen. The amylases can be derived from
several sources such as plants, animals and microbes. The major advantage of using
microorganisms for production of amylases is in economical bulk production capacity and
microbes are also easy to manipulate to obtain enzymes of desired characteristics [1]. The
microbial amylases meet industrial demands a large number of them are available
commercially; and, they have almost completely replaced chemical hydrolysis of starch in starch
processing industry Although many microorganisms produce this enzyme. the most
commonly used for their industrial application are Bacillus licheniformis,: amyloliquifaciens
and Aspergillus niger. The use of the submerged culture is advantageous because of the ease of
sterilization and process control is easier to engineer in these systems. Depending on the strain
and the culture conditions, the enzyme can be constitutive or inducible, showing different
production pattern. The purpose of this work was to study the production of amylase by Bacillus
sp., in submerged cultures and optimized the cultural conditions for the production of amylase
Material and Method
Microorganism:
Bacillus spp was isolated from environment and maintained on nutrient agar slants and for every
10 days
Inoculum and Fermentation Medium:
The inoculum was prepared by the addition of sterile distilled water in the freshly grown
nutrient ager from this 0.5 ml of cell suspension was inoculated in to 100 ml of sterilized
fermentation medium and incubated at 35°C for 10 hrs. The composition of the fermentation
medium was [g/l] 6.0 g Bacteriological peptone; 0.5 g MgSO4 .7H O; 0.5 g KCl; 1.0 g Starch-,
pH 7. For inoculum media we use glucose instead of starch.
For 60ml we use these compositions of fermentation media
Bacteriological peptone = 3.6 g/60 ml

MgSO4 .7H O = 0.3 g/60 ml
KCl = 0.3 g/60 ml
Starch = 0.6 g/60 ml
Extraction of Amylase from the Fermentation Medium:
After incubation the fermentation medium was harvested by centrifugation at 5000 rpm for 20
minutes at 4°C. The supernatant was collected and subjected to estimate the amylase activity
Amylase Activity:
The enzyme activity is determined by measuring the reducing sugars released as a result of the
action of α-Amylase on starch.so we follow Dinitrosalicylic Acid Method (DNS).
Dinitrosalicylic Acid Method (DNS):
In the dinitrosalicylic acid method, We prepared different concentration of stock solution of
glucose like (0.5%,1%,1.5%,2% ) Followed by 10 min of incubation at 50C, DNS reagent is
added to the test tube and the mixture is incubated in a boiling water bath for 5 min. After
cooling to room temperature, the absorbance of the supernatant at 540 nm is measured. The
A540 values for the substrate and enzyme blanks are subtracted from the A540 value for the
analyzed sample (0.5%,1%,1.5%,2% ) and note the absorbance of fermentation media .In a study
on alkalophilic α-Amylase from Bacillus strain the enzyme assay was done by measuring the
reducing sugars by DNS method and the activity was found to be a maximum of 0.75 U ml-
1 after incubation of 24 h
Calculation of Amylase Activity:
Absorbance of fermentation media = 0.208
As,
0.171 Absorbance is due to = 1%
1 Absorbance is due to = 1 / 0.171
0.208 Absorbance is due to = 1 / 0.171 x 0.208
= 1.21g
For micro gram,
= 1.21 x 1000000
= 1210000 ug

Activity of Amylase = ug of glucose
Molecular weight x Incubation time
Activity of Amylase = 1210000 ug
180 x 10
= 672.22 ug / ml

Acknowledgments

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