DATE:

Experiment No. 1
AIM: Estimation of Ascorbic Acid (Vitamin C) by titration method.
APPARATUS/GLASSWARE:
Burette, volumetric flask (100 ml), pipette, conical flask (100 ml), analytical
balance, funnel, Whatman No. I filter paper etc.
PRINCIPLE:
Ascorbic acid is oxidized by the colored dye 2-6-dichlorophenol-indophenol to
dehydroascorbic acid. The dye is reduced to a colorless compound so the end
point of the titration is easily detected.
The decolorization of dye by ascorbic acid is not very specific, but specificity
may be increased by carrying out the reaction in an acidic solution.

REAGENTS REQUIRED:
1) Indophenol solution - dye:
To 50 ml distilled water in a 150 ml beaker, add and stir to dissolve 42 mg
(0.042 g) sodium bicarbonate, and then add and stir to dissolve 50 mg (0.05 g)
2,6-dichloroindophenol sodium salt. Dilute the mixture to 200 ml with distilled
water. Filter through fluted filter paper into an amber bottle. Close the bottle
with a stopper or lid and store refrigerate until used.
2) Ascorbic acid standard solution: (prepare only at time of use)
Accurately weigh (on an analytical balance) approximately 50 mg (0.05 g) of
ascorbic acid. Record this weight. Transfer to a 50-ml volumetric flask. Dilute
to volume immediately before use with the metaphosphoric acid acetic-acid
solution.
3) Metaphosphoric acid-acetic acid solution:
To a 250 ml beaker, add 100 ml add water then 20 ml acetic acid. Add and stir
to dissolve 7.5 g metaphosphoric acid. Dilute the mixture to 250 ml with

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distilled water. Filter through fluted filter paper into a bottle. Close the bottle
with a stopper or lid and store refrigerated until used.
4) Sample: Fruit Juice
Weight the sample given (lemon) and then squeeze all the juices and after that
make an equivalent concentration of fruit juice with metaphosphoric acid
making the volume 100ml.
PROCEDURE:
Standardization of Dye:
1) Pipette 2 ml metaphosphoric acid-acetic acid solution into each of three 50ml
Erlenmeyer flasks.
2) Add 2.0 ml ascorbic acid standard solution to each flask.
3) fill the burette with the indophenol solution (dye) and record the initial burette
reading.
4) Place the Erlenmeyer flask under the tip of the burette. Slowly add indophenol
solution to standard ascorbic acid solution until a light but distinct rose-pink
color persists for >5 s (takes about 15-17 ml). Swirl the flask as you add the
indophenol solution.
5) Note the final burette reading and calculate the volume of dye used. Repeat
Steps 3-5 for the other two standard samples. Record the initial and final
burette readings and calculate the volume of dye used for each

(Note: If the sample is colored, then the detection of the endpoint becomes difficult. In such cases, 1 ml of
chloroform is added to the titration mixture, and the appearance of pink color in the organic layer indicates the
endpoint).

The standard should also be titrated with chloroform. If the presence of SO₂ is likely
in fruit juices, the titration should be carried out in the presence of 2.5 ml of acetone
which forms a Bisulphate-acetone complex sample.

Analysis of Juice Samples:

1) Pipette out into each of three 50 ml Erlenmeyer flasks 5 ml Metaphosphoric
acid acetic acid solution and 2 ml lemon juice.
2) Titrate each sample with the indophenol dye solution (as you did above, Steps
3-5) until a light but distinct rose-pink color persists for >5 s.
3) Record the initial and final readings and calculate the difference to determine
the amount of dye used for each titration.

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OBSERVATION TABLE:

Solution taken in flask (ml) Burette reading dye

Sr. No. (metaphosphoric acid - Std. Ascorbic acid
acetic acid) + Ascorbic acid

1 2 16

2 2 16.6

3 2 16.8

Solution taken in flask (ml) Burette reading dye sample

Sr. No. (metaphosphoric acid - (Lemon)
acetic acid) +Lemon juice

1 3 11

2 3 10.2

3 3 9.8

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FORMULA:
1
Dye factor =
𝑇𝑖𝑡𝑟𝑒(𝑅𝑒𝑎𝑑𝑖𝑛𝑔 𝑓𝑜𝑟 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑎𝑠𝑐𝑜𝑟𝑏𝑖𝑐 𝑎𝑐𝑖𝑑)

𝑡𝑖𝑡𝑟𝑒 × 𝑑𝑦𝑒 𝑓𝑎𝑐𝑡𝑜𝑟 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑚𝑎𝑑𝑒 𝑢𝑝 × 100

Ascorbic acid mg/100gm =
𝑎𝑙𝑖𝑞𝑢𝑜𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑡𝑎𝑘𝑒𝑛 𝑓𝑜𝑟 𝑒𝑠𝑡𝑖𝑚𝑎𝑡𝑖𝑜𝑛 × 𝑤𝑡.𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

CALCULATION:

RESULT: In sample, the ascorbic acid is found to be mg/100gm.

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Experiment No. 2
AIM: Estimation of proteins by biuret assay.
APPARATUS: Test tubes, Pipettes, Colorimeter, etc.
MATERIALS:
Protein standard (500mg albumin (BSA) /250 ml) Prepare fresh.
Biuret reagent: Dissolve 1.5 gm of copper sulphate (CuSO4.5H2O) and 4.5gm
of sodium potassium tartrate (Rochelle salt) in 500ml of 0.2N sodium
hydroxide (NaOH); add 2.5gm of potassium iodide (KI) and make up to 1 lit,
with 0.2N sodium hydroxide.
PRINCIPLE:
Proteins can be estimated by the biuret method. Alkaline CuSO4 reacts with
compounds containing two or more peptide bonds (-CO-NH-) to form a violet
color complex. The depth of color formed is a measure of peptide bonds
present in the protein. The absorbance is read at 540 nm against a suitable
blank. The reaction is not specific to peptide bonds since any compound
containing two carbonyl groups is linked through nitrogen which gives positive
reaction. Biuret protein assay based on the binding of copper ions to peptide
bonds under alkaline conditions, producing a purple color.
Weakness - Not very sensitive and requires a large amount of protein
(0.04mg-2mg of BSA "Bovine Serum Albumin "protein per reaction volume)
PROCEDURE:
1) Pipette out 0.0, 0.5, 1.0, 1.5, 2.0 and 1 ml of working standard in to the series
of labeled test tubes.
2) Pipette out 1 ml of the given sample in another test tube.
3) Now add 3 ml of Biuret reagent to all the test tubes including the test tubes
labelled 'blank' and 'unknown'.
4) Make up the volume to 6 ml in all the test tubes.
5) Mix the contents of the tubes by vertexing/shaking the tubes and warm at 37°C
for 10 min.
6) Now cool the contents to room temperature and record the absorbance at 540
nm against blank.
7) Then plot the standard curve by taking concentration of protein along X- axis
and absorbance (Optical Density) at 540 nm along Y-axis.
8) Then from this standard curve calculate the concentration of protein in the
given sample.

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Fig: Colour intensity increases with increase in concentration

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OBSERVATION TABLE:

Protein
Concentration
Sample Distilled Biuret Optical
Sr. of
(ml) Water Solution Density
No. Protein
2mg/ml (ml) (ml) (540nm)
(mg/ ml)

1 0.0 3.0 3.0

2 0.5 2.5 3.0

3 1.0 2.0 3.0

4 1.5 1.5 3.0

5 2.0 1.0 3.0

Sample
Unknown 1.0 2.0 3.0
(milk)

From the graph sample solution contain ____ mg/ml of protein. Hence, in 100ml it
contains _____ mg of protein.

CALCULATION:
𝑦2 −𝑦1
Slope = =
𝑥2 −𝑥1

𝑂.𝐷
Unknown Sample (milk) = =
𝑆𝑙𝑜𝑝𝑒

RESULT: The give sample contains mg of protein per 100ml of solution.

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Experiment No. 3
AIM: To estimate the protein by the Folin - Lowery method
APPARATUS: Test tubes, Pipettes, Colorimeter, etc.
PRINCIPLE:
Protein reacts with Folin-Ciocalteau to give a coloured complex. The colour so
formed is due to the reaction of the alkaline copper with the protein as in the
biuret test and the reduction of phosphomolybdate by tyrosine and tryptophan
present in the protein.
REAGENTS:
1) Alkaline sodium carbonate solution: (Reagent A) Prepare 2% sodium
carbonate (Na2CO3) in 0.1 N NaOH.
2) Copper sulphate in sodium potassium tartrate solution: (Reagent B)
Prepare 0.1% CuSO4 in 1% sodium potassium tartrate (KNaC4H4O6.4H2O).
Prepare fresh by mixing double-strength stock solutions.
3) Alkaline solution: (Reagent C)
Prepare on the day of use by mixing 50 ml (1) and 1.0 ml (2)
4) Folin-Ciocalteau Reagent: Into 1000 ml of volumetric flask, 100 gm of
sodium tungstate, 25 gm of sodium molybdate, 700 ml water, 50 ml of 85%
phosphoric acid and 100 Conc. HCI is taken and refluxed gently for 10 Hrs.
150 gm of lithium sulphate, 50 ml water and a few drops of bromine are added.
The mixture is boiled for 15 min without a condenser to remove excess
bromine. It is then cooled and diluted to 1 lit. And filtered. Care is taken that
the reagent does not have any greenish tinge. We have to dilute the commercial
reagent of (20 ml) with an equal volume of H₂O on the day of use. (1:1)
5) Standard Protein Solution: Prepare 0.6 mg/ml of albumin solution. Prepare
fresh
PROCEDURE:
1) Add 5 ml of the alkaline solution to 0.5 ml of the test solution, Mix thoroughly
and allow to stand at room temperature for 10 minutes or longer.
2) Then add 0.5ml of dilute Folin- ciocaltenu reagent rapidly with the immediate
mixing.
3) Then lastly add water to make the total volume of 10ml. After 30 minutes, read
the extinction against the appropriate blank at 650nm, and estimate the protein
concentration of an unknown solution after preparing a standard curve.

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OBSERVATION TABLE:

Floin-
Alkaline Sol.
Std. Ciocaltea Distilled
Na2C03
Sr. No. Protein Reagent Water
(ml)
Sol. (ml) (ml) (ml)

Blank
0.0 5.0 0.5 4.5

1 0.5 5.0 0.5 4.0

2 1.0 5.0 0.5 3.5

3 1.5 5.0 0.5 3.0

4 2.0 5.0 0.5 2.5

5 2.5 5.0 0.5 2.0

Sample
1.0 5.0 0.5 3.5
(milk)

OBSERVATION:

Sr. Conc. of protein Optical Density

No. (mg/ml) (630 nm)

Blank

Sample
(milk)

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From the graph, Sample solution contains mg/ml of protein. Hence, in 100ml it
contains mg of protein.

CALCULATION:
𝑦2 −𝑦1
Slope = =
𝑥2 −𝑥1

𝑂.𝐷
Unknown Sample = =
𝑆𝑙𝑜𝑝𝑒

RESULT: The given sample containing mg of protein per 100ml of solution.

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Experiment No. 4
AIM: The determination of iron in given food sample (red kidney beam)
THEORY:
Iran in food is determine by converting the iron to ferric from using oxidizing
agent like potassium persulphate or hydrogen peroxide and treating thereafter
with potassium thiocyanate to form the red ferric thiocyanate which is measure
calorimetrically at 480 nm
REAGENTS:
1. Conc. H2SO4 (iron free)
2. Saturated Potassium Persulphate (𝐊 𝟐 𝐒𝟐𝐎𝟐 ) Solution: Shake 7 to 8g of
reagent grade iron-free potassium persulphate with 100ml of water in a
glass stopperd bottle. The undissolved excess settles to the bottom and
compensate for loss by decomposition. Shake briefly before using. Keep
the reagent in the refrigerator.
3. 0.1 N Potassium Thiocyanate (KSCN) Solution: Dissolve 146g of
reagent grade potassium thiocyanate in water and dilute to 500ml. Filter if
turbid. Add 20ml of pure acetone to improve the keeping quality.
4. Standard Iron Solution: Dissolve 0.702g of reagent grade crystalline
ferrous ammonium sulphate [FeSO4 , (NH)2 SO4 6H₂O) in 100ml of water.
Add 5ml of conc., H2 SO4 warm slightly, and add cope potassium
permanganate solution drop by drop until one drop produces produces a
one-liter volumetric flask, rime with water and mike up to volume.
PROCEDURE:
Use the ash solution of the sample prepared by dry ashing for colors
development. Into three separate stoppered measuring cylinders, pipette the
solution as given below.
DRY ASHING PROCESS FOR ESTIMATION OF MINERALS
1) Weigh accurately a suitable quantity of the well mixed sample in a tared silica
dish. Sample used for the determination of moisture may be taken for ashing
2) Heat first over a low Bunsen flame to volatilize as much of the organic matter
(until no more of smoke is given out by the material) as possible.
3) Transfer the dish to a muffle furnace at temperature 550° C
4) Generally, 5 to 7 hr. are sufficient to ash most of the fruits or vegetables or their
products. If it is suspected that all the carbon has not been oxidized, remove the
dish from the muffle, allow to cool, add 1 to 2 ml of conc. HNO., evaporate to
dryness and heat in the muffle for another hour or so.
5) Remove from the muffle furnace, allow to cool and note the weight of the ash.

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6) Cover the dish with watch glass and add gently 20-30 ml of dilute HCI (1+1)
with the help of a pipette.
7) Heat over a water bath for 30 min, remove the cover and rinse. Continue
heating for another 30 min to dehydrate silica.
8) Add another 10ml of HCI (1+1) and water to dissolve soluble salts.
9) Filter into a 100-ml volumetric flask using No. 44 Whatman filter paper. Wash
the residue in the basin once or twice using dilute HCI.
10) Make up to volume with water.
11) Use aliquots of this dry ash solution for estimation of minerals

Blank (ml) Standard (ml) Sample (ml)

Std. iron solution
0.0 1.0 0.0
(1ml=0.1mg of fe)
Sample ash solution 0.0 0.0 5.0
Water 5.0 4.0 0.0
Conc.H2SO4 0.5 0.5 0.5
Pot. Persulphate 1.0 1.0 1.0
Pot. Perthiocyanate 2.0 2.0 2.0
In each of the above cases, make up the volume to 15ml with water. Measure the color
at 480nm setting the blank at 100% transmission.
OBSERVATION TABLE:

Solution Optical density

Sample 0.04
Standard Solution 0.14
Blank 0.03
CALCULATION:
𝑶. 𝑫 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 × 𝟎. 𝟏 × 𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆𝒐𝒇 𝒂𝒔𝒉 𝒔𝒐𝒍 × 𝟏𝟎𝟎
𝐈𝐫𝐨𝐧 𝐦𝐠/𝟏𝟎𝟎 𝐦𝐥 =
𝑶. 𝑫 𝒐𝒇 𝒔𝒕𝒅 × 𝟓 × 𝒘𝒕 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝒕𝒂𝒌𝒊𝒏𝒈 𝒂𝒔𝒉

RESULT: The amount of iron in given food sample (red kidney beans) was found to
be ______ mg/100gm.

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Experiment No. 5
AIM: Determination of calcium of a given food sample.
THEORY:
Calcium is precipitated as calcium oxalate. The precipitate is dissolved in hot
dilute H2SO4 and titrates with standard potassium permanganate.
REAGENTS:
1) Ammonium Oxalate-saturated solution.
2) Methyl Red Indicator: Dissolve 0.5g methyl red in 100ml of 95% alcohol.
3) Dilute Acetic Acid-1+4.
4) Dilute Ammonium Hydroxide - 1+4.
5) Dilute Sulphuric Acid-1+4: Add acid to water slowly and with constant stirring.
Cool and make up to volume.
6) 0.1N Potassium Permanganate (KMnO4).
7) 0.01N Potassium Permanganate working standard. Dilute 10ml of 0.1N
KMnO4, solution to 100ml with water (1ml-0.2mg of Ca). Prepare fresh
solution before using.

PROCEDURE:
Pipette an aliquot (20 to 100ml) of the ash solution obtain by dry ashing to a
250ml beaker. Add 25 to 50ml of water, if necessary. Add 10ml of saturated
ammonium oxalate solution and two drops of methyl red indicator. Make the
solution slightly alkaline by the addition of dilute ammonia and then slightly
acid with a few drops of acetic acid until color is faint pink (pH 0.5). Heat the
solution to the boiling point. Allow to stand at room temperature for at least
4hrsor preferably overnight. Filter through whatman no.42 paper and wash with
water till the filtrate is oxalate free. (Since IICI has been used for preparing the
original ash solution it is convenient to lest for the absence of chloride using
AgNO3). Break the point of filter paper with potassium wire or pointed
glassrod. Wash the precipitate first using hot dilute H₂SO₄ (1+4) from wash
bottle in to the beaker in which the Ca was precipitated, then wash with hot
water and titrate while still hot (temp. 70 to 80°c) with 0.01N KMnO4, to first
permanent pink color. Finally add filter paper to solution and complete the
titration.

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CALCULATION:
Titere × 0.2 × Total volume of ash sol × 100
Calcium mg/100gm =
volume taken for estumation × wt. of sample taking for ash

8 × 0.2 × 50 × 100
=
20 × 4.8
= 83.33

RESULT: The given sample of Almond contains 83.33 mg/100gm of calcium.

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Experiment No. 6
AIM: Determination of AG-Activity in dextroenzyme by the method of PNPG
method.
APPARATUS: Spectrophotometer measuring at 400 nm Water bath at 300 c
Automatic
syringes of 2ml and 3ml respectively Test tube (16×160 mm)
REACTION CONDITIONS :
Temperature: - 30.0+ or -0.10C
Reaction time: - 20 min pH: -4.3-4.5 (0.1 M Acetate buffer) PNPG conc.: -
0.067% approximate 2x10-3 M
Enzyme conc.: 0.2-1 AG/ml
REAGENTS:
1) 0.1 m acetate buffer, pH 4.3
CH3COOH, 100% - 4.05 gm.
CH3COO Na3.H2O -4.44gm.
Deionized water up to 1000 mm
0.1 m borax solution

2) Na2B4O7.H2O - 38.1gm .
Deionized water up to 1000 mm.

3) PNPG substrate 4-nitro phenol-alpha-d-glucopyranoside Sigma, Art No. N

1377-0.1 g 0.1 m acetate buffer, pH 4.3 up to 100 ml (Maximum advisable
storage time: 2 weeks at 440 C)
PRICIPLE:
Amyl glucosidase (glucoamylase or y amylase) reacts with the colorless p-nitro
phenyl-alpha-D-glucopyranosides (PNPG) under the formation of glucose & p-
nitrophenol. In an alkaline liquid the latter will cause the development of
yellow colour which can be measured spectrophotometrically at 400 nm.

UNIT DEFINATION:
One amyl glucosidase unit (AG) is the amount of enzyme which under the
reaction conditions stated will cleave the same amount of PNPG as 1 AG unit
determined according to the Novo Method No. 22/2.

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PROCEDURE:
1) Dilute the sample with 0.1 M acetate buffer, pH 4.3 to contain approximate 0.5
AG/ml.
2) From the AG std solution and from the dilutions mentioned above as well as
from the applied buffer take out and transfer to 16 x 160 mm test tubes 1ml as
sample (s) and 1 ml as blank (B).
3) The samples are placed in a 300C water bath, and when the temperature
equilibrium is restored, add (e.g. at 10 sec. intervals) 2ml PNPG substrate by
means of the automatic syringe and shake vigorously after each addition to
obtain thorough and quick mixing. After 20 min stop the reaction by adding
3ml 0.1M Borax solution by means of the automatic syringe and shake
thoroughly.
This procedure is to follow the same order of succession and observe the same
time intervals as the PNPG addition.

4) Prepare the blanks by adding the reagents in the opposite orders i.e. start with
the 3ml 0.1M Borax solution and then the 2ml PNPG substrate. The addition
can take place at room temperature and independent of time intervals provided
that vigorous shaking is perform after each condition.
5) Measure the sample and blank values spectrophotometrically at 400nm using
water as the reference. These measurements should be carried out as quickly as
possible as PNPG is unstable in an alkaline liquid. The extinctions must be
within the OD-range covered by the std. curve, and the sample and blank
values of pure buffer must not exceed 0.05 and not differed among themselves
by more than 0.01.
SAMPLE: - 1.66×106 AMG/ml
OBSERVATION TABLE:

ml of Vol. of 0.1N Borax

O.D.
Sr. No. enzyme PNPG Acetate buffer Reagent AG/ml
(400 nm)
solution pH (4.3) ml

1 2 2 8 3 0.2 0.15

2 4 2 6 3 0.4 0.30

3 6 2 4 3 0.6 0.45

4 8 2 2 3 0.8 0.67

5 10 2 0 3 1.0 0.75

Sample 2 2 8 3 0.65 0.5

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CALCULATION
Activity (AG/gm) = SF
Where,
S = Reading on the std. curve scale AG/ml
F = The dilution factor of the sample ml/gm.
S = 0.65AG/gm & F = 10000ml/g
Activity (AG/g) = 0.65 x 10AG/g
= 65AG/g
= 6.5 x 104 x AG g

RESULT: Thus, AG - Activity of dextroenzyme by means of the PNPG method was

found to be 6.5 x 104 AG/g

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MICROBIOLOGY

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Experiment No. 1
TITLE: Characterization of Micro-organisms. Title: Basic Laboratory Techniques for
Isolation, Cultivation and Cultural
PURPOSE:
To become familiar with,
1) The type of laboratory equipment and culture media needed to develop and
maintain pure culture.
2) 2. The concept of sterility and the procedure necessary for successful sub
culturing of microorganisms
3) 3. Streak plate and spread plate inoculations for separation of microorganism in
a mixed microbial population for subsequent pure culture isolation
4) Cultural and morphological characteristics of microorganisms grown in pure
culture.
INTRODUCTION:
Microorganisms are ubiquitous. They are found in soil, air, water, food, sewage
and on body surface. In short, every area of our environment is replaced with
them. The microbiologist separates these mixed populations into individual
species for study. A culture containing a single unadulterated species of cells is
called a pure culture. To isolate and study microorganisms in pure culture, the
micro-biologist requires basic laboratory apparatus and the application of
specific techniques.
MEDEIA:
The survival and continued growth of micro-organism depend on an adequate
supply of nutrients and a favorable growth environment. Most microbes must
use soluble low molecular weight substances that are frequently derived from
the enzyme degradation of complex nutrients. A solution containing these
nutrients is a culture medium. Basically, all culture media are liquid, semi-solid
medium. A broth medium supplemented with a solidifying agent called agar
results in a solid or semi-solid medium. Agar is an extract of seaweeds; a
complex carbohydrate compared mainly galactose and is without nutritional
value. Agar serves as an excellent solidifying agent because it liquefies at
100°C and solidifies at 40°C. Because of these properties, organisms especially
pathogens, can be cultivated at temp. Of 37.5°C or slightly higher without fear
of the medium liquefying. A completely solid medium requires an agar
concentration of about 1.5% to 1.87%. A concentration of less than 1% agar
results in a semi-solid medium.

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Fig. 1.1 Laboratory Apparatus and Culture Technique

A solid medium has the advantage that it presents a hardened sure on which
microorganism can be grown using specialized technique for the isolation of
discrete colonics. Each colony is a cluster of cells that originates from the
multiplication of the single cell and represents the growth of single species of
microorganism. Such a defined and well isolated colony is pure culture. Also,
while in the liquefied state, solid media can be placed in test tubes, which are
then allowed to cool and hardened in a slanted position, producing agar glands.
These are useful for maintaining pure cultures. Similar tubes that following
preparation, are allow to harden in the upright position are designated as agar
deep tubes.

Fig. 1.2 Forms of solid (agar) media

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Agar deep tubes are used primarily for the study of the gaseous retirement of
microorganisms. However, they may be liquefied in a boiling water bath and
poured to Petri dishes. Producing agar plates, which provide large surface area
illustrated in fig 1.2. In addition to nutritional needs, the environmental factor
for isolation and study of microorganisms. The various forms of solid media
are must also be regulated, including proper pH, temperature, gaseous
requirements. and osmotic pressure. A more detailed explanation is presented in
part 4, the section dealing with cultivation of microorganism. For now, you
should simply bear in mind that numerous type of media are available.

STERLIZATION:
Sterility is the hallmark of successful work in the microbiology laboratory. To
achieve sterility, it is mandatory that you use sterile equipment and sterile
techniques. Sterilization is the process of rendering a medium or material free
of all forms of life. Although a more detailed discussion is presented in part 9,
the section dealing with control of microorganism fig 1.3 is brief outline of
routine techniques used in microbiology laboratory.
CULTURE TUBES and PETRI DISHES:
Glass test tubes and glass or plastic Petri dishes are used to cultivate
microorganism. A suitable nutrient medium in the form of broth or agar may be
added to the tubes, while only a solid medium is used in Petri dishes. A sterile
environment is maintained in culture tubes by various types of closures.
Historically, the first type of cotton plug was developed by sehroeder and von
dusch in the nineteenth century. Today most laboratories use sleeve like caps
made up of metal, such as stainless steel, heat resistant plastics. The advantages
of the seclosures over the cotton plug are that they are labour-saving and, most
of all slip on and off the test tubes easily. Petri dishes provide a larger surface
area for growth and cultivation. They consist of a bottom dish portion that
contains the medium and a larger top portion that serves as a loose cover. Petri
dishes are manufactured in various sizes to meet different experimental
requirements. For routine purposes, dishes approximately 15 cm diameter is
used. The sterile agar medium is dispensed to previously sterilized dishes from
molten agar deep tubes containing 15 to 20 ml of medium, or from a molten
sterile medium prepared in bulk and contained in 250 ml to 500 ml flasks.
When cooled to 40° C, the medium will solidify.

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Remember that after inoculation, petri dishes are incubated in an inverted position
(top down) to prevent condensation that forms on the cover during solidification from
dropping down onto the surface of the hardened agar. Figure 1.4 illustrates some of
the culture vessels used in the laboratory.
TRANSFER INSTRUMENT:
Microorganisms must be transferred from one vessel to another or from stock cultures
various media for maintenance and study. Such a transfer is called
sub culturing and must be carried out under sterile conditions to prevent possible
contamination. Wire loops and needles are made from inert metal such as nichrome or
platinum and are inserted in to metal shaft that serve as handles. They are extremely
durable instruments and easily sterilized by incineration in the blue (hottest) portion of
the Bunsen burner flame. A pipette is another instrument used for sterile transfer.
Pipettes are similar in function to straws that is, they draw up liquids. They are made
of glass or plastic drawn out to a tip at one end and one with a mouthpiece forming the
other end. They are calibrated to deliver different volumes depending on requirements.
Pipettes may be sterilized in bulk inside canisters or they may be wrapped individually
in brown paper and sterilized in an autoclave or dry heat oven, Fig 1.5 illustrates these
transfer instruments.
Note- pipetting by mouth is not permissible pipetting is to be performed with the aid of mechanical
devices. The proper procedure for the use of pipettes will be demonstrated by your instructor.

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CULTIVATION CHAMBERS: -
The specific temperature requirements for growth are discussed in details in part I
however; a prime requirement for the cultivation of microorganism is that they be
grown at their optimum temperature. An incubator is use to maintain optimum
temperature during the necessary growth period. It resembles an oven and is
thermostatically controlled so that temperature can be varied depending on the
requirements of specific microorganism. Most incubator use dry heat. Moisture is
supplied by placing a beaker of water in incubator during the growth period. A moist
environment retards dehydration of the medium and there by avoids spurious
experimental results.
A thermostatically controlled shaking water bath is another piece of apparatus use to
cultivate microorganism. It advantage is that it provides a rapid and uniform transfer
of heat to the culture vessel, and its agitation provides increased aeration, resulting in
acceleration of growth. The single disadvantages of this instrument are that it can be
used only for cultivation of organism in a broth medium.

REFRIGERATER:
A refrigerator is used for a wide variety of purpose such as maintenance and storage of
stock culture between sub culturing period and storage of sterile media to prevent
dehydration. It is also used as repository for thermo labile solutions, antibodies,
serums, and biochemical reagents.

RESULT: Thus, we have prepared media successfully.

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Experiment No. 2
TITLE: Simple Staining.
OBJECTIVES OF SIMPLE STAINING:
1) To perform a simple staining procedure.
2) To compare the morphological shapes and arrangements of bacterial cells.
PRINCIPLE:
In simple staining, the bacterial smear is stained with a single reagent, which
produces a distinctive contrast between the organism and its background. Basic
stains with a positively charged chromogen are preferred because bacterial
nucleic acids and certain cell wall components carry a negative charge that
strongly attracts and binds to the cationic chromogen. The purpose of simple
staining is to elucidate the morphology and arrangement of bacterial cells. The
most commonly used basic stains are methylene blue, crystal violet, and carbol
fuchsin.
MATERIALS:
24-hour nutrient agar slant cultures of Escherichia coli and Bacillus cereus, and
a 24-hour nutrient broth culture of Staphylococcus aureus or Curd.
REAGENTS:
Methylene blue, crystal violet, and carbol fuchsin.
EQUIPMENTS:
Bunsen burner, inoculating loop, staining tray, microscope, lens paper, bibulous
paper, and glass slides.
PROCEDURE
1) Prepare separate bacterial smears of the organism following the procedure
described.
Note: All smears must be heat-fixed prior to staining.
2) Place a slide on the staining tray and flood the smear with one of the indicated
stains, using the approximate exposure time for each carbol fuchsin (15 to 30
seconds); crystal violet (20 to 60 seconds) and methylene blue (1 to 2 minutes).
3) Wash the smear with tap water to remove excess stains. During this step, hold
the slide parallel to the stream of water, in, this way, you can reduce the loss of
organisms from the preparation.
4) Using bibulous paper, blot dry but do not wipe the slide.
5) Repeat this procedure with the remaining two organisms, using a different stain
for each.
6) Examine all stained slides under oil immersion.

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OBSERVATION & RESULT:

1) Draw a representative field for each organism.
2) Describe the morphology of the organism concerning its shapes (bacilli,
cocci, spirilli) and arrangements (chains, clusters, pairs).

Reagent Methylene Blue

Drawing of a
representative field

Lactobacillus

Bulgaricus,
Organism
Steptococcus
lactis

Cell morphology
Bacilli, cocci
Shape

Arrangement Chains and clusters

Cell colour
Blue

RESULT:
Lactobacillus bulgaricus and Steptococcus lactis are observed under the
microscope in curd sample.

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