Department of Microbiology

Faculty name: Mr. Rajeev R. Potadar

SEMESTER – VI
PRACTICAL - PAPER – II: -- FOOD AND INDUSTRIAL MICROBIOLOGY

Contents
Expt. Title Page No.
No.
1. Bacterial examination of milk by SPC and DMC methods.
1.a Direct Microscopic examination of milk
1.b Standard Plate Count
2. Methylene Blue Reduction test (MBRT) for quality assessment of milk
3. Estimation of Lactic acid from curd samples
4. Isolation of microorganisms from spoiled fruits, vegetables, canned foods
5. Preparation of Wine
6. Estimation of specific gravity and percentage of alcohol
7. Production and estimation of citric acid
8. Production and estimation of enzyme protease
9. Immobilization of whole cells. Immobilization of yeast cell by using
sodium alginate
 SEMESTER – VI
PRACTICAL - PAPER – II: -- FOOD AND INDUSTRIAL MICROBIOLOGY
Experiment No.1. Bacterial examination of milk by SPC and DMC methods.
Qualitative analysis of milk
Aim
To determine the number of bacteria present in the given, milk sample by using direct
microscopic method and standard plate count method.
Background Information
Milk because of its high nutritive value is one of the most important food products. Not only
milk gives nutrient to human beings but also forms an excellent medium for microbes. Unless
the animal is diseased the udder milk is virtually sterile. Hence the contamination is usually
exogenous. Milk from animals with mastitis characteristically has large number of WBC and
bacteria
Possible Pathogens from Milk
Alkaligens viscolyticus, Mycobacterium tuberculosis, Mycobacterium bovis, Brucella sp.,
Toxoplasma sp., Rickettsia., Leptospira., Staphylococcus aureus.
Normal Value of Milk bacteria
Aseptically drawn milk - 500-1000 CFU/ mL
Bulk milk tank - 5000-20000CFU/ mL
Milk pail or milking machine - 1000-10000CFU/mL
Experiment No.1.a Direct Microscopic examination of milk
Aim
To count microbial population of milk.
Principle
In this experiment a measured volume of milk is spreaded over an area of a slide, fixed, stained
and examined microscopically. Since the area of a single microscopic field and volume of milk
examined are known, the number of microscopic field can be determined and total number of
bacteria / mL is computed.
Materials Required
Milk sample, microscope, methylene blue stain, clean microscopic slide.
Procedure
1. Mark 1 cm2 area on a clean glass slide. Spread 0.01mL of milk uniformly over this area.
Air dry the smear.
2. Immerse smear in xylene solution to remove fat materials for 1 minute. Wash with ethyl
alcohol to remove xylene.
3. Stain with methylene blue for 1-2 minutes. Examine the slide under oil immersion
microscope.
4. Count the number of microorganisms / field.
Calculations
Diameter of one oil immersion field = 0.6mm
Area of microscopic field = 7π r2
π = 3.14
r2 = 0.3mm2
= 3.14 x 0.09
= 2.83
Note: 1 cm2 =10mm2
That is, 100/2.83 = 35 fields / 1 cm2 =35 fields/cm2 l/100mL was spreaded over lcm2
Each microscopic field has l/100 x area 1 = 1/3,500 Total number of cells in one mL of
milk
= Number of cells in one field x 3500
= Answer
Result
Methylene blue stained bacterial cells are counted and enumerated. Compare the cells present
in one mL of milk with normal microbial flora of milk and interpretated as good quality / poor
quality.
Experiment No.1.b Standard Plate Count
Aim: To enumerate microbialpopulation of milk.
Materials Required
Milk sample, plate count agar, petriplates, pipettes etc.
Procedure:
1. Dilute one mL of milk sample up to 1:100000 using sterile saline under aseptic condition
2. Plate one mL diluted sample from 1:100, 1:1000 and 1:10000 on plate count agar by using
pour plate method.
3. Duplicate should be maintained during the performance of this technique.
4. Incubate all plates at specific environment for 24 hours
5. Observe the plates and interpretate the results.
Observation and Result
Table 1.1
Sl.No. Technique & medium Dilution No of colonies
Original Duplicate Average
1 Plate count agar
Pour plate 1:100
Spread plate 1 :1000
1 :10000
Result
Result of milk quality is interpretated after comparing normal value. Result will be given as
good or poor quality.
 Experiment No.2.Methylene Blue Reduction test (MBRT) for quality assessment of milk
Methylene Blue Reductase Test (MBRT)
Aim
To determine the quality of the given milk sample by Methylene blue reductase test.
Principle
Large number of bacteria present in milk indicates poor methods of production or handling.
From the time the milk leaves the udder, until it is dispensed into containers, everything which
comes in contact with potential source of contamination. The bacterial content can be reduced
by strict sanitary practice. Bacteria present in the milk utilize the oxygen present in the sample
thus lowering oxidation-reduction potential due to the exhaustion of dissolved oxygen when
methylene blue is mixed with contaminated milk; it loses its colour. The colour disappearance
of methylene blue is directly proportional to the number of bacteria present in the milk sample.
Materials Required
Procedure:
1. Collect raw milk sample in a sterile conical flask.
2. Transfer 10mL milk sample to sterile screw cap tube.
3. Add 1mL of methylene blue solution to all screw capped tubes.
4. Close the tubes with rubber stoppers and gently inverted thrice to mix all contents.
5. Control tubes with and without dye also maintained.
6. Incubate all tubes in water bath at 35 ° C for up to eight hours.
7. Observe colour change every half an hour.
8. Quality of milk sample is assessed on the basis of standard given in

Fig. 2.1 Test tubes containing test sample

inoculated with dye and control.
 Table 2.1: Quality standard of milk by methylene blue reductase test
Class Interpretation
I Excellent - not discolourised upto 8hours.
II Good - discolourise after 6 hours but within 8 hours.
III Fair - discolourise after 2 hours but within 6 hours.
IV Poor - discolourise within 2 hours.

Table 2.2: Observable record

Sl. Sample Time of discolouration in minutes Milk

No. 3 0 6 0 9 0 120 150 180 210 240 270 300 330 360 390 420 450 480 quality
1 A
2 B
3 C
4 D
5 Control-1
6 Control -2

Result
The given milk is found to be excellent/ good / fair/ poor
 Experiment No. 3.Estimation of Lactic acid from curd samples.
Introduction:
Fermentation is initiated by Leuconostoc mesenteroides. It produces lactic and acetic acids
which rapidly lower the pH thereby inhibiting the growth of spoilage microorganisms. It also
produces C02 which helps to generate anaerobic conditions within the product and prevent the
oxidation of vitamin C and loss of colour. Fermentation is then continued by Lactobacillus
brevis and L. plantarum, the latter being mainly responsible for the final high levels of acidity
The finished product contains a total acidity of 1.6 to 1.8% of which lactic acid represents 1.0
to 1.3%.
Procedure:
1. Total acidity: expressed as % lactic acid by performing a titration.
2. Pour 10ml of the juice/curds and 10ml distilled water into an Erlenmeyer 100ml flask.
3. Boil to remove the C02.
4. Allow to cool the contents.
5. Add 5 drops of 1% phenolphthalein into the flask.
6. Titrate with 0.1 N NaOH for the appearance of pink colour.
7. Calculate the % lactic acid by applying the formula.
% Lactic acid = Vo1.of alkali used x normality of alkali x 9
Vol. of sample taken (i.e. 10 ml)
Tabulations:

Result: % lactic acid content in the given sample is ______

 Experiment No. 4.Isolation of microorganisms from spoiled fruits, vegetables, canned
foods
Aim
To check the quality of fruits and vegetables.
To identify the isolated microbes.
To know kinds of microorganisms present in fruits and vegetables.
Background Information
Microorganisms present on the surface of freshly harvested fruits and vegetables include not
only those of the normal surface flora but also those from soil, water and perhaps plant
pathogens. Vegetables and fruits may be dried, frozen, fermented, pasteurized and canned.
Generally fruits and vegetables have low number of microbes. During transportation and
processing mechanical damage may increase contamination. Precooling of the product and
refrigeration during transport may reduce the growth of contaminated microorganisms.
Possible sources of contamination are trays, tanks, tables, aprons and filters.
Possible Isolates
Vegetables - Bacteria: Pseudomonas sp., Alkaligens sp., Erwinia sp., Xanthomonas Sp.,
Micrococci, Coryneforms., Bacillus sp., and Other enterobacteriaceae members.
Fungi: Fusarium; Altemaria; Aureobasidium; Penicilliutn; Rhizopus
Fruits- Bacteria : Pseudonwnas sp., Alkaligens sp., Erwinia sp., Xanthomonas sp.,
Micrococci sp., Bacillus sp.,
Fungi: Fusarium; Altemaria; Aureobacidium; Penicillium., Rhizobus.,
Cladosporium; Trichoderma; etc.,
Normal range of mould in fruits and vegetables 1000-10000 cells /g
Table – 4.1 Range of bacteria in fruits and vegetables

Materials Required:
Sample : Vegetable, fruits or its by products.
Media: Nutrient agar, Gram Negative Broth, SS agar, Mannitol Salt agar, EMB agar,
EC broth, Azide dextrose broth, Violet Red Bile Agar, KF streptococcus agar, TCBS
 agar, Alkaline peptone water.
Others: Water / Saline blank, Test tubes, Petriplates, Conical flasks, etc.,
Procedure:
1. Preparation of sample
• Weigh 10g of sample and blend with 90mL of sterile saline in a sterilized jar
2. Total viable count of bacteria
• Dilute the sample up to 1:100000 (l05)
• Add 1mL of diluents from 1:1000,1:10000 and 1:100000 dilutions to sterile,
petriplates
• Pour 20mL of sterile nutrient agar or plate count agar
• Gently rotate the plates in clockwise and anti clockwise direction and allow solidify
• Incubate all plates at 35 ° C for 18 -24 hours
• Examine the plates for its number and nature of colonies
3.Coliform isolation
• Add lmL of sample from 1:100 and 1:1000 dilutions to Violet Red Bile Agar plates &
perform pour plate method
• Observe the plates after 24 hours of incubation at 37° C for its red coloured colonies
4. Faecal coliform isolation
• Inoculate 1mL of sample from 1:10 dilution to EC and Azide Dextrose Broth tubes
and incubates at 44° C for 24 hours
• Inoculate a loop full of culture from EC broth and Azide Dextrose Broth into EMB
and KF streptococcus agar respectively and incubated at 37° C for 24 hours
• Observe plates for its specific colony morphology
5. Isolation of Staphylococcus aureus
• Spread 0.1 mL of sample on the surface of Baired Parker agar and incubate at 37 °C
for 24 hours. Observe plates for black coloured colonies
6. Isolation of Salmonella
• Transfer 1 mL of blended sample to lOmL of gram negative broth and incubate at 37 °
C for 4-6 hours
• Streak a loop full of culture from gram negative broth on SS agar and incubated at
37°C for 24 hours
7. Isolation of Vibrio
• Inoculate 1 mL of blended sample in to 10mL of alkaline peptone water and incubate
at 37° C for 18 hours
• Streak a loop full of culture from Alkaline peptone water on TCBS agar and incubate
at 37° C for 24 hours
8. Isolation of yeast and moulds
 • Inoculate 1mL of sample from 1:100 dilution into the Rose Bengal chloramphenicol
agar as pour plate method and incubated at 28-30 ° C for 48 hours. Examine plates for
the growth of eucaryotic microorganisms.*
Table 4.2 - Observable Results
Sl.No. Sample Test Result Interpretation
1 Fruits Total viable count
2 Coli form count
3 E. coli
4 Staphylococcus ,
5 Salmonella
6 Yeast
7 Mould
8 Vibrio
Sl. Sample Test Result Interpretation
1 Vegetables Total viable count
2 Coli form count
3 E.coli
4 Staphylococcus
5 Salmonella
6 Yeast
7 Mould
8 Vibrio

Results
1. Given vegetable is found to be poor/ good
2. Given fruit is found to be poor/ good
 Experiment No. 5. Preparation of Wine
Introduction:
Wine is traditionally any alcoholic beverage arising from the fermentation of grape juice.
Industrially, wine is the fermented product of the grape juice by the action of the yeast
Saccharomyces cerevisiae var. ellipsoideus (S. ellipsoideus) or with the strains of yeast
naturally occurring on grapes. The former method is common in the USA and the latter in
Europe. Grape juice contains water, carbohydrate (glucose, fructose and pentose etc.),
nitrogenous compounds as proteins and protein split products, acids (tartaric and malic acid),
minerals, pigments and enzymes. It is the concentration of these that determines the
characteristic taste and texture of different wines.
Grape wines are of two types: white and red. White wines are prepared either from white
skinned grapes or from red skinned grapes that have had the skin removed. Red wine comes
from the red or purple skinned varieties and the crushed grapes are fermented with their skin
to allow the extraction of their colour into the juice. Juice containing 20-30% sugar will yield
wines with an alcohol content of 10-15%.
Principle:
During wine production, the yeast convert glucose and fructose present in the juice
enzymatically first to acetaldehyde and then to ethyl alcohol. Wine production, or the science
of enology (Gr. oinos = wine + ology = the science of), starts with the collection of grapes.
Protocol for wine making includes: preparation of must, fermentation, storage and aging.
Production of fermentable substrate called must is done by crushing the grapes and the
separation of the liquid. The must is sterilized by pasteurization by the addition of Potassium
metabisulphite or S02 fumigant to retard the growth of acetic acid bacteria, molds and wild
yeasts that act as/spoilage agents. Saccharomyces ellvpsoideus (S. cerevisiae var. ellipsoideus)
is added to the pasteurized must and incubated for 3 to 5 days in red wines and for 7 to 14 days
in white wines, under aerobic conditions, at temperature varying between 20°C and 28°C. This
is followed by anaerobic incubation period for anaerobic alcoholic fermentation.
The fermented juice (raw wine) is decanted and transferred to large beds to settle and clarify.
The wine is then aged for a period of one to five years in wooden casks or aging tanks. During
aging, non – microbial changes produce aromas and flavours due to the production of volatile
esters. The clarified product is filtered, pasteurized at 60°C for 30 minutes and finally bottled.
The final product i.e. the wine may contain 10-17.5 % alcohol because concentration of alcohol
above 17% inhibit the metabolism of the yeast.
Red wine becomes red due the extraction of the grape skin colour. At one week intervals, the
wine produced in the laboratory will be examined for taste, odour, clarity, total acidity, volatile
acidity and percent alcohol.
 Fig. 5.1 Pathway for the production of alcohol from glucose by the yeasts.
Requirements:
• 48-hour white grape juice broth culture of Saccharomyces ellipsoideus incubated at 25°C
- 50 ml.
• Pasteurized white grape juice -500 ml
• Sucrose (60 gm)
• 1% Phenolphthalein solution
• 0.1N Sodium hydroxide
• 1-liter Erlenmeyer flask
• Rubber stopper fitted with a 2-inch glass tube plugged with cotton
• 10ml graduated cylinder
• Ebulliometer (or alcoholometer)
• Burette or pipette for titration
• Spatula
Procedure:
1. Pour 500ml of the pasteurized white grape juice (must) into a 1-liter
sterilized Erlenmeyer flask.
2. Add 20g of sucrose into the must.
3. Now add 50ml of the S. ellipsoideus grape juice broth culture (i.e., 10 %
starter culture) into the flask.
4. Close the flask with sterilized stopper containing cotton plugged air vent for producing aerobic
conditions to the yeast.
5. Incubate the inoculated fermenting wine flask at 25°C for 21 days.
6. Add 20g of sucrose to the fermenting wine after two days and then after four days of
inoculation.
Total acidity
(i) Take 10 ml of the grape juice (must) in a 100 ml conical flask.
(ii) Add 10 ml of distilled water and 5 drops of phenolphthalein solution (1%).
(iii) Mix the contents well.
(iv) Titrate to the first persistent pink colour with 0.1N NaOH.
 Formula:

Observations and results:

(i) Perform the following tests on the uninoculated white grape juice and samples taken from
the fermenting wine at seven-day intervals for the taste, aroma and clarity.
(ii) Determine total acidity Expressed as % tartaric acid and volatile acidity expressed as
acetic acid by the titration test for both the un-inoculated juice and fermenting
wine as described.
 Experiment No.6. Estimation of specific gravity and percentage of alcohol.
Principle
The density ρ (g/mL or g/cm3) means the mass per unit volume,
and the relative density means the ratio of the mass of a sample
specimen to that of an equal volume of a standard substance.
The relative density is also called the specific gravity.
The specific gravity, at , means the ratio of the mass of the
sample specimen at t′ °C to that of an equal volume of water
(H2O) at t °C.
Procedure
1. A pycnometer is a glass vessel with a capacity of usually 10mL to 100mL, having a
ground-glass stopper with a capillary for liquid to overflow.
2. Weigh a pycnometer, previously cleaned and dried, to determine its mass, W1.
3. Remove the stopper and fill the pycnometer with the test solution and slowly place the
stopper so as to overflow excess solution through capillary.
4. Keeping them at a slightly lower temperature by 1°C to 4°C than the specified
temperature t′°C, and stopper them, taking care not to leave bubbles.
5. Raise the temperature gradually, and when the thermometer shows the specified
temperature, wipe the overflow solution with tissue paper, cap the side tube, and wipe the
outside surface thoroughly.
6. Measure the mass W2 of the pycnometer filled with the test solution (Wine).
7. Perform the same procedure, using the same pycnometer containing water, and note
the mass W3 at the specified temperature t °C.
8. The specific gravity can be calculated by use of following equation.
Observations:
Weight of Empty specific gravity bottle + stopper = W1 gm

Weight of specific gravity bottle filled with test solution + stopper = W2 gm

Weight of specific gravity bottle filled with distilled water + = W3 gm

stopper
 Table 6.1 Estimation of Percentage of alcohol sample using American Official
Analytical Chemist (AOAC) Chart
Figures given as percent by volume @ 60°Farenheit, As per United States Bureau of
Standards.
Alcohol Specific Alcohol Specific Alcohol Specific Alcohol Specific
Percent gravity Percent gravity Percent gravity Percent gravity
1 1.0000 26 0.9698 51 0.9323 76 0.8746
2 0.9985 27 0.9687 52 0.9303 77 0.8720
3 0.9970 28 0.9676 53 0.9283 78 0.8693
4 0.9956 29 0.9665 54 0.9263 79 0.8666
5 0.9928 30 0.9653 55 0.9242 80 0.8638
6 0.9915 31 0.9642 56 0.9221 81 0.8610
7 0.9902 32 0.9630 57 0.9200 82 0.8553
8 0.9890 33 0.9617 58 0.9178 83 0.8528
9 0.9878 34 0.9604 59 0.9156 84 0.8524
10 0.9866 35 0.9591 60 0.9134 85 0.8494
11 0.9854 36 0.9577 61 0.9112 86 0.8464
12 0.9843 37 0.9563 62 0.9089 87 0.8434
13 0.9832 38 0.9548 63 0.9066 88 0.8402
14 0.9821 39 0.9533 64 0.9043 89 0.8371
15 0.9810 40 0.9518 65 0.9020 90 0.8338
16 0.9800 41 0.9502 66 0.8997 91 0.8305
17 0.9789 42 0.9486 67 0.8973 92 0.8270
18 0.9779 43 0.9469 68 0.8949 93 0.8235
19 0.9769 44 0.9452 69 0.8924 94 0.8198
20 0.9760 45 0.9435 70 0.8900 95 0.8160
21 0.9750 46 0.9417 71 0.8875 96 0.8121
22 0.9740 47 0.9399 72 0.8850 97 0.8079
23 0.9729 48 0.9381 73 0.8824 98 0.8036
24 0.9719 49 0.9362 74 0.8799 99 0.7989
25 0.9708 50 0.9343 75 0.8773 100 0.79389

Note:
1. Figures are accurate only for readings taken at 60°Farenheit.
2. For readings at other temperatures, use following correction table.

Step 1:
Estimate the approximate percentage of water in the sample to determine correction factor listed
below.
 Table 6.2 Correction table
Percentage Correction Percentage Correction Percentage Correction
factor factor factor
0 – 5% =0.00049 68% =0.00035 82% =0.00019
6 – 16% =0.00048 69% =0.00034 83% =0.00018
17 – 29% =0.00047 70% =0.00033 84% =0.00017
30 – 37% =0.00046 71% =0.00032 85% =0.00016
38 – 43% =0.00045 72% =0.00031 86% =0.00015
44 – 49% =0.00044 73% =0.00030 87% =0.00014
50 – 53% =0.00043 74% =0.00029 88% =0.00013
54 – 56% =0.00042 75% =0.00028 89% =0.00012
57 – 58% =0.00041 76% =0.00027 90 – 91% =0.00011
59 – 61% =0.00040 77% =0.00026 92 – 93% =0.00010
62% =0.00039 78% =0.00025 94 – 95% =0.00009
63 – 64% =0.00038 79% =0.00023 95 – 100 % =0.00008
65% =0.00037 80% =0.00022
66 – 67% =0.00036 81% =0.00020

Step 2:
Multiply the correction factor by the number of degrees greater or lesser than 60°Farenheit.

Step 3:
If temperature is greater than standard, add the correction to the specific gravity reading to
obtain the temperature corrected reading. If the temperature is less than standard, the
correction should be subtracted from the reading.

Results:
1. The specific gravity of the given solution is ______
2. The percentage of alcohol content in given sample as per AOAC chart is ___ %
7. Production and estimation of citric acid.
a) Production of Citric acid
Introduction:
Citric acid is an important organic acid and it was initially being extracted from citrus fruits.
Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used
in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is
the most commonly used species for the production of citric acid. Most strains of Aspergillus
niger which are mutants cannot oxidize citric acid and hence accumulate in culture medium.
The composition of the culture medium is critical for obtaining high yield of citric acid. It is
essential to limit the growth of the fungus, so that high yield of citric acid accumulates in the
medium. This can be accomplished by keeping trace metal deficiency in the medium. Acid is
added to achieve low pH of 3.5. Sucrose serves as a carbon source for the production of citric
acid. Ammonium nitrate is used to prevent the fermentation of oxalic acid glutamic acid.
Fermentation is aerobic and can be carried out by submerged culture method.
Citric acid is an important organic acid and it was initially being extracted from citrus fruits.
Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used
in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is
the most commonly used species for the production of citric acid.
Citric Acid Production Principle:
Most strains of Aspergillus niger which are mutants cannot oxidize citric acid and hence
accumulate in culture medium. The composition of the culture medium is critical for obtaining
high yield of citric acid. It is essential to limit the growth of the fungus, so that high yield of
citric acid accumulates in the medium. This can be accomplished by keeping trace metal
deficiency in the medium. Acid is added to achieve low pH of 3.5. Sucrose serves as a carbon
source for the production of citric acid. Ammonium nitrate is used to prevent the fermentation
of oxalic acid & glutamic acid. Fermentation is aerobic and can be carried out by submerged
culture method.
Table 7.1Citric Acid Production Medium
Component Quantity (g/ L)
Sucrose - 150 gm
Ammonium nitrate - 2.5 gm
Potassium Dihydrogen Orthophosphate - 1.0 gm
Magnesium sulphate heptahydrate - 0.25 gm
Distilled water - 1L
pH - 3.5
Procedure
Culturing Aspergilus niger
1. Prepare the citric acid medium & dispense about 50ml in 250ml conical flask.
2. Autoclave and allow it cool.
3. Inoculate the medium with spores of Aspergillus niger & incubate it on a shaker water
bath at 25oC with gentle shaking for 3-5 days.
4. After Incubation, filter the mycelium using double layered muslin cloth & measure the
amount of citric acid in the filtrate by colorimetric and titrimetric methods.
b) Quantitative estimation of Citric acid.
Estimation Citric Acid by Titrimetric Method
The filtrate obtained is titrated against an alkali of known strength using phenolphthalein as
indicator. The end point is the formation of pale pink colour. The volume of alkali used for
neutralization is used to find the normality and the percentage of acid in the sample.
Materials required: -
• A laboratory burette of 25 or 50ml capacity or an automatic burette is used. A 10ml
pipette, beaker (250ml), a filter (muslin cloth or fine filter) and an extractor or
homogeniser.
• A bottle of distilled water.
• Sodium Hydroxide (NaOH): The Standard Laboratory solution of 0.1M which is used in
the actual titration is considered to be dilute, and can readily be purchased in this form.
• Phenolphthalein: This is a 1% w/v solution of phenolphthalein in 95% v/v ethanol which
is flammable and toxic if ingested. This is only required for the method using a coloured
indicator. -
• Indicator stripes To check the exact point of neutrality an indicator stripe should be used.
Not necessary if pH Meter is used.
Procedure:
1. Add 3 drops of phenolphthalein to the juice/ culture filtrate solution in each beaker from a
dropping pipette which is specifically kept for that purpose.
2. Ensure the tap on the burette is shut and using a funnel pour the 0.1M solution of NaOH
into the burette until it reaches the zero mark. Do not spill the solution onto the skin.
3. Slowly titrate the NaOH into the juice/ culture filtrate solution (with a 25ml burette or an
automatic burette).
4. While titrating care must be taken to continually swirl the solution in the beaker to keep it
thoroughly mixed. This is essential, particularly when the solution nears neutrality.
5. The phenolphthalein indicator changes very rapidly from colourless to pink. It is important
therefore that towards the end of the titration the NaOH is added a drop at a time.
6. Using phenolphthalein as an indicator, the point of neutrality is reached when the indicator
 changes from colourless to pink. The indicator colour must remain stable (persisting for 30
seconds) and be light pink when viewed over a white background. However, the shade can
vary depending on the concentration of citric acid in juice/ culture filtrate being tested.
7. Read off the amount of the amount of NaOH used (titre) on the burette and record this figure.
- Re-fill the burette for each subsequent test. -
8. Clean the equipment thoroughly and rinse with distilled water.
Calculation of the Acid Content (Percentage or g/L)
Some products may contain more than one type of acid; it is the primary acid that is tested. A
list of these acids and multiplication factors are found below.
Factor for:
- Citric acid : 0.0064 (Source - Citrus fruit)
- Malic acid : 0.0067 (Source - Apples)
- Tartaric acid : 0.0075 (Source - Grapes)
Note: Using citric acid as an example, 1ml 0.1M NaOH is equivalent to 0.0064g citric
acid.

Results expressed as percentage acid:

% of Citric acid = Titre x acid factor x 100
10 (ml juice/ culture filtrate)
This formula can be simplified to: Percentage Citric Acid = Titre x 0.064

Results expressed as percentage acid:

% of Citric acid = Titre x acid factor x 100
10 (ml juice/ culture filtrate)
This formula can be simplified to: Percentage Citric Acid = Titre x 0.064
Experiment No. 8.Production of enzyme protease.
Aim: To Produce Protease from microorganisms
Background Information
Proteases and metalloprotease constitute one of the most important groups of industrial
enzymes, accounting for about 60% of the total enzyme market. Among the various proteases,
bacterial proteases are the most significant, compared with animal and fungal proteases and
among bacteria, Bacillus sp. are specific producers of extra-cellular proteases. These enzymes
have wide industrial application, including pharmaceutical industry, leather industry, and
manufacture of protein hydrolisates, food industry and waste processing industry.
Thermostable proteases are advantageous in some applications because higher processing
temperatures can be employed, resulting in faster reaction rates, increase in the solubility of
nongaseous reactants and products, and reduced incidence of microbial contamination by
mesophilic organisms. Proteases secreted from thermophilic bacteria are thus of particular
interest and have become increasingly useful in a range of commercial applications.
Materials required
Autoclave, Microwave oven, Nutrient Agar, Casein, Gelatin, Balance, Shaker,
Spectrophotometer, Water bath, disposable spoons, micro pipettes, sterile water, Petri dishes,
Inoculation loop, Dissecting needle, Bunsen burner matches, Glass spreader, 95% ethanol.
Protease production Media (g/L of distilled water):
MgSO4-0.5, K2HPO4-2.0, KCl-0.3, NH4NO3-10.0, Peptone-1.0. Trisodium citrate-10.0.
pH is adjusted to 6.9-7.0 with 1.0 M NaOH and this basal medium is sterilized by autoclaving
at 121°C for 15 min. Peptone is sterilized separately and aseptically added to the flasks
containing the liquid medium, after cooling.
Procedure:
Isolation of Protease producers:
1. Suspend about 10 grams of soil in 100mL sterile distilled water, mix properly (10-1)
2. Pipette 10mL of the above and transfer to another 90mL of water (10-2)
3. Dilute further in two more 90mL sterile water blanks (10-3 and 10-4)
4. Spread 0.1mL of the diluted samples (10-3 and 10-4) on Gelatin hydrolysis medium
(4plates) and incubate at 30°C for 24 hours
5. Gelatin utilizing colonies will have an area of clearing around them.
6. It is confirmed by flooding plates with Mercuric chloride solution.
7. Transfer distinguishable, protease-producing bacteria, streak on a fresh
plate of Nutrient Agar containing 1 % gelatin.
Protease production Seed culture:
1. Prepare protease production medium, sterilize properly.
2. Inoculate loopful of protease producing Bacillus sp.,
 3. Incubate for 24 hours at 37°C
Fermentation
1. Prepare 250mL of protease production medium in 500mL conical flask.
2. Sterilize at 121°C for 15 minutes.
3. Inoculate the above medium with 1ml of an overnight culture and incubated 37°C for
24 hours in a rotary shaker operated at 150 rpm. At time intervals, the turbidity of the
cultures is determined by measuring the increase in optical density at 470 nm with a
spectrophotometer
4. Withdrew culture and subject to extraction of enzyme
Extraction of Enzyme from bacteria
1. Pour the bacterial culture into centrifuge tubes, and spin for 20 minutes at 5000rpm.
2. Decant the supernatant in to a sterile beaker, which is the crude enzyme extract.
Enzyme Activity:
i. Pipette 1 mL of culture extract "enzyme" into a test tube
ii. Add 1 mL of 1 % soluble casein in citrate-phosphate buffer (pH6.5)
iii. Incubate in a water bath at 40°C for 30minutes
iv. Set up a blank consisting of 2mL of the enzyme extract that has been boiled for 20 minutes
(boiling inactivates the enzyme), added to the casein solution and treated with the same
reagent as the experimental tubes.
v. Estimate protein content by using FCR method.
Enzyme activity may be defined as the amount of casein produced per mL in the reaction
mixture per unit time.
Result:
Isolated soil bacteria produced protease enzyme by utilizing protein and quantity of protease
estimated, compared with standard graph and interpretated as unit of enzyme at the particular
time.
 Experiment No.9. Immobilization of whole cells. Immobilization of yeast cell by using
sodium alginate
Aim
To entrap the live cells in a solid matrix and analyze its activity.
Background information
Whole cell immobilization has been defined as "the physical confinement or localization of
intact cells to a certain defined region of space with preservation of some desired catalytic
activity" (Karel et al., 1985). Many microorganisms own the capability to adhere to different
kinds of surfaces in nature on which way are in close proximity to nutrients and easy realize a
food supply. Therefore, we can say that these biological systems
in their natural state are immobilized. However, many biotechnological process needs to be
carried out using immobilization of biocatalysts. Thereby several different techniques and
support materials have been proposed for the cell immobilization in vitro.
These techniques can be divided into four major groups based on the physical mechanism
causing immobilization: physical entrapment within a porous matrix, attachment or adsorption
to a pre-formed carrier, self aggregation by floculation (natural) or crosslinking agents
(artificially induced) and cell contained behind barrier.
All of these methods have the same purpose: to retain high cell concentrations within "a certain
defined region of space" such as a bioreactor giving increased volumetric productivity of a
system.
During the last thirty years a numerous different kinds of cell-supporting materials for
immobilization have been developed, such as polymeric matrices (alginate, agar, gelatin, k-
karrageenan, chitosan, pectin, polyacrylamide, epoxy resin, silica gel) and porous and non-
porous preformed support materials (wood chips, stainless steel, volcanic rock, cotton cloth,
porous glass particles, DEAE cellulose, porous silica, porous ceramics, diatomaceous earth).
The choice of cell-supporting material for any specific application is related to the following
points: bioreactor configuration, high carrier activity, availability of the carrier in commercial
qualities, low cost of immobilization, immobilization, easy and controllable, ease of operating scale-
up, excellent mechanical strength during long-time operation, physiological and environmental
safety of the materials used and low affinity to contaminations.
Principle
This technique involves a reaction between sodium alginate and calcium chloride forming
calcium alginate. With the optimum concentrations of the reactants, the product particles
coagulates resulting in the formation of beads. This property could be used as a suitable
technique to immobilize microbial cells in the form of beads. Entrapment in insoluble Ca
alginate gel is recognized as a rapid, nontoxic, inexpensive, versalite and the most often used
method for immobilization of cells (more than 80% of cell immobilization processes are still
carried out using alginate). Immobilization offers many potential advantages over free cell
systems, such as Higher cell densities and cell loads, Increased volumetric productivity, Shorter
overall reaction times, Smaller fermenter sizes which may lower capital costs, The reuse of the
same biocatalysts for prolonged period of time due to constant cell regeneration, A continuous
process which may be performed beyond the nominal washout rate, Improved substrate
utilization, Reduced risk for microbial contamination, Process design simplified, Constant
product quality and Improved tolerance or protection of cells from substrate and end-product
inhibition.
Materials required
Sodium alginate- 5.5gm; Cacl2- 4%
Culture: any one type of yeast / Seed culture for fermentation.
Medium: Potato dextrose broth, Growth medium, fermentation medium
Procedure
Preparation of seed culture
1. Prepare Growth medium and sterilize at 110 ° C for 10 minutes
2. Inoculate a loop full of yeast culture from slant or 1 mL of culture from broth.
3. Incubate at 37°C for 18 hours.
Preparation of immobilized cells
1. Prepare sodium alginate slurry by adding 5.5g sodium alginate tol50mL of 0.1 %
sodium chloride solution with continuous stirring.
2. Allow the slurry to stand for 6-8 hours and add 15mL of seed culture.
3. Prepare the beads by controlled drop wise addition of slurry to the 4% calcium
chloride solution.
4. Keep the beads in calcium chloride solution about 30 minutes for gelation.
Activation of immobilized beads
1. Perform activation of immobilized beads with the help of Growth medium.
2. Inoculate required volume of beads into the rich medium and kept it for 30 minutes
3. Note the movements of beads.
Fermentation of alcohol using immobilized beads
1. Add 25 mL of beads to 250 mL of fermentation medium and carryout the fermentation
under static condition at room temperature.
2. Estimate alcohol content through chromic acid method
Observation
Spherical shaped immobilized beads are prepared and activated in Growth medium.
Result
Saccharomyces cerevisiae cells were immobilized and further can used to produce alcohol,
which can estimated through chromic acid method.