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Food Safety Lab Manual
Food Safety Lab Manual
Food Safety Lab Manual
Department of Microbiology
St Aloysius College, Mangalore-575003.
(Autonomous)
Re -accredited by NAAC “A” Grade –CGPA3.62
LABORATORY MANUAL
CERTIFICATE COURSE
IN
LIST OF EXPERIMENTS
Materials Required:
Reagents:
Procedure:
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
3
with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.
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.
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.
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.
Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.
5
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.
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.
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The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of
staining and observing fungi and is simple to prepare. The preparation has three components:
Lactophenol Cotton Blue Solution is a mounting medium and staining agent used in the
preparation of slides for microscopic examination of fungi. Fungal elements are stained
intensely
8
blue.
MATERIALS REQUIRED:
Glass slide
Coverslips
Needle
REAGENTS REQUIRED:
Lactophenol Cotton blue
12. Before the alcohol dries out add one or at most two drops of the stain. A common fault is to
add too much to the preparation. Holding the coverslip between your index finger and
thumb, touch one edge of the drop of stain with the edge of the coverslip.
13. Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is
now ready for examination.
14. Make the initial examination using a low power objective lens. The thinner parts of the
preparation, generally around the edges of the mounted material, will yield the best images.
15. Switch to a higher power 40X objective for more detailed examination of spores and other
structures.
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FUNGI
Learning Objective
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13
Yeasts are non filamentous unicellular fungi. Yeast cultures resemble bacteria when grown on
the surface
of artificial laboratory media; however, they are 5 to 10 times larger than bacteria.
Microscopically, yeast cells may be ellipsoidal, spherical, or in some cases, cylindrical . Unlike
molds, yeast do not have aerial hyphae and supporting sporangia. Yeast reproduce asexually by
budding or by fission. In budding, an outgrowth from the parent cell (a bud) pinches off,
producing a daughter cell . Fission occurs in certain species of yeast, such as those in the genus
Schizosaccharomyces. During fission, the parent
cell elongates, its nucleus divides, and it splits evenly into two daughter cells. Some yeast may
also undergo sexual reproduction when two sexual spores conjugate, giving rise to a zygote, or
diploid cell. The nucleus of this cell divides by meiosis, producing four new haploid nuclei
(sexual spores), called ascospores, contained within a structure called the ascus When the
ascus ruptures, the ascospores
are released and conjugate, starting the cycle again. Yeasts are important for many reasons.
Saccharomyces cerevisiae is referred to as baker’s yeast and is used as the leavening agent in
dough.
Two major strains of yeast, Saccharomyces carlsbergensis and Saccharomyces cerevisiae, are
used for brewing. The wine industry relies on wild yeast (present on the grape) for the
fermentation of grape juice, which is supplemented with Saccharomyces ellipsoideus to begin
the fermentation. Also, the high vitamin content of yeasts makes them particularly valuable as
food supplements. As useful as some yeasts are, there are a few species that can create
problems in the food industry or are harmful to humans. Undesired yeast must be excluded
from the manufacture of fruit juices, such as grape juice or apple cider, to prevent the
fermentation of fruit sugars to alcohol. The contamination of soft cheese by some forms of
yeast will destroy the product.
Materials
Cultures
14
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The survival and growth of microorganisms depend on available and a favourable growth
environment. Culture media are nutrient solutions used in laboratories to grow
microorganisms. For the successful cultivation of a given microorganism, it is necessary to
understand its nutritional requirements and then supply the essential nutrients in the proper
form and proportion in a culture medium. The general composition of a medium is as follows:
15
2. Composition
Chemically-defined (or synthetic) media are composed only of pure chemicals
with defined quantity and quality.
Complex (or non-synthetic) media are composed of complex materials, e.g. yeast
extract, beef extract and peptone (partially digested protein), therefore their
chemical composition is poorly defined. On the other hand, these materials are
rich in nutrients and vitamins.
3. Function
All-purpose media do not contain any special additives and they aim to support
the growth of most bacteria.
Selective media enhance the growth of certain organisms while inhibit others
due to the inclusion of particular substrate(s).
Differential media allow identification of microorganisms usually through their
unique (and visible) physiological reactions. In the detection of common
pathogens, most practical media are both selective and differential.
Enrichment media contain specific growth factors that allow the growth of
metabolically fastidious microorganisms. An enrichment culture is obtained with
selected media and incubation conditions to isolate the microorganisms of
interest.
Object of study:
distilled water
measuring cylinder
17
flask
bacteriological chemicals
laboratory scales
chemical spoons
1N NaOH solution
1N HCl solution
pH indicator paper or pH meter
cotton gloves
dispenser
test tubes
test tube caps
test tube basket
slanting stage
autoclave
incubator
PROCEDURE
1. Measure the components of the medium (e.g. TSA or nutrient, see Appendix) into a flask
containing 9/10 volume of the solvent. Use a clean chemical spoon for every
measurement. Dissolve the solid components and fill with the remaining solvent up to
final volume. If the medium contains heat sensitive components (like sugars), they must
be separately sterilised in solution (e.g. by filter sterilisation), and then mixed with the
already sterilised and cooled agar medium.
2. Close the flask with cotton plug and cover with aluminium foil, put into the autoclave
and start a sterilisation cycle . This cycle could be intermitted when the internal
temperature has reached 121°C, at that temperature every component (e.g. agar-agar)
will be dissolved correctly.
3. Check the pH of the medium with an indicator paper or with a pH meter and adjust to
the proper value with NaOH or HCl solution.
18
4. Pour the 60-70°C medium into the dispenser. Add 5-6 mL medium to each test tube,
close them with caps and place them into a test tube basket.
5. Place the tubes into the autoclave and complete a whole sterilisation cycle for 20 min at
121°C .
6. Put the test tubes onto a slanting stage to let the medium solidify in the test tubes.
7. Label the slants according to the type of the medium and perform a sterility test:
incubate the test tubes at 28°C for 24 hours, and check for sterility.
8. The prepared media can be stored for 1-2 weeks at 12-15°C, or longer in a refrigerator.
(Do not store medium containing agar-agar under 4-5°C as it destroys its structure!)
Object of study:
distilled water
measuring cylinder
flask
bacteriological chemicals
laboratory scales
chemical spoons
1N NaOH solution
1N HCl solution
pH indicator paper or pH meter
cotton gloves
sterile, empty Petri dishes
Bunsen burner
autoclave
incubator
19
Practise:
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The pour plate technique can be used to determine the number of microbes/ml or
microbes/gram in a specimen. It has the advantage of not requiring previously prepared plates,
and is often used to assay bacterial contamination of foodstuffs. Each colony represents a
"colony forming unit" (CFU). For
optimum accuracy of a count, the preferred range for total CFU/plate is between 30 to 300
colonies/plate.
PRINCIPLE
The number of bacteria in a given sample is usually too great to be counted directly. However,
if the sample is serially diluted and then plated out on an agar surface in such a manner that
single isolated bacteria form visible isolated colonies , the number of colonies can be used as a
measure of the number of viable (living) cells in that known dilution.
However, keep in mind that if the organism normally forms multiple cell arrangements, such as
chains, the colony-forming unit may consist of a chain of bacteria rather than a single
bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing
the plate count technique, we generally say we are determining the number of Colony-Forming
Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to
calculate the number of CFUs in the original sample.
Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation,
the number of colonies on a dilution plate showing between 30 and 300 colonies are
determined.
Series of dilution used in SPC.
A plate having 30-300 colonies is chosen because this range is considered statistically
significant. If there are less than 30 colonies on the plate, small errors in dilution technique or
the presence of a few contaminants will have a drastic effect on the final count. Likewise, if
there are more than 300 colonies on the plate, there will be poor isolation and colonies will
have grown together.
Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find
this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of
times the original ml of bacteria was diluted (the dilution factor of the plate counted). For
21
example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150
colonies, then 150 represents 1/1,000,000 the number of CFUs present in the original ml.
Therefore the number of CFUs per ml in the original sample is
found by multiplying 150 x 1,000,000 as shown in the formula below:
Number of CFUs per ml of sample =Number of colonies × Dilution factor of the plate counted
ml of sample plated
In the case of the example above,150 x 1,000,000 = 150,000,000 CFUs per ml.
For a more accurate count it is advisable to plate each dilution in duplicate or triplicate and
then find an average count.
One disadvantage of pour plates is that embedded colonies will be much smaller than those
which happen to be on the surface, and must be carefully enumerated so that none are
overlooked. Also, obligate aerobes may grow poorly if deeply embedded in the agar.
MATERIALS REQUIRED
Cultures: 18-24 hour old nutrient agar slant or nutrient broth cultures of E.coli
Reagents: Sterile Dilution blanks (containing 9ml of 0.9% NaCl2 ), Plate count agar (standard
methods)
Equipment and glassware: Petri dishes, glass or plastic (at least 15 x 90 mm), Pipettes with
pipette aids, Pipette and petri dish containers, Incubator, Colony counter, blender or stomacher
(for food sample), autoclave
PROCEDURE
E. Coli Culture
1. Label the bottom of six petri plates 1-6. Label four tubes of saline 10-2,10-4, 10 -6 and 10-8.
2. Using aseptic technique, the initial dilution is made by transferring 1 mlof E. coli sample to a
9ml sterile saline blank (Figure 5.1). This is a1/100 or 10-2 dilution.
3. The 10-2 blank is then shaken by grasping the tube between the palms of both hands and
rotating quicklyto create a vortex. This serves to distribute the bacteria and break up any
clumps.
4. Immediately after the 10-2 blank has been shaken, uncap it and aseptically transfer 1ml to a
second 9ml saline blank. Since this is a 10-2 dilution, this second blank represents a 10-4
dilution of the original sample.
22
5. Shake the 10-4 blank vigorously and transfer 1ml to the third 9ml blank. This third blank
represents a 10-6 dilution of the original sample. Repeat the process once more to produce a
10-8 dilution.
6. Shake the 10-4 blank again and aseptically transfer 1.0 ml to one petri plate and 0.1 ml to
another petriplate. Do the same for the 10-6 and the 10-8 blanks.
7. Remove one agar pour tube from the 48 to 50oC water bath. Carefully remove the cover
from the 10-4 petri plate and aseptically pour the agar into it. The agar and sample are
immediately mixed gently moving the plate in a figure-eight motion or a circular motion while it
rests on the tabletop. Repeat this process for the remaining five plates.
8. After the pour plates have cooled and the agar has hardened, they are inverted and
incubated at 37oC for 24 hours.
9. At the end of the incubation period, select all of the petri plates containing between 30 and
300 colonies. Plates with more than 300 colonies cannot be counted and are designated too
many to count (TMTC). Plates with fewer than 30 colonies are designated too few to count
(TFTC) . Count the colonies on each plate. A Quebec colony counter should be used.
10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the
number of colonies by the dilution factor multiplied by the amount of specimen added to
liquified agar.
Number of bacteria per ml = Number of colonies dilution × amount plated
11. Record your results.
Food Samples
1. Using separate sterile pipettes, prepare decimal dilutions of 10-2, 10-3, 10-4, and others as
appropriate, of food homogenate. (For food homogenate. Add 450 ml phosphate-buffered
dilution water to blender
jar or stomacher sterile bag containing 50 g analytical food sample and blend for 2 min. This
results in a dilution of 10-1.)
2. Make dilutions of original homogenate promptly, using pipettes that deliver required volume
accurately.
23
3. Prepare all decimal dilutions with 9 ml of sterile diluent plus 1 ml of previous dilution, unless
otherwise specified, by transferring 1 ml of previous dilution to 9 ml of diluent.
4. Pipette 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes.
5. Add 12-15 ml plate count agar (cooled to 45 ± 1°C) to each plate within 15 min of original
dilution.. Pour agar and dilution water control plates for each series of samples.
6. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate
rotation and back-and-forth motion of plates on flat level surface.
7. Let agar solidify. Invert solidified petri dishes, and incubate promptly for 24 ± 2 h at 37°C.
8. Repeat step 9, 10 and 11 from culture procedure above.
OBSERVATIONS
1. Choose a plate that appears to have between 30 and 300 colonies.
2. Count the exact number of colonies on that plate using the colony counter (as demonstrated
by your instructor).
3. Calculate the number of CFUs per ml of original sample as follows:
The number of CFUs per ml of sample = The number of colonies (30-300
plate) × ml of dilute sample plated
____________ = Number of colonies
____________ = Dilution factor of plate counted
____________ = Number of CFUs per ml
RESULTS
Record your results and find the average number of cfus/ ml by adding the
results from all of your plates and dividing by the number of plates.
PRECAUTIONS
1. Avoid sampling foam.
2. Do not deliver less than 10% of total volume of pipette. For example, do not use pipette with
capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use
pipette with capacity higher than 1.0 ml.
3. Do not stack plates when pouring agar or when agar is solidifying.
24
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EXPERIMENT NO:7-WATER EXAMINATION: MULTIPLE TUBES METHOD. WATER EXAMINATION
I. Set up three series consisting of three groups , a total of 15 tubes per series, in a test tube
rack, for each tube label the water source and volume of sample to be inoculated.
ii. Flame bottle and then, using a 10ml pipette , transfer 10 ml aliquots of water sample to the
five tubes labeled LB2X-10 ml.
iii. Flame bottle and then, using a 1ml pipette , transfer 1 ml aliquots of water sample to the
five tubes labeled LB1X-1 ml.
iv. Flame bottle and then, using a 0.1ml pipette , transfer 0.1 ml aliquots of water sample to
the five tubes labeled LB1X-0.1 ml.
v. Incubate all tubes for 48 hours at 37ᴏ C.
PART-B-CONFIRMED TEST.
AIM: To confirm the presence of coliform bacteria in a water sample for which the
presumptive test was positive.
PRINCIPLE:
The presence of a positive or doubtful presumptive test immediately suggests that the water
sample is non potable. Confirmation of these results is necessary , since positive presumptive
tests may be the result of organisms of non coliformorigin that are not recognized as indicators
of faecal pollution.
The confirmed test requires that selective and differential media such as Eosine Methylene Blue
(EMB) or Endo agar be streaked from a positive lactose broth tube obtained from the
presumptive test.
Eosine Methylene Blue contains the dye methylene blue , which inhibits the growth of gram-
positive organisms. In the presence of an acid environment ,EMB forms a complex that
precipitates out into the coliform colonies, producing dark centres and a green metallic sheen.
This reaction is characteristic for Escherichia coli, the major indicator of faecal pollution.
Endo agar is a nutrient medium containing the dye fuchsion, which is present in the decolorized
state. In the presence of acid produced by the coliform bacteria, fuchsion forms a dark pink
complex that turns the E.coli colonies and the surrounding medium pink.
MATERIALS REQUIRED:
Cultures
26
One 24 hour –old positive lactose broth culture from each of the three series from the
presumptive test.
MEDIA:
Eosine Methylene Blue agar plates or Endo agar plates.
EQUIPMENT:
Bunsen burner, label and inoculation loop.
PROCEDURE:
I. Label the covers of the three EMB plates or Endo agar plates with source of water sample.
ii. Using a positive 24 hour lactose broth cultures from the presumptive test ,streak the surface
of one EMB agar or one Endo agar plate to obtain discrete colonies.
iii. Incubate all plate cultures in an inverted position for 24 hours at 37 ᴏ C.
PART-C-COMPLETE TEST
AIM: To confirm the presence of coliform bacteria in a water or if necessary, to confirm a
suspicious but doubtful result of the previous test.
PRINCIPLE:
The complete test is the final analysis of the water sample. It is used to examine the coliform
colonies that appeared on the EMB or Endo agar plates used in the confirmed test..An isolated
colony is picked from the confirmatory test plate and inoculated into a tube of lactose broth
and streaked on a nutrient agar slant to perform a Gram stain. Following inoculation and
incubation, tubes showing acid and gas in the lactose broth and presence of gram-negative
bacilli on microscopic examination are further confirmation of the presence of E.coli and they
are indicative of a positive completed test.
MATERIALS REQUIRED:
Cultures
One 24 hours coliform positive EMB or Endo agar culture from each of the three series of
confirmed test.
MEDIA:
Nutrient agar slants and Lactose fermentation broths.
REAGENTS:
27
PART-B-CONFIRMED TEST
COLONIES ON EMB PLATE FROM PRESUMTIVE POSITIVE TEST
VOLUME OF SAMPLE 1 2 3 4 5
E.COLI COLONIES
1Oml 2X LTB
1ml 1XLTB
0.1ml 1X LTB
VOLUME OF SAMPLE 1 2 3 4 5
GROWTH/ GAS
1Oml 2X LTB
30
1ml 1XLTB
0.1ml 1X LTB
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EXPERIMENT NO:8 METHYLENE BLUE TEST AND RESAZURIN TEST FOR MILK
Introduction
This test is based on the work done by Wilson (1935) and Milk Regulations 1963. This test is
used to check the contamination of bacteria in the sample of milk. It tells us about the viable
count of bacteria that may be present in the milk. So by this particular test we can have a clue
about the quality of the milk we have that whether it is invaded by bacteria or not and in which
quantity these are present.
Principle
This test is based on the principle that if viable bacteria are present in the milk, they will reduce
the metylene blue dye and decolourize the sample if kept for sometime in a dark place. The
methylene blue is reduced due to depletion of oxygen in the milk as it is consumed by bacteria.
Methylene Blue is a dye and it is an aromatic chemical compound having the molecular formula
C16H18N3SCl. Its molar mass is 319.85 g/mol. It is a dark green powder at room temperature and
when it is dissolved in water, it forms a blue solution.
31
The bacteria in the milk ferment lactose to lactic acid. During this process electrons are
released, which are taken up by Methylene Blue and results in its reduction and ultimately
decoloration. So, more bacteria will produce more electrons, in turn it would be faster
reduction and rapid decolouration.
Requirements
Following apparatus and reagents are required for the Methylene Blue Test.
Procedure
Mix the sample well that has to be analyzed and then pour 10 ml of that milk into a
clean and sterilized test tube.
With the help of pipette add 1 ml of Methylene Blue Solution into the milk sample.
The test tube is then closed with a rubber stopper and invered slowly so that all the
contents have been mixed.
Then this test tube is places in a water bath at 370C for half an hour making sure that
water level is upto sufficient height of the test tube. Covered with lid.
Then the milk is examined after 30 minutes for checking the decoloration.
Maintain the control tubes: one with milk boiled at 100 C for 3 minutes and another
control with 1 ml tap water in place of methylene blue.
32
Interpretation
If the viable bacteria decolourize the milk within 30 minutes then the test is positive and milk is
of unsatisfactory quality.
If the milk is not decolourized within 30 minutes then the test is negative or milk is of good
quality.
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EXPERIMENT NO:9- RESAZURIN TEST FOR MILK
Relevant Information:
The majority of the organisms in milk are capable of reducing and decolorizing the resazurin dye. When
bacteria grow in the milk they utilize oxygen, the rate of remove or reduction is proportional to the
keeping quality. This test is also based on the same principles as M.B.R. (Methylene Blue Reduction) test,
but dye is Resazurin which is much more sensitive that the Methylene blue. For this reason this test
provides a rapid measure of the keeping quality of milk.
During incubation, the dye undergoes reduction very largely through the metabolic activity of the
organisms present. The greater the number of organisms present in milk, the more quickly the dye is
reduced. The reduction takes place in two distinct stages. Resazurin is blue at the reaction of milk. In the
first stage dye is changed to pink colour and in second stage pink colour is changed to colourness. The
cells present in the milk may also influence the reduction of Resazurin and for that reason; the test may
also measure physiologically or pathologically abnormal milk.
Precaution:
Material Required:
i Milk sample
ii. Resazurin colour/solution 0.05%.
34
Apparatus:
Procedure:
vii. Tubes are examined and classified at the end of an hour in the "one-hour test" or at the end of three
successive hourly intervals in the "triple reading test.".
35
5. White: Bad
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This is a method in which the number of bacteria or yeasts in a sample (e.g. broth culture, cell
suspension, milk) may be determined by direct microscopic examination. Since the staining
procedure does not differentiate between living and dead organisms, the bacterial count
obtained is known as a "total count". Any suitable staining technique may be used (e.g. simple
stain or Gram's method). In the case of samples of milk and other foods with a high fat content
it is necessary first to defat (e.g. with xylene). Newman's stain conveniently combines both
defatting and staining processes although not allowing the determination of the Gram's
staining reaction. The Breed's smear method is also used for counting the animal cells -
predominantly leucocytes -which are found in much larger numbers in mastitis milk (see page
179) than in milk from healthy animals and so can be diagnostic of mastitis.
It is essential in this procedure that the microscope is first calibrated so that the area of the
microscopic field is known. A known volume (0'01 ml) of the sample or an appropriate dilution
is then spread over a known area (I cm2), on a glass slide. An alternative procedure often used
which gives the same sample concentration on the slide is to draw two straight lines across the
width of the slide to enclose a length of 2 cm. Since the standard microscope slide is 2.5 cm
wide, this marks off an area of 5 cm2, over which 0.05 ml of sample is spread. The sample is
allowed to dry, then stained and examined microscopically. The average number of bacteria or
cells per field is determined and it is then possible to calculate the number of bacteria or cells
per ml of original sample.
37
1. Set up the microscope and, using the X 10 eye-piece and low power objective, adjust the
stage micrometer so that the graduated scale (I mm divided into 100 units of 10ILm each) is in
the centre of the field.
2. Place a drop of immersion oil on the stage micrometer and focus with the oil-immersion
objective. Determine the diameter of the microscopic field in ILm, using the micrometer scale,
adjusting the tube length of the micro- scope slightly if necessary. In subsequent observations
this same tube length must be maintained.
3. Calculate the area of the microscopic field in mm for the oil-immersion lens and X 10 eye-
piece using the formula r2= Area of field r2where r = radius of field (0.08). Knowing the area
of the microscopic field, it is then possible to determine the microscope factor (MF) by calculation:
Preparation of smear
I. Thoroughly mix the sample to be examined. Prepare dilutions as necessary -to produce
smears with no more than 20 organisms per field. (If pure cultures are being examined a barely
detectable turbidity will be given by a suspension containing about 106 cells per mi.)
2. Deliver 0.01 ml of sample onto a clean glass slide and spread over an area of I cm2 using
either a guide card or a marked slide. The sample may be delivered either (0) by means of a
capillary pipette calibrated to deliver 0.01 ml or (b) by using a wire loop. A closed loop of 4 mm
38
internal diameter will hold a drop of approximately 0.01 ml of sample, if withdrawn with the
plane of the loop perpendicular to the surface.
3. Dry the smear immediately by placing on a warm level surface or in an incubator at 55°C.
Drying should be complete within 5 min so as to prevent possible bacterial multiplication.
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EXPERIMENT NO: 11 – LACTIC ACID ESTIMATION IN MILK
Determination of acidity The acidity in milk is measured, for example by titration with a 0.1 n
NaOH solution, and indicates the consumption of NaOH necessary to shift the pH-value from
6.6 ± 0.1 (corresponding to fresh milk) to a pH-value of 8.2 - 8.4 (phenolphthalein). Lactic acid
is an organic acid with one carboxylic acid, CH3-CHOH-COOH, having a molecular weight of
90. One ml 0.1 n NaOH therefore corresponds to: 90 x 0.1 / 1000 = 0.009 g of lactic acid If the
titration requires e.g. 14.5 ml 0.1 n NaOH, the result is often expressed as: 14.5 x 0.009 ≈
0.13% lactic acid The titratable acidity is expressed as % lactic acid and is determined by
titration of a known amount of reconstituted milk with 0.1 N NaOH using phenolphthalein as
indicator. Acidity Expressed in °SH – (Soxhlet-Henkel Degrees)
The titrable acidity test is employed to ascertain if milk is of such a high acidity as to reduce its
keeping quality and heat stability. The acidity of milk is of two kinds.
i. Natural acidity which is due to citrates and phosphates present in the milk and dissolved CO2
during the process of milking and thereafter.
39
ii. Developed acidity which is due to lactic acid produced by the action of bacteria on lactose in
milk.
Generally the acidity of milk means the total acidity (Natural + developed) or titrable acidity. It
is determined by titrating a known volume of milk with standard alkali to the point of an
indicator like phenolphthalein.
The titrable acidity test measures the amount of alkali which is required to change the pH of
milk from its initial value of about 6-6 to 6.8, to the pH of the colour change of phenolphthalein
added to milk to indicate the end point (pH 8.3). In fact, the method measures the buffering
capacity of milk and not the true acidity.
Procedure:
1. Fill the burette with N/10 NaOH solution.
2. Mix the milk sample thoroughly by avoiding incorporation of air.
3. Transfer 10 ml milk with the pipette in conical flask.
5. Add equal quantity of glass distilled water.
6. Add 3-4 drops of phenolphthalein indicator solution* and stir with glass rod.
7. Take the initially reading of the alkali in the burette at the lowest point of meniscus.
8. Rapidly titrate the contents with N/10 NaOH solution continue to add alkali drop by the drop
and stirring the content with glass rod till first definite change to pink colour which remains
constant for 10 to 15 seconds.
9. Complete the titration within 20 seconds.
10. Note down the final burette reading.
Calculation:
No of ml. of 0.1 N NaOH solutions
required for neutralization x 0.009
% Lactic acid = ---------------------------------------------------------------------- x 100
Weight of sample
(Weight of sample = Volume of milk x specific gravity)
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PRINCIPLE: Sugars such as glucose , lactose maltose are oxidized in presence of sodium
hydroxide and iodine is removed fom the solution as sodium iodide ( NaI) . Excess of iodine can
be titrated against 0.1 N sodium thiosulfate using starch as indicator. The difference in the titre
value of blank titration and sample titration is used to calculate the percentage of lactose in the
milk.
PROCEDURE:
iii) Mix the contents well and keep aside for 20 minutes.
iv) Add 4ml of dilute H2SO4 .The solution becomes dark brown in color.
v) Titrate against standard sodium thiosulfate (Na2S2O3).
vi) When the solution becomes pale yellow ,add 1ml of freshly prepared starch as indicator.
Solution turns to dark blue.
vii) Titration is continued slowly till the blue colour disappears and solution turns colourless.
viii) A blank titration is also carried out with 10 ml of water instead of filtrate. Difference in
burette reading is used to calculate percentage of lactose.
Calculations: Percentage of Lactose = 0.171 (B-A) x N V1/V2 x 100/W
Where:
B=Burette Reading of blank titration
A= Burette Reading of sample titration
V1= 250ml
V2 =25ml
N=Normality of Na2S2O3 =0.1
W= Original volume of milk sample=10ml
1gm mol of Iodine = 1 gm mol of Lactose=342g
1gm eq.Wt of Iodine = 171g
1000ml of 1N Iodine =171 g of lactose
1ml of 1N Iodine = 0.171g of lactose.
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products which results in the formation of a blue colour. The blue colour is due to the
formation of an inclusion complex between iodine and the amylose. The amylase coils into the
spiral and the iodine molecule aligns within the centre of this spiral and causes light absorption
which gives a blue colour.
Procedure
Take 5 ml of milk in a test-tube and add 5 ml of p-dimethyl amino benza]dehyde reagent to it.
The appearance of a distinct yellow colour indicates the presence of added urea. The
appearance of slight yellow colour may be ignored which may be because of the presence of
natural urea in milk.
Detection of Sodium Chloride in Milk
Lactometer is a simple device which can be used to detect adulteration of milk with water.
Addition of water
alters the density of milk which can be read on the Iactometer scale. Hence, to increase the
density of milk,
adulterant like sodium chloride is added.
Procedure
Take about 2 g of milk product in a test-tube and add Take 2 ml of milk in a test-tube and add
O.I ml of
about 5 ml water to it. Boil for a few minutes on the 5 percent potassium chromate solution
and 2 ml of
gas burner. COOI and add iodine solution to the test- 0.1 N silver nitrate to it. The appearance
of a yellow
tube. The formation of a blue coloration shows the colour indicates the presence of added
sodium chloride
in the milk while the appearance of a brick red precipitate indicates the absence of added
sodium.
Detection of Metanil Yellow in Sweets
To make a sweet attractive, various colours are added to sweet preparations. As per the PFA
Act, onlinatural
43
food colours or permitted artificial food colours can be used in sweets. Metanil yellow is a
cheap textile
dye which is commonly used in colouring sweets which amounts to adulteration. The metanil
yellow is
carcinogenic.
Procedure
Take about 5 g of the sweet sample in a test-tube and add hot water to bring the sample in
pure solution.
Separate the coloured matter by filtration and add few drops of concentrated hydrochloric
acid. The appearance of a pinkish red coiour shows the presence of metanil yellow.
Detection of Mashed Potatoes and Other Starches in Ghee/Butter
General
Mashed potatoes and edible starches are one of the adulterants in ghee/butter as these,
without hampering
the colour appeal of ghee/butter will add to the weight of ghee/butter.
Procedure
Take some ghee/butter in a test-tube and warm it. Add few drops of iodine solution to the
sample.
The appearance of a blue colour will indicate the presence of mashed potatoes and edible
starches to
gheelbutter.
Detection of Chalk Powder in Bura Sugar/ Jaggery/Common Salt
General
Chalk powder is added to food items like bura sugar, jaggery and common salt as an adulterant
to increase
the weight. Chalk powder is carbonates of calcium which is insoluble in water and if consumed
is injurious
to health.
Procedure
44
There are two methods for detection of chalk powder in adulterated foods like bura
sugar(jaggery/common
salt.
Method 1
Dissolve about 10-15 gof a sample in water in a beaker. Allow to settle for 5 min. The
unadulterated samples
will dissolve completely while the presence of any sediment at the bottom of the beaker will
indicate the
presence of chalk powder in the sample.
Method II
Take some sample of the food item to be examined in a test-tube and add few drops of 1:1
hydrochloric acid.
The appearance of bubbles (effervescence) will indicate the presence of chalk powder in the
sample.
45
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