Exp. No. 1 Isolation of Bacteria and Fungi from spoiled food
Introduction
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.
Aim
To check the quality of fruits and vegetables. To identify the isolated
microbes. To know kinds of microorganisms, present in fruits and vegetables.
Materials
and Methods
Media: Nutrient agar, Gram Negative Broth, SS agar,
Baired parker 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, Petri plates, Conical flasks,
etc.,
Procedure
1.
Preparation of sample
10g of
given food sample was weighed and blended with 90mL of sterile saline in a
sterilized jar.
2. Total
viable count of bacteria
The sample was diluted up to 1:100000 (10-5).
1ml of diluent from 1:1000 (10-3), 1:10000 (10-4)
and 1:100000 (10-5) dilutions were added into sterile petri plates. 20 mL of molten sterile
nutrient agar was added in each plate. The plates were gently rotated in clockwise
and anti-clockwise directions and allowed to solidify. All the plates were
incubated at 35°C for 18-24 hours. The plates were examined for its number and
nature of colonies.
3.Coliform
isolation
1mL of sample from
1:100 and 1:1000 dilutions were inoculated onto Violet Red Bile Agar plates using
pour plate method. The plates were observed for red colored colonies after
24 hours of incubation at 37° C.
4. Faecal coliform isolation
1 mL of sample from 1:10 dilution was inoculated onto Escherichia coli
and Azide Dextrose Broth tubes and incubated at 44 °C for 24 hours. A loop full of culture from Escherichia coli broth
and Azide Dextrose Broth were inoculated into EMB and KF streptococcus agar
respectively and incubated at 37°C for 24 hours. The plates were observed for
its specific colony morphology.
5.
Isolation of Staphylococcus aureus
0.1 mL
of sample was inoculated on the surface of Baired Parker agar and incubated at
37 °C for 24 hours. The plates were incubated for black colored colonies.
6.
Isolation of Salmonella
1 mL of blended sample to 10mL of gram-negative broth and incubated at
37°C for 4-6 hours. A loop full of culture from gram negative broth was
streaked on SS agar and incubated at 37°C for 24 hours.
7.
Isolation of Vibrio
1 mL of blended sample was inoculated in to 10mL of alkaline peptone
water and incubated at 37°C for 18 hours. A loop full of culture was streaked from
Alkaline peptone water on TCBS agar and incubated at 37°C for 24 hours.
8.
Isolation of yeast and moulds
1 mL of sample from 1:100 dilution was inoculated into the Rose Bengal
chloramphenicol agar as pour plate method and incubated at 28-30 °C for 48
hours. The plates were incubated for the growth of eucaryotic microorganisms.
Result
Based on the growth in the selective medium, the spoiled food
contains the organisms as follows.
1.------------------- sp.,
2. --------------------- sp.,
3.---------------------- sp.,
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