This document summarizes the microbial examination of raw milk and fruits (apples and oranges). For milk, it describes the standard plate count method to determine total viable bacteria present. Key microorganisms found in milk include lactic acid bacteria like Lactobacillus and pathogens like E. coli. Fruits normally carry a surface microflora including Lactobacillus, Staphylococcus, and fungi like Penicillium. The document outlines procedures to analyze fruits for total plate count, yeast/mold count, and coliforms using serial dilution and agar plating. Common microbes identified on apples include Aspergillus and E. coli, while oranges often contain acid-tolerant yeasts like Candid

1. MICROBIOLOGICAL
EXAMINATION OF NORMAL
FOOD (MILK AND FRUITS)
PRESENTED BY:-
JUHI
L-2019-BS-307-M

2. MICROBIOLOGICAL EXAMINATION OF RAW MILK
1. INTRODUCTION
• WHITE LIQUID PRODUCED BY MAMMARY GLAND OF MAMMALS.
• PRIMARY SOURCE OF NUTRITION FOR YOUNG ONES BEFORE THEY CAN DIGEST OTHER
FOOD.
• IT HAS 30-35 gm OF PROTEIN/LITRE WHICH HAS 80% ARRANGED IN FORM OF CASEIN
MICELLES.
• DIFFERENT CARBOHYDRATES LIKE LACTOSE, GLUCOSE AND GALACTOSE ARE PRESENT.
• VARIOUS CATIONS LIKE Ca+2, Mg+2 AND VITAMINS LIKE A, D, C AND K ARE ALSO PRESENT.
• MILK IN THE MAMMARY GLAND IS NORMALLY STERILE. ONCE THE MILK LEAVES THE
UDDER, MICROFLORA FROM EXTERIOR OF UDDER, COAT OF THE ANIMAL, ATMOSPHERE,
UTENSILS AND WORKERS EASILY PASS INTO MILK.

3. • Pasteurization of milk is the process in which milk is treated with mild heat, usually to
less than 100 degrees to effectively destroy organisms like Mycobacterium tuberculosis
and Coxiella burnetii.
• Milk quality is determined by its composition and hygienic levelexercised during milking,
such as, cleanliness of the milking utensils,condition of storage, manner of transport as
well as the cleanliness ofthe udder of the individual animal.
• Production of milk and various milk products under unsanitary conditions and poor
production practices can exert both a public health and economic constraints.
• Analysis for microbial quality of raw milk involved count of Lactobacillus species, total
coliform, yeast and moulds and common milk-borne pathogens namely producing
Escherichia coli, Salmonella spp., Staphylococcus aureus, Streptococci, Brucella spp. and
Mycobacteria.

4. STANDARD PLATE COUNT
• It is an estimate of the total
number of viable bacteria
present in milk.
• The samples were serially
diluted and appropriate dilutions
( 10-1 and 10-2 for milk ) will be
used for spread plating.

5. • Incubate the plates at 35oC for
24 hours.
• Count the colonies in the plate
having a count between 30-300
colonies and record the
observations.
• If the count< 2 x 104 = Grade A
milk.

6. 1. LACTIC ACID BACTERIA
• Lactic acid bacteria (LAB) are an order of gram-positive, low-GC,
acid-tolerant, generally nonsporulating, nonrespiring, either rod-
shaped (bacilli) or spherical (cocci) bacteria.
• These bacteria, usually found in milk and milk products, produce
lactic acid as the major metabolic end product of carbohydrate
fermentation.
• The genera that comprise the LAB are at its core Lactobacillus
(Lactobacillus bulgaricus), Leuconostoc, Pediococcus, Lactococcus,
and Streptococcus (Streptococcus lactis)
• Lactobacilli MRS medium is based on the formulation of deMan,
Rogosa and Sharpe. It supports luxuriant growth of all Lactobacilli
from dairy products and other sources.
• Lactobacilli appear as large clear colonies after 24-72 hours
incubation at 35ºC as white, mucoid colonies.

7. 2. Escherichia coli and other coliforms
• Escherichia coli also known as E. coli is a Gram-negative, facultative
anaerobic, rod-shaped, coliform bacterium.
• The presence of E. coli is an indication there is a much greater risk of
infections because of the concurrent risk of other pathogens like
Salmonella and Brucella being present.
• So from a positive E. coli result, further testing might be conducted to
see whether other pathogens are present.

8. Organisms Growth
• Escherichia coli ------ Blue-black bulls eye (greenmetallic sheen)
• Pseudomonas aeruginosa ------ Colorless
• Enterobacter aerogenes ------- Good growth; pink, without sheen
• Klebsiella pneumoniae ----- Pink, mucoid colonies
• Proteus mirabilis ------ Luxuriant growth; colorless colonies
• Salmonella Typhimurium ------ Luxuriant growth; colorless colonies

9. OTHER MICRO ORGANISMS
• Proteolytic bacteria which degrade protein and cause bitterness and
putrefaction.
• Pseudomonas which are psychrotrophic
• Bacillus which form heat stable spores and survive pasteurization
• Lipolytic bacteria which degrade fats and produce lipolytic rancidity.
Again, the most common example in milk is the genus Pseudomonas.
Several psychrotrophic species of Pseudomonas produce heat stable
lipases as well as proteases.
• Gas forming bacteria which includes Clostridium perfringes and
Clostridium butyricum

10. • PATHOGENIC MICROBES Includes
• Bacillus cereus (can be isolated on Bacillus cereus selective agar)
• Listeria monocytogenes(can be isolated on Brain heart infusion agar)
• Yersinia enterocolitica (can be isolated on CIN Agar)
• Salmonella spp.(can be isolated on SS Agar)
• Campylobacter jejuni(can be isolated on charcoal based selective
medium)
• SPOILAGE ORGANISMS INCLUDE
• Clostridium, proteus, achromobacter

11. MICROBIAL ANALYSIS OF FRUITS (APPLE AND
ORANGE)
• INTRODUCTION
• Fresh fruits normally carry a surface flora of micro organisms, which
play an important role in spoilage.
• Microbial analysis of these fruits are important as they are consumed
raw.
• An examination for the presence of food poisoning organisms or for
the indicators of faecal contamination is required.
• The micro organism on the surface of fruit include not only the
surface microflora but also those from soil, water and plant pathogen
which includes- Lactobacillus, Staphylococcus, streptococci,
Penicillium, Aspergillus, Saccharomyces

12. • REQUIREMENTS
• Nutrient agar, yeast and mold agar,
EMB agar, sterile water blanks, petri
dishes, fruit sample.
• PROCEDURE
• The serial dilution agar plate method
is a commonly used procedure to
isolate and enumerate bacteria,
fungi, yeast and actinomycetes.
• This method is based upon the
principle that material containing
microbes when cultured will develop
into a colony, hence the number of
colonies appearing in the plate will
correspond to number of micro
organisms.

13. • 0.1 ml from the dilutions of 10-2, 10-3 and 10-4 will be used for spread
plating on the solidified agar plates.
• Sample will be analysed to study density of micro-organisms by:--
1. STANDARD PLATE COUNT(SPC)
2. YEAST AND MOLD COUNT(yeast and mold agar)
3. COLIFORM COUNT(EMB Agar)
• Plates will be incubated at 25-30O C for three days in an inverted
position.
• OBSERVATIONS
• The number of colonies appearing on the plates after incubation are
counted, averaged and multiplied by dilution factor to find number of
cells/gram of sample.
• m/o per ml or gm of sample=avg num of colonies/(amt platedxdil fac)

14. Apple
• Microbes like Aspergillus,
Alternaria, Candida,
Saccharomyces and bacillus
might be present.
• If faecal contamination had
occured, presence of E coli will
be evident on EMB plates.
yeast and mold agar

15. ORANGE
• The high acid content prevents
growth of pathogenic microbes
while allowing certain acid-
tolerant bacteria, yeasts and
molds to flourish.
• microbes like Candida, Pichia,
Saccharomyces, Rhodotorula,
Rhizopus and Mucor are mainly
present.
yeast and mold agar

16. Thank you