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Page 1
A Microbial Study of
Locally Produced
Pasteurised Milk
Presented by:
Yujna Devi Banjeet
ID: 1115365
Course: BSc
Microbiology (Year 3)
Date: 09/06/2014
Page 2
OUTLINE
Introduction
Aims of the study
Methodology
Results
Discussion
References

Page 3
INTRODUCTION
Milk is:
A highly nutritious food.

An ideal medium for microbial growth.

Easily contaminated by pathogenic and
non-pathogenic microorganisms.
Page 4
INTRODUCTION
Pathogenic microorganisms in milk:
Salmonella spp

Listeria monocytogenes

Staphylococcus aureus

Pathogenic strains of Escherichia coli
Page 5
INTRODUCTION
Non-pathogenic microorganisms:
Non-pathogenic strains of Escherichia coli

Enterobacteriacea such as Klebsiella spp
and Citrobacter spp

Psychrotrophs such as Pseudomonas
spp.
Page 6
INTRODUCTION
Milk is pasteurised:
Safer for consumption
Eliminate pathogenic and non-pathogenic
microorganisms
Increase the shelf life of milk
Pasteurised milk can get contaminated
Post pasteurisation contamination

Page 7
AIMS OF THE STUDY
To test the presence of the potential non-
pathogenic, spoilage microorganisms.

To test if the microbial load increases as
the expiry date approaches.

To test if the presence of these
microorganisms affected the components
in milk
Page 8
METHODOLOGY
Samples:
3 different brands used (Brand V, SC and
M)
A total of 45 samples were used
5 samples of each brand were analysed
weekly
Sample collection:
Samples were collected from 3 different
outlets
Page 9
METHODOLOGY
Experimental design:
All the microbiological tests were carried
out in duplicates
A tenfold dilution was carried out up to a
concentration of 10
-4
.

Figure 1: The steps involved in serial dilution
Page 10
METHODOLOGY
1. Total Viable Count (Pour plate method and Milk Plate Count agar)
2. Coliform and Staphylococcal count (Spread plate method; MacConkey and
Baird Parker agar.)
3. Further detection of coliforms (Brilliant Green Bile Lactose Broth and Eosin
Methylene Blue agar.)
4. Physico-chemical and biochemical tests of milk such as peroxidase test.
5. Identification (Gram stain and a set of biochemical tests such as IMViC
tests and Catalase test)
6. Confirmatory molecular analyses (DNA extraction and Polymerase Chain
Reaction with a set of specific primers)
7. Samples giving no positive PCR result (amplified with 16S primer and
sequenced)
Page 11
METHODOLOGY
The primers used for each microorganism
are shown in Table 1.

Table 1: The primers used for each microorganism
Microorganisms Primers Sequences
Escherichia coli TEcol553
Tecol 754
5-TGGGAGCGAAAATCGTG-3

5-CAGTACAGGTAGACTTCTG-
3
Klebsiella
pneumoniae
KP(F)
KP(R)
5-CAACGGTGTGGTTACTGACG-3

5-
TCTACGAAGTGGCCGTTTTC-3
Staphylococcus
aureus
GSEBR-1
GSEBR-2
5-GTATGGTGGTGTAACTGAGC-3

5-
CCAAATAGTGACGAGTTAGG-3
Page 12
RESULTS
Total Viable Count:
Brand V: 1.51x10
2
4.74x10
3
CFUs/ml

Brand SC: 0 6.82x10
2
CFUs/ml

Brand M: 1X10
2
3.69X10
3
CFUs/ml

Page 13
RESULTS
Coliform count:
Brand V: 1x10
2
3.75x10
5
CFUs/ml

Brand SC: 0 2x10
4
CFUs/ml

Brand M: 5.55x10
3
4.15x10
4
CFUs/ml
Page 14
RESULTS
Staphyloccus count:
Colonies were formed on plates inoculated
with the mother solution (MS) only.
Brilliant Green Bile lactose Broth:
All three brands gave similar results:
MS: Cloudy + Gas formation
10
-1
, 10
-2
: Cloudy only

Figure 2: Test-tubes of BGBLB
showing turbidity
Page 15
RESULTS
Morphological characteristics:
MacConkey agar:
Small, circular, raised, pink and non-
mucoid (Escherichia coli)
Large, dome-shaped, pink
mucoid (Klebsiella spp)
Few yellow round (Shigella)

Figure 3:Pink and yellow colonies formed
on MacConkey agar

Page 16
RESULTS
Morphological characteristics:
Baird Parker agar:
Black, shiny and convex colonies with an
opaque zone
Eosin Methylene Blue agar:
Pink mucoid (Klebsiella spp)
Green with a metallic sheen
(Escherichia coli)
Figure 4:The colonies formed on EMB
E.coli (left) and Klebsiella (right).

Page 17
RESULTS
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6
l
o
g

c
f
u

Replicate number
Brand V
4 days before
expiration
1 day before
expiration
2 days before
expiration
0
0.5
1
1.5
2
2.5
0 2 4 6
l
o
g

c
f
u

Replicate number
Brand SC
1 day before
expiration
3 days before
expiration
4 days before
expiration
0
0.5
1
1.5
2
2.5
3
0 2 4 6
l
o
g

c
f
u

Replication number
Brand M
3 days before
expiration
6 days before
expiration
5 days before
expiration
The graphical representations below give an indication of the changes in
the log CFU of the TVC of the three different brands.
Figures 5,6&7: The changes in the log cfu of the different replicates of brand V, SC
and M respectively
Page 18
RESULTS
Statistical results:
Kruskal Wallis test:
Very small difference in the TVC of the three
brands over the three weeks.

Tukeys test (5% significance level):
Significant difference in the coliform count
between V and the two others.
Page 19
RESULTS
Fat content:
V: 3.4%, SC: 3.8%, M:
2.4%
Titratable acidity:
V: 0.167, SC: 0.171, M:
0.142
pH:
V: 6.91, SC: 6.76, M: 6.72
Peroxidase test:
All gave negative results
0
0.5
1
1.5
2
2.5
3
3.5
4
Week 1
Week 2
Week 3
Figure 8: The difference in the average fat content of
the three brands
Physicochemical and biochemical results:
Page 20
RESULTS
Biochemical tests results:
IMViC results for coliforms
The results are depicted in Table 2.

Biochemical test Results
Indole 44.4% (+), 55.6% (-)
Methyl Red 55.6% (+), 44.4% (-)
Voges Proskauer 88.9% (-)
Citrate 55.6% (+), 44.4% (-)
Catalase results for Staphylococcus
spp:
They all gave positive results.
Table 2: The percentage of samples which gave positive or negative results for
each biochemical test.
Page 21
RESULTS
Table 3: The samples present in the
labelled lanes and the band sizes
Lane Sample Size of
band
Size of
the
amplicon
H SC
1
R
1
400 bp 212 bp
I SC
2
R
2
400 bp 212 bp
N M
1
R
1
500 bp 212 bp
S M
3
R
2
500 bp 212 bp
PCR results for suspected E.coli:
Figure 9: The gel electrophoresis for the
PCR result of the suspected E.coli
Page 22
RESULTS
PCR results for suspected Klebsiella
spp:
Presence of bands
in all the lanes.

Size of amplicon:
108 bp

Sizes of bands:
200 bp-500 pb
Page 23
RESULTS
PCR results for suspected
Staphylococcus spp:
No bands with specific primers (GSEBR-1
&GSEBR-2)
Sequencing results:
The microorganism identified as being
either an Enterobacter spp or
Lactobacillus plantarum

Page 24
DISCUSSION
Physico-chemical changes observed:
Fat content:
Brand SC and V had a normal fat content.
Brand M had lowest fat content- milk was diluted
with water.
Titratable acidity and pH:
They were within the required range for the three
brands.
Biochemical test of milk:
Peroxidase test:
All negative results; Milk was heated properly.

Page 25
DISCUSSION
PCR identification of suspected E.coli:
Primers successfully used to identify
environmental E.coli.
Primers bind to tuf gene; amplicon of size
212 bp.
Bands were of different sizes; E.coli
contain more than 1 tuf gene.

Page 26
DISCUSSION
PCR identification of suspected
Klebsiella spp:
The primers used to identify Klebsiella
pneumoniae.
Primers amplify the rpoB with a product
size of 108 bp.
The bands were of different sizes; the
strains were not K.pneumoniae.

Page 27
DISCUSSION
Microbiological analysis of milk samples:
Brand V the most contaminated one.
The number of microorganisms do not increase
as expiry date approaches.
The predominating coliform isolated was
Klebsiella spp.
Presence of microorganisms:
Use of unclean equipment
Unethical practices
Unhygienic handling
Page 28
CONCLUSION
Regular verification of processing plants.
Microbiological analysis of raw material
and end-products.
Tests on environmental samples.
Operators have to follow the principles of
the HACCP system.

Page 29
REFERENCES
Alam, K., Dwipayan, S., Hossain, T. 2010. Chemical and Microbiological Quality
Assessment of Raw and Processed Liquid Market Milks of Bangladesh. Journal of
Dairy Sciences. 4(4), 28-34.
Albanell, E., Caja, G., Casals, R., Rovai, M., Salama, A., Such, X. 2003.
Determination of Fat, Protein, Casein, Total Solids and Somatic Cell Count in Goats
Milk by Near-Infrared Reflectance Spectroscopy. Journal of AOAC International. (86),
746-753.
Mirkena, A. 2010. Microbiological Safety of Pasteurized and Raw Milk from Milk
Processing Plants in and around Addis Ababa. Thesis (MSc). Addis Ababa
University. [online]. Available at:
http://etd.aau.edu.et/dspace/bitstream/123456789/3306/1/ABERRA%20ASSEFA%20
MIRKEMA.pdf [Accessed 20th December 2013].
Park, Y.K., Koo, H.C., Kim, S.H., Hwang, S.Y., Jung, W.K., Kim, J.M., Shin, S., Kim,
R.T. and Park, Y.H. 2007. The Analysis of Milk Components and Pathogenic Bacteria
Isolated from Bovine Raw Milk in Korea. Journal of Dairy Science. [Website:
http://www.journalofdairyscience.org/article/S0022-0302(07)72013-1/fulltext]
[Accessed 18th August 2013]
Murphy, S. 1996. Sources and causes of high bacterial count in raw milk. Cornell
University. New York. Pp 32-34.

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