Bibliography PUCTIYAO, FLORENCE G. ...
Bibliography
PUCTIYAO, FLORENCE G. April2012. Bacterial Isolation from Repacked Milk Powder
sold at Baguio City Public Market. Benguet State University, La Trinidad, Benguet.
Adviser: Marietta Q. Amatorio, DVM
Abstract
A total of 15 samples were collected from randomly selected stalls and grocery stores
selling repacked milk powder at Baguio City Public Market and were examined to determine the
microbial load and the presence of coliform organism.
The coliform bacteria isolated and their frequencies are: Enterobacteraerogenes, 33%;
Escherichia coli, 47%; Klebsiellapneumoniae, 47%;Proteus mirabilis,13%. Further, Standard
Plate Count results range from 1.6M-9.3M .The repacked milk samples have microbial load
exceeding the maximum limit.
TABLE OF CONTENT
Page
Bibliography.................................................................................................... i
Abstract........................................................................................................... i
Table of Content.............................................................................................
ii
CHAPTER No .I. INTRODUCTION............................................................ 1
Background of the Study....................................................................
1
Importance of the Study......................................................................
3
Objectives of the study.......................................................................
4
Place and Time of the Study.............................................................. 4
CHAPTER No. II.REVIEW OF LITERATURE...........................................
5
CHAPTER No.III. MATERIALS AND METHOD....................................... 8
CHAPTER No .IV.RESULTS AND DISCUSSION...................................... 14
CHAPTER No. V.SUMMARY, CONCLUSION AND DISCUSSION..........
22
CHAPTER No.VI. LITERATURE CITED....................................................
23
APPENDICES................................................................................................ 25
BIOGRAPHICAL SKETCH..........................................................................
43
Chapter No. I
INTRODUCTION
Background of the Study
Milk is considered as nature's most complete food because of its unique
composition. It’s fairly suitable proportion of protein, carbohydrates, vitamins and
minerals and other substances enhance and supplement the nutrient in poor quality foods.
However, the Philippine dairy industry remains in its infancy stage despite the effort of
the government and private sectors to uplift the local milk production. It comprises only
2% of the total domestic supply. Thus to meet the local demand, the dairy industry
heavily relies on importation. The consumption also varies according to the form of the
product. By volume, milk powder accounted for 41% of the importation. These Imported
dairy products are often sold as reprocessed and repacked goods. Over the year, milk
consumption has been increasing. Based on food consumption surveys of the Food and
Nutrition Research Institute (FNRI), per capita milk consumption averaged 8.7 kg/year
for the period of 1958-1969. This figure increased to 12.6 kg in 1978 and 16.0 kg in
1982(PCARRD, 1991).
Powdered milk is also a common item in UN food aid supplies, fallout shelters,
warehouses, and wherever fresh milk is not a viable option. It is widely used in many
developing countries because of reduced transport and storage costs (reduced bulk and
weight, no refrigerated vehicles). As with other dry foods, it is considered nonperishable,
And is favored by survivalists, hikers, and others require nonperishable, easy-to-prepare
food (Anonymous, 2012).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
2
Perhaps the most difficult problem in handling and processing dairy products is
their highly perishable nature. There have been a number of food borne illnesses resulting
from the ingestion of dairy products made with milk that was not properly pasteurized or
was poorly handled causing post-processing contamination. In order to avoid excessive
spoilage, many dairy products were developed as a result of attempts to prolong storage
life. These include Milk powder, evaporated milk, condensed milk, and cheeses. All have
prolonged storage life but still subject to contamination (Anonymous, 2011). Further,
Brock, 1978 stated that Milk contains relative few bacteria when it leaves the udder of a
healthy cow.Since milk is such a good growth medium, it is better to sterilize or
pasteurize before drinking, because such bacteria rapidly multiply. Though bacteria can
be retarded or killed during pasteurization, consumers should be aware of contacting
disease brought by improper food processing that leads to microbial contamination that
cause spoilage, unpleasant odors and tastes and a food poisoning outbreak in a
community due to ingestion of the bacteria and or their toxins present in the
food(Quinn,1994).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
3
Importance of the Study
Powdered milk is a convenient form of nutritious milk that doesn't require
refrigeration. It is easily reconstituted and also an easily transported and stored dairy
ingredient. It is an economical source of dairy solids. They are required for the
manufacture of infant formulae and recipes for baked goods where adding liquid would
render the product too thin. Powdered milk is also widely used in various sweet and
certain products such as milk candy, chocolates, caramel candy, pulvoron, pastillas,
snacks, soup, ice-cream, ice crumble toppings, coffee creamer, sauces and etc.
(Anonymous, 2011).
Downeset al., 2001 stated that powdered milk must be considered sensitive
products from a public health aspect because they are often consumed after reconstitution
without additional heating. It is well established that dried milk can be a source of
foodbourne illness because of contamination. Probably the two most significant sources
of contamination are the dairy utensils or the milk-contact surfaces. Unaseptic packaging
from workers hands, Equipments such as fillers used in handling and processing as well
as storage of these products may also serves as possible source of contamination and
pathogen. Normally the packaging material contributes very little to the total microbial
load in the finished product. If the dairy utensils or the milk-contact surfaces are
inadequately cleaned, sanitized and dried, bacteria may develop in large number. Under
very poor conditions these sources may increase the bacterial content of milk by millions
per millilitre. Undesirable bacteria from these sources include coliform bacteria, lactic
Streptococci, and thermodurics, those which survives pasteurization. These bacteria grow
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
4
well in milk hence endanger its keeping quality (Frazier and Westhoff, 1988).
It is therefore important to determine the bacterial load of the product to evaluate
the safetiness of the food for the consumer.
Objectives of the Study
The study was conducted to:
1. determine the microbial load in repacked milk powder, and
2. determine presence of coliform organism.
Place and Time of the Study
Fifteen samples were collected from three different stalls and two grocery stores
selling repacked milk powder at Baguio City Public Market and were brought to the
Microbiology Laboratory Room, College of Veterinary Medicine, Benguet State
University, La Trinidad, and Benguet for processing from August 2011 to March 2012.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No. II
REVIEW OF LITERATURE
Microorganisms are associated with most of the food we eat. These
microorganisms can cause spoilage and lend up unpleasant odours and flavours to the
milk. Also, milk can harbour pathogenic microorganism which produces disease if
microbial populations are permitted to proliferate extensively. Certain microorganism
produces toxic substances which results in food poisoning outbreak (McCuenet al.,
1988).
Davis, 1962 reported that Bacterial standards for milk varies from place to place,
some areas require that milk must contain not over 100,000 bacteria per ml, however,
United State public health suggested a certain regulation of not over 200,000 bacteria per
ml. Federal regulation defined in the pasteurized milk ordinance mandate that milk
standard plate count should not be greater than or equal to 10 4 .While in the Philippines,
the Standard Microbial Plate Counts shall not exceed 50,000 cfu/ml and coliform count
should be less than or equal to ten cfu/ml (Anonymous,2007).
Wholesome, high quality milk and hygienic control of milk is indeed necessary,
these involves many carefully controlled steps, regardless of whether the end result is
filled milk or powdered milk. These hygienic controls involves such matter as clean
milking operation and equipment, hygienic processing of the milk and proper storage and
delivery to the end consumer. The genera comprising the coliform bacteria are principally
E.coli, Enterobacter and klebsiella. Test for this bacteria are designed to measures the
quality of the procedures for the handling and processing of the milk after the
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
6
pasteurization process (McCuen, 1988).
Coliforms are facultative anaerobes with an optimum growth at 37° C. Coliforms
are indicator organisms; they are closely associated with the presence of pathogens but
not necessarily pathogenic themselves. They also can cause rapid spoilage of milk
because they are able to ferment lactose with the production of acid and gas, and are able
to degrade milk proteins. Mabesaet al., 1989 as cited by Dulay, 2008 reported that
Coliform group of microorganism has been used as index of the sanitary quality of foods
and sometimes as an indicator of sanitary conditions existing in environment or kitchen
during handling and processing.Further, Enumeration of these organisms in heat
pasteurized foods can be used to assess the adequacy of heating process designed to
inactivate bacteria and in assessment of overall quality of food and hygienic conditions
present during food processing (Downeset al., 2011).
Escherichia coli is a natural inhabitant of the large intestine and small intestine of
all mammals(Quinn et al.,1994).It causes coliform mastitis in cow and sow In dairy
cows, the source of infection is faecal contamination of the skin of the mammary gland
and relaxation of the teat sphincter following milking increases vulnerability to infection.
In man it produces fatal intestinal infections in infants.E.coli is excreted in the faeces and
can survive in faecal particles, dust and water for week or months. Because of its
prominence as a normal intestinal bacterium in most mammals, most strains of E.coli are
easy to detect in food and water and currently one of the indicator bacteria to monitor in
water, foods and dairy products (Nester, 2004).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
7
Enterobacteraerogenes is very common normal faecal flora and can be isolated
from wound. It causes coliform mastitis in cattle and occasionally part of the mastitis-
detritus-agalactia (MMA) syndrome in sows (Quinn et al., 1994).
Klebsiellapneumoniae is normally found in the normal flora of the mouth, skin
and intestine. It is present in the environment, vegetation, soil and in faeces. It also causes
urinary tract and wound infection, otitis, meningitis and pneumonia in man (Hyde, 1989).
Proteus mirabilis is found in faeces of mammals and environment. It causes
urinary tract infections in man, dogs and horses. Also associated with otitis externa in
dogs and diarrhoea in young mink and lambs, calves, goats and pups (Hyde, 1989).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.III
MATERIALS AND METHOD
Materials
Materials used in the study were: 15 milk powder samples from different stalls
and grocery stores in Baguio City Public Market, Trypticase Soy Broth(TSB),phosphate
Buffer, Xylose Lysine Deoxycholate Agar(XLD), Triple Sugar Iron Agar, Simmon Citrate
agar, Indole Broth, Methyl Red Broth,Vogues-Proskauer Broth, Ehrlich’s Reagents,
Methyl Red Indicator, Crystal Violet, Gram’s Iodine ,Acetone Alcohol, Safranin, mineral
oil, glass slides, microscope, sterilizing oven, autoclave, electric stoves, Quebec colony
counter, bacteriological incubator, weighing balance, denatured alcohol, alcohol lamp,
Erlen Meyer flasks, graduated cylinder, inoculating loops and needles beaker, petridishes
,pipettes, spatulas, stirring rods, test tubes, test tubes racks, test tube brush, wire baskets,
distilled water, staining can, marking pen and masking tapes.
Methodology
Pre-experimental phase
Three stalls and two grocery stores selling repacked milk powder at Baguio
CityPublic Market were identified as collection sites (Figure No.1). In each stall, one
fourth kilogram of milk powder sample was collected in each collection time with five
days interval until a total of 15 samples were completed.Each sample was properly
labelled.The materials used in this study were autoclaved and sterilized.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
E
C
Danes
Bakery
.
1
.
D
g
st.2
n
g
st.3
n
g
st
rd
y
an
Ka
kaya
Kaya
Abanao
ZanduetaSTt
Fish and
p
meat sec
Maharlika
Hillto
B
A
Magsaysay Rd
Figure No.1 Schematic Diagram of the sites of Collection of Samples
*A,B,C,D,E-Sites of collection
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
10
The media were prepared based on the manufacture’s specification(Appendix D).
Experimental Phase:
Six grams of each sample in powdered form was reconstituted in 10millilitres
(ml) phosphate buffered- distilled water at PH of 7.2. Using a pipette, one ml of the
reconstituted milk powder was poured in Trypticase Soy Broth that served as cultivation
medium for isolating bacteria and was incubated for 24 hours at 37oC.
Standard Plate Count (Figure No.2)
A series of dilution was made on a five sterile test tubes containing nine ml of
distilled water as diluents. One ml from the incubated sample in TSB was delivered to
test tube one containing nine ml of sterile distilled water thereby giving a dilution of
1:100.It was mixed by swirling. Another one ml from test tube one was pipetted and
delivered to test tube two containing nine ml distilled water giving a dilution of 1,000.It
was then mixed by swirling. From test tube two, one ml was transferred to test tube three
containing nine ml of distilled water giving a 1:10,000 dilutions. The same procedure was
repeated to test tube four and five with a dilution of 1:100, 000, and 1:1,000,000
respectively.0.1 ml from each test tube was delivered to a sterile plate containing 20ml of
melted nutrient agar tempered at 43-45 0C. It was gently swirled to mix and then allowed
to cool and solidify. It was placed in an upside down position inside the incubator at 37c
for 24 hours. The plate was examined for visible microbial growth. Counts were made
from each plate using a Quebec colony counter. The average count from all plates were
calculated and recorded to obtain the microbial count per ml of sample.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Milk Sample
1 ml
Sterile Distilled Water
1 ml
1 ml
9m
1 ml
1 ml
99 m
99 m
9ml
9ml
9ml
9ml
0.1ml
1:100
1:1,000
1:10,000
1:100,000
1:1,000,000
*Number of bacteria per ml= number of colonies x dilution of sample/number of plates
used.
Figure no.2 Standard Plate Count Method
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
12
Test for the Presence of Coliform Organism (Figure No.3)
A loopful of samples from TSB was inoculated to XLD and was incubatedat 370C
for 24 hours. Colony from XLD suggestive of coliform was touch using an inoculating
needle then was streaked to triple sugar iron and incubated at 37OC for 16 hours. This
was later subjected to gram staining and to biochemical tests such as Indole test, methyl
red test, vogues-proskauer test, citrate utilization test.
Milk powder sample reconstituted with
Phosphate buffer
Inoculated to TSB at 37ºc for 24 hours
Nutrient Agar Plate (SPC)
Xylose Lysine Deoxycholate (XLD)
Colony characteristic Tipple sugar Iron (TSI)
Gram staining
Biochemical tests
MR
VP
Citrate test
Indole test
Figure no.3 Schematic Diagram for the Bacterial Isolation in Milk Powder
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
13
Data Gathered
The Data gathered were the following:
1. Source of samples: Milk powder samples were taken from different stalls selling
Repacked Milk Powder at Baguio city public market (Figure no.1)
2. Mean Number of Bacteria Per ml of the Sample. This was computed by
multiplying the colonies counted on each individual plate by their
corresponding dilutions. The mean total number of the colonies will be
divided by the number of plate used where the count will be made.
3. Colony Characteristic of Coliform Bacteria. The growth of coliform bacteria was
observed according to their characteristics appearance on XLD (Appendix
A).
4. Biochemical Tests. These were based on their reaction in MR, VP, and Indole
And Citrate (Appendix A).
5. Identity of coliform organism. This was based on the colony characteristics on
XLD agar, gram staining reaction and biochemical test (Appendix B).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.1V
RESULTS AND DISCUSSION
Table No. 1 shows the mean number of bacteria per ml of repacked milk powder
sold at Baguio City Public Market
TABLE No.1 Mean number of microorganisms per ml of sample, n=15
Sample No.
Mean
count
(1x107)
1
3.9
2 2.3
3 7.9
4 4.2
5 2.3
6 8.4
7 1.8
8 2.2
9 2.7
10
1.6
11
5.4
12
3.6
13
9.3
14
6.5
15
8.3
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
15
It shows that the Mean Number of bacteria per ml of the sample ranged from
1.6 x 107 to 9.3 x107.This indicates that all the samples are contaminated and have
exceeded the maximum count of United State Public Health Services which is 200,000
per ml (Davis,1962) and the Philippines standard which is 50,000 cfu/ml of milk sample
(Anonymous, 2007).
The very high load of Microorganism in the sample can be attributed to the
improper repacking and storage of the product .It was observed that the samples were not
properly sealed and the packaging materials were dusty and too vague. Samples were
even stored in places accessible to pest infestation such as mice and fliesand arefreely
exposed to air contaminants and passersby. Downes, 2011, reported that improper
processing and unaseptic packaging lead to contamination. Further, Frazier and
Westhoff, 1988 stated that very poor condition of the environment may increase bacterial
count of milk by millions per ml.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
16
Plate No.1. Microbial Growth in Standard Plate Count at 1x 106 Dilutions
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
17
Table No.2 shows the identity of the coliform bacteria isolated from
repacked milk powder.
Table No.2 Identity and frequency of coliform Organism in repacked milk powder,
n=15
Identity of Gram XLD TSI Indole MR VP Citrate Frequency
Micro-
stain colony
(%)
Organism
E.aerogenes gram yellow, mucoid Y/Y
+ - + + 33%
Negative and glistening
E.coli
gram yellow, large
Y/Y, + + - -
47%
Negative flat, smooth
CO2
and glistening
K.pneumonia gram yellow, mucoid,
Y/Y - +/- +/- +
47%
Negative large and
Smooth
P.mirabilisgram yellow with
R/Y, - + -/+ +/- 13%
Negative black centre
H2S
It shows that the Coliforms isolated and their frequencies
are:Enterobacteraerogenes, 33%; E.coli, 47%; Klebsiella pneumonia, 47%.And Proteus
mirabilis, 13%.
These coliform organisms thrive in the environment and in faecal particles. It was
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
mentioned earlier the condition of the samples and their storage.McCuen,
1988
reportedthat these organisms are normal flora of
f faeces. Presence of it in water and
food
18
indicates faecal contamination. All coliforms are killed by pasteurization. Presence in the
samplepost pasteurization can be attributed to improper handling, from equipments used
in handling milk, from workers hands, and during storage of
f the product as mentioned by
McCuen, 1988 and Frazier and Westhoff, 1988. Further, Pelczar, 1986 mentioned that
foods are always subject to growth of any organism considering the method by which it is
handled during processing.
Plate no.2. Growth of coliform bacteria in XLD (A) colony with black centre and
(B) Yellow colony
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
19
Plate no.3.Triple Sugaar Iron Test. A.R/R (non-coliform) B.Y/Y (typ
pical coliform)
C. R/Y/H2S (P.mirabilis)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
20
Plate no.4.Vooges-Proskauer Tests A (+) and B (-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
21
Plate No.5 Methyl Red Tests A (+) and B (-)
Plate No.6. Indole Tests A (+) and B (-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Plate no.7.Simmon Citrate Test A (+)
Chapter No. V
SUMMARY, CONCLUSION AND RECOMMENDATION
Fifteen samples were collected from randomly selected stalls and grocery stores
selling repacked milk powder at Baguio City Public Market to determine the microbial
load and the presence of coliform organism in each sample. Each sample were
reconstituted in phosphate buffer and then inoculated to TSB. One ml of each sample
from TSB was inoculated to nutrient agar for standard plate count. Further, loopful of
sample from TSB were inoculated to XLD. Growth suggestive of coliform was streaked
to TSI agar. Then subjected to Gram staining and Biochemical tests.
Results showed that the isolated coliform and their frequency were:
Enterobacteraerogenes, 37%; Escherichia coli, 47%; Klebsiellapneumoniae, 47% and
Proteus mirabilis,13%.And the mean number of microorganism per ml of sample ranged
from 1.6x107-9.3x107.
It is concluded therefore that the repacked milk powder samples are heavily
contaminated with faecal coliform.
It is recommended that repacked powdered milk should be studied further on
the presence of Gram positive bacteria, molds, yeast, most especially Staphylococcus
Sppand Salmonella where poisoning have been recorded.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.VI
LITERATURE CITED
ANONYMOUS.2011.How to Prepare Phosphate Buffers
eHow.comhttp://www.ehow.com/how_6319495_prepare-phosphate-
buffers.html#ixzz1Uyqhey1T
ANONYMOUS.2011.How to Reconstitute Dry Milk Powder | eHow.com
http://www.ehow.com/how_5753380_reconstitute-dry-milk-
powder.html#ixzz1Uyt47rxP
ANONYMOUS.1994.FLUKA biochemika, Microbiology-culture media for general
Microbiology, for food analysis, for environmental analysis and for molecular
biology.Flukachemie AG, Buchs.Pp 16
ANONYMOUS.2007. Guidelines for microbiologicalExamination of foods....The
Standard Plate Count (SPC), also referred to as the aerobic plate count or the
total...www.foodstandards.gov.au/.../guidelinesformi.
BROCK, T.D. 1978.Basic Microbiology with Aplication.2nd edition, prentice
Hall, Inc.EnglewoodCliffs, New Jersey.Pp.500-501.
DAVIS R.F. 1962.Modern Dairy Cattle Management.prentice- hall, inc.
Englewood Cliffs, New Jersey.pP.223-224.
DOWNES, F.P. 2001.Compendium of methods for the microbiological
examination of foods .Google Books Result chapter 8.Enterobacteriaceae,
coliforms and E.coli as quality and safety
indicator.books.google.com.ph/books?isbn=087553175X. American Public
Health Association - - Medical \\
DULAY, F.O. 2008.Bacterial isolation from “Boneless Bangus” Sold at Baguio City
Public Market.Undergraduate Thesis. Benguet State University, Philippines.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
FENG, P., S.D. WEAGANT and M.A. Grant.1998 Bacteriological Analytical Manual,
8th Edition, Revision A,Chapter 4 Enumeration of Escherichia coli and the
Coliformbacteriawww.fda.gov/.../BacteriologicalAnalyticalManualBAM/
default.htm
24
FRAZIER, W.C, and D.C.WESTHOFF.1988.Food microbiology.4th edition.
McGRAW HILL Company. SingaporePp 227
NESTER, E.W., D.G. ANDERSON, C.E.ROBERTS JR., N.N.PEARSHALL and
M.T.NESTER. 2004.Microbioogy: A Human Perspective.4th Edition. McGraw-
Hill Companies.United State of America.Pp 282-283.
PEARCE, K.N. 2012."Milk Powder", Food Science Section, New Zealand Dairy
Research Institute. nzic.org.nzen.wikipedia.org/wiki/Powdered milk
PELCZAR, M.J. 1986.Microbiology 5th Edition, McGraw-hill book company, United
State of America.pp 618.
POWER,D.A and P.J. McCUEN.1988.Manual of BBL products and Laboratory
procedure.6thEdition. United State of AmericaPp.4,15-16,62-63,224,214,274,
288,307,269.
QUINN, P.J., M.E.CARTER, G.R.CARTER, and B.K MARKEY.1994. Clinical
Veterinary Microbiology.Wolfe Publishing.Spain.Pp 54 and Pp 221.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
25
Appendix A
Expected Colonial Characteristic in XLD, TSI and Biochemical Test of
Coliform Organisms.
Identity
Colonial characteristic in
TSI Biochemical
Tests
XLD
MR VP I C
Escherichia coli
Yellow colony, flat, small,
Y/Y w/
+ - +
-
smooth and glistening,
CO2
circular in outline
Enterobacteraerogenes Yellow colony, spreading
Y/Y
- +
+
+
moist, medium in size and
glistening
Klebsiellapneumoniae
Large mucoid, Yellow colony,
+
+
- +
Y/Y
raised, and entire in outline
(-)
(-)
Proteus mirabilis
Yellow colony with black
R/Y w/
+ -
- +
centre
H2S
(+)
(-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
26
Appendix B
Morphological Characteristic, TSI and Biochemical Test Reactions and Identity of
Coliform Bacteria
Sample TSB
XLD
Gram
TSI MR VP
SC
I Organism
stain
identified
1 turbid
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
2 turbid
Yellow
spreading
negative Y/Y - + + - Enterobacter
moist, and
rod
(+) aerogenes
glistening
Yellow,
Negative Y/Y + + + - Klebseillapneu
mucoidlarge,raised, rod
moniae
and entire in outline
3 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebsiellapneu
round and entire in rod
moniae
outline
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
4 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebseillapneu
mcoid and entire in rod
moniae
outline
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
27
5 turbid
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
6 turbid
Yellow
colonies
Negative R/Y + +
+ -
Proteus
with black center
rod
(-) (-)
mirabilis
Tiny
translucent,
Positive R/R Non coliform
raised, circular
cocci
7 turbid
Tiny
translucent,
Positive Y/Y
Non coliform
raised, circular
cocci
8 turbid
Yellow,
flat, Negative Y/Y + - +
-
Escherichia
smooth, glistening rod
(-)
coli
and circular in
outline
9
turbid
No
growth
No
growth
No
growth
10 turbid
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent, Positive
R/R Non
coliform
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
raised, circular
cocci
11. turbid
Yellow,large,raised, Negative Y/Y + - +
- Klebsiellapneu
mucoid and entire rod
monia
in outline
Yellow,flat,smooth, Negative Y/Y + - - + Escherichia
glistening and
rod
coli
circular in outline
12 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebsiellapneu
mucoid and entire rod
(+) moniae
in outline
Yellow,
flat, Negative Y/Y + -
- + Escherichia
smooth, glistening rod
(+)
coli
and circular in
outline
28
13 turbid
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
Yellow,large,raised, Negative Y/Y + +
+ - Klebsiellapneu
mucoid and entire rod
(-)
moniae
in outline
14
turbid Yellow, flat,
Negative Y/Y + +
- + Escherichia
smooth, glistening rod
(-)
coli
and circular in
outline
Yellow,large,raised, Negative Y/Y + +
-
-
Klebsiellapneu
mucoid and entire rod
(-) (+)
moniae
in outline
Yellow
colonies
Negative R/Y + +
+ - Proteus
with black centre
rod
(-)
mirabilis
due to h2s
production
15 turbid
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
Yellow
spreading Negative Y/Y - + + + Enterobactera
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
moist, and
rod
erogenes
glistening
29
Appendix C
Gram Staining, TSI and Biochemical Test Performed
A. Gram staining
This is the differential colouring solution that distinguishes gram negative bacteria
from gram positive organisms.
Procedure:
1. Put a little amount of the colony in a dry and clean glass slide.
2. Place a small drop of distilled water at the centre of the slide and spread it evenly using
a sterile wire loop.
3. Air dry and heat fix on a flame and cool.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
4. Flood with crystal violet for 2 minute then wash with running water.
5. Drop grams iodine and let it stand for 1 minute and then rinse with running water.
6. Apply grams acetone decolourizer for 10 seconds then rinse.
7. Counter stain with safranin for 30 seconds and then rinse
8. Dry the stained smear and examine under microscope.
B.Tripple sugar iron test
Isolated colony is touched with an inoculating needle. A tube of TSI agar is stab
inoculated in the middle of the agar to within 5mm from the bottom of the tube. On the
withdrawal of the straight wire, the entire slant is streaked (right to the top) the tubes are
incubated at 37 for 16 hours.
30
C.INDOLE TEST
A loopful of the test organism will be inoculated into Indole broth for 24
hours.Ehrlich’s reagent will be added. A positive result is indicated by a Pink/Red layer
forming on top of the liquid.
D.METHYL RED TEST
Methyl broth will be inoculated with pure culture from the plate and incubated for
24-48 hour at 37 0C.Five drops of Methyl Red Indicator will be added to the broth after
incubation. A change in colour from yellow to red indicates a positive result.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
E.VOGUES-PROSKAUER TEST
Vogues-proskauer broth will be inoculated with pure culture from the plate and
will be incubated at 37 OC for 48 hours. After incubation, 0.6ml of 5%Naphtol in absolute
ethyl alcohol 0.2ml of 40% potassium hydroxide containing 2% creatine will be added.
Shake well and keep the broth undisturbed for three to ten minutes. A positive result will
appear as bright red to orange in colour.
F.CITRATE UTILIZATION TEST
A slant of Simmon’s Citrate Agar is streaked with a pure culture stock and
incubated for 24 to 48 hours at 37c.The PH indicator Bromothymol Blue, changes the
slant from green to blue if the growth and utilization occurs. It is negative if the medium
31
remains green and is positive if it turns blue.
Appendix D
Culture Media, Broth and Reagent Preparation
1. Phosphate buffer
Ingredients
Potassium dihydrogen phosphate 26.22g
Sodium carbonates
7.78
Final ph 7.2+/-0.1
Direction:
Prepare a stock solution by dissolving 34g in 1 L distilled water. Dispense and
sterilize, if desired. Store under refrigeration. Prepare a working solution by adding 1.25
ml of the stock solution to distilled water and make up to 1 L. Dispense in bottles or other
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
appropriates quantities and sterilize by autoclaving at 121OC for 15 minutes.
2. Trypticase soy broth
Ingredients:
Grams
per
litre
Pancreatic digest of casein
17.0g
Papaic digest of soybean meal
3.0
Sodium
chloride
5.0
Dipotassium
phosphate 2.5
Dextrose
2.5
Direction;
Suspend 30g of the powder in 1 litre of distilled water. Mixed thoroughly and
warm gently until solution is completed. Dispense and sterilize by autoclaving at 121C
for 15minutes.
32
3. Nutrient Agar
Standard formula
Ingredients;
grams per litre
Peptic digest of animal tissue
5
Beef
extract
1.50
Yeast
extract
1.50
Sodium
chloride
5.00
Agar
15
Final
ph
at
25c
7.4+/-0.2
Direction:
Suspend 28 grams in 1000 ml distilled water. Heat to dissolve the medium
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
completely. Sterilize by autoclaving at 15 pounds pressure (121c) for fifteen minutes.
4. Xylose Lysine Deoxycholate Agar (XLD agar)
Standard formula
Ingredient
Grams/Litre
Yeast
extract
3.0
L-lysine 5.0
Lactose 7.50
Sucrose 7.50
Xylose
3.50
NaCl
5.00
Na
Deoxycholate
2.50
Na
Thiosulphate
2.50
Ferric ammonium Citrate
0.80
Phenol
Red
0.80
Agar
15.0
Final
ph 7.4+/-0
Direction:
Suspend 56.68 grams in 1000 ml distilled water. Heat with frequent agitation until
the medium boils. Do not autoclave or over heat. Transfer immediately to a water bath at
50 0C. After cooling, pour into sterile plates. It is advisable not to prepare large volume
33
which will require prolonged heating.
5. Triple sugar iron test
Ingredients:
Grams
per
litter
Peptic digest of animal tissue
10g
Pancreatic digest of casein
(casein enzymatic hydreolysate)
10
Sodium
chloride
5
Lactose 10
Dextrose
10
Ferrous ammonium sulphate
0.2
Sodium
thiosulfate
0.2
Phenol
red
O.O25
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Agar
13.0
Direction:
Suspend 50.4 g of the powder in 1L of distilled water. Heat to boiling to dissolve
the medium completely.mix well and distribute into test tubes. Sterilize by autoclaving at
121C for 15 minutes. Cool in slanted position such that deep butts (1 inch long) are
formed.
6. METHYL RED-Vogues Proskauer Medium (buffered glucose broth)
Standard formula
Ingredients
gram/Litre
Buffered
peptone
7.00
Dextrose
5.00
Dipotassium
phosphate 5.00
DIRECTION:
Suspend 17.0 grams in 1000ml distilled water. Distribute in test tubes in10ml
amounts and sterilize by autoclaving at 15 pounds pressure (121c) for 15 minutes.
34
6A.METHYL red reagents
Ingredients
Methyl
red
(Mallinckrodt)
0.1
Ethyl
alcohol
300ml
Distilled
water
200ml
DIRECTION;
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Dissolve methyl red in alcohol before adding distilled water. Store in a colored
bottle in a cool and dry place.
7. Vogues-proskauer (VP) Reagent A
INGREDIENTS:
A-naphtol (sigma chemical Co)
5g
Ethyl alcohol (absolute)
100ml
DIRECTION:
Dissolve a-Naphtol in a small amount of ethyl alcohol and bring the volume to
100ml in a volumetric flask or cylinder. The alcohol should be almost colourless. Store in
a colored bottled and store in a dry place.
8. Vogues-proskauer (VP) reagent b
Ingredients
Potassium hydroxide (KOH)
40g
Distilled
water
100ml
35
DIRECTION:
Weigh out the KOH very quickly, since it is hygroscopic and will become caustic
when moist. Add less than 100ml water to the pellets in a flask in a cold water bath to
prevent overheating. Bring the volume to 100ml in a volumetric flaks or cylinder. Store
this reagent in the refrigerator in a polyethylene bottle or one that has been socially
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
treated for storage chemical.
9. Simmon’s citrate agar
Standard formula
Ingredients
grams/liter
Magnesium
sulphate
0.20
Ammonium dihydrogen phosphate
1.00
Dipotassium
phosphate 1.00
Sodium
citrate
2.00
Sodium
chloride
5.00
Bromothymol
blue
5.00
Agar
15.00
Final
ph
(at
25C)
6.8+/-0.2
DIRECTIONS:
Dissolve ingredients. Distribute to tubes or vials. Before medium solidifies,
incline tubes to obtain 4-5 cm slant and 2-3cm butts. Sterilize by autoclaving at 15
pounds pressure (121c) for 15 minutes. Final ph, 6.8+0.2.
36
10. Indole test:
Ingredients;
gram
Pancreatic digest of casein
20.g
Disodium
phosphate
2
Dextrose
1
Agar
1
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Potassium
nitrate
1
Direction
Suspend 254g of the powder in 1 L distilled water. Add 2 g of agar for use as a
motility medium.mix thoroughly. Heat and boil for 1 minute until completely dissolved.
Dispense in regular testubes, filling them half full. Sterilise by autoclaving at 118 to 121C
for 15 minutes.
Kovacs reagent for indole test
Amyl
alcohol
22.5
ml
P-dimethylaminobenzaldehyde 1.5g
Hcl
7.5ml
Ehrlich’s reagent for Indole test
Ingredients
Paradimethylaminobenzaldehyde
2
grams
Ethyl
alcohol
190ml
Hydrochloric
acid
40ml
Directions:
Dissolve paradimethylaminobenzaldehyde in 190 ml of 95% ethyl alcohol first.
37
Then gently add hydrochloric acid to the moisture by pouring on the sides of the
Volumetric flask.Appendix E
Program of Activities of Thesis Performance
Bacterial Isolation from Repacked Milk Powder Sold at Baguio City Public Market
Date
Activity
October 15, 2011
Purchase of Reagents and Chemicals. Barrow instruments
and washes them. Sterilization of Glassware.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
October 16, 2011
First collection of sample and preparation of TSB and
phosphate buffer stock solution.
October 17, 2011
The sample will be reconstituted with phosphate buffer
then inoculated to TSB.Incubate for 24 hours at 37 OC.
Preparation of XLD.
October
18,
2011
Loopful of mixture from TSB will be streak
d into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mixed
with nutrient agar for standard plate count.
Preparation
of
TSI.
October 19, 2011
Reading and recording of results from XLD ad nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of media for biochemical tests.
October 20, 2011
Reading and Recording of results from TSI.
Gram
staining
Inoculation for biochemical test
2nd collection of sample
October 21, 2011
reading of results from Biochemical test
Preparation
of
TSB
and Phosphate buffer
October 22, 2011
the sample will be reconstituted with phosphate buffer then
inoculated to TSB.incubate for 24 hours at 37 OC.
Preparation of XLD.
October 23, 2011
Loopful of mixture from TSB will be streaked into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mix
with nutrient agar for standard plate count.
38
Preparation
of
TSI.
October 24, 2011
Reading and recording of results from XLD ad nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of media for biochemical tests.
October 25, 2011
Reading and Recording of results from TSI.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Gram
staining
Inoculation
for
biochemical tests
October 26, 2011
reading of results from Biochemical test
Preparation
of
TSB
and Phosphate buffer
3rd collection of sample
October 27, 2011
the sample will be reconstituted with phosphate buffer then
inoculated to TSB.Incubate for 24 hours at 3OC.Preparation
of XLD.
October 28, 2011
Loopful of mixture from TSB will be streaked into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mix
with nutrient agar for standard plate count.
Preparation
of
TSI.
October 29, 2011
Reading and recording of results from XLD and nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of Media for Biochemical tests.
October 30, 2011
Reading and Recording of Results from TSI.
Gram
staining
Inoculation
for
Biochemical
Test
October
31,
2011
Reading of Results from Biochemical tests
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Appendix F1
Appendix F2
Appendix G1
APPLICATION FOR ORAL DEFENSE
Benguet State University
COLLEGE OF VETERINARY MEDICINE
LaTrinidad, Benguet (2601)
APPLICATION FOR FINAL ORAL DEFENSE
Name: PUCTIYAO, FLORENCE G.
Student Number: 0608051
Thesis Title: BACTERIAL ISOLATION FROM REPACKED MILK POWDER
SOLD AT BAGUIO CITY MARKET
Date and Time of Examination: February, 29 2012, 9:00 AM
Place of Examination: CVM Accreditation Room
MEMBERS OF THE EXAMINING COMMITTEE
Name
of
Member
Signature
CRISELDA S. BATTAD, DVM, MS
___________________
JOSEPH A. DIANSO, DVM
___________________
EDLYN MAE N. CIANO, DVM
___________________
Recommending
Approval:
MARIETTA
Q.AMATORIO
Adviser
Approved:
JOSEPH
A.
DIANSO,
DVM
Dean
Appendix G2
Benguet State University
COLLEGE OF VETERINARY MEDICINE
LaTrinidad, Benguet (2601)
REPORT OF RESULTS OF FINAL ORAL DEFENSE
Name: PUCTIYAO, FLORENCE G.
Student Number: 0608051
Thesis Title: BACTERIAL ISOLATION FROM REPACKED MILK POWDER
SOLD AT BAGUIO CITY MARKET
Date of Examination: February, 29 2012
Place of Examination: CVM Accreditation Room
Name and Signature of Examinees:
Remarks
MARIETTA Q.AMATORIO, DVM
Adviser
Date
JOSEPH A. DIANSO DVM
Panel
Date
EDLYN MAE NABUSANCIANO,DVM
Panel
Date
CRISELDA SILVESTRE-BATTAD, DVM,MS
Department chairman
Date
JOSEPH A. DIANSO DVM
Dean
Date
BIOGRAPHICAL SKETCH
Name:
FLORENCE GOLWINGON PUCTIYAO
Date of Birth:
October 05, 1989
Home Address: Lower Lubo, TanudanKalinga
E-mail Address: fp_akawilma@yahoo.com
Education:
Elementary:
Name of School:
Lubo Elementary school
Address:
LuboTanudan, Kalinga
Year Graduated:
2002
Secondary:
Name of School:
St.William’s Academy
Address:
Bulanao, Tabuk, Kalinga
Year Graduated:
2006
College: Name of School:
Benguet State University
Address:
La Trinidad Benguet
Degree and
Year of Graduation:
Doctor of Veterinarry Medicine
2012
Organization and Extra-curricular Activities:
Rodeo Club Philippines (Batch STEROIDS 2006) 2010-2011: Whips
2011-2012: Examiner
BSU-Highland Cowboys/Cowgirls:
2010-present
Clinicians Club
2010-2012
Girl Scout of the Philippines
2009-2010
PUCTIYAO, FLORENCE G. April2012. Bacterial Isolation from Repacked Milk Powder
sold at Baguio City Public Market. Benguet State University, La Trinidad, Benguet.
Adviser: Marietta Q. Amatorio, DVM
Abstract
A total of 15 samples were collected from randomly selected stalls and grocery stores
selling repacked milk powder at Baguio City Public Market and were examined to determine the
microbial load and the presence of coliform organism.
The coliform bacteria isolated and their frequencies are: Enterobacteraerogenes, 33%;
Escherichia coli, 47%; Klebsiellapneumoniae, 47%;Proteus mirabilis,13%. Further, Standard
Plate Count results range from 1.6M-9.3M .The repacked milk samples have microbial load
exceeding the maximum limit.
TABLE OF CONTENT
Page
Bibliography.................................................................................................... i
Abstract........................................................................................................... i
Table of Content.............................................................................................
ii
CHAPTER No .I. INTRODUCTION............................................................ 1
Background of the Study....................................................................
1
Importance of the Study......................................................................
3
Objectives of the study.......................................................................
4
Place and Time of the Study.............................................................. 4
CHAPTER No. II.REVIEW OF LITERATURE...........................................
5
CHAPTER No.III. MATERIALS AND METHOD....................................... 8
CHAPTER No .IV.RESULTS AND DISCUSSION...................................... 14
CHAPTER No. V.SUMMARY, CONCLUSION AND DISCUSSION..........
22
CHAPTER No.VI. LITERATURE CITED....................................................
23
APPENDICES................................................................................................ 25
BIOGRAPHICAL SKETCH..........................................................................
43
Chapter No. I
INTRODUCTION
Background of the Study
Milk is considered as nature's most complete food because of its unique
composition. It’s fairly suitable proportion of protein, carbohydrates, vitamins and
minerals and other substances enhance and supplement the nutrient in poor quality foods.
However, the Philippine dairy industry remains in its infancy stage despite the effort of
the government and private sectors to uplift the local milk production. It comprises only
2% of the total domestic supply. Thus to meet the local demand, the dairy industry
heavily relies on importation. The consumption also varies according to the form of the
product. By volume, milk powder accounted for 41% of the importation. These Imported
dairy products are often sold as reprocessed and repacked goods. Over the year, milk
consumption has been increasing. Based on food consumption surveys of the Food and
Nutrition Research Institute (FNRI), per capita milk consumption averaged 8.7 kg/year
for the period of 1958-1969. This figure increased to 12.6 kg in 1978 and 16.0 kg in
1982(PCARRD, 1991).
Powdered milk is also a common item in UN food aid supplies, fallout shelters,
warehouses, and wherever fresh milk is not a viable option. It is widely used in many
developing countries because of reduced transport and storage costs (reduced bulk and
weight, no refrigerated vehicles). As with other dry foods, it is considered nonperishable,
And is favored by survivalists, hikers, and others require nonperishable, easy-to-prepare
food (Anonymous, 2012).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
2
Perhaps the most difficult problem in handling and processing dairy products is
their highly perishable nature. There have been a number of food borne illnesses resulting
from the ingestion of dairy products made with milk that was not properly pasteurized or
was poorly handled causing post-processing contamination. In order to avoid excessive
spoilage, many dairy products were developed as a result of attempts to prolong storage
life. These include Milk powder, evaporated milk, condensed milk, and cheeses. All have
prolonged storage life but still subject to contamination (Anonymous, 2011). Further,
Brock, 1978 stated that Milk contains relative few bacteria when it leaves the udder of a
healthy cow.Since milk is such a good growth medium, it is better to sterilize or
pasteurize before drinking, because such bacteria rapidly multiply. Though bacteria can
be retarded or killed during pasteurization, consumers should be aware of contacting
disease brought by improper food processing that leads to microbial contamination that
cause spoilage, unpleasant odors and tastes and a food poisoning outbreak in a
community due to ingestion of the bacteria and or their toxins present in the
food(Quinn,1994).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
3
Importance of the Study
Powdered milk is a convenient form of nutritious milk that doesn't require
refrigeration. It is easily reconstituted and also an easily transported and stored dairy
ingredient. It is an economical source of dairy solids. They are required for the
manufacture of infant formulae and recipes for baked goods where adding liquid would
render the product too thin. Powdered milk is also widely used in various sweet and
certain products such as milk candy, chocolates, caramel candy, pulvoron, pastillas,
snacks, soup, ice-cream, ice crumble toppings, coffee creamer, sauces and etc.
(Anonymous, 2011).
Downeset al., 2001 stated that powdered milk must be considered sensitive
products from a public health aspect because they are often consumed after reconstitution
without additional heating. It is well established that dried milk can be a source of
foodbourne illness because of contamination. Probably the two most significant sources
of contamination are the dairy utensils or the milk-contact surfaces. Unaseptic packaging
from workers hands, Equipments such as fillers used in handling and processing as well
as storage of these products may also serves as possible source of contamination and
pathogen. Normally the packaging material contributes very little to the total microbial
load in the finished product. If the dairy utensils or the milk-contact surfaces are
inadequately cleaned, sanitized and dried, bacteria may develop in large number. Under
very poor conditions these sources may increase the bacterial content of milk by millions
per millilitre. Undesirable bacteria from these sources include coliform bacteria, lactic
Streptococci, and thermodurics, those which survives pasteurization. These bacteria grow
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
4
well in milk hence endanger its keeping quality (Frazier and Westhoff, 1988).
It is therefore important to determine the bacterial load of the product to evaluate
the safetiness of the food for the consumer.
Objectives of the Study
The study was conducted to:
1. determine the microbial load in repacked milk powder, and
2. determine presence of coliform organism.
Place and Time of the Study
Fifteen samples were collected from three different stalls and two grocery stores
selling repacked milk powder at Baguio City Public Market and were brought to the
Microbiology Laboratory Room, College of Veterinary Medicine, Benguet State
University, La Trinidad, and Benguet for processing from August 2011 to March 2012.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No. II
REVIEW OF LITERATURE
Microorganisms are associated with most of the food we eat. These
microorganisms can cause spoilage and lend up unpleasant odours and flavours to the
milk. Also, milk can harbour pathogenic microorganism which produces disease if
microbial populations are permitted to proliferate extensively. Certain microorganism
produces toxic substances which results in food poisoning outbreak (McCuenet al.,
1988).
Davis, 1962 reported that Bacterial standards for milk varies from place to place,
some areas require that milk must contain not over 100,000 bacteria per ml, however,
United State public health suggested a certain regulation of not over 200,000 bacteria per
ml. Federal regulation defined in the pasteurized milk ordinance mandate that milk
standard plate count should not be greater than or equal to 10 4 .While in the Philippines,
the Standard Microbial Plate Counts shall not exceed 50,000 cfu/ml and coliform count
should be less than or equal to ten cfu/ml (Anonymous,2007).
Wholesome, high quality milk and hygienic control of milk is indeed necessary,
these involves many carefully controlled steps, regardless of whether the end result is
filled milk or powdered milk. These hygienic controls involves such matter as clean
milking operation and equipment, hygienic processing of the milk and proper storage and
delivery to the end consumer. The genera comprising the coliform bacteria are principally
E.coli, Enterobacter and klebsiella. Test for this bacteria are designed to measures the
quality of the procedures for the handling and processing of the milk after the
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
6
pasteurization process (McCuen, 1988).
Coliforms are facultative anaerobes with an optimum growth at 37° C. Coliforms
are indicator organisms; they are closely associated with the presence of pathogens but
not necessarily pathogenic themselves. They also can cause rapid spoilage of milk
because they are able to ferment lactose with the production of acid and gas, and are able
to degrade milk proteins. Mabesaet al., 1989 as cited by Dulay, 2008 reported that
Coliform group of microorganism has been used as index of the sanitary quality of foods
and sometimes as an indicator of sanitary conditions existing in environment or kitchen
during handling and processing.Further, Enumeration of these organisms in heat
pasteurized foods can be used to assess the adequacy of heating process designed to
inactivate bacteria and in assessment of overall quality of food and hygienic conditions
present during food processing (Downeset al., 2011).
Escherichia coli is a natural inhabitant of the large intestine and small intestine of
all mammals(Quinn et al.,1994).It causes coliform mastitis in cow and sow In dairy
cows, the source of infection is faecal contamination of the skin of the mammary gland
and relaxation of the teat sphincter following milking increases vulnerability to infection.
In man it produces fatal intestinal infections in infants.E.coli is excreted in the faeces and
can survive in faecal particles, dust and water for week or months. Because of its
prominence as a normal intestinal bacterium in most mammals, most strains of E.coli are
easy to detect in food and water and currently one of the indicator bacteria to monitor in
water, foods and dairy products (Nester, 2004).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
7
Enterobacteraerogenes is very common normal faecal flora and can be isolated
from wound. It causes coliform mastitis in cattle and occasionally part of the mastitis-
detritus-agalactia (MMA) syndrome in sows (Quinn et al., 1994).
Klebsiellapneumoniae is normally found in the normal flora of the mouth, skin
and intestine. It is present in the environment, vegetation, soil and in faeces. It also causes
urinary tract and wound infection, otitis, meningitis and pneumonia in man (Hyde, 1989).
Proteus mirabilis is found in faeces of mammals and environment. It causes
urinary tract infections in man, dogs and horses. Also associated with otitis externa in
dogs and diarrhoea in young mink and lambs, calves, goats and pups (Hyde, 1989).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.III
MATERIALS AND METHOD
Materials
Materials used in the study were: 15 milk powder samples from different stalls
and grocery stores in Baguio City Public Market, Trypticase Soy Broth(TSB),phosphate
Buffer, Xylose Lysine Deoxycholate Agar(XLD), Triple Sugar Iron Agar, Simmon Citrate
agar, Indole Broth, Methyl Red Broth,Vogues-Proskauer Broth, Ehrlich’s Reagents,
Methyl Red Indicator, Crystal Violet, Gram’s Iodine ,Acetone Alcohol, Safranin, mineral
oil, glass slides, microscope, sterilizing oven, autoclave, electric stoves, Quebec colony
counter, bacteriological incubator, weighing balance, denatured alcohol, alcohol lamp,
Erlen Meyer flasks, graduated cylinder, inoculating loops and needles beaker, petridishes
,pipettes, spatulas, stirring rods, test tubes, test tubes racks, test tube brush, wire baskets,
distilled water, staining can, marking pen and masking tapes.
Methodology
Pre-experimental phase
Three stalls and two grocery stores selling repacked milk powder at Baguio
CityPublic Market were identified as collection sites (Figure No.1). In each stall, one
fourth kilogram of milk powder sample was collected in each collection time with five
days interval until a total of 15 samples were completed.Each sample was properly
labelled.The materials used in this study were autoclaved and sterilized.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
E
C
Danes
Bakery
.
1
.
D
g
st.2
n
g
st.3
n
g
st
rd
y
an
Ka
kaya
Kaya
Abanao
ZanduetaSTt
Fish and
p
meat sec
Maharlika
Hillto
B
A
Magsaysay Rd
Figure No.1 Schematic Diagram of the sites of Collection of Samples
*A,B,C,D,E-Sites of collection
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
10
The media were prepared based on the manufacture’s specification(Appendix D).
Experimental Phase:
Six grams of each sample in powdered form was reconstituted in 10millilitres
(ml) phosphate buffered- distilled water at PH of 7.2. Using a pipette, one ml of the
reconstituted milk powder was poured in Trypticase Soy Broth that served as cultivation
medium for isolating bacteria and was incubated for 24 hours at 37oC.
Standard Plate Count (Figure No.2)
A series of dilution was made on a five sterile test tubes containing nine ml of
distilled water as diluents. One ml from the incubated sample in TSB was delivered to
test tube one containing nine ml of sterile distilled water thereby giving a dilution of
1:100.It was mixed by swirling. Another one ml from test tube one was pipetted and
delivered to test tube two containing nine ml distilled water giving a dilution of 1,000.It
was then mixed by swirling. From test tube two, one ml was transferred to test tube three
containing nine ml of distilled water giving a 1:10,000 dilutions. The same procedure was
repeated to test tube four and five with a dilution of 1:100, 000, and 1:1,000,000
respectively.0.1 ml from each test tube was delivered to a sterile plate containing 20ml of
melted nutrient agar tempered at 43-45 0C. It was gently swirled to mix and then allowed
to cool and solidify. It was placed in an upside down position inside the incubator at 37c
for 24 hours. The plate was examined for visible microbial growth. Counts were made
from each plate using a Quebec colony counter. The average count from all plates were
calculated and recorded to obtain the microbial count per ml of sample.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Milk Sample
1 ml
Sterile Distilled Water
1 ml
1 ml
9m
1 ml
1 ml
99 m
99 m
9ml
9ml
9ml
9ml
0.1ml
1:100
1:1,000
1:10,000
1:100,000
1:1,000,000
*Number of bacteria per ml= number of colonies x dilution of sample/number of plates
used.
Figure no.2 Standard Plate Count Method
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
12
Test for the Presence of Coliform Organism (Figure No.3)
A loopful of samples from TSB was inoculated to XLD and was incubatedat 370C
for 24 hours. Colony from XLD suggestive of coliform was touch using an inoculating
needle then was streaked to triple sugar iron and incubated at 37OC for 16 hours. This
was later subjected to gram staining and to biochemical tests such as Indole test, methyl
red test, vogues-proskauer test, citrate utilization test.
Milk powder sample reconstituted with
Phosphate buffer
Inoculated to TSB at 37ºc for 24 hours
Nutrient Agar Plate (SPC)
Xylose Lysine Deoxycholate (XLD)
Colony characteristic Tipple sugar Iron (TSI)
Gram staining
Biochemical tests
MR
VP
Citrate test
Indole test
Figure no.3 Schematic Diagram for the Bacterial Isolation in Milk Powder
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
13
Data Gathered
The Data gathered were the following:
1. Source of samples: Milk powder samples were taken from different stalls selling
Repacked Milk Powder at Baguio city public market (Figure no.1)
2. Mean Number of Bacteria Per ml of the Sample. This was computed by
multiplying the colonies counted on each individual plate by their
corresponding dilutions. The mean total number of the colonies will be
divided by the number of plate used where the count will be made.
3. Colony Characteristic of Coliform Bacteria. The growth of coliform bacteria was
observed according to their characteristics appearance on XLD (Appendix
A).
4. Biochemical Tests. These were based on their reaction in MR, VP, and Indole
And Citrate (Appendix A).
5. Identity of coliform organism. This was based on the colony characteristics on
XLD agar, gram staining reaction and biochemical test (Appendix B).
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.1V
RESULTS AND DISCUSSION
Table No. 1 shows the mean number of bacteria per ml of repacked milk powder
sold at Baguio City Public Market
TABLE No.1 Mean number of microorganisms per ml of sample, n=15
Sample No.
Mean
count
(1x107)
1
3.9
2 2.3
3 7.9
4 4.2
5 2.3
6 8.4
7 1.8
8 2.2
9 2.7
10
1.6
11
5.4
12
3.6
13
9.3
14
6.5
15
8.3
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
15
It shows that the Mean Number of bacteria per ml of the sample ranged from
1.6 x 107 to 9.3 x107.This indicates that all the samples are contaminated and have
exceeded the maximum count of United State Public Health Services which is 200,000
per ml (Davis,1962) and the Philippines standard which is 50,000 cfu/ml of milk sample
(Anonymous, 2007).
The very high load of Microorganism in the sample can be attributed to the
improper repacking and storage of the product .It was observed that the samples were not
properly sealed and the packaging materials were dusty and too vague. Samples were
even stored in places accessible to pest infestation such as mice and fliesand arefreely
exposed to air contaminants and passersby. Downes, 2011, reported that improper
processing and unaseptic packaging lead to contamination. Further, Frazier and
Westhoff, 1988 stated that very poor condition of the environment may increase bacterial
count of milk by millions per ml.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
16
Plate No.1. Microbial Growth in Standard Plate Count at 1x 106 Dilutions
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
17
Table No.2 shows the identity of the coliform bacteria isolated from
repacked milk powder.
Table No.2 Identity and frequency of coliform Organism in repacked milk powder,
n=15
Identity of Gram XLD TSI Indole MR VP Citrate Frequency
Micro-
stain colony
(%)
Organism
E.aerogenes gram yellow, mucoid Y/Y
+ - + + 33%
Negative and glistening
E.coli
gram yellow, large
Y/Y, + + - -
47%
Negative flat, smooth
CO2
and glistening
K.pneumonia gram yellow, mucoid,
Y/Y - +/- +/- +
47%
Negative large and
Smooth
P.mirabilisgram yellow with
R/Y, - + -/+ +/- 13%
Negative black centre
H2S
It shows that the Coliforms isolated and their frequencies
are:Enterobacteraerogenes, 33%; E.coli, 47%; Klebsiella pneumonia, 47%.And Proteus
mirabilis, 13%.
These coliform organisms thrive in the environment and in faecal particles. It was
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
mentioned earlier the condition of the samples and their storage.McCuen,
1988
reportedthat these organisms are normal flora of
f faeces. Presence of it in water and
food
18
indicates faecal contamination. All coliforms are killed by pasteurization. Presence in the
samplepost pasteurization can be attributed to improper handling, from equipments used
in handling milk, from workers hands, and during storage of
f the product as mentioned by
McCuen, 1988 and Frazier and Westhoff, 1988. Further, Pelczar, 1986 mentioned that
foods are always subject to growth of any organism considering the method by which it is
handled during processing.
Plate no.2. Growth of coliform bacteria in XLD (A) colony with black centre and
(B) Yellow colony
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
19
Plate no.3.Triple Sugaar Iron Test. A.R/R (non-coliform) B.Y/Y (typ
pical coliform)
C. R/Y/H2S (P.mirabilis)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
20
Plate no.4.Vooges-Proskauer Tests A (+) and B (-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
21
Plate No.5 Methyl Red Tests A (+) and B (-)
Plate No.6. Indole Tests A (+) and B (-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Plate no.7.Simmon Citrate Test A (+)
Chapter No. V
SUMMARY, CONCLUSION AND RECOMMENDATION
Fifteen samples were collected from randomly selected stalls and grocery stores
selling repacked milk powder at Baguio City Public Market to determine the microbial
load and the presence of coliform organism in each sample. Each sample were
reconstituted in phosphate buffer and then inoculated to TSB. One ml of each sample
from TSB was inoculated to nutrient agar for standard plate count. Further, loopful of
sample from TSB were inoculated to XLD. Growth suggestive of coliform was streaked
to TSI agar. Then subjected to Gram staining and Biochemical tests.
Results showed that the isolated coliform and their frequency were:
Enterobacteraerogenes, 37%; Escherichia coli, 47%; Klebsiellapneumoniae, 47% and
Proteus mirabilis,13%.And the mean number of microorganism per ml of sample ranged
from 1.6x107-9.3x107.
It is concluded therefore that the repacked milk powder samples are heavily
contaminated with faecal coliform.
It is recommended that repacked powdered milk should be studied further on
the presence of Gram positive bacteria, molds, yeast, most especially Staphylococcus
Sppand Salmonella where poisoning have been recorded.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Chapter No.VI
LITERATURE CITED
ANONYMOUS.2011.How to Prepare Phosphate Buffers
eHow.comhttp://www.ehow.com/how_6319495_prepare-phosphate-
buffers.html#ixzz1Uyqhey1T
ANONYMOUS.2011.How to Reconstitute Dry Milk Powder | eHow.com
http://www.ehow.com/how_5753380_reconstitute-dry-milk-
powder.html#ixzz1Uyt47rxP
ANONYMOUS.1994.FLUKA biochemika, Microbiology-culture media for general
Microbiology, for food analysis, for environmental analysis and for molecular
biology.Flukachemie AG, Buchs.Pp 16
ANONYMOUS.2007. Guidelines for microbiologicalExamination of foods....The
Standard Plate Count (SPC), also referred to as the aerobic plate count or the
total...www.foodstandards.gov.au/.../guidelinesformi.
BROCK, T.D. 1978.Basic Microbiology with Aplication.2nd edition, prentice
Hall, Inc.EnglewoodCliffs, New Jersey.Pp.500-501.
DAVIS R.F. 1962.Modern Dairy Cattle Management.prentice- hall, inc.
Englewood Cliffs, New Jersey.pP.223-224.
DOWNES, F.P. 2001.Compendium of methods for the microbiological
examination of foods .Google Books Result chapter 8.Enterobacteriaceae,
coliforms and E.coli as quality and safety
indicator.books.google.com.ph/books?isbn=087553175X. American Public
Health Association - - Medical \\
DULAY, F.O. 2008.Bacterial isolation from “Boneless Bangus” Sold at Baguio City
Public Market.Undergraduate Thesis. Benguet State University, Philippines.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
FENG, P., S.D. WEAGANT and M.A. Grant.1998 Bacteriological Analytical Manual,
8th Edition, Revision A,Chapter 4 Enumeration of Escherichia coli and the
Coliformbacteriawww.fda.gov/.../BacteriologicalAnalyticalManualBAM/
default.htm
24
FRAZIER, W.C, and D.C.WESTHOFF.1988.Food microbiology.4th edition.
McGRAW HILL Company. SingaporePp 227
NESTER, E.W., D.G. ANDERSON, C.E.ROBERTS JR., N.N.PEARSHALL and
M.T.NESTER. 2004.Microbioogy: A Human Perspective.4th Edition. McGraw-
Hill Companies.United State of America.Pp 282-283.
PEARCE, K.N. 2012."Milk Powder", Food Science Section, New Zealand Dairy
Research Institute. nzic.org.nzen.wikipedia.org/wiki/Powdered milk
PELCZAR, M.J. 1986.Microbiology 5th Edition, McGraw-hill book company, United
State of America.pp 618.
POWER,D.A and P.J. McCUEN.1988.Manual of BBL products and Laboratory
procedure.6thEdition. United State of AmericaPp.4,15-16,62-63,224,214,274,
288,307,269.
QUINN, P.J., M.E.CARTER, G.R.CARTER, and B.K MARKEY.1994. Clinical
Veterinary Microbiology.Wolfe Publishing.Spain.Pp 54 and Pp 221.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
25
Appendix A
Expected Colonial Characteristic in XLD, TSI and Biochemical Test of
Coliform Organisms.
Identity
Colonial characteristic in
TSI Biochemical
Tests
XLD
MR VP I C
Escherichia coli
Yellow colony, flat, small,
Y/Y w/
+ - +
-
smooth and glistening,
CO2
circular in outline
Enterobacteraerogenes Yellow colony, spreading
Y/Y
- +
+
+
moist, medium in size and
glistening
Klebsiellapneumoniae
Large mucoid, Yellow colony,
+
+
- +
Y/Y
raised, and entire in outline
(-)
(-)
Proteus mirabilis
Yellow colony with black
R/Y w/
+ -
- +
centre
H2S
(+)
(-)
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
26
Appendix B
Morphological Characteristic, TSI and Biochemical Test Reactions and Identity of
Coliform Bacteria
Sample TSB
XLD
Gram
TSI MR VP
SC
I Organism
stain
identified
1 turbid
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
2 turbid
Yellow
spreading
negative Y/Y - + + - Enterobacter
moist, and
rod
(+) aerogenes
glistening
Yellow,
Negative Y/Y + + + - Klebseillapneu
mucoidlarge,raised, rod
moniae
and entire in outline
3 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebsiellapneu
round and entire in rod
moniae
outline
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
4 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebseillapneu
mcoid and entire in rod
moniae
outline
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
27
5 turbid
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
6 turbid
Yellow
colonies
Negative R/Y + +
+ -
Proteus
with black center
rod
(-) (-)
mirabilis
Tiny
translucent,
Positive R/R Non coliform
raised, circular
cocci
7 turbid
Tiny
translucent,
Positive Y/Y
Non coliform
raised, circular
cocci
8 turbid
Yellow,
flat, Negative Y/Y + - +
-
Escherichia
smooth, glistening rod
(-)
coli
and circular in
outline
9
turbid
No
growth
No
growth
No
growth
10 turbid
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent,
Positive R/R Non
coliform
raised, circular
cocci
Tiny
translucent, Positive
R/R Non
coliform
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
raised, circular
cocci
11. turbid
Yellow,large,raised, Negative Y/Y + - +
- Klebsiellapneu
mucoid and entire rod
monia
in outline
Yellow,flat,smooth, Negative Y/Y + - - + Escherichia
glistening and
rod
coli
circular in outline
12 turbid
Yellow,large,raised, Negative Y/Y + + + - Klebsiellapneu
mucoid and entire rod
(+) moniae
in outline
Yellow,
flat, Negative Y/Y + -
- + Escherichia
smooth, glistening rod
(+)
coli
and circular in
outline
28
13 turbid
Yellow
spreading
Negative Y/Y - + + + Enterobactera
moist, and
rod
erogenes
glistening
Yellow,large,raised, Negative Y/Y + +
+ - Klebsiellapneu
mucoid and entire rod
(-)
moniae
in outline
14
turbid Yellow, flat,
Negative Y/Y + +
- + Escherichia
smooth, glistening rod
(-)
coli
and circular in
outline
Yellow,large,raised, Negative Y/Y + +
-
-
Klebsiellapneu
mucoid and entire rod
(-) (+)
moniae
in outline
Yellow
colonies
Negative R/Y + +
+ - Proteus
with black centre
rod
(-)
mirabilis
due to h2s
production
15 turbid
Yellow,
flat, Negative Y/Y + - - + Escherichia
smooth, glistening rod
coli
and circular in
outline
Yellow
spreading Negative Y/Y - + + + Enterobactera
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
moist, and
rod
erogenes
glistening
29
Appendix C
Gram Staining, TSI and Biochemical Test Performed
A. Gram staining
This is the differential colouring solution that distinguishes gram negative bacteria
from gram positive organisms.
Procedure:
1. Put a little amount of the colony in a dry and clean glass slide.
2. Place a small drop of distilled water at the centre of the slide and spread it evenly using
a sterile wire loop.
3. Air dry and heat fix on a flame and cool.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
4. Flood with crystal violet for 2 minute then wash with running water.
5. Drop grams iodine and let it stand for 1 minute and then rinse with running water.
6. Apply grams acetone decolourizer for 10 seconds then rinse.
7. Counter stain with safranin for 30 seconds and then rinse
8. Dry the stained smear and examine under microscope.
B.Tripple sugar iron test
Isolated colony is touched with an inoculating needle. A tube of TSI agar is stab
inoculated in the middle of the agar to within 5mm from the bottom of the tube. On the
withdrawal of the straight wire, the entire slant is streaked (right to the top) the tubes are
incubated at 37 for 16 hours.
30
C.INDOLE TEST
A loopful of the test organism will be inoculated into Indole broth for 24
hours.Ehrlich’s reagent will be added. A positive result is indicated by a Pink/Red layer
forming on top of the liquid.
D.METHYL RED TEST
Methyl broth will be inoculated with pure culture from the plate and incubated for
24-48 hour at 37 0C.Five drops of Methyl Red Indicator will be added to the broth after
incubation. A change in colour from yellow to red indicates a positive result.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
E.VOGUES-PROSKAUER TEST
Vogues-proskauer broth will be inoculated with pure culture from the plate and
will be incubated at 37 OC for 48 hours. After incubation, 0.6ml of 5%Naphtol in absolute
ethyl alcohol 0.2ml of 40% potassium hydroxide containing 2% creatine will be added.
Shake well and keep the broth undisturbed for three to ten minutes. A positive result will
appear as bright red to orange in colour.
F.CITRATE UTILIZATION TEST
A slant of Simmon’s Citrate Agar is streaked with a pure culture stock and
incubated for 24 to 48 hours at 37c.The PH indicator Bromothymol Blue, changes the
slant from green to blue if the growth and utilization occurs. It is negative if the medium
31
remains green and is positive if it turns blue.
Appendix D
Culture Media, Broth and Reagent Preparation
1. Phosphate buffer
Ingredients
Potassium dihydrogen phosphate 26.22g
Sodium carbonates
7.78
Final ph 7.2+/-0.1
Direction:
Prepare a stock solution by dissolving 34g in 1 L distilled water. Dispense and
sterilize, if desired. Store under refrigeration. Prepare a working solution by adding 1.25
ml of the stock solution to distilled water and make up to 1 L. Dispense in bottles or other
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
appropriates quantities and sterilize by autoclaving at 121OC for 15 minutes.
2. Trypticase soy broth
Ingredients:
Grams
per
litre
Pancreatic digest of casein
17.0g
Papaic digest of soybean meal
3.0
Sodium
chloride
5.0
Dipotassium
phosphate 2.5
Dextrose
2.5
Direction;
Suspend 30g of the powder in 1 litre of distilled water. Mixed thoroughly and
warm gently until solution is completed. Dispense and sterilize by autoclaving at 121C
for 15minutes.
32
3. Nutrient Agar
Standard formula
Ingredients;
grams per litre
Peptic digest of animal tissue
5
Beef
extract
1.50
Yeast
extract
1.50
Sodium
chloride
5.00
Agar
15
Final
ph
at
25c
7.4+/-0.2
Direction:
Suspend 28 grams in 1000 ml distilled water. Heat to dissolve the medium
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
completely. Sterilize by autoclaving at 15 pounds pressure (121c) for fifteen minutes.
4. Xylose Lysine Deoxycholate Agar (XLD agar)
Standard formula
Ingredient
Grams/Litre
Yeast
extract
3.0
L-lysine 5.0
Lactose 7.50
Sucrose 7.50
Xylose
3.50
NaCl
5.00
Na
Deoxycholate
2.50
Na
Thiosulphate
2.50
Ferric ammonium Citrate
0.80
Phenol
Red
0.80
Agar
15.0
Final
ph 7.4+/-0
Direction:
Suspend 56.68 grams in 1000 ml distilled water. Heat with frequent agitation until
the medium boils. Do not autoclave or over heat. Transfer immediately to a water bath at
50 0C. After cooling, pour into sterile plates. It is advisable not to prepare large volume
33
which will require prolonged heating.
5. Triple sugar iron test
Ingredients:
Grams
per
litter
Peptic digest of animal tissue
10g
Pancreatic digest of casein
(casein enzymatic hydreolysate)
10
Sodium
chloride
5
Lactose 10
Dextrose
10
Ferrous ammonium sulphate
0.2
Sodium
thiosulfate
0.2
Phenol
red
O.O25
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Agar
13.0
Direction:
Suspend 50.4 g of the powder in 1L of distilled water. Heat to boiling to dissolve
the medium completely.mix well and distribute into test tubes. Sterilize by autoclaving at
121C for 15 minutes. Cool in slanted position such that deep butts (1 inch long) are
formed.
6. METHYL RED-Vogues Proskauer Medium (buffered glucose broth)
Standard formula
Ingredients
gram/Litre
Buffered
peptone
7.00
Dextrose
5.00
Dipotassium
phosphate 5.00
DIRECTION:
Suspend 17.0 grams in 1000ml distilled water. Distribute in test tubes in10ml
amounts and sterilize by autoclaving at 15 pounds pressure (121c) for 15 minutes.
34
6A.METHYL red reagents
Ingredients
Methyl
red
(Mallinckrodt)
0.1
Ethyl
alcohol
300ml
Distilled
water
200ml
DIRECTION;
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Dissolve methyl red in alcohol before adding distilled water. Store in a colored
bottle in a cool and dry place.
7. Vogues-proskauer (VP) Reagent A
INGREDIENTS:
A-naphtol (sigma chemical Co)
5g
Ethyl alcohol (absolute)
100ml
DIRECTION:
Dissolve a-Naphtol in a small amount of ethyl alcohol and bring the volume to
100ml in a volumetric flask or cylinder. The alcohol should be almost colourless. Store in
a colored bottled and store in a dry place.
8. Vogues-proskauer (VP) reagent b
Ingredients
Potassium hydroxide (KOH)
40g
Distilled
water
100ml
35
DIRECTION:
Weigh out the KOH very quickly, since it is hygroscopic and will become caustic
when moist. Add less than 100ml water to the pellets in a flask in a cold water bath to
prevent overheating. Bring the volume to 100ml in a volumetric flaks or cylinder. Store
this reagent in the refrigerator in a polyethylene bottle or one that has been socially
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
treated for storage chemical.
9. Simmon’s citrate agar
Standard formula
Ingredients
grams/liter
Magnesium
sulphate
0.20
Ammonium dihydrogen phosphate
1.00
Dipotassium
phosphate 1.00
Sodium
citrate
2.00
Sodium
chloride
5.00
Bromothymol
blue
5.00
Agar
15.00
Final
ph
(at
25C)
6.8+/-0.2
DIRECTIONS:
Dissolve ingredients. Distribute to tubes or vials. Before medium solidifies,
incline tubes to obtain 4-5 cm slant and 2-3cm butts. Sterilize by autoclaving at 15
pounds pressure (121c) for 15 minutes. Final ph, 6.8+0.2.
36
10. Indole test:
Ingredients;
gram
Pancreatic digest of casein
20.g
Disodium
phosphate
2
Dextrose
1
Agar
1
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Potassium
nitrate
1
Direction
Suspend 254g of the powder in 1 L distilled water. Add 2 g of agar for use as a
motility medium.mix thoroughly. Heat and boil for 1 minute until completely dissolved.
Dispense in regular testubes, filling them half full. Sterilise by autoclaving at 118 to 121C
for 15 minutes.
Kovacs reagent for indole test
Amyl
alcohol
22.5
ml
P-dimethylaminobenzaldehyde 1.5g
Hcl
7.5ml
Ehrlich’s reagent for Indole test
Ingredients
Paradimethylaminobenzaldehyde
2
grams
Ethyl
alcohol
190ml
Hydrochloric
acid
40ml
Directions:
Dissolve paradimethylaminobenzaldehyde in 190 ml of 95% ethyl alcohol first.
37
Then gently add hydrochloric acid to the moisture by pouring on the sides of the
Volumetric flask.Appendix E
Program of Activities of Thesis Performance
Bacterial Isolation from Repacked Milk Powder Sold at Baguio City Public Market
Date
Activity
October 15, 2011
Purchase of Reagents and Chemicals. Barrow instruments
and washes them. Sterilization of Glassware.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
October 16, 2011
First collection of sample and preparation of TSB and
phosphate buffer stock solution.
October 17, 2011
The sample will be reconstituted with phosphate buffer
then inoculated to TSB.Incubate for 24 hours at 37 OC.
Preparation of XLD.
October
18,
2011
Loopful of mixture from TSB will be streak
d into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mixed
with nutrient agar for standard plate count.
Preparation
of
TSI.
October 19, 2011
Reading and recording of results from XLD ad nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of media for biochemical tests.
October 20, 2011
Reading and Recording of results from TSI.
Gram
staining
Inoculation for biochemical test
2nd collection of sample
October 21, 2011
reading of results from Biochemical test
Preparation
of
TSB
and Phosphate buffer
October 22, 2011
the sample will be reconstituted with phosphate buffer then
inoculated to TSB.incubate for 24 hours at 37 OC.
Preparation of XLD.
October 23, 2011
Loopful of mixture from TSB will be streaked into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mix
with nutrient agar for standard plate count.
38
Preparation
of
TSI.
October 24, 2011
Reading and recording of results from XLD ad nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of media for biochemical tests.
October 25, 2011
Reading and Recording of results from TSI.
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Gram
staining
Inoculation
for
biochemical tests
October 26, 2011
reading of results from Biochemical test
Preparation
of
TSB
and Phosphate buffer
3rd collection of sample
October 27, 2011
the sample will be reconstituted with phosphate buffer then
inoculated to TSB.Incubate for 24 hours at 3OC.Preparation
of XLD.
October 28, 2011
Loopful of mixture from TSB will be streaked into XLD.
Preparation of Nutrient Agar.1 ml from TSB will be mix
with nutrient agar for standard plate count.
Preparation
of
TSI.
October 29, 2011
Reading and recording of results from XLD and nutrient
Agar.
Growth Suggestive of coliform will be streaked to TSI
Preparation of Media for Biochemical tests.
October 30, 2011
Reading and Recording of Results from TSI.
Gram
staining
Inoculation
for
Biochemical
Test
October
31,
2011
Reading of Results from Biochemical tests
Bacterial Isolation from Repacked Milk Powder sold at Baguio City
Public Market / Florence G. Puctiyao. 2012
Appendix F1
Appendix F2
Appendix G1
APPLICATION FOR ORAL DEFENSE
Benguet State University
COLLEGE OF VETERINARY MEDICINE
LaTrinidad, Benguet (2601)
APPLICATION FOR FINAL ORAL DEFENSE
Name: PUCTIYAO, FLORENCE G.
Student Number: 0608051
Thesis Title: BACTERIAL ISOLATION FROM REPACKED MILK POWDER
SOLD AT BAGUIO CITY MARKET
Date and Time of Examination: February, 29 2012, 9:00 AM
Place of Examination: CVM Accreditation Room
MEMBERS OF THE EXAMINING COMMITTEE
Name
of
Member
Signature
CRISELDA S. BATTAD, DVM, MS
___________________
JOSEPH A. DIANSO, DVM
___________________
EDLYN MAE N. CIANO, DVM
___________________
Recommending
Approval:
MARIETTA
Q.AMATORIO
Adviser
Approved:
JOSEPH
A.
DIANSO,
DVM
Dean
Appendix G2
Benguet State University
COLLEGE OF VETERINARY MEDICINE
LaTrinidad, Benguet (2601)
REPORT OF RESULTS OF FINAL ORAL DEFENSE
Name: PUCTIYAO, FLORENCE G.
Student Number: 0608051
Thesis Title: BACTERIAL ISOLATION FROM REPACKED MILK POWDER
SOLD AT BAGUIO CITY MARKET
Date of Examination: February, 29 2012
Place of Examination: CVM Accreditation Room
Name and Signature of Examinees:
Remarks
MARIETTA Q.AMATORIO, DVM
Adviser
Date
JOSEPH A. DIANSO DVM
Panel
Date
EDLYN MAE NABUSANCIANO,DVM
Panel
Date
CRISELDA SILVESTRE-BATTAD, DVM,MS
Department chairman
Date
JOSEPH A. DIANSO DVM
Dean
Date
BIOGRAPHICAL SKETCH
Name:
FLORENCE GOLWINGON PUCTIYAO
Date of Birth:
October 05, 1989
Home Address: Lower Lubo, TanudanKalinga
E-mail Address: fp_akawilma@yahoo.com
Education:
Elementary:
Name of School:
Lubo Elementary school
Address:
LuboTanudan, Kalinga
Year Graduated:
2002
Secondary:
Name of School:
St.William’s Academy
Address:
Bulanao, Tabuk, Kalinga
Year Graduated:
2006
College: Name of School:
Benguet State University
Address:
La Trinidad Benguet
Degree and
Year of Graduation:
Doctor of Veterinarry Medicine
2012
Organization and Extra-curricular Activities:
Rodeo Club Philippines (Batch STEROIDS 2006) 2010-2011: Whips
2011-2012: Examiner
BSU-Highland Cowboys/Cowgirls:
2010-present
Clinicians Club
2010-2012
Girl Scout of the Philippines
2009-2010
Document Outline
- Bacterial Isolation from Repacked Milk Powdersold at Baguio City Public Market
- Bibliography
- TABLE OF CONTENT
- INTRODUCTION
- REVIEW OF LITERATURE
- MATERIALS AND METHOD
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATION
- LITERATURE CITED