The Effects of PH Water Level to the Microflora of Spoiled Infant Formula

__________

Submitted to

Centro Escolar University – Makati

In partnership with

Centro Escolar Integrated School

__________

A thesis submitted in partial fulfillment of the

Requirements for the Inquiries, Investigations, and Immersion

__________

Lupac, Mhykel Anghelo B. *

Ferrer, Karen Gail J.

Atienza, Louella Jean L.

Devara, Eunice Gabrielle B.

Sibayan, Marc Vincent I.

Valencia, Ardis Faye M.

January 12, 2018

 CHAPTER I

Background of the Study

This Chapter present the different essential elements: The introduction which contains the

rationale, the literature of the study, and foundation, the general and specific problem, the scope

and delimitations that identifies the major variable the sub-variables and the indicators,

significance of the study which states the beneficiary of the further study and the corresponding

benefits, and lastly is the notation.

Introduction

Milk is the nutrient liquid secreted by the mammary glands of female mammals, which the

female’s ability to produce milk is one of the defining characteristics of mammals. The early

lactation milk is known as colostrum and carries the maternal antibodies to the baby. It can help

reduce the risk of various diseases in both the mother and baby that could benefit them in long

term. (USDA National Nutrient Data Base for standard references, 2005).

Exclusive Breastfeeding is recommended to mothers worldwide for the child's first six

months to achieve optimal growth, development and health. Thereafter, they should be given

nutritious complementary foods and continue breastfeeding up to the age of two years or beyond

(WHO, 2011). Although experts believe breast milk is the best nutritional choice for infants,

breastfeeding may not be possible for all women. For many, the decision to breastfeed or

formula feed is based on their comfort level, lifestyle, and specific medical situations which for

mothers who are unable to breastfeed or who decide not to, infant formula is a healthy alternative

that Formula Milk also provides babies with the nutrients they need to grow and thrive. (Ben-

Joseph, E. P., 2015).

 Infant Formula Milk is a manufactured food designed and marketed for feeding to babies

which is based on a human mother’s milk nutrition. However, there are significant differences in

the nutrient of the Human Milk and the Infant Formula, but with that deficiencies, manufacturers

often innovate and add new ingredients to infant formulas in an attempt to mimic the

composition or performance of human milk (Wells, J.C.K., 1996). The popularity of Infant

Formula Milk increased because it has a longer shelf life than liquid milk and does not need to be

refrigerated due to its low moisture content and it can be accessible and convenient to mothers

(USDA National Nutrient Data Base for standard references, 2005).

Food spoilage, and food poisoning are caused by numerous types of microorganisms that

are influenced by many factors, that includes environment for which food was produced and

acquired, the microbiological quality of the food in its processed state, the sanitary conditions

under which the product was handled and processed and the adequacy of subsequent packing,

handling, and storage conditions in maintaining the microflora at low level (Jay, 1986).

Food spoilage can be defined as “any sensory change (tactile, visual, olfactory or flavor)”

which the consumer considers to be unacceptable. Spoilage may occur at any stage along food

chain. Spoilage may arise from insect damage, physical damage, indigenous enzyme activity in

the animal or plant tissue or by microbial infections. Most natural foods have a limited life.

Perishable foods such as fish, meat and bread have a short life span. Other food can be kept for a

considerably longer time but decomposes eventually. Enzymes can bring about destruction of

polymers in some foods while chemical reactions such as oxidation and rancidity decompose

others but the main single cause of food spoilage is invasion by microorganisms such as moulds,

yeast and bacteria. In case of mould spoilage a furry growth covers the food and it becomes soft
and often smells bad. Bacterial contamination is more dangerous because very often food does

not look bad even though severely infected, it may appear quite normal. The presence of highly

dangerous toxins and bacterial spores is often not detected until after an outbreak of food

poisoning, laboratory examination uncovers the infecting agent (Seema, R., 2015).

Chemical reactions that cause offensive sensory changes in foods are mediated by a

variety of microbes that use food as a carbon and energy source. These organisms include

prokaryotes (bacteria), single-celled organisms lacking defined nuclei and other organelles, and

eukaryotes, single-celled (yeasts) and multicellular (molds) organisms with nuclei and other

organelles. Some microbes are commonly found in many types of spoiled foods while others are

more selective in the foods they consume; multiple species are often identified in a single spoiled

food item but there may be one species (a specific spoilage organism, SSO) primarily

responsible for production of the compounds causing off odors and flavors. Within a spoiling

food, there is often a succession of different populations that rise and fall as different nutrients

become available or are exhausted. Some microbes, such as lactic acid bacteria and molds,

secrete compounds that inhibit competitors (Gram, L., 2002).

 Statement of Problem

This study aims to identify the differences of the Microflora of different Spoiled Infant

Formula subjected to different PH Water Level. This study is to be conducted during the second

semester of S.Y 2017-2018 in Centro Escolar Integrated School-Makati.

With regard to this, the researchers came up with following questions to be answered:

Specific:

I. What are the bacterial species can be found in Spoiled Infant Formula?

II. Are there any significant differences to the Microflora of Spoiled Infant Formula which are

subjected to different PH Water Level in terms of:

a. Colony Shape

b. Colony Elevation

c. Colony Margin

d. Colony Size

e. Colony density?

III. Which among the PH Water Level have the most bacterial growth?

Hypothesis

There are significant differences among the Microflora of Spoiled Infant Formula

subjected to different PH Water Level in terms of bacterial growth in terms of:

a. Colony Shape

b. Colony Elevation

c. Colony Margin

d. Colony Size
e. Colony density

Scope and Delimitation

This study would primarily deal with the differences of the Effects of the PH Water Level

to the Microflora of Spoiled Infant Formula.

The selection of Infant formula would be based on the availability of the product in the

Philippine market, and the recommended age intake of Infant Formula from 6-12 months. The

brand of infant formula would be 'Lactum 6-12 months' . The infant formula would be prepared

in a controlled environment for the sake of limiting the effects of extraneous variables.

For the selection of Water sample, researchers would also base it on the availability of the

products in the Philippine Market. The Water sample would be Fresh Tap Water, Boiled Tap

Water, Distilled Water, and Sterilized Water. These Water would be tested with PH Meter to

indicate the variation of the PH Level.

For the culture media that would be used for isolation of bacterial species are Potato

Dextrose Agar, Standard Method Agar, Nutrient Agar, and Violet Red Bile salts Agar would be

acquired from Asiagel Corporation that are prepared under sterile and controlled conditions

following Laboratories Conda Spain procedures, standards, and protocols. Furthermore, these

microflora would be then subjected to purification to attain a more reliable and accurate bacterial

growth.

Identification of isolated bacteria would be crucial because this is also the part of gathering

of data. Several tests such as Gram Staining Technique, Motility Test, Coagulase Test, Catalase

Test, Oxidase Test, Indole Test, Voges-proskauer Test, Glucose-Gas Fermentation, Lactose
Fermentation, and Sucrose Fermentation. These tests would help the researchers identify the

biochemical characteristics of the bacterial species.

This study aimed to prove if the use of different PH Water Level would have effects on the

growth of bacterial species in the Spoiled Milk Formula.

Significance of the Study

Every parent heed about their health and feeding. The best source of babies feeding is

their mother’s milk thru breastfeeding. But in few cases the mother naturally fails to fulfill the

breast feeding requirement of baby due to disease factor or hormonal imbalance. But in some

cases the mother itself is not interested in breastfeeding, reasons manifested, the working females

can not provide proper feeding to their baby. In rare cases modern world females do not feed

their babies just to maintain their apparently beauty. So they follow the infant formula that is

available in market and suggested by their nutritionist or doctor. In this generation, the Infant

milk powders are generally considered as product of good microbiological quality with no risk of

spoilage, but several factors may contribute to change its physical and chemical properties which

reduce shelf-life and thus its commercial value (Cousins et al., 1987). Although the micro-

organisms in infant milk cannot grow due to its low moisture content, microbes can grow during

the preparation of the milk formula, which is then subjected to different external factors with

different effects such as temperature, the water used, time, and others.

The study would be beneficial to mothers in order to carefully assess the condition of

every formula milk that they would be giving to their baby. In this case they would be limiting

their exposure of the formula milk outside the environment and also lessening the exposure of

their young to pathogenic bacterias that could cause diseases.

 The future researchers will also be benefited by the results of the study which could be

used as a core areas of researching in the discipline of food microbiology that could be reiterated

using distinctive variables that would result to an implicit research topic to their own study.

Furthermore, enrichment of the variables is recommended for them to have revolutionized results

to their study.
 CHAPTER II

Review of Related Literature

Food spoilage is defined as any change that makes a product unacceptable for human

consumption. It can result from different causes involving physical-chemical and biochemical

changes and also microbial growth and activity (Huis in’t Veld, 1996). Spoilage microbes are

often common inhabitants of soil, water, or the intestinal tracts of animals and may be dispersed

through the air and water and by the activities of small animals, particularly insects. It should be

noted that with the development of new molecular typing methods, the scientific names of some

spoilage organisms, particularly the bacteria, have changed in recent years and some older names

are no longer in use. Many insects and small mammals also cause deterioration of food but these

will not be considered here.

Yeasts

Yeasts are a subset of a large group of organisms called fungi that also includes molds

and mushrooms. They are generally single-celled organisms that are adapted for life in

specialized, usually liquid, environments and, unlike some molds and mushrooms, do not

produce toxic secondary metabolites. Yeasts can grow with or without oxygen and are well

known for their beneficial fermentations that produce bread and alcoholic drinks. They often

colonize foods with a high sugar or salt content and contribute to spoilage of maple syrup,

pickles, and sauerkraut. Fruits and juices with a low pH are another target, and there are some

yeasts that grow on the surfaces of meat and cheese.

 There are four main groups of spoilage yeasts: Zygosaccharomyces and related genera

tolerate high sugar and high salt concentrations and are the usual spoilage organisms in foods

such as honey, dried fruit, jams and soy sauce. They usually grow slowly, producing off-odors

and flavors and carbon dioxide that may cause food containers to swell and burst. Debaryomyces

hansenii can grow at salt concentrations as high as 24%, accounting for its frequent isolation

from salt brines used for cured meats, cheeses, and olives. This group also includes the most

important spoilage organisms in salad dressings (Mandrell, R.E., et. Al. 2006). Saccharomyces

spp. are best known for their role in production of bread and wine but some strains also spoil

wines and other alcoholic beverages by producing gassiness, turbidity and off- flavors associated

with hydrogen sulfide and acetic acid. Some species grow on fruits, including yogurt containing

fruit, and some are resistant to heat processing (Elez-Martinez, P., et. Al., 2004)

Candida and related genera are a heterogeneous group of yeasts, some of which also

cause human infections. They are involved in spoilage of fruits, some vegetables and dairy

products (Casey, G.D., 2003). Dekkera and Brettanomyces are principally involved in spoilage of

fermented foods, including alcoholic beverages and some dairy products. They can produce

volatile phenolic compounds responsible for off-flavors (Couto, J.A., et. Al., 2005)

Molds

Molds are filamentous fungi that do not produce large fruiting bodies like mushrooms.

Molds are very important for recycling dead plant and animal remains in nature but also attack a

wide variety of foods and other materials useful to humans. They are well adapted for growth on

and through solid substrates, generally produce airborne spores, and require oxygen for their

metabolic processes.
Most molds grow at a pH range of 3 to 8 and some can grow at very low water activity levels

(0.7–0.8) on dried foods. Spores can tolerate harsh environmental conditions but most are

sensitive to heat treatment. An exception is Byssochlammys, whose spores have a D value of 1–

12 minutes at 90 degrees centigrade . Different mold species have different optimal growth

temperatures, with some able to grow in refrigerators. They have a diverse secondary

metabolism producing a number of toxic and carcinogenic mycotoxins. Some spoilage molds are

toxigenic while others are not (Pitt, J.I., 2006)

Spoilage molds can be categorized into four main groups: Zygomycetes are considered

relatively primitive fungi but are widespread in nature, growing rapidly on simple carbon sources

in soil and plant debris, and their spores are commonly present in indoor air. Generally they

require high water activities for growth and are notorious for causing rots in a variety of stored

fruits and vegetables, including strawberries and sweet potatoes. Some common bread molds

also are zygomycetes. Some zygomycetes are also utilized for production of fermented soy

products, enzymes, and organic chemicals. The most common spoilage species are Mucor and

Rhizopus. Zygomycetes are not known for producing mycotoxins but there are some reports of

toxic compounds produced by a few species.

Penicillium and related genera are present in soils and plant debris from both tropical and

Antarctic conditions but tend to dominate spoilage in temperate regions. They are distinguished

by their reproductive structures that produce chains of conidia. Although they can be useful to

humans in producing antibiotics and blue cheese, many species are important spoilage organisms,

and some produce potent mycotoxins such as patulin, ochratoxin, citreoviridin, penitrem.

Penicillium spp. cause visible rots on citrus, pear, and apple fruits and cause enormous losses in
these crops. They also spoil other fruits and vegetables, including cereals. Some species can

attack refrigerated and processed foods such as jams and margarine. A related genus,

Byssochlamys, is the most important organism causing spoilage of pasteurized juices because of

the high heat resistance of its spores.

Aspergillus and related molds generally grow faster and are more resistant to high

temperatures and low water activity than Penicillium spp. and tend to dominate spoilage in

warmer climates. Many aspergilla produce mycotoxins: aflatoxins, ochratoxin, territrems,

cyclopiazonic acid. Aspergilli spoil a wide variety of food and non-food items (paper, leather,

etc.) but are probably best known for spoilage of grains, dried beans, peanuts, tree nuts, and

some spices.

Other molds, belonging to several genera, have been isolated from spoiled food. These

generally are not major causes of spoilage but can be a problem for some foods. Fusarium spp.

cause plant diseases and produce several important mycotoxins but are not important spoilage

organisms. However, their mycotoxins may be present in harvested grains and pose a health risk.

Bacteria

Spore-forming bacteria are usually associated with spoilage of heat-treated foods because

their spores can survive high processing temperatures. These Gram-positive bacteria may be

strict anaerobes or facultative which is capable of growth with or without oxygen. Some spore-

formers are thermophilic, preferring growth at extreme temperatures with as high as 55 degrees

centigrade. Some anaerobic thermophiles produce hydrogen sulphide (Desulfotomaculum) and

others produce hydrogen and carbon dioxide (Thermoanaerobacterium) during growth on

canned/ hermetically sealed foods kept at high temperatures, for example, soups sold in vending

machines.

Other thermophiles (Bacillus and Geobacillus spp.) cause a flat sour spoilage of high or

low pH canned foods with little or no gas production, and one species causes ropiness in bread

held at high ambient temperatures (Boor, K., 2006). Mesophilic anaerobes, growing at ambient

temperatures, cause several types of spoilage of vegetables (Bacillus spp.); putrefaction of

canned products, early blowing of cheeses, and butyric acid production in canned vegetables and

fruits (Clostridium spp.); and "medicinal" flavors in canned low-acid foods (Alicyclobacillus)

(Chang & Kang, 2003). Psychrotolerant sporeformers produce gas and sickly odors in chilled

meats and brine-cured hams (Clostridium spp.) while others produce off-odors and gas in

vacuum-packed, chilled foods and milk (Bacillus spp.).

Lactic acid bacteria (LAB) are a group of Gram-positive bacteria, including species of

Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, some of which are useful in

producing fermented foods such as yogurt and pickles. However, under low oxygen, low

temperature, and acidic conditions, these bacteria become the predominant spoilage organisms

on a variety of foods. Undesirable changes caused by LAB include greening of meat and gas

formation in cheeses (blowing), pickles (bloater damage), and canned or packaged meat and

vegetables. Off-flavors described as mousy, cheesy, malty, acidic, buttery or liver-like may be

detected in wine, meats, milk, or juices spoiled by these bacteria. LAB may also produce large

amounts of an exopolysaccharide that causes slime on meats and ropy spoilage in some

beverages.
 Pseudomonas and related genera are aerobic, gram-negative soil bacteria, some of which

can degrade a wide variety of unusual compounds. They generally require a high water activity

for growth (0.95 or higher) and are inhibited by pH values less than 5.4. Some species grow at

refrigeration temperatures (psychrophilic) while other are adapted for growth at warmer, ambient

temperatures. Four species of Pseudomonas (P. fluorescens, P. fragi, P. lundensis, and P.

viridiflava), Shewanella putrefaciens, and Xanthomonas campestris are the main food spoilage

organisms in this group. Soft rots of plant-derived foods occur when pectins that hold adjacent

plant cells together are degraded by pectic lyase enzymes secreted by X. campestris, P.

fluorescens and P. viridiflava. These two species of Pseudomonas comprise up to 40% of the

naturally occurring bacteria on the surface of fruits and vegetables and cause nearly half of post-

harvest rot of fresh produce stored at cold temperatures. P. fluorescens, P. fragi, P. lundensis, and

S. putrefaciens cause spoilage of animal-derived foods (meat, fish, milk) by secreting lipases and

proteases that cause formation of sulfides and trimethylamine (off-odors) and by forming

biofilms (slime) on surfaces (55;73). Some strains are adapted for growth at cold temperatures

and spoil these foods in the refrigerator.

Enterobacteriaceae are gram-negative, facultatively anaerobic bacteria that include a

number of human pathogens (Salmonella, E. coli, Shigella, Yersinia) and also a large number of

spoilage organisms. These bacteria are widespread in nature in soil, on plant surfaces and in

digestive tracts of animals and are therefore present in many foods. Erwinia carotovora is one of

the most important bacteria causing soft rot of vegetables in the field or stored at ambient

temperatures.
 Biogenic amines are produced in meat and fish by several members of this group while

others produce off-odors or colors in beer (Obesumbacterium), bacon and other cured meats

(Proteus, Serratia), cheeses (several genera), cole slaw (Klebsiella), and shell eggs (Proteus,

Enterobacter, Serratia). Temperature, salt concentration, and pH are the most important factors

determining which, if any, of these microbes spoil foods.

Many Gram-negative bacteria, including pseudomonads and enterobacteriaceae, secrete

acyl homoserine lactones (AHLs) to regulate the expression of certain genes, such as virulence

factors, as a function of cell density. These AHL quorum-sensing signals may regulate

proteolytic enzyme production and iron chelation during spoilage of some foods (Rasch, M., et.

Al., 2005)

Other bacteria are associated with spoilage of chilled, high protein foods such as meat,

fish, and dairy products. They may not be the predominant spoilage organisms but contribute to

the breakdown of food components and may produce off-odors.Most species are aerobic

although some grow at low oxygen levels and may survive vacuum packaging,and one

(Brochothrix) is a facultative anaerobe. Some examples include: Acinetobacter and

Psychrobacter, which are predominant bacteria on poultry carcasses on the processing line and

have been isolated from a variety of spoiled meat and fish. Acinetobacter grows at a pH as low

as 3.3 and has been detected in spoiled soft drinks. These two genera do not produce extracellular

lipases, hydrogen sulfide, or trimethylamine (fishy odor) and so are considered to have a low

spoilage potential.

Alcaligenes is a potential contaminant of dairy products and meat and has been isolated

from rancid butter and milk with an off-odor. These bacteria occur naturally in the digestive tract
of some animals and also in soil and water. Flavobacterium is found widely in the environment

and in chilled foods, particularly dairy products, fish, and meat. It uses both lipases and proteases

to produce disagreeable odors in butter, margarine, cheese, cream, and other products with dairy

ingredients. Moraxella and Photobacterium are important constituents of the microflora on the

surface of fish. Photobacterium can grow and produce trimethylamine in ice-stored, vacuum-

packaged fish. Brochothrix has been isolated from meat, fish, dairy products and frozen

vegetables. During spoilage, it produces odors described as sour, musty, and sweaty (Russo, F.,

et. Al., 2006)

Spoilage of dairy products

Milk is an excellent medium for growth for a variety of bacteria (Deeth, H.C., 2008).

Spoilage bacteria may originate on the farm from the environment or milking equipment or in

processing plants from equipment, employees, or the air. LAB are usually the predominant

microbes in raw milk and proliferate if milk is not cooled adequately. When populations reach

about 106 cfu/ml, off-flavors develop in milk due to production of lactic acid and other

compounds. Refrigeration suppresses growth of LAB and within one day psychrophilic bacteria

(Pseudomonas, Enterobacter, Alcaligenes and some spore-formers) grow and can eventually

produce rancid odors through the action of lipases and bitter peptides from protease action

(Dogan, B., 2003).

Pasteurization kills the psychrophiles and mesophilic bacteria (LAB), but heat-tolerant

species (Alcaligenes, Microbacterium, and the sporeformers Bacillus and Clostridium) survive

and may later cause spoilage in milk or other dairy products. Immediately following

pasteurization, bacterial counts are usually <1000 cfu/ml. However, post-pasteurization

contamination of milk, particularly with Pseudomonas and some Gram-positive psychrophiles

does occur (Stevenson, R.G., et. Al., 2003). Spoilage problems in cheese can sometimes be traced

to low quality milk but may also result from unhygienic conditions in the processing plant. Hard

and semi-hard cheeses have a low moisture content (<50%) and a pH ~5.0, which limits the

growth of some microbes. Some coliforms and Clostridium spp. that cause late gas blowing can

grow under these conditions as can several species of molds. Other psychrotrophs produce

biogenic amines, particularly tyramine, during storage of cheese (Novicka et al., 2003). Soft

cheeses with a higher pH of 5.0–6.5 and a moisture content of 50–80% may be spoiled by

Pseudomonas, Alcaligenes, and Flavobacterium. Clostridium sporogenes has been found in

spoiled processed cheese, where it produces gas holes and off-flavors (Meyer, J.D., et. Al.,

2003).Yeasts and molds are the main spoilage organisms found in cultured milks (yogurt, sour

cream and buttermilk) because the higher acidity in these products inhibits many bacteria

(Mayoral, M.B., et. Al., 2005). Pseudomonas, yeasts and molds can spoil butter and “light”

butters. Since the light butters have higher moisture content than butter, they can support more

microbial growth. Cream may become rancid when populations of Pseudomonas and

Enterobacter proliferate.
 CHAPTER III

Methodology

This chapter details out the research methodology for the present study. It explains the

research objectives and a suitable methodology to achieve those objectives. The objective of this

study is to identify the Microflora of Spoiled Infant Formula subjected to different PH Water

Level. This chapter discusses the kind of research, the research design that researchers would use,

and materials and the methods in this research.

Kind of research

The researchers will use an experimental research to identify the Microflora of Spoiled

Infant Formula subjected to different PH Water Level. This refers to a research in which one

variable is manipulated by the researcher to determine any impact on other variable. The element

being manipulated by the researchers is known as the independent variable, whereas the change

resulting from implementation of the independent variable is the dependent variable (Tierrey, S.,

2008).

Researchers identified the PH Water Level as independent variable which could have an

impact to overall result of the dependent variable . The Spoiled Infant Formula is the dependent

variable in terms of bacterial growth.

Research design of the study

After identifying the variables in the problem situation, the next step is to design the

research in a way that the requisite data can be gathered and analyzed to arrive at a result. A

proper planned research design is important in order to ensure the accuracy, and confidence of

the study.
 This experimental research will use the Randomized Block Experimental Design

specifically Within-Subject Design. In this design the PH Water Level would be classified into

subgroups called blocks. These blocks would homogenized the results to have an accurate and

unbiased data.

BLOCK I BLOCK II BLOCK III BLOCK IV

Legend:

I. PH Water Level: Spoiled Infant Formula 1

II. PH Water Level: Spoiled Infant Formula 1

III. PH Water Level: Spoiled Infant Formula 1

IV. PH Water Level: Spoiled Infant Formula 1

Materials and Methods of the Research

Culture Media

The following culture media would be used during the study:

I. Violet Red Bile Agar with Lactose

 Violet Red Bile Agar with Lactose is a selective medium used for the isolation,

detection, and enumeration of coli-aerogenes bacteria in water, milk, other dairy

products, and also from clinical samples. The coliform group consists of several

genera of bacteria belonging to the family Enterobacteriaceae. The appearance

of the prepared petri dish is reddish purple and slightly opalescent gel. Numerous

bacteria could be cultured in this petri dish such as Enterobacter aerogenes,

Escherichia coli, Salmonella enteriditis, and Staphylococcus aureus.

II. Standard Method Agar

Standard Method Agar or Plate Count Agar is recommended for the determination of

plate counts of microorganisms in food, dairy, water, waste water and also from

clinical samples. The appearance of the prepared petri dish is slightly light yellow.

Numerous bacteria can be cultured in this petri dish such as Bacillus subtilis,

Enterococcus faecalis, Escherichia coli, Lactobacillus casei, and Staphylococcus

aureus.

III. Potato Dextrose Agar

Potato Dextrose Agar is recommended for the isolation and enumeration of yeasts

and molds from dairy and other food products. The appearance of the prepared petri

dish is light amber. Numerous bacteria can be cultured in this petri dish such as

Candida albicans, Aspergillus brasiliensis, Aspergillus fumigatus, and

Saccharomyces cerevisiae.

IV. Nutrient Agar

 Nutrient Agar is used for the cultivation of less fastidious microorganisms. The

appearance of the prepared petri dish is light yellow. Numerous bacteria can be

cultured in this petri dish such as Escherichia coli, Pseudomonas aeruginosa,

Salmonella Typhi, Staphylococcus aureus, and Streptococcus pyogenes.

Collection of the Specimen

A total of 4 samples would be collected from the Spoiled Infant Formula which would be

transferred immediately in sterile containers for bacteriological investigation.

Methods of Isolation, and Identification of Bacteria

I. Primary Isolation

All samples would be cultured in different agar plates with the use of sterile standard

loop and to be incubated to a standard temperature of 37 degrees centigrade.

II. Examination of Cultures

Examination of all cultures on solid media was performed for detection of growth,

pigmentation, colonial morphology as well as changes in the media. Plates which showed visible

growth were subjected to further bacteriological tests while those which did not show visible

growth were incubated for further 48 hours and then discarded if no growth was detected.

III. Purification of Cultures

The primary isolates would be subcultured in Nutrient Agar in order to attain pure

colonies for the accuracy and reliability of the bacteria growth.

IV. Identification of Isolated Bacteria

Gram Stain Technique

Gram Stain Technique is the most important step in identification of bacteria. This

differentiate bacteria into two types which are the Gram Positive and the Gram Negative. In
examining the bacteria, Gram Positive bacteria would appear violet, while the Gram Negative

bacteria would appear pink. (Al-mohanna, M. T., 2016)

Motility Test

This test is done to help differentiate species of bacteria that are motile from non-motile.

Basically means that Motile bacterias have the ability to propel themselves through liquids by

means of flagella. Motility media contains tryptose, sodium chloride, agar, and color indicator.

(Al-Mohanna, M. T., 2016)

Coagulase Test

Coagulase Test is used to differentiate positive Staphylococcus aureus to negative

Staphylococci S. aureus which produces two form of coagulase, the bound and free. Bound

coagulase or clumping factor, is bound to the bacterial cell wall and reacts directly with

fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in

fibrinogen of the plasma to precipitate on the staphylococcal cells, causing the cells to clump.

Free coagulase however, produced extra-cellularly by the bacteria that causes the formation of a

clot when S. aureus colonies are incubated with plasma (Pharm, M., Kavitha, R., 2016)

Catalase Test

This test demonstrate the presence of enzyme catalase in the organism. The enzyme

catalase mediates the breakdown of hydrogen peroxide in to oxygen and water. The presence of

the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen

peroxide, and the rapid effervescence of oxygen bubbles occur (Pharm, M., Kavitha, R., 2016).

Oxidase Test

Oxidase test is used to determine the presence of bacterial cytochrome oxidase enzyme

using the oxidization of substrate tetramethyl-p-phenylenediamine dihydrochloride to indophenol

a dark purple colored end product. A positive test indicates a dark purple color, and for the

negative test no color would be visible (Al-Mohanna, M. T., 2016).

Indole Test

Indole Test is performed to determine the ability of the organism to split triptophan

molecule into indole. Indole is one of the metabolic degradation product of the amino acid

tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and

deamination tryptophan with the production of indole, pyruvic acid, and ammonia. This test help

to differentiate species of the family Enterobacteriaceae (Pharm, M., Kavitha, R., 2016).

Voges-proskauer Test

Voges-proskauer test helps to determine the ability of the organisms to produce neutral

trsl end product acetyl-methyl-carbinol or acetoin from glucose fermentation. Positive test would

appear pinkish red color, and for the Negative test, it would appear Yellowish in color (Al-

Mohanna, M. T., 2016).

Lactose Fermentation

Lactose fermentation identifies the ability of the bacteria to ferment lactose as a carbon

source which also helps to differentiate species of the family Enterobacteriaceae. A Positive test

would indicate a yellowish color, and for the Negative test, there would be no change in color or

somewhat reddish (Pharm, M., Kavitha, R., 2016).

Sucrose Fermentation

Sucrose fermentation is done to help differentiate species of the family

Enterobacteriaceae. This tests the bacteria's ability to ferment sucrose and the production of acid

end-product. A Positive result would appear yellowish color, and for the Negative result, there
would be no change in color or between the tone of reddish to none (Pharm, M., Kavitha, R.,

2016).

Glucose-Gas Fermentation

Glucose-Gas fermentation helps to determine species of the family Enterobacteriaceae

which tests the ability of the bacteria to ferment glucose and produce gas and or an acid end-

product. A Positive test would indicate a yellow result, and for the Negative test, there would be

no color change or little to reddish color (Pharm, M., Kavitha, R., 2016).

Statistical Treatment

One-way ANOVA would be used to determine the significant differences among the

samples, which would be backed up with the p-value to rationalize the significance of the results.

The researchers will compute for the mean of the number of samples that inhibits growth of

different bacterial species to find the value to be indicated each block.

Bibliography