I

BACTERIAL CONTAMINATION OF POWDER MILK

By: Ragia Hussein Elhussein Mohammed

B.Sc. Faculty of Science Al Neelain University

Supervisor: Dr. Khalid Mohammed Suleiman

A thesis submitted to the University of Khartoum in 

Partial Fulfillment of the Requirements for Master of Science in Microbiology 

  

Department of Microbiology Faculty of Veterinary Medicine 

University of Khartoum August 2007 

  

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DEDICATION

To Soul of my Father

To my dear Mother

To my dear Husband

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IV

Abstract 

  The present study was carried out to isolate, identify and

to determine the extent of contamination of milk powder. Sixty

powered milk samples were collected from retail shops in

Alkalakla, Alsagana, Alhag Yousif, Ombeda, Dar Alsalama and

Shambat.

Samples were cultured on blood and MacConkeys agar

and isolated bacteria were identified by primary and

secondary methods. Bacterial growth was demonstrated in

(56.67%) powdered milk samples.

Results of present investigation showed that powdered

milk is contaminated with numerous species of spore- forming

aerobic bacilli which include Bacillus cereus (7.5%) and

B.amyloiquefacients (50%), B.mycoides. B.firmest,

B.pantothenicus, B.pumilus, B.megaterium (2.5%) B.licheniformis

(12.5%) and B.thuringiensis (10%).

The highest rate of bacterial isolation was documented in

powdered milk samples collected from Dar Alsalam (35%) and

the least was detected in samples obtained from Ombada

(2.5%).

The viable bacterial counts of powdered milk sampled

from three area ranged between 1.2x102 – 3.5 x 1010 CFU/gm.

V

ةـــــــــــــــالخلاص

. المجففنأجريت الدراسة لعزل وتعريف الأنواع البكتيرية التي تلوث اللب

لات التجارية الصغيرة في عينة من اللبن المجفف من المح) 60(تم جمع

.مناطق الكلاكلة، السجانة، الحاج يوسف، أم بده، دار السلام و شمبات

م تزريع هذه العينات في وسط اجار الدم واجار الماكونكي و عرفت البكتريا ت

عينات اللبن . المعزولة باستخدام الصفات المزرعية والاختبارات الكيموحيوية

).%56.67(المجفف التي نمت بها البكتريا

نتائج البحث أوضحت أن اللبن المجفف ملوث بعدد من السلالات البكتيرية

-:يات والتي شملت الأنواعلجنس الصو

B.cereus (7.5%), B.amyloiquefacients(50%), B.mycoides. B.firmest, B.pantothenicus, B.megaterium (2.5%) B.licheniformis (12.5%) and B.thuringiensis (10%).

عينات التي اعلي معدل لعزل البكتريا من عينات اللبن تم توضيحها في ال

بة سجلت في العينات من منطقة واقل نس%) 35(جمعت من منطقة دار السلام

%).2.5(بده ام

نتائج العد البكتيري الحي أوضحت أن عينات اللبن المجفف تحتوي على أعداد

وحدة مكونة لمستعمرة 1.2x102 – 3.5 x 1010مقدرة من البكتيريا تتراوح بين

.في الجرام

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VII

VIII

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Introduction

The world is faced with a problem of food shortage and milk products

are considered as a partial solution for this problem in developing countries.

But these products are vulnerable to spoilage by certain microorganisms,

some of which are beneficial and others are harmful to human beings.

The number and type of microorganisms present in foods are influenced

by many factors, that include the general environment for which food was

obtained, the microbiological quality of the food in its processed state, the

sanitary conditions under which the product was handled and processed and

finally the adequacy of subsequent packing, handling and storage conditions

in maintaining the flora at low level (Jay, 1986).

Food spoilage and food poisoning by microbes have been known since

1880 ( Jay,1970) , and there were many food borne diseases that have been

referred to as food poisoning , such as brucellosis , scarlet fever , typhoid

fever , diphtheria and others were associated with consumption of milk .

Banwart (1981) reported that for contamination control of the food, the

microbial load must kept as low as possible and the main sources of

contamination must be importantly known.

Numerous studies have documented that powder milk could

contaminated by bacteria. In national institute of public health it was

reported that on spray drying of milk artificially contaminated with Bacillus

cereus spores (In't veld, 1993).

Study performed in New Zealand for sample of milk powder revealed

that it's contaminated by Bacillus licheniformis and Bacillus subtilis

(Ronimus et al., 2006).

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Anthoer study of sample from different countries were examined, the

dominated isolates was Anoxybacillus flevithermus followed by Bacillus

licheniformis (Rucket et al., 2004). A previous study also demonstrated that

Bacillus cereus isolated from powdered milk produce diarrhoeal enterotoxin

(Reves et al., 2007).

The objectives of this study to isolate and identify the bacterial species

contaminating powder milk and to determine the extent of this

contamination by estimating the viable bacterial count.

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CHAPTER ONE

LITRATURE REVIEW

1.1 Milk:

1.1.1 Definitions:

Milk is the nutrient liquid secreted by the mammary glands of female

mammals. The female 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 new born. It can the reduce the risk

of many diseases in both the mother and new born (USAD National Nutrient

Data Base for standard references, 2005).

The milks of all species are complex biological fluids ـ containing a wide

variety of different constituents and possessing unique physical

characteristics (Robinson and Phil, 1985)

1.1.2 Milk Nutrition:

Milk is the source of high quality protein ـ calcium - Vitamins A and D ـ

riboflavin ـ other B vitamins and phosphorus (USAD National Nutrient Data

Base for standard references, 2005).

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1.2 Powdered milk:

One of the methods of preserving various foodstuffs is by drying them,

and this by depriving microorganisms of the water necessary for their

growth (Bylund, 1995).

Powdered milk has a far longer shelf life than liquid milk and does not need

to be refrigerated due to its low moisture content (USAD National Nutrient

Data Base for standard references, 2005).

1.2.1 History:

Powdered milk was first made in 1802 by Russian Osip Krichevsky. It

is found abundantly in many developing countries because of reduced

transport and storage costs. Like other dry foods it is considered

nonperishable and is favored by survivalists and other people in need of

nonperishable easy to prepare foodstuffs (USAD National Nutrient Data

Base for standard references, 2005).

1.2.2 Use of dry milk:

Powdered milk was first produced as an infant food. It’s use has spread

into every industry manufacturing food products in which normal milk is

used. It is used in the baking industry in candy and ice cream manufacture

(Eckles and Combs, 1980).

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1.2.3 Processing the powdered milk:

Drying means that the water in a liquid product (milk) is removed, so

that the product acquires a solid form. The water content of milk powder

ranges between 2-5 and 5% and no bacteria growth occurs at such low water

content. Drying extends the shelf life of the milk, simultaneously reducing

its weight and volume and thus reduces the cost of transport and storage of

the product (Bylund, 1995).

Commercial methods of drying are based on heat being applied to the

product, the water is evaporated and removed as vapor and the residue is the

milk powder.

Two principal methods are used for drying milk in the dairy industry,

the spray drying and roller drying.

In spray drying the milk is first concentrated by evaporation and then

dried in spray tower. During the first stage of drying the excess water in free

form between the particles of the dry solids is evaporated in the final stage,

the water in pores and capillaries of the solid particles is also evaporated.

In roller drying the product will be significantly affected by the heat,

if this drying is carried out in such a way that milk particles are in contact

with the hot heat transfer surface, the powder may then contain charred

particles which impair its quality (Bylund, 1995).

1.2.4 Micro flora of dried milks: In the case of roller dried milk, micrococci , aerobic spore formers and

Sarcina spp. predominate in the mesophilic range , whilst aerobic spore

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formers make up the microbial population in the thermophilic range . The

severe heating, which the milk receives during the drying operation in the

rollers, reduces the bacterial content to a low level and only heat resistant

spores survive the process (Robinson and Phil, 1985).

In spray dried milk the microflora made up of thermoduric

micrococci, thermoduric streptococci and corynebacteria .

Recently, there has been a move towards the ultra ـ high temperature

heat treatment of milk for processing into powdered milk, the results is near

sterile powder with only causal post heat treatment contaminants being

present. In addition, inlet air temperatures in the spray drying operation have

risen, but even temperatures above 200º C fail to remove all spore formers

(Robinson and Phil, 1985).

1.3 Bacterial contaminating powdered milk:

1.3.1 Genus Bacillus:

1.3.1.1 Description:

Species of the genus Bacillus are mainly Gram-positive rods motile

(some non motile forms occur) and non acid fast. They produce heat

resistant spores under aerobic conditions (Gordon et al ., 1973 ) .

The term bacillus in a general sense has been applied to cylindrical or

rods like bacteria. The largest species are about 2µm across by 7µm long

and frequently occur in chains (Darland and Brock, 1971).

Most bacilli are aerobic, some species are facultatively anaerobes, and

usually oxidase variable, and catalase positive. Species of the genus differ in

the manner in which they attack sugars (Clause and Berkeley, 1986).

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Bacillus species are widely distributed in the environment mainly

because of their highly resistant endospores. In soil endospores of Bacillus

anthracis, prototype of the genus, can survive for more than 50 years able to

tolerate extremely adverse conditions such as desiccation and high

temperatures (Quinn et al., 2002).

Most strains of Bacillus are not pathogenic for humans and only infect

them incidentally. A notable exception is Bacillus anthracis , which causes

anthrax in humans and domestic animals and Bacillus thuringientsis causes

disease in insects (Terranova and Blake , 1978 ) .

Bacillus cereus is known to cause a variety of nongastrointestinal

diseases as well as two different types of food poisoning (kotiranta et al.,

2000).

Many of the earlier reports of systemic disease caused by B. subtilis

are difficult to interpret because of incorrect species identification and many

of these isolates were probably B. cereus. Occasional reports have

implicated B. thuringiensis, B. alvei, B. circulans, B. licheniformis , B.

macerans,, B. pumilus, B. sphaericus and B. subtilis in systemic and

gastrointestinal disease. B. coagulans, B. sphaericus, B. macerans and B.

subtilis have caused septicemia (Turnbull et al ., 1990).

1.3.1.2 Habitat:

Bacillus species are ubiquitous, inhibiting soil, water, and airborne

dust (Schnepf et al .,1998). Thermophilic and psychrophilic members of the

genus can grow at temperatures ranging between (5 – 58) º C and can

flourish at extremes of acidity and alkalinity ranging pH 2–10 (Gordon et al

., 1973), therefore , Bacillus species can be recovered from a wide variety of

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ecologic niches . Some species may be part of the normal intestinal

microbiota of humans and other animals (Elmer et al ., 1997).

Most Bacillus species encountered in the laboratory are saprophytic

contaminants or members of the normal flora (Smith et al., 1952).

1.3.1.3 Bacillus cereus:

It is an aerobic spore forming rod normally present in soil, dust, water

and spices (Wagner and Jr., 2000). It's optimum temperature for growth is

30º C, with minimum temperature of 10º C and maximum of 49º C the pH

range for growth is 4.9 to 9.3 (Frazier and Westhoff , 1978).

Bacillus cereus is a widely spread saprophyte that infects bovine

udder causing gangrenous mastitis (Carter, 1975). Numerous surveys on

food and food ingredients have indicated a high percentage of samples

contain B. cereus. It is widely distributed in nature and food supply (Frazier

and Westhoff, 1978). Extremely large numbers of viable cell of B. cereus

must be ingested to develop signs and symptoms of food borne disease. The

mechanism of the pathogenicity involves the lyses of the cell in the intestinal

tract and the resulting release of toxin (Frazier and Westhoff, 1978). It is

suggested that the food environments influenced enterotoxin production by

B. cereus ( Raevuori et al ., 1978 ) . B. cereus toxin caused food poisoning,

the toxin is produced during sporulation (Chessburgh, 2000; Turnbull,

1986), but the relation between sporulation and enterotoxin production is

unclear (Turnbull, 1986). Spore forming and psychrotrophic properties

enable B. cereus to survive pasteurization as well as to grow in milk at

refrigerated storage conditions (Coghill and Juffs, 1979).

Food poisoning strains of B. cereus produce four different

enterotoxins two protein complexes , hemolysin BL (HBL) and

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nonhemolytic enterotoxin and two enterotoxic proteins (Lund et al ., 2000).

Hemolysin BL has been shown to lyse sheep erythrocytes and elicit a

vascular permeability reaction in rabbits (Glatz et al., 1974). It has been

suggested that hemolysin BL is responsible for the diarrheal food poisoning

syndrome (Beecher et al., 1995). Reasntly Awed alkareem (2007)

demonstrated that 80% of B. cereus isolated from minced meat in Khartoum

state were enterotoxigenic as shown by the rabbit deal loop test.

1.3.1.3.1 Pathogenicity of Bacillus cereus: Food brone gastroenteritis syndrome is caused by the emetic and the

diarrheal entrotoxin. The diarrheagenic toxin is heat labile and its production

is favored over the pH range 6-8.5, and in one study no toxin was found at

pH 5. The toxin is sensitive to trypsin and pepsin and induces diarrhea (Jay,

2000).

The emetic enterotoxin (vomiting type) is heat and pH stable and it is

sensitive to trypsin and induced vomiting (Jay, 2000).

Diarrheal type is rather mild, with symptoms developing within 6 to12

h and the symptoms consist of nausea (with vomiting being rare) cramps like

abdominal pains, tenesmus and watery stool. Fever is generally observed

(Jay, 2000) and the symptoms usually last for 12 to 24 h ( Rhodehamel and

Harmon, 2001) . The similarity between this syndrome and that of

Clostridum perfringens food poisoning has been noted (Jay, 2000).

The emetic type is characterized by an acute attack of nausea and

vomiting occurs within 1 to 5 h after consumption of contaminated foods.

Diarrhea is not a common feature in this type of illness (Rhodehamel and

Harmon, 2001).

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The symptoms of this type of food poisoning paralled those caused by

Staphylococcus aureus food borne intoxication (Todar, 2005).

Many foods can be expected to contain small number of B. cereus

because it is such a common environmental contaminant. Harrigan and

McCance (1976) reported that at intermediate concentration of B. cereus in

food may represent a potential hazard because some proliferation has

occurred.

In outbreak setting, diagnosis is confirmed by performing quantitative

culture with selective media to estimate the number of organisms present in

the suspected food. Generally more than 105 organisms per gram of the

incriminated food are required to preduce symptom of B. cereus food

poisoning. Diagnosis is also confirmed by isolation of organisms from the

stool of two or more ill persons and not from stools of controls. Enterotoxin

testing is valuable but may not be widely available (Konuma et al ., 1998).

Mode of transmission through ingestion of food that has been kept at

ambient temperature after cooking, permitting multiplication of the

organisms. Outbreaks associated with vomiting have been most commonly

associated with cooked rice that has subsequently been held at ambient room

temperatures before reheating (Jay, 2000).

1.3.1.3.2 Contamination of powdered milk with B. cereus:

Dairy products are all susceptible to microbial spoilage because of

their chemical composition and nutritional value (Jay, 1986).

Bacillus cereus is a widely distributed organism in the environment

and undoubtedly the most important of the aerobic spore forming species

found in milk (Ahmed et al., 1983) .

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Bacillus cereus was the causative agent of four outbreaks of food poisoning

involving 600 person which were reported in Oslo, Norway (Hauge, 1955).

Holmes et al . (1981) reported an outbreak in which eight people

developed acute food poisoning symptoms after the consumption of a

macaroni and cheese dish. The epidemiological investigation resulted in the

incrimination of the macaroni and cheese based upon the identification of

high levels of B. cereus (108 – 109 organisms / g) within the food that was

not served. Bacteriological analysis of the ingredients identified the powder

milk as the source.

Schmitt et al . (1976) reported two cases of poisoning that resulted

from the consumption of powdered milk products and Bacillus cereus like

organisms were identified in the milk products.

1.3.1.4 Bacillus mycoides: Bacillus mycoides is one of the Bacillus cereus group (B. cereus, B.

mycoides and B. thuringiensis). Colonies of B. mycoides, after overnight

growth on Nutrient Agar plate , are large and have hairy , rhizoid , root like

out growth from the colony margin that spread over the surface of the agar

(formerly B. cereus var. mycoides) which is a non motile species (Gordon et

al ., 1973 ; Sneath , 1986) .

Bacillus mycoides is common contamination in milk and dairy products

(Coghill and Juffs, 1979; Ahmed et al., 1983).

The organism cause spoilage (Overcast and Atmaram, 1974), may

produce toxins (Gilbert et al., 1981).

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Like B. cereus, B. mycoides strains from milk have been shown to

produce diarrheagenic enterotoxin in 9 days at temperatures between 6º C to

21º C (Griffiths, 1990).

Bacillus mycoides is distinguished from B. cereus by the rhizoid colony

morphology (Claus and Berkeley, 1986).

1.3.1.5 Bacillus thuringiensis: Bacillus thuringiensis is a Gram-positive bacteria, which produces

insecticidal parasporal inclusions, is widely distributed in the soil of various

regions of the world (Delucca et al ., 1981) . This suggests that the soil is the

primary habitat of B. thuringiensis in nature, but the organism also occur in

other environments.

Previous workers reported that high-density populations of B.

thuringiensis are often retained in stored-product environments (Norris,

1969; Burges and Hurst, 1977), in diseased silkworm larvae (Shaikh et al.,

1986), in animal feed milk (Meadows et al., 1992) and on phylloplanes

(Smith and Couche, 1991).

Bacillus thuringiensis is also one of the B. cereus groups. Colonies are

large very rough and flat. Large cell with (> 1µm) that produce terminal

ellipsoid or cylindrical spores (Koneman et al., 1997). It is common

contamination in milk and dairy products (Coghill and Juffs, 1979; Ahmed

et al., 1983). The organism cause spoilage (Overcast and Atmaram, 1974),

may produce toxins (Gilbert et al., 1981).

Bacillus thuringiensis is found enterotoxin producers (Griffiths, 1990). It

has been isolated from foods, and it apparently produces a vero-cell active

toxin (Damgaard et al., 1996).

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1.3.1.6 Bacillus licheniformis: Bacillus licheniformis , a common contaminant of dairy products is a

spore former and likely to survive all industrial processing of milk, such as

the manufacture of milk powder and whey concentrate (Salkinoja-Salonen et

al ., 1999) .

Cases of B. Licheniformis food poisoning present a clinical picture

similar to that of Clostriduim perfringens food poisoning and the diarrhea

syndrome caused by B. cereus and B. subtilis, in contrast it produces an

acute onset of emetic syndrome, although a significant minority also suffer

diarrhea (Kramer and Gilbert, 1989). The toxic threshold of the B.

licheniformis extracts was ≥100 times higher than those observed for emetic-

toxin-producing B. cereus strains (Andersson et al., 1998).

Ruckert et al. (2004) examined 28 milk powder samples and found

that the dominant isolate was Anoxybacillus flavithermus followed by

Bacillus lichenifoemis.

1.3.1.7 Contamination of powdered milk with other Bacillus

spp.:

Relatively few researchers have reported the presence of food borne

illness associated with Bacillus spp. other than B. cereus. However, due to

the high degree of phylogenetic relatedness among members of this genus, a

variety of species were considered to be potentially enterotoxigenic

including Bacillus thuringiensis (Damgaard et al., 1996).

Beattie and Williams et al . (1999) reported that different Bacillus

species isolated from raw milk and from the farm environment may have the

potential to produce diarrheal enterotoxins, included Bacillus circulans,

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Bacillus Lentimorbis , Bacillus mycoides , Bacillus subtilis , Bacillus

licheniformis , Bacillus cereus and Bacillus thuringiensis .

Rowan et al . (2001) reported that different clinical and food isolates of

B. cereus, B. licheniformis, B. circulans and B. megaterium could express

diarrheal enterotoxins, hemolysins and B. cereus entrotoxins T after growth

in reconstituted infant milk formulae .

It was reported that isolates of many Bacillus spp. from veterinary

samples associated with serious nongastrointestinal infections in animals,

may carry diarrheagenic entrotoxin genes traditionally harbored by toxigenic

B. cereus and have expressed hemolysin BL enterotoxins (Rowan et al .,

2003).

Study of enterotoxin genes by PCR demonstrated the ability of B.

amyloliquefaciens , B. cereus , Bacillus circulans , Bacillus Lentimorbis ,

Bacillus pasteurii and Bacillus thuringiensis to produce toxins in a model

food system ( Phelps and Mckillip, 2002).

Consumption of entrotoxigenic Bacillus spp. at high cell densities

results in symptoms of diarrhea, with possible vomiting from a separate

heat-stable emetic toxin (Agata et al ., 1995) . Symptoms may appear 10 to

40 h following ingestion of foodstuffs contaminated with enterotoxigenic

strains. Food most often implicated in the diarrheal syndrome includes

poultry, cooked meats soups, desserts and fluid and dry milk products

(Koneman et al ., 1997). The infective dose is high (> 106 CFU / g) because

symptoms rely on the ingestion of the viable cells or spores not the

preformed toxin in criminated foods (Granum, 1997).

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1.3.2. Staphylococcus spp.:

Staphylococcus food poisoning is an intoxication caused by

enterotoxins produced by strain of Staph. aureus in preformed toxin (Payne

and Wood, 1974 ) .

This is a small, round organism that thrives everywhere in our

environment and is a leading cause of food poisoning. Staphylococci caused

food poisoning is often associated with starchy food, cheese ect, from

improper handling techniques. Staphylococci produces the toxin as a by

product of its growth (O’connell, 2002).

Staphylococcus strains must produce sufficient enterotoxin in foods to

result in food poisoning (Pereira et al., 1991). The staphylococcal food

poisoning (food intoxication syndrome ) was first studied in 1894 by Denys

and later in 1914 by Barker who produced it in himself , the signs and

symptoms of the disease were reproduced experimentally by consuming

milk that had been contaminated with a culture of Staphylococcus aureus

(Jay , 1986 ).

Stophylococcal food poisoning is one of the main food borne disease

of food borne intoxication (Todd, 1978). In Spain, statistics showed that it is

the second most common type of food – borne disease (Anoa, 1985).

Staph. aureus, Staph. hyicus , Staph. hycius subsp. hyicus , Staph.

hyicus subsp. chromogenes , and Staph. intermedius are of interest in food

microbiology (Jay, 1986). Staphtlococcal food – poisoning follows the

consumption of the food heavily contaminated with certain staphylococci,

that produce a poisonous or toxic substance in the food which when ingested

(Hobbs and Gilbert, 1979) by man and after 4 – 6 hrs results in severe

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vomiting, diarrhea, abdominal pain and cramps some times followed by

collapse but recovery is rapid (Hobbs and Gilbert, 1979 ; Jay, 1986).

The symptoms usually last one or two days and is rarely a serious threat to

otherwise healthy adults (O’connell, 2002). On the other hand, a common

type of food poisoning is staphylococcal enterotoxin (Jawez and Adel,

1990).

In 1958 and 1965, the outbreaks of staphylococcal food poisoning

occurred in the United States due to cheese (The International Commission

of Microbiological Specifications for Foods, 1980).

Sanchez (1991) reported that from 112 powdered milk samples found that 31

samples contaminated with Staph. aureus.

1.3.3. Salmonella spp.: Several infections caused by certain species of salmonella are important

as a cause of food poisoning in humans and of several diseases in domestic

animals. The term salmonellosis has been used for two main kinds of

gastrointestinal diseases in human, enteric fevers (including typhoid and

paratyphoid fevers) and gastroenteritis (Fraizer and Westhoff, 1978). The

latter is caused primarily by S. typhimurium and S. enteritidis, infection

occurs following ingestion bacteria on food, drink, fingers and other objects

(Cheesburgh, 2000).

Contamination is mainly from two sources of food products from

diseased poultry and cattle, and whole some food subsequently exposed to

infected feacal matter during food storage (mice and rats) and during food

preparation (human handlers) by hands that are not washed after using the

toilet (Davis and Samuel, 1997).

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The onset of the disease is sudden and sometime severe, producing

nausea, vomiting, diarrhea, and slight fever occur 12-30 hours after eating

infected food (Cheesburgh, 2000). Salmonella bacteria thrive at temperature

between 40 -140 º F, they are readily destroyed by cooking to 165º F and

don’t grow at refrigerator or freezer temperatures (Davis and Samuel, 1997).

Usera et al. (1996) reported that the out break of salmonella occurred in

forty-eight cases involving children due to powdered infant formula.

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CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials:

2.1.1 Media:

For the isolation and identification of bacteria contaminating milk

powder different types of media were used, including solid, semisolid and

liquid media.

2.1.1.1 Solid media:

2.1.1.1.1 Nutrient agar (Oxoid):

The medium was prepared by dissolving 28 grams of powder in 1 liter

of distilled water by boiling. The medium was sterilized by autoclaving

(121ºC for 15 minutes), cooled to 55 º C and then distributed into sterile petri

dishes 20 ml in each.

2.1.1.1.2 Blood agar:

Hundred ml of fresh, sterile, defibrinated blood was added aseptically

to 900ml of melted sterile nutrient agar at 55 º C, mixed and distributed into

sterile petri dishes 20ml in each dish.

2.1.1.1.3 MacConkeys agar (Oxoid):

Fifty two gram of the medium was dissolved in 1 liter of distilled

water by boiling. The pH adjusted to 7.4, after which the medium was

sterilized by autoclaving at 121ºC for 15 minutes, cooled to 55 º C and

distributed into sterile petri dishes 20 ml in each.

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2.1.1.1.4 Urea agar (Oxoid):

The medium was prepared by dissolving 2.4 grams of the powder in

95 ml distilled water by boiling. After sterilization by autoclaving at 115 º C

for 20 minutes the base medium was cooled to 50 º C and aseptically 5 ml of

sterile 40% urea solution were added. The pH was adjusted to 6.8 and

distributed into screw – capped bottles 10 ml each and then was allowed to

set in the slope position.

2.1.1.1.5 Simmon's citrate agar (Oxoid):

Twenty-three grams of powder were dissolved in 1000 ml distilled

water by boiling. The pH was adjusted to 7.0, and the medium was sterilized

by autoclaving at 121 º C for 15 minutes, distributed into sterile screw-caped

bottles and allowed to solidify in a slope position.

2.1.1.1.6 Starch agar:

Fifty gram of starch were triturated with water to smooth cream, and

added to the molten nutrient agar. The mixture was sterilized at 115 º C for

15 minutes and distributed into sterilized Petri dishes 20ml each.

2.1.1.1.7 Casein agar (milk agar):

Whole fresh milk was stored overnight in refrigerator. The creamy

layer was removed, and the milk was steamed for one hour and cooled in

refrigerator. Sufficient litmus solution was added to give a bluish-purple

colour and the medium was sterilized at 115 º C for 10 minutes. It was then

cooled to about 50 º C , added to double strength nutrient agar which was

melted and cooled to 50-55 ºC, mixed and distributed in to petri-dishes .

20

2.1.1.1.8 Ammonium salt sugars (ASS):

This medium consisted of ammonium phosphate (1.0 g), potassium

chloride (0.2 g), magnesium sulphate (0.2 g), yeast extract (0.2 g), agar

(2og) and bromoeresol purple (0.04 ml) . It was prepared according to

Barrow and Feltham (1993) by adding the solids to 1000 ml distilled water,

dissolved completely by boiling and sterilized at 115 º C for 20 minutes. The

medium was allowed to cool to about 55 º C and the appropriate sugar was

added as a sterile solution to give a final concentration 1%. The medium was

mixed and distributed aseptically into sterile tubes and allowed to set in the

slope position.

2.1.1.1.9 Plate count agar (Oxoid):

Seventeen point five grams of dehydrated mediums was suspended in

1 liter distilled water and boiled with frequent stirring. The medium was

sterilized by autoclaving at 121 º C for 15 min after adjusting the pH to 7.4

then distributed into petri-dishes.

2.1.1.2 Semi-Solid Media:

2.1.1.2.1 Hugh and Leifson's (O.F) medium :( Oxoid)

The medium was prepared by dissolving 10.3 grams of solids in 1 liter

of distilled water by heating, and the pH was adjusted to 7.1. Filtered

bromothymol blue (0.2% aqueous solutions) was added and then sterilized at

115 º C for 20 minutes. Sterile solution of glucose was added aseptically to

give final concentration 1%, mixed and distributed aseptically into sterile

tubes.

21

2.1.1.2.2 Motility medium: (Oxiod)

Thirteen gram of dehydrated nutrient broth was added to 4 gram of

agar and dissolved in 1 liter of distilled water by boiling , the pH was

adjusted to 7.4 , distributed in 5 ml amounts in tests tubes containing Craig-

tubes and sterilized by autoclaving at 121ºC for 15 minutes.

2.1.1.3 Liquid media:

All liquid media were prepared according to Barrow and Feltham

(1993).

2.1.1.3.1 Nutrient broth:

This medium was prepared by dissolving 13g of the medium in 1 liter

of distilled water. The pH was adjusted to 7.4, distributed into screw-capped

bottles 5 ml each and sterilized at 121 º C for 25 minutes.

2.1.1.3.2 Glucose phosphate broth (M.R-V.P medium):

Five grams of peptone and 5g of potassium phosphate were

dissolved in 1 liter of distilled water by steaming. The pH was adjusted to

7.5 and 5g of glucose was added and mixed. The medium was distributed

into test tubes 5 ml each and sterilized by autoclaving at 110 º C for 10

minutes.

2.1.1.3.3 Peptone water sugars:

Nine hundred ml of peptone water was prepared and pH was adjusted

to 7.1-7.3 before 10 ml of Andrade's indicator was added. Ten gram of the

appropriate sugar was added to the mixture, distributed into tubes 5 ml in

22

each one. The peptone water was sterilized by autoclaving at 110 º C for 10

minutes.

2.1.1.3.4 Nitrate broth:

One gram of nitrate was dissolved in 1 liter of nutrient broth,

distributed into tubes and sterilized by autoclaving at 115 º C for 15 minutes.

2.1.2. Reagents:

2.1.2.1 Hydrogen peroxide:

Hydrogen peroxide was prepared as 3% aqueous solution and used for

catalase test.

2.1.2.2 Kovac's reagent:

This reagent composed of paradimethyl-aminobenzaldehyde,

amylalcohol and concentrated hydrochloric acid. After preparation the

reagent was stored in the refrigerator at 4 º C.

2.1.2.3 Potassium hydroxide:

Potassium hydroxide was prepared as 40 % solution and used for

Voges-Proskaur (V.P) test.

2.1.2.4 Alphanaphthol solution:

It was prepared as 1% aqueous solution and also used for V.P test.

2.1.2.5 Oxidase reagent:

Tetramethyl p-phenylene diamine dihydrochloride was prepared as

1% aqueous solution and used for oxidase test.

23

2.1.2.6 Andrade's indicator:

This was prepared by dissolving 5g of acid fuchsin in 1 liter of

distilled water, and then 150 ml of alkali solution (NaOH) was added. It was

used in peptone sugar medium.

2.1.2.7 Bromothymol blue solution:

This was prepared as 0.2% w/v by dissolving 0.2g of bromothymol

blue powder in 100 ml distilled water. It was used for oxidation fermentation

(O.F) test.

2.1.2.8 Bromocresol purple solution:

Bromocresol purple solution was prepared as 0.9% solution, it was

used in ammonium salt sugar (ASS).

2.1.2.9 Physiological saline:

This was prepared by dissolving 8.5g of sodium chloride in 1000

distilled water.

2.1.2.10 Nitrate test reagent:

This reagent was composed of two solutions:

• Solution A: Sulphsnilic acid 0.33% in 5N-Acetic

acid dissolved by gentle heat.

• Solution B: Dimeyhyl-α naphthylamine 0.6% in

5N-acetic acid.

The complete reagent was used to detect nitrate

reduction.

24

2.2 Methods:

2.2.1 Sterilization:

2.2.1.1 Autoclaving:

Screw-capped bottles, rubber caps, media solution, normal saline, etc.

were sterilized in autoclave at 121 º C for 15 minutes and 110 º C for 10

minutes was used in case of sugar media .

2.2.1.2 Hot-air oven:

Glassware such as petri dishes, tubes, flasks and glass rods were

sterilized in hot air oven at 160 º C for one hour.

2.2.1.3 Disinfection:

Solution of 70% alcohol and phenolic disinfection were used for

bench sterilization.

2.2.2 Collection of samples:

Sixty samples of milk powder were collected from ten retail shops in

each of six areas which included Alklakla , Alsagana , Alhag yousif ,

Ombada , Dar Alsalam and shambat . Samples were collected in sterile

containers and plastic bags and immediately transferred to the laboratory for

bacteriological investigation.

2.2.3 Cultural methods:

2.2.3.1 Primary isolation:

One gram of each milk powder sample was suspended in 10 ml sterile

normal saline. Using sterile standard loop, 0.05 ml of each suspension was

25

striked into blood and Maconkeys agar. All plates were incubated at 37 º C

for 24 hours.

2.2.3.2 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.

2.2.3.3 Purification of cultures:

The primary isolates were subcultured on nutrient agar and blood agar.

The subculture was repeated several times until pure colonies were obtained.

2.2.4 Identification of isolated bacteria:

Identification was carried out according to the procedure described by

Barrow and Feltham (1993).

2.2.4.1 Primary Identification:

2.2.4.1.1 Preparation of smears:

Smears were prepared by emulsifying small inoculums of the bacterial

coloney in a drop of sterile normal saline on clean slide and spreading it. The

smears were allowed to dry and then fixed by gentle heating.

2.2.4.1.2 Gram's technique:

This was done as described by Barrow and Feltham (1993).

26

2.2.4.1.3 Microscopic examination of isolates:

All isolated microorganisms were subjected to microscopic

examination under oil immersion tens and the shape, arrangement and

Gram's reaction were recorded.

2.2.4.1.4 Catalase test:

This test is used to identify bacteria which produce the enzyme

catalase (Cheesbrough, 1987). A portion of the tested colony was placed on

a drop of 3% hydrogen peroxide on a clean slide using a wooden stick.

Production of air bubbles indicated a positive result.

2.2.4.1.5 Oxidase test:

A portion of the tested colony was picked using sterile bent glass rod

and rubbed on a filter paper saturated with oxidase reagent. The

development of dark purple colour within 10 seconds indicated a positive

result.

2.2.4.1.6 Oxidation – Fermentation test (O.F):

Two tubes of Hugh and Leifson's medium were inoculated with tested

organism, one of them was covered with a layer of sterile paraffin. Tubes

were incubated at 37ºC and examined daily for seven days. Fermentative

organisms produced a yellow colour on both tubes while oxidative

organisms produced a yellow colour only in the tube with out paraffin.

2.2.4.1.7 Motility test:

Motility was tested by stabbing the isolated bacteria with straight loop

wire in a semi solid medium (Craige tube method). The medium was then

27

incubated for up to 3 days at 37º C together with un inoculated media as

control. The growth of a non-motile organism was confined to the stab,

while that of a motile one spreaded out side the craige tube.

2.2.4.1.8 Sugar fermentation test:

The sugar media were inoculated with 24 hours culture of tested

organism. They were incubated at 37 º C and examined daily for up to seven

days. Acid production was indicated by the development of a pink color in

the medium and gas production was indicated by trapped air in the Durham's

tube.

2.2.4.2 Secondary Identification:

2.2.4.2.1 Indole test:

The tested organism was inoculated in peptone water and incubated at

37 º C for 48 hours. Two to three drops of Kovac's reagent were added to

culture and shaked well. Production of pink colour on the upper layer of the

reagent was considered for indole production.

2.2.4.2.2 Voges-Proskauer test (V.P):

This test was performed to detect the production of acetylmethyl

carbinol. Glucose phosphate broth was inoculated with tested organism and

incubated at 37 º C for 48 hours. 0.6 ml of alpha-naphthol solution followed

by 0.2 ml of 40% potassium hydroxide solution were added to 1 ml of

culture, mixed well and examined after 15 minutes and one hour.

Development of a bright red colour indicated a positive result.

28

2.2.4.2.3 Urease test:

The tested organisms were inoculated on a slope of urea agar medium

and incubated at 37 º C for up to 5 days. The change of colour of the medium

to red or pink indicated a positive result.

2.2.4.2.4 Citrate utilization test:

Simmon's citrate medium was inoculated with the tested organism,

incubated at 37 º C and examined daily for up to seven days. The

development of a blue colour in the medium was considered as a positive

result.

2.2.4.2.5 Starch hydrolysis test:

Starch agar plate was inoculated with test organism and incubated at

37 º C for 24 hours. The plate was flooded with lugol's iodine solution and

starch hydrolysis was indicated by clear colour less zones around colonies.

Starch which was not hydrolyzed give a blue colour.

2.2.4.2.6 Casein hydrolysis test:

Casein agar plate was inoculated with test organism and incubated at

37 º C for 24 – 48 hours. A clear zone around the colonies indicated casein

hydrolysis.

2.2.4.2.7 Nitrate reduction test:

The tested organism was grown in nitrate broth and incubated at 37 º C

for 5 days. One ml of nitrate reagent A was added followed by 1 ml of

reagent B. Development of a deep red colour was considered as a positive

29

reaction. Zinc powder was added to tubes which did not show red colour,

development of red colour in these tubes indicated that nitrate was present

and tested organism did not reduce it.

2.2.4.2.8 Viable count:

The Miles and Misra (1938) method was used to determine the

number of a viable bacteria in powder milk samples. Examined samples

were collected from three areas, Shambat, Alklakla , and Dar Alsalam . One

gram of milk powder was suspended in 9 ml of normal saline and 1ml of

suspension was serially ten – fold diluted in sterile normal saline .

30

CHAPTER THREE

RESULTS

3.1 Sampling and Bacteriology:

A total of sixty powdered milk samples were collected from Alkalakla,

Alsagana, Alhag Yousf, Ombada, Dar Alsalam and Shambat Alhela.

Samples were cultured on blood and Macconkeys agar and the cultures

were identified by primary and secondary biochemical methods. Thirty four

samples (56.67 %) showed bacterial growth.

The highest rate of bacterial isolation was documented in samples

collected from Dar Alsalam, and the least was detected in samples obtained

from Ombada (Table: 1) and (Fig: 1).

All bacterial isolates belonged to the genus bacillus, the most frequent

species was B. amyloliquefaciens (50 %), and the lest frequent were B.

mycoides, B. firmus, B. pantothenticus, B. pumilus and B. megaterium (2.5

%) (Table: 2) .

Results of the viable count indicated that the powder milk was

contaminated the ranged between 1.2×102 -3.5×103 CFD/gm. The highest

bacterial count of powder milk samples in Dar Alsalam (Table: 3).

3.2. Biochemical test:

Results of the biochemical methods used to identify isolates are shown in

table (4).

31

Table 1. Number of bacterial growth and isolates and percentage in

each source

Source No. of

samples

No. of

bacterial

growth

No. of

bacterial

isolates

Percentages

Alkalakla 10 4 4 40%

Shambat 10 7 9 70%

Alsagana 10 5 5 50%

Ombada 10 3 1 30%

Alhag yousif 10 7 7 70%

Dar Alsalam 10 8 14 80%

Total 60 34 40 56.67%

32

Table 2. Bacillus spp. isolated from powdered milk

Bacillus spp. No. of isolate Percentage

B.amyloliquefaciens 20 50 %

B.cereus 3 7.5 %

B.mycoides 1 2.5%

B.firmus 1 2.5%

B.pantothenticus 1 2.5%

B.pumilus 1 2.5%

B.licheniformis 5 12.5 %

B.coagulans 3 7.5 %

B.megaterium 1 2.5 %

B.thuringiensis 4 10 %

33

Table 3. Bacterial counts of powdered milk samples

Sample

A P C

Alkalakla

Shambat

Dar Alsalam

Sample 1 2.0×102 6.5×102 1.5×103

Sample 2 1.4×103 3.5×102 1.05×103

Sample 3 5.0×102 4.0×102 6.5×103

Sample 4 1.2×102 1.4×103 7.5×102

Sample 5 _ 5.0×102 3.5×103

APC = Aerobic Plate Count.

_ = No growth.

34

Table 4. Biochemical properties of Bacillus spp. isolated from powdered milk Test B.cereus B.mycoides B.thuringiensis B.firmus B.megaterium

Gram reaction + + + + +

Catalase + + + + +

Oxidase + + + _ _

OF F F F F F

Glucose + + + + +

Motility + _ + + +

Carbohydrates acid from Ass:-

glucose + + + + +

galactose _ ND _ _ +

raffiose _ _ _ _ _

salicin + + + _ +

xylose _ _ _ _ +

Utilization of citrate + + + _ +

Urease + + _ _ _

Vp ND ND ND _ _

Nitrate reduction + + + + _

Casein hydrolysis + + + + +

Starch hydrolysis + + + + +

35

Test B.pumilus B.licheniformis B.amiloliquefaciens B.coagluans B.pantothenticus

Gram reaction + + + + +

Catalase + + + + +

Oxidase _ _ _ _ +

OF F F F F F

Glucose + + + + +

Motility + + + + +

Carbohydrates acid from Ass:-

glucose + + + + +

galactose + + + ND _

raffiose + + + + _

salicin + + + + _

xylose + + + + _

Utilization of citrate + + + _ _

Urease _ + _ _ _

Vp + + + _ _

Nitrate reduction _ + + + +

Casein hydrolysis + + + + +

Starch hydrolysis _ + + + _

- = Negative reaction + = Positive reactio ND = Not done

Continued: table (4)

36

Fig. (1): Percentage of bacterial growth in each source

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Percentages

AlkalaklaShambatAlsaganaOmbadaAlhag yousifDar Alsalam

37

Fig. (2): Colonies of Bacillus mycoides on nutrient agar

38

Fig. (3): Colonies of Bacillus cereus on nutrient agar

39

Fig. (4): Colonies of Bacillus thuringiensis on nutrient agar

40

CHAPTER FOUR

DISCUSSION

In the present study powdered milk samples were collected from retail

shops in six areas in Khartoum state and were investigated bacteriologically

for the presence of bacterial contaminants.

Bacterial growth was demonstrated in 56.67% of the samples. All the

bacterial isolates belonged to the genus Bacillus and this is a reflection to

their wide spread and survival in the environment due to their ability to form

resistant endospores.

The Bacillus species identified in this study included B.

amyloliquefaciens , B. licheniformis , B. cereus, B. thuringienss, B.

coagulans , B. mycoides , B. firmus , B. pantothenticus, B. pumilus , and B.

megaterium . Similar findings were reported by Vaisanen et al. (1991) who

isolated a great numbers of Bacillus strains from various diary products. The

majority of their isolates (90 %) were members of B. cereus group (B.

cereus, B. mycoides , B. thuringiensis ). Results of this study also

substantiated the previous works of Coghill and Juffs (1979); Ahmed et al.

(1983) that Bacilli, especially members of the Bacillus cereus group, are

common contaminant of milk and dairy products , as well as products based

on dried milk (Singh et al ., 1980) .

Most strains of bacilli isolated from dairy products were able to grow at

or below 10º C, which is the temperature for storage of dairy products in

retail stores. Obviously cold in dairy products has favored the colonization

of the product by psychrotrophic (Vaisanen et al., 1991). Similarly, the

bacilli isolated in powdered milk samples in the present investigation might

41

be explained by the fact bacilli initially contaminated the milk powder,

which is a good medium for bacterial growth, from the environment and

under favorable humidity and temperature bacterial multiplication occurred.

The Bacillus cereus is widely distributed in nature and foodstuffs, and

was reported to be the main cause of food intoxication (Frazier and

Westhoff, 1978). In this study 7.5 % of isolated bacilli were B. cereus in this

fact might constitute a public health hazard since the resistant spores might

germinate under appropriate conditions and liberate substantial amounts of

toxins. This finding is in agreement with those of Reves et al. (2007) who

isolated this species from dried milk and also consistent with the findings of

Holmes et al. (1981) who reported the powder milk was a source of B.

cereus in an outbreak of food borne illness.

Bacillus mycoides isolated in this investigation constituted 2.5% of the

isolates. This species was shown capable of producing diarrheagenic

enterotoxin (Griffiths, 1990) hence its presence in milk powder investigated

in the present study could be hazardous. This species was isolated from raw

milk by Johnston and Bruce (1982) and from hands and nose of food

handlers by Mohamad (2007).

Study of enterotoxin genes demonstrated the ability of B.

amloliquefaciens and B. thuringiensis to produce toxin (Phelps and Mckillip,

2002). In the present study 50 % and 10 % of the isolates were B.

amyloliquefaciens and B. thuringiensis respectively but both of them have

not been reported in the available literature concerning bacterial

contaminating powder milk, however, B. amyloliquefaciens was isolated

from air by Phelps and Mckillip (2002) and B. thuringiensis was isolated

from soil by Terranova and Blake (1978), consequently powder milk might

be contaminated from these source.

42

Bacillus licheniformis extracts was found to be ≥100 times toxic than the

emetic-toxin-producing B. cereus strains (Andersson et al., 1998). 12.5 % of

the isolates was B. licheniformis and this level of contamination plus that of

B. cereus (7.5%) might pose a serious risk to the public. This species was

also isolated by Ruckert et al. (2004) from powder milk samples and by

Ronimus et al. (2006) from factory of powder milk in New Zealand.

The highest rate of bacterial contamination of milk powder was recorded

in samples obtained from Dar Alsalam, refugee camp located in Elhaj

Yousif, and this might possibly be explained by fact that poor hygienic

measures usually prevail in such camps, in addition others factors such as

prolonged storage under suboptimal condition might also contributed in

level of contamination.

Results of the viable bacterial counts of sampled milk powder was found

to range from 1.2 × 102 to 3.5 × 103 CFU/gm of powder and this count was

less than the levels proposed by the Sudanese Standards and Metrology

Organization (2006) which is in the order 2 ×104 -3 ×105 CFU/gm. The

counts were also less than the recommended levels in the USA (2001) (104 –

105 CFU/gm).

In conclusion , our study indicated that the powdered milk is

contaminated with numerous species of spore forming aerobic bacillus and

the viable bacterial counts was found ranging between 1.2×102 -3.5×103

CFU/gm.

43

Recommendation

Based on the results of this study, we recommend the following:

• Improvement of personal hygiene of sellers.

• The use of small packages of powder milk in order to reduce the

duration of exposure and hence decrease chance of contamination.

• Further studies should investigate the effect of boiling and hot

water upon the survival of spore forming bacilli in milk powder.

44

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