Infectious Microbes Found In Cows Milk Biology Essay

Milk is a healthy and nutritionary nutrient, ( pH 6.7A±0.09 ) . It is an aqueous solution of proteins, fats and saccharides with presence of many minerals and vitamins.. Milk contains comparatively few bacteriums when it is secreted from the bag of a healthy animate being. However, during milking operations it gets contaminated from the assorted countries, dairy utensils, milking machines, the custodies of the dairy cattles, from the dirt and dust. In this manner, bacteriums, barms and casts get into the milk and forms the normal vegetations of milk. The figure of contaminations added from assorted beginnings depends on the attention taken to avoid taint

Infectious bugs found in cow ‘s milk

Bacillus Cereus: These bacteriums produces toxin that causes diarrhoea and emesis. Bacillus Cereuss have heat-resistant spores and may even last pasteurisation.

Brucella: A bacterial bug normally found in unpasteurised dairy merchandises. Since there is regular return of febrility associated with the disease Brucella infection, or Brucellosis, has besides been called “ Undulatory Fever ” .

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Campylobacter jejuni: It is the most common bacteriums that causes diarrhoeal disease and the opportunity of disease increses when natural milk consumed, because the basic pH of milk neutralizes the sourness of the tummy.

Coxiella burnetii: . This bug is immune to heat and drying and can be found in cow ‘s milk. Infection causes Q febrility, a high febrility that may last up to 2 hebdomads.

E. coli O157: H7: This peculiar strain causes bloody diarrhoea and has been associated with a figure of food-borne eruptions. Normally associated with dairy cowss, microbic taint of natural milk and soft cheeses can ensue in disease.

Listeria monocytogenes: A common bacterial pathogen found in soft cheeses and unpasteurised milk. Survives below stop deading temperatures and hence withstand infrigidation. Persons who have weakened immune systems are peculiarly in unsafe, including pregnant adult females, AIDS patients, and the really immature and really old.

Mycobacterium avium races paratuberculosis: This strain of Mycobacteria can defy pasteurisation and has been associated with inflammatory intestine syndrome or Crohn ‘s disease. However, its really infection in worlds remains controversial.

Mycobacterium TB: Mycobacteria bovis is in association with ingestion of natural milk. M. bovis causes TB in cattles that passes to worlds via unpasteurised cow ‘s milk and causes a disease that is really similar to M. TB.

Salmonella: Several eruptions in recent old ages have occurred due to taint of natural milk and milk merchandises with Salmonella. Diseases symptoms include diarrhoea and high febrility.

Staphylococcus aureus: A toxin bring forthing bug that causes explosive emesis. The disease is a effect of existent nutrient poisoning from merely devouring the toxin, instead than from an existent infection.

Yersinia enterocolitica: This bug is associated with natural milk and ice pick, among other nutrients. A dislocation in sanitation and sterilisation techniques at dairy processing installations can take to taint.

Beneficial micro-organism found in cow ‘s milk.

Raw milk and other milk-based merchandises besides contain good bacteriums. Lactic acid bacteriums such as Lactococcuss, Streptococcus lactis, Lactobacillus casei and L.acidophilus, Pediococcus and Leuconostoc aid bring forth yoghurt and other dairy nutrients which have high nutritionary value play an of import function in advancing GI wellness in humans..

In Sikkim, milk is normally consumed either after boiling or fermented. Milk production is dominated by smallholder husbandmans, including landless agricultural workers, who rely chiefly on household labour to roll up and present milk to consumers and markets. So during conveyance of milk from rural countries to urban countries opportunities of taint additions.

Harmonizing to the Sikkim Cooperative Milk Producers Union Ltd. the entire milk production in the province is estimated to 31.00 M.T. ( 1000 ) per annum, The native cowss of Sikkim are Siri and Siri-type but unreal insemination and placing of alien genteelness bulls in Veterinary dispensaries and Veterinary infirmaries has influenced the native type into the crossbred. About 50 % of the population is crossbred the remainder are Siri and non-descript type. The hybrids present in province are Cattle Jersey, Jersey crossbred, Brown Swiss crossbred, Mixed-cross, Siri, Holstein.

Therefore, microbiological analysis of milk is of import to happen the grade of taint with the enunciations and numbering of index beings i.e. the coliform group of bacteriums which is defined as the index ( fecal coliform ) of suitableness of milk for imbibing ( Standard Method Committee, 1981 ) . Coliform peculiarly Escherichia coli are largely used as an index in the microbiological analysis of nutrient because these coliform bacteriums are able to turn good in a assortment of substrates and are able to use a figure of saccharides and some other organic compounds as nutrient for energy.

Therefore this survey is done to find the presence of E.coli in natural milk sample of Sikkim and their susceptibleness to assorted antibiotics.

Literature overview

Milk and dairy merchandises is an of import point in our nutrient. But these merchandises are really suited for microbic growing, therefore it becomes necessary to cognize the chemical science of milk, its spoilage, method of saving, and different dairy merchandises where bugs play a positive instead than negative function. Fresh milk is impersonal or somewhat alkalic but on turning becomes acid because of the lactic acid formed by bacterial action on milk sugar. It has a H2O content of 88 % and 12 % of solids which constitute of 4.8 % sugars, 3.5 % fats, 3.1 % protein and 0.6 % salts ( Stewart, 1978 ) . With respect to old studies it has been found that natural milk appears to be one of the favourite medium for Escherichia coli to be ( Massa et al 1997 )

MATERIALS AND METHODS

CULTURE MEDIA

NUTRIENT AGAR

Peptone 5g

Beef infusion 3g

Agar 15g

pH 7.0

ENDO AGAR

Peptone 10 g

Lactose 10 g

Dipotassium phosphate 3.5 g

Sodium sulphite 2.5 g

Basic fuchsin 0.4 g

Agar 15 g

PH 7.5

LACTOSE FERMENTATION BROTH 1X AND 2X* ( 1000 milliliter )

Beef infusion 3.0 g

Peptone 5.0 g

Lactose 5.0 g

pH 6.9

*for 2x stock usage twice the concentration of the ingredients.

BRILLIANT GREEN LACTOSE BROTH ( BGLB )

Peptone 10.0 g

Lactose 10.0 g

Bile salt 20.0 g

Brilliant green 0.0133g

PH 7.0

NUTRIENT BROTH

Peptone 5.0 g

Beef infusion 3.0 g

PH 7.0g

TRYPTONE BROTH

Tryptone 10g

Calcium chloride ( reagent ) 0.01-0.03m

Sodium chloride 5.0g

METHYL RED – VOGES PROSKAUER BROTH ( MR-VP BROTH )

Peptone 7.0g

Dextrose 5.0g

Potassium phosphate 5.0g

pH 6.9

Reagents

KOVAC ‘S REAGENT ( for sensing of indole )

p- Dimethylaminobenzaldehyde 5.0g

Amyl alcohol 75.0ml

Hydrochloric acid ( concentrated ) 25.0ml

METHYL RED SOLUTION ( for sensing of acid )

Methyl red 0.1g

Ethyl alcohol 300.0ml

Distilled H2O 200.0ml

BARRITT ‘S REAGENT ( for sensing of acetylmethylcarbinol )

Solution A:

I±- napthol 5.0g

95 % ethyl intoxicant 100ml

Solution Bacilluss:

Potassium hydrated oxide 40.0g

Distilled H2O 100.0ml

Method

Milk Sampling

For this survey entire 14 samples of natural milk were straight collected from different topographic points of Sikkim. The volume of the sample was non less than 100ml and these samples were collected aseptically in unfertile Durham bottles, sealed, labelled and stored in 4A°C.

Microbial analyses:

The microbic trials considered were standard home base count and most likely figure trial to find the presence of coliform bacteriums in Sikkim milk.

Coliform trial

To find the presence of coliform in milk sample a statistical appraisal method called the most likely figure ( MPN ) trial was followed. The most likely figure ( MPN ) trial is a multi-step check comprising of the three stairss i.e. presumptive trial, confirmed trial and completed trial. Five tubings of dual strength lactose stock and five tubings of individual strength lactose stock were inoculated with milk samples mensurating 10 milliliter, 1 milliliter, and 0.1 milliliter severally. During incubation, coliform beings produce gas. Depending upon which tubes from which milk samples display gas, an MPN index tabular array was consulted and a statistical scope of the figure of coliform bacterium was determined.

Antimicrobial susceptibleness trial

Disk diffusion method ( Bauer-Kirby method ) was used to prove the sensitiveness of E.coli that was isolated from the samples. This trial was performed against eight antibiotics and their concentrations were: Erythrocin 15mcg, amoxicillin 30mcg, ampicillin 25mcg, Chloromycetin 30mcg, tetracycillin 30mcg, penicillinG 10units, streptomycin 25mcg, nalidixic acid 30mcg ( Himedia ) .

The agar home bases were allowed to solidify. 0.1ml sample from each liquid stock were transferred to the home bases and with a aid of a spreader, civilization was uniformly spread over the agar surface. Using a unfertile forceps the antibiotic susceptibleness phonograph record was placed over the agar surface and each phonograph record was pressed gently to guarantee that the phonograph record adhere to the surface of the agar and the home bases were incubated at 37a‚’C for 48hrs. Inhibition zone and bacterial sensitiveness against the peculiar antimicrobic agent i.e. either immune or susceptible or intermediate based on zone size interpretive chart was observed and recorded.

Word picture of bacterial isolates

Gram staining

It is a differential staining method of distinguishing bacterial species into two big groups ( Gram-positive and Gram-negative ) based on the chemical and physical belongingss of their cell walls. A vilification of 24hrs old liquid civilization was made on a lubricating oil free slide. It was so air fixed, heat- fixed, flooded by crystal violet discoloration for1min, and washed for 5 sec with H2O. The vilification was treated with few bead of Gram ‘s Iodine and allowed to move for a minute. The slide was once more washed in H2O and so decolorized in 95 % ethyl alcohol. After the vilification was decolorized, it was washed with H2O and the vilification was eventually treated with few beads of counterstain such as saffranine.

Biochemical trial

Indole production trial

10 beads of Kovac ‘s reagent was added to all the civilization stock and the civilization were agitated gently. The coloring material of the reagent bed in each civilization was examined after 15-20 mins and recorded in the chart. Appearance of distinguishable pink or ruddy coloring material in the upper bed shows positive trial.

Methyl ruddy trial

( Note: Inoculate tubings of MR-VP stock and incubate for 24hrs at 35A°C for Methyl ruddy trial and Voges-Proskauer trial. The civilization stock is divided for the above stated two trial )

3-5 beads of methyl ruddy solution was added to the civilization stock and the alteration in the coloring material was observed and recorded.

Voges-Proskauer trial

10 beads of Barritt ‘s reagent ‘A ‘ were added and the civilizations were shaken in every 3-4 proceedingss. 15 mins after the add-on of Barritt ‘s reagent ‘B, the coloring material of the civilization was examined and recorded

Consequence

Coliform trial:

MPN- Presumptive Test for the coliform:

Depending upon the MPN ( most likely figure ) index ; Table ( 1 ) shows the consequence for the presumptive trial. The milk sample codification R1, R2, R4, R6 and R7 shows all the 15 tubings with positive consequence therefore holding the highest chance for the presence of coliforms while R3 and R5 milk sample codification shows 12 tubings and 14 tubings with positive consequences severally. All the tubings with positive consequence showed gas production in the Durham tubings.

Table ( 1 ) : presumptive trial for coliform in milk sample

Sample codification

No. of tubings with positive consequences

MPN index

per 100ml

Range 95 % chance

Five of 10ml each

Five of 1ml each

Five of 0.1ml each

Lower

Upper

R1

5

5

5

a‰?2400

700

R2

5

5

5

a‰?2400

700

R3

5

4

3

280

100

710

R4

5

5

5

a‰?2400

700

R5

4

5

5

R6

5

5

5

a‰?2400

700

R7

5

5

5

a‰?2400

700

2 ) MPN-Confirmed trial for Escherichia coli:

For the confirmed trial, from each gassing positive tubings of lactose broth 1ml of the sample was suspended in Brilliant green stock and incubated for 48hrs. Table ( 2 ) shows that milk sample codification R1 had all BGLB tubings with positive consequence while R4, R5 and R6 had nine BGLB tubings with positive consequence. Sample codification R7 & A ; R3 showed six BGLB positive tubings while R2 showed merely five tubings with positive consequence. It was observed that there was lessening in figure of tubings with positive consequence. All these tubings with positive consequence had gas production and coloring material of superb green stock was changed from green to yellow.

Table ( 2 ) : confirmed trial for Escherichia coli in milk sample

Sample codification

Five of 1ml each

Five of 0.1ml each

R1

5

5

R2

5

Nothing

R3

4

2

R4

4

5

R5

5

4

R6

5

4

R7

4

2

MPN-Completed trial for Escherichia coli:

A loopful sample from each positive BGLB tubing was taken and streaking was done on Endo agar for isolation. All samples showed dark centered, level settlements with or without green metallic shininess. Further these settlements were transferred and suspended in lactose stock which showed positive consequence with gas production. Colony from Endo agar was transferred in alimentary stock and incubated for 24 hour at 35A°C and was subsequently subjected to Gram staining. The consequence showed that all civilization appeared gram negative non-spore organizing rods. All the civilizations looking gram negative rod-shape was tested for IMViC trial and consequence showed that indole and Methyl red ( MR ) trial was positive whereas Voges-Proskauer ( VP ) trial was negative. Table ( 3 ) shows the consequence for the completed trial.

Table ( 3 ) : completed trial for Escherichia coli in milk sample

Sample Code

Gram Stain

( morphology

/reaction )

Endo-agar Plate

Lactose Broth

Indole Production Test

Methyl Red Test

Voges-Proskauer Trial

R1

Rod shaped & A ; gram negative

+

+

+

+

R2

Rod shaped & A ; gram negative

+

+

+

+

R3

Rod shaped & A ; gram negative

+

+

+

+

R4

Rod shaped & A ; gram negative

+

+

+

+

R5

Rod shaped & A ; gram negative

+

+

+

+

R6

Rod shaped & A ; gram negative

+

+

+

+

R7

Rod shaped & A ; gram negative

+

+

+

+

Antimicrobial susceptibleness trial:

From tabular array ( 4 ) it can be observed that isolates from sample codification R1, R2, , R4 and R5 is opposition to Erythromycin ( E15 ) while isolate from sample codification R6 and R7 are holding intermediate susceptibleness to E15. Merely isolate demoing intermediate consequence is R3B5 while R3A3 is immune.

For Nalidixic acid ( NA30 ) all the isolates from sample R1, R2, R3, R4, R5, R6 and R7 are susceptible with larger zone of suppression.

Maximal sample are susceptible to Tetracycline ( T30 ) while merely R1B4 shows resistant and R2A4 with little suppression zone, R5A3 is intermediate.

All the sample shows resistant to Amoxycillin ( Am30 ) as no zone of suppression was observed except one R2A1 which shows intermediate susceptibleness with really little zone of suppression.

( Note: Bacterias with intermediate susceptibleness may be considered reasonably susceptible and they may react to the peculiar antimicrobic agent with broad safe dose scope. )

From tabular array ( 5 ) it can be observed that all the isolates from each sample is immune to Penicillin G ( P10 ) because no zone of suppression was observed.

Large zone of suppression shows that all the sample are extremely susceptible to Chloramphenicol ( C30 ) . They besides may be susceptible to Streptomycin ( S25 ) while resistant or intermediate to Ampicillin ( A25 ) when suppression zone is compared.

From both the tabular array ( 4 ) and table ( 5 ) it is observed that isolates are extremely susceptible to four antibiotics i.e. Nalidixic acid ( NA30 ) , Tetracycline ( T30 ) , Chloramphenicol ( C30 ) and Streptomycin ( S25 ) while resistant to Erythromycin ( E15 ) , Amoxycillin ( Am30 ) , Penicillin G ( P10 ) . For Ampicillin ( A25 ) isolates may be immune or intermediate.

Table ( 4 ) : Antimicrobial susceptibleness of isolates

Sample codification

Isolates code

Diameter of zone of suppression in millimeter w/r to Antibiotic

E15

NA30

T30

Am30

R1

R1A2/

26mm

22mm

Sensitivity

Roentgen

Second

Second

Roentgen

R1B4

20mm

14mm

Sensitivity

Roentgen

Second

Roentgen

Roentgen

R2

R2A1

27mm

19mm

21mm

Sensitivity

Roentgen

Second

Second

I

R2A4

22mm

18mm

11mm

Sensitivity

Roentgen

Second

I

Roentgen

R3

R3A3

26mm

20mm

Sensitivity

Roentgen

Second

Second

Roentgen

R3B5

15mm

26mm

20mm

Sensitivity

I

Second

Second

Roentgen

R4

R4A2

29mm

21mm

Sensitivity

Roentgen

Second

Second

Roentgen

R4B4

27mm

20mm

Sensitivity

Roentgen

Second

Second

Roentgen

R5

R5A3

23mm

16mm

13mm

Sensitivity

Roentgen

Second

I

Roentgen

R5B5

20mm

35mm

Sensitivity

Roentgen

Second

Second

Roentgen

R6

R6A1

15mm

21mm

24mm

Sensitivity

I

Second

Second

Roentgen

R6B5

18mm

22mm

24mm

Sensitivity

I

Second

Second

Roentgen

R7

R7A1

13mm

27mm

21mm

Sensitivity

I

Second

Second

Roentgen

R7B5

13mm

23mm

24mm

Sensitivity

I

Second

Second

Roentgen

Table ( 5 ) : Antimicrobial susceptibleness of isolates

Sample codification

Isolates code

Diameter of zone of suppression in millimeter w/r to Antibiotic

P10

A25

S25

C30

R1

R1A3

10mm

26mm

28mm

Sensitivity

Roentgen

Second

R1B5

10mm

26mm

28mm

Sensitivity

Roentgen

Second

R2

R1A2

12mm

26mm

28mm

Sensitivity

Roentgen

Second

R1A3

18mm

28mm

34mm

Sensitivity

Roentgen

Second

R3

R3A4

22mm

29mm

Sensitivity

Roentgen

Second

R3B1

22mm

28mm

Sensitivity

Roentgen

Second

R4

R4A5

28mm

32mm

Sensitivity

Roentgen

Second

R4B3

26mm

34mm

Sensitivity

Roentgen

Second

R5

R5A2

13mm

17mm

28mm

Sensitivity

Roentgen

Second

R5B4

30mm

22mm

Sensitivity

Roentgen

Second

R6

R6A2

22mm

28mm

Sensitivity

Roentgen

Second

R6B3

26mm

28mm

Sensitivity

Roentgen

Second

R7

R7A2

10mm

25mm

33mm

Sensitivity

Roentgen

Second

R7B3

11mm

26mm

31mm

Sensitivity

Roentgen

Second

Decisions

In this survey it has been found that

Standard Method Committee for H2O and waste H2O analysis. ( 1981 ) . J. Ame. Public Health Association Washington.DC.

Cappuccino JG, Sherman N ( 2007 ) . A laboratory manual in microbiology. 7th edition. Pearson Education Inc. and Dorling Kindersley Publishing, Inc.161-165.

Massa, S. , Altiri, C. , Quaranta, V. and De Pace, R. 1997. Survival of Escherichia coli O157: H7 in yogurt during readying and storage at 4C.Letters in Applied Microbiology 24, 347-350.

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