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 ” .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 5gBeef infusion 3gAgar 15gpH 7.0
ENDO AGAR
Peptone 10 gLactose 10 gDipotassium phosphate 3.
5 gSodium sulphite 2.5 gBasic fuchsin 0.4 gAgar 15 gPH 7.5
LACTOSE FERMENTATION BROTH 1X AND 2X* ( 1000 milliliter )
Beef infusion 3.0 gPeptone 5.0 gLactose 5.0 gpH 6.9*for 2x stock usage twice the concentration of the ingredients.
BRILLIANT GREEN LACTOSE BROTH ( BGLB )
Peptone 10.0 gLactose 10.0 gBile salt 20.0 gBrilliant green 0.0133gPH 7.
0
NUTRIENT BROTH
Peptone 5.0 gBeef infusion 3.0 gPH 7.
0g
TRYPTONE BROTH
Tryptone 10gCalcium chloride ( reagent ) 0.01-0.03mSodium chloride 5.0g
METHYL RED – VOGES PROSKAUER BROTH ( MR-VP BROTH )
Peptone 7.0gDextrose 5.0gPotassium phosphate 5.0gpH 6.
9
Reagents
KOVAC ‘S REAGENT ( for sensing of indole )p- Dimethylaminobenzaldehyde 5.0gAmyl alcohol 75.0mlHydrochloric acid ( concentrated ) 25.0mlMETHYL RED SOLUTION ( for sensing of acid )Methyl red 0.1gEthyl alcohol 300.0mlDistilled H2O 200.0mlBARRITT ‘S REAGENT ( for sensing of acetylmethylcarbinol )Solution A:I±- napthol 5.0g95 % ethyl intoxicant 100mlSolution Bacilluss:Potassium hydrated oxide 40.
0gDistilled 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 thatStandard 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.
