Pathogenic /Nonpathogenic Bacteria

Pathogenic bacteria are microorganisms that have the capability to cause various diseases in their host organisms. They can harm their host by releasing toxins, invading tissues, and disrupting normal physiological processes. Pathogenic bacteria can cause a wide range of illnesses, from mild to severe.

1. INTRODUCTION
•Milkiscompulsorypartofdailydietfortheexpectant
motheraswellasgrowingchildrenmilkbeingnutrition
foodforhumanbeing,alsoservesasagoodmediumfor
thegrowthofmanymicroorganismspeciallyKlebsiella,
Enterobacter,Pseudomonas,Bacillussp.Bacterial
contaminationofrawmilkcanoriginateformofdifferent
sourcesuchasaair,milkingequipment,feedsoil,faeces,
andgrass.
•Milkacomplexbiologyfluidanditsnature,agood
growthmediumformanymicroorganismbecauseofthe
itsphysicochemicalandpropertiesmilkisagoodgrowth
mediaformicroorganismsandpronetomicrobial
contaminationeasily.

2. •Milkareafollowwater87.8%andtotalsolid12.0%.Thetotal
solidcontentinthemasspercentageofthesubstanceinthe
milkcomprisingprotein3.3%,fat3.3%,lactose4.7%,ash0.7%,
calcium0.13%,phosphorus0.10%,potassium0.14%,chlorine
0.11%,megnesium0.01%,sodium0.05%,componentofthe
milk.
Buffalomilkismorethanthickerandcontainslightlymore
vitaminandmineralsthancowmilk.cowmilkbeingeasily
digestiblemorebeneficialtopatientthanbuffalomilk.

3. COMPOSITION OF MILK
COMPONENT INSIDE MILK
Water
Fat
Non fat solid
8.8%
Cow milk: low fat content(3.66%)
87.3%
3.9%
Buffalo milk: High fat content(7.44%)

4. AIM AND OBJECTIVES
•AimofthestudyistoperformMicrobialanalysisofraw
milkandmilkproducts.Tofulfilltheaimfollowing
objectiveswerecarriedout:
Objectives:
•Sample collection and labeling.
•Isolation of bacteria from collected samples.
•Pure culture preparation.
•Strain identification through Biochemical characterization
and molecular characterization.

5. MATERIALS &
METHODOLOGY

6. SAMPLE COLLECTION AND SAMPLE
AREA:
•Thetwentyeight(28)rawmilksamplewascollectform
ofmilkingbucketsterilecontainerwithcover.
Timings = 9AM-11AM
•ThestudyoftheconducedatthestateofUttarPradesh,
DistrictBareillyformMaytoAugust2023.thecollection
pointofrawmilkwereDelapeer,Cantt,SanjayNagar,
Badaun,etcareaofBareillydairyformsofrawmilkand
milkproductcollectionformthebucketusingsterile
containerwithcover.

7. SAMPLE COLLECTION:
PASTEURISED MILK

8. MEDIA AND INSTRUMENTS
•Mediaareinclude:MacConkeyagar,MannitolSaltAgar
(MSA),EosinMethyleneBlueagar(EMB),demanrogosa
agar,Cetrimideagar,Brilliantgreenagar,Nutrientagar
etc.
•INSTRUMENTS:
•Laminarairflow,Autoclave,Hotairoven,Incubator,
Waterbathcentrifuge,refrigerator,Testtube,Testtube
strand,Breaker,etc.

9. ISOLATION OF BACTERIA OF RAW MILK
•SERIAL DILUTION OF MILK:
Number of colonies * dilution factor
COLONY FORMING UNIT= volume of culture plate
= 3.57*106
1
= 357000000
= 3.57*107

10. STREAKING

11. Biochemical
Characterization
1-Gram staining
2-Catalase test
3-Oxidase test
4-Indole test
5-Methyl Red(MR) test
6-Voges Proskauer(VP) test
7-Citrate test
8-Triple sugar iron (TSI)
9-Urease test
Molecular
characterization
1-DNA Isolation
2-Agarose gel
electrophoresis
3-Polymerase chain
reaction(PCR)
4-DNA Sequencing

12. GRAM STANING
•Gramstaining,isamethodofstainingusedtoclassify
bacterialspeciesintotwolargegroup.
•Gram(+)=Positive(purplebacteria)
•Gram(-)=Negative(pinkbacteria)

13. CATALASE TEST
Thetestisusedtoidentifyorganismsthatproduceenzyme
Catalase.Theenzymesdetoxifieshydrogenperoxideby
breakingitdownintobubblesresultingfromproductionof
oxygengasclearlyindicatesacatalasepositiveresults.

14. OXIDASE TEST
•The Oxidase test is use to determine if an organisms if an
organisms process the cytochromes oxidase enzymes.
•Oxidase test performed two methods.
1.Filter paper test methods
2.Oxidase dish

15. INDOLE TEST
•Nutrient broth 5ml+sample culture inoculate( incubation
period 24 hours after) +add 3 drop of kovac’sregents.
Positive result=
junction red ring
Negative result=
junction yellow
ring

16. METHYL RED(MR) TEST
•Peptonebroth5ml+samplecultureinoculate(incubation
period24hoursafter)+add3-6dropsofMRsolution.
Positive result=
Red yellow
Negative
result= yellow

17. VOGES-PROSKAUER(VP) TEST
•Peptonebroth5ml+samplecultureinoculate(incubation
period24hoursafter)+add3-3dropsofalphaNaphthol
andpotassiumhydroxide.
Positive result= red/pink
Negative result= green

18. CITRATE TEST
•Simmonscitrateagarslant+samplecultureinoculate
(incubationperiod24hours)
Positive result= Blue
Negative result= green

19. TRIPLE SUGAR IRON (TSI) TEST
•Triplesugarironagarisusedforthepresumptive
identificationofEnterobacteriaceaebasedonthe
fermentationofglucose,lactose,sucrose,andthe
productionofgasandH2O.
•TSIcontainsthreecarbohydrate(glucose,lactoseand
sucrose).
TSI= Black (H2)Positive, bubbles or creaks (gas)positive,

20. UREASE TEST
•Ureaagarwasdevelopbychristensenin1946forthe
differentiationof.entericbacilli,theUreasetestisused
fordeterminetheabilityofanorganismstospliturea,
thoughtheproductionoftheenzymesoftheenzymes
urease.
Ureasetest positive(pink), negative (yellow)

21. Quality of
the milk
sample
1-Clot and
blot test
3-Alcohol
test
4-Fehling’s
solution test
2-Methylene
Blue
reduction test
5-standard
plate count

22. CLOT AND BLOT TEST
METHYLENE BLUE REDUCTASE TEST(MBRT)
Sample showing precipitated
Practical are recorded as C.O.B.
Positive
Blue color (near to good quality)

23. ALCOHOL TEST
•FEHLING’S SOLUION TEST:
Alcohol test (coagulation , Precipitation)
AReddishbrownprecipitateappearance
indicatesapositiveresultsandthatthe
resumingsugarinpresent

24. ISOLATION OF BACTERIAL GENOMIC
DNA
Step 1= sample preparation
step 2= cell Lyses 1-TE BUFFER(T=TRIS,E= EDTA)
2-10% SDS( Sodium dodecylacetate)
3-Proteins K
Step 3= Purification of DNA(Phenol: Choloroform: IAA)
Step 4= Precipitation of DNA(DNA(Sodium acetate+100%
Chilled ethanol)
Step 5= Washing (70% ethanol)
Step 6= storage (TE buffer)
Step 7= Agarose gel electrophoresis)

25. 1.5 ml culture
Centrifuge 10,000 ,10
min
Water bath 60°c, 1 hour
Transfer in upper layer in
fresh MCI+CH3COONA
TE Buffer+10%SDS+Protiaase
k
Phenol:
Choloform: IAA
CENTRIFUGEStep5=70% ethanol
TE buffer
Step= 2 Cell Lysis
Step3 =Purification of
DNA
Step4= Precipitation
of DNA
step6
stora
ge
Step 1 sample
preparation
DNA Isolation

26. Agarose gel electrophoresis
Electro means(electronic field) Phoresis(Separation)
Separation of Biomoleculesin the presence of electric field

27. POLYMERASE CHAIN REACTION(PCR)
Polymerase chain reaction(PCR)
Reaction(technique)
Invitro(out side cell)amplification (copies)of DNA
INSTRUMENTS:
Thermal (temperature)cycler(cycle)
Step 1= Denaturation( 94°C)
Step 2= Annealing (55°C)
Step 3= Extension(72°C)

28. POLYMERASE CHAIN REACTION(PCR)
800kb

29. DNA SEQUENCING
Forward:
CACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAA
GCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGG
AGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGACTGACAC
TGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTA
GCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCA
AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGC
GAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACAC
AGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC
Reverse:
GTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAA
CCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGT
CGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGAAGGTCT
TCGGATCGTAAAACTCTGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTACCTAATCA
GAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTAGACTGGGATAACTTC
GGGAAACCGGAGCTAATACCGGATAATATTTTGAACCGCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCAC
TTATAGATGGATCCGCGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAGCCGCGGTAATACGGAGGGTGC
AAGCGTTAATCGGAATTACTGGGCGTAAAGCGAACACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGG
GCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCCTTTAAGTTGGGAGGAAGGGCAGTAAGTT
AATA

30. query
OQ615324.1:737-1303Pseudomonasaeruginosa strainSI116S
GU991527.1:131-703Pseudomonasaeruginosa strainARA116S
OQ615324.1:510-625PseudomonasaeruginosastrainSI116S
OQ615324.1:300-509PseudomonasaeruginosastrainSI116S55
OQ651983.1:12-54Pseudomonas sp. strain RSR-1 16S
OQ875739.1:404-436Pseudomonasaeruginosa strainVITARK5 16S
OQ615324.1:424-456PseudomonasaeruginosastrainSI116S
99
OQ875739.1:280-489Pseudomonasaeruginosa strainVITARK5 16S
55
18
OR253809.1:167-282Pseudomonassp.strainB2W6/NITRR 16S
OQ875739.1:490-605Pseudomonasaeruginosa strainVITARK5 16S
29
OQ642290.1:20-56Pseudomonas sp. strain RSR-1 16S
17
30
86
84
84
OQ651983.1:167-733Pseudomonas sp. strain RSR-1 16S
60
OQ875739.1:717-1283PseudomonasaeruginosastrainVITARK5 16S
97
OR253809.1:394-960Pseudomonassp.strainB2W6/NITRR 16S
PHYLOGENIC TREE FOR PSEUDOMONAS

31. ANTIBIOTIC SENSITIVITY TEST

32. AntibioticCode Concen.ResistanceSusceptible Interpretation
AmpicillinAMP 10mcg 12 mm 18mm 14-16
Chlorampheni
col
C 30 mcg12 mm 18 mm 13-17
TetracyclineTE 30 mcg10 mm 15mm 12-14
Pencillin P 10 U 14 mm 17 mm 14-16
Meropenam MRP 10 mcg18mm 23 mm 20-22
ErythromycinE 10 mcg13mm 23mm 14-22
AzithromycinAZM 15 mcg14mm 18mm 14-17
Amoxyclav AMC 30 mcg13mm 19mm 14-17
CefotaxineCX 30/10mcg21 mm 24mm 23-25
CiprfloxacinCIP 5 mcg 20 mm 25mm 22-24
ClindamycinCD 10 mcg14 mm 21mm 15-20
GentamicinGEN 10 mcg16mm 18 mm 15-17
NitrofurationNIT 300mcg14mm 15mm 15-16
Doxycycline
hydrochloride
DO 10 mcg12mm 15mm 13-15
Zone of inhibition: Pseudomonas

33. RESULTS

34. PURE CULTURE ON SLANT
•PURE CULTURE ON PETRI PLATE:

35. s.no s.nam
e
palaceMedia
.
Col.ch
.
G.strai
n.
Catala
se.
Oxida
se.
Indole
.
MR. VP. CitrateTSI Ureas
e.
Bac.na
me
1 c.m1G.N1Mac.Fer.(+)(+)(+)(-) (-) (+)(+)H-G-(-) Bacill.
2 Cu.s1D.B1Mac.Fer.(-) (+)(+)(+)(-) (-) (+)H+G-(+)N.I.
3 B.M1G.N2MSAYell.c.(+)(+)(-) (-) (-) (-) (+)H+ (+)Step.
4 G.M1G.N5MSAYell.c.(+)(+)(-) (-) (+)(+)(+)H+ (+)Stap.
5 Mal1D.B.Mac.Fer.(-) (+)(-) (-) (-) (+)(+)H-G+(+)Kleb.
6 Bum1D.B.Mac.Fer.(-) (+)(-) (-) (-) (-) (+) (-) N.I.
7 Boil1H.M.EMBFer.(-) (+)(-) (-) (-) (+)(+)H-G+(+)Kleb.
8 GheeH.M.EMBN.Gro- - - - - - - - - -
9 Butt1D.B.EMBN.Gro- - - - - - - - - -
10 Cu.s2G.N2MRSWhi.c(+)(+)(+)(-) (-) (-) (+)A/A(+)Pedio
cocc.
11 C.m2D.B.MRStrans.(-) (+)(+)(-) (-) (+)(+)H-G-(+)Pseu.
12 C.m3CanttMac.Fer.(-) (-) (-) (+)(+)(-) (+)H-G+(+)N.I.
13 B.m2Bada.Mac.Fer.(-) (+)(-) (+)(+)(-) (+)H-G+(+)N.I.
14 B.m3Bada.Mac.Fer.(-) (+)(+)(+)(+)(+)(+)H- (+)Citro.

36. s.noGs.nam
e
palaceMedia
.
Col.ch
.
Gram.
stain
Catala
se.
Oxida
se.
Indole
.
MR. VP. CitrateTSI Ureas
e.
Bac.na
me
15 Pane.S.N.EMBFer.(+)(+)(+)(+)(+)(+)(+)H-G-(+)N.I.
16 C.M4Dur.nEMBFer.(-) (+)(-) (-) (-) (+)(+)H-G+(+)Enter.
17 B.M4D.B.EMBFer.(-) (+)(+)(-) (-) (-) (+)H-G-(+)Pseu.
18 Mal2D.B.Mac.C.L.C.(-) (+)(+)(-) (-) (-) (+)H-G-(+)Pseu.
19 Cu.s3S.N.MRS.N.G.- - - - - - - - - -
20 B.M5S.N.Mac.Fer.(-) (-) (-) (-) (-) (+)(-) H-G+(+)Kleb.
21 C.M5G.N.7Mac.Fer.(-) (+)(-) (+)(-) (+) (+)H-G+(+)Kle.o
x
22 Che.sD.B.Mac.Fer.(+)(-) (-) (-) (-) (+)(+)H+G-(+)N.I.
23 B.M6Dela.Mac.C.L.C.(-) (+)(+)(-) (-) (-) (+)H-G-(+)Pseu.
24 Cu.s4D.B.Mac.Fer.(-) (-) (-) (+)(-) (-) (+)H+G-(+)N.I.
25 ArlysBada.BGAN.G.- - - - - - - - - -
26 Mad.Bada.BGAN.G.- - - - - - - - - -
27 Pya.Bada.BGAN.G.- - - - - - - - - -
28 P.M.BadaBGAN.G.- - - - - - - - - -

37. SUMMARY
•To asses the quality of raw milk and detect bacteria in a
milk and milk products:
•Collect the sterile container representative milk sample.
Detects in a lab , plate a milk sample on specific agar
plate for isolate of bacteria and incubate in 37°C.
,followed by confirmation tests.
BACTERIA TOTAL SAMPLE
Bacillus cereus 1
Streptococcus 1
Staphylococcus aureus 1
Klebsiella 4
Enterobacter 1
Citrobacter 1
Pseudomonas 4

38. THANK YOU