Different microbiological tests applied to detect the presence of some pathogens that transmitted to human via consumption of raw milk or dairy products processed from raw milk and how to isolate them.

1. Isolation of Some
Pathogens from
Milk
Presented by: Dr. Zeinab I. Ali
Lecturer of Food Hygiene and Control
Faculty of Veterinary Medicine
Cairo University

2. Outlines
1.1. Staining
1.2. Animal inoculation
1.3. Culture method
Mycobacterium Tuberculosis
01
2.1. Methods of Detection
2.2. Methods of Isolation
Brucella
02
3.1. Pre-enrichment
3.2. Selective enrichment broth
3.3. Selective agar media
Salmonella and Shigella
03
4.1. Isolation of coagulase positive staph. Aureus
4.2. isolation of haemolytical staphylococci and streptococci
Staphylococcus aureus
04
5.1. Primary enrichment
5.2. Secondary enrichment
5.3. Selective agar media
Listeria monocytogenes
05

10. Name Here
DOCTOR

11. Global TB Facts
Each year, nearly 9
million people around
the world become sick
with TB
Each year, there are
almost 2 million TB-
related deaths worldwide
One of the world’s
deadliest diseases
One third of the world’s
population are infected
with TB
TB

12. Methods of detection and isolation of
Mycobacterium tuberculosis
02
Microscopically
Examination
Animal
Inoculation
Method
Culture Method

13. Microscopically Examination
3000 rpm/ 30 min

15. Animal inoculation method
100 ml. of milk
centrifuged at 3000
r.p.m/ 30 min.
.
Milk
Cream or sediment
or mixture is treated
with equal volume of
6% HCL or 5%
oxalic acid (to kill all
associated
organisms)
Acid
leave at 37°C/15-20
min then Re-
centrifuge to get ride
of acid
.
Re-centrifugation
Neutralized by
diluted NaOH with
bromothymol blue
0.1% as indicator
(end point is grassy
green color).
Neutralize
Centrifuge the tubes
then wash the cream
& sediment with
sterile saline
Suspend the treated
material in equal vol.
sterile saline sol
Centrifuge
Inoculate (0.5-1 ml) S/C
6 weeks
later

16. Animal inoculation method
P.M
• Lung
• Liver
• Spleen
Film
• Acid Fast
Bacilli with
Z.N. stain
Culture
• L.J.
• Dorset Egg.
• Petragnani.
The animals are killed
after 6 weeks. on killed
animals (G.pigs)

17. Direct Culture method
Streak Treated milk samples (previously
described) on the specific media / incubate at
37°C ∕3-5 weeks
Egg-based media, Contain malachite-green which inhibits growth of
contaminating bacteria and fungi.
Lowenstein Jensen (L.J.)
Agar-based media, contain antibacterial and antifungal agents to
control the contaminants.
Middlebrook 7H10 and 7H11
3 Recommended, increased recovery of mycobacteria and
decreased time of detection compared to solid media
Liquid Media (e.g. Middlebrook 7H9 broth)
2
1
M. tuberculosis is rough and non-pigmented Culture is examined microscopically using Z.N. stain

20. RB51 is resistant to
rifampin

21. Methods of detection and isolation of Brucella organism
Animal
Inoculation
method
Culture
Method
Serological
Method
Brucellosis Human
Disease:
Malta Fever
Undulant Fever
Mediterranean Fever
Rock Fever of Gibraltar
Gastric Fever
Brucellosis Animal
Disease
Bang’s Disease
Enzootic Abortion
Epizootic Abortion
Slinking of Calves
Ram Epididymitis
Contagious Abortionver

22. Animal inoculation method
20 ml. of milk
centrifuged at 3000
r.p.m/ 30 min.
.
Milk
Mixture of sediments
and cream is
suspended in sterile
saline solution.
suspension
injected (0.5 1ml) in
3 male G.pigs S/C in
the inner side of the
thigh.
Injection
Apply The blood
serum agglutination
test (Titre of 1/40 or
more is positive)
Serum
agglutination test
Killing animals
2 weeks 6 weeks
P.m. examination
Microscopical examination
Culture

23. Following centrifugation of milk, Cream and Sediment are streaked on a specific media for isolation
of Brucellla organism
Incubated 37°C ∕4-6 days with10% CO2
suspect colonies
Culture Method
small grey non hemolytic(small, transparent, 1-2 mm. in diameter)

24. Examine the growth microscopically with modified ZN,
Hansen or gram stain.(-ve bacilli).

25. Rapid agglutination test
Serological
m e t h o d s
A.B.R.T
M.R.T.
Slow agglutination test
On milk, blood,serum

26. Abortus Bang Ring Test (A.B.R.T)
Milk Ring Test (MRT)
MRT is a screening test to determine the presence of brucellosis
in a dairy herd.
The test is carried on bulk milk, then individual milk to detect the
infected animal.
+ =
Brucella Ab
(Lipophylic)
Stained Ag
Stained Ag-Ab
complex

27. 1ml milk
One drop
(30µl stained
brucella Ag)
Mix gently & incubate in water bath 37°C∕45 - 60 min.

28. judgment Color of milk
column
Color of cream layer
Strong positive Completely white Deeply blue colored
Doubtful The same color (blue)
Negative Deeply blue colored White
Strong positive
Doubtful

29. Milk Rapid agglutination test
On a clean glass slide put Two drops of the examined milk and one
drop of stained Brucella Ag. Mix well and check for agglutination

30. Slow agglutination test (Blood
serum agglutination Test)SAT
Add One drop of brucella antigen to each tube & mix
incubate at 37°C ∕24hours .

32. Isolation of Salmonella and Shigella

33. Indirect method
pre-
enrichment
• Aseptically add 10 ml of milk
• 100 ml. of non-selective broth
• Thoroughly mix and incubate at 37oC ∕ 24 hours.
Selective
enrichment
• 10 ml. of incubated pre-enrichment broth
• 100 ml. of selective broth (incubation acc.to
media type)
Selective
plating
• Streak loop-full from the enrichment broth on
selective media
• Incubate 37oC ∕ 24-48 hours

34. non-selective broth:
Tryptic soy broth.
Buffered peptone water.
Lactose broth
selective broth:
Selenite F broth (37oC ∕ 16-18 hr).
Tetrathionate broth (37oC ∕18-24 hr).
Rappaport Vassiliadis broth (41oC ∕18-24 hr).
Salmonella Shigella agar (SS agar)

35. Direct method
3000 rpm/10 min
Sediment

37. Coagulase positive staphylococci
Incubation 37oC ∕ 24-48hr

38. Inoculate Pure culture in 5 ml. of B.H.I. broth at 37oC ∕ 24 hours
Coagulase test
Transfer 0.1 ml. of incubated B.H.I. broth to coagulase tube containing
0.3 ml. rabbit plasma incubate at 37oC ∕4-6 hours.

44. Isolation of Listeria
Plating
Secondary enrichment
Primary enrichment

45. Incubation at 30°C/24 hours
Transfer 0.1 ml. of incubated half fraser
broth into tube containing 10 ml.
Fraser broth
Incubated at 37°C/48 hours

46. streak Loop-full from incubated Fraser broth on one of Listeria selective media

47. confirmation
Listeria