RAHI KRISHNA COLLEGE OF VETERINARY & ANIMAL SCIENCES MANNUTHY

1. MICROBIOLOGICAL
EXAMINATION OF FOODS
(CONVENTIONAL METHOD)
RAHI KRISHNA
16-MSVM-014

2. MICROBIOLOGICAL EXAMINATION
• Culture media & methods
• Microbial staining & motility
• Dye reduction tests
• Bio chemical tests
Conventional
methods
• Nucleic acid based method
• Immunological based method
• Biosensor methodRapid method

3. CULTURE MEDIA
Sugar
media

5. Simple media / basal media:
• Most common in routine diagnostic laboratories
• Eg: nutrient broth, nutrient agar

6. COMPLEX MEDIA
• Media other than basal media.
• Added complex ingredients (Yeast extract) Provide special nutrients
SYNTHETIC OR DEFINED MEDIA
* Prepared from pure chemical substances
* Used for special studies,
Eg. Metabolic requirements

7.  SPECIAL MEDIA
ENRICHED MEDIA
• Substances like blood, serum, egg are added to the basal medium.
Eg: Blood agar, Chocolate agar

8. ENRICHMENT MEDIA
• Liquid media used to isolate pathogens from a mixed culture.
• Stimulate growth of desired bacterium inhibit growth of unwanted bacterium
Selenite F Broth – for the isolation of Salmonella, Shigella
Tetrathionate Broth – inhibit coliforms
Alkaline Peptone Water – for Vibrio cholerae

10. SELECTIVE MEDIA
• The inhibitory substance is added to a solid media
• Increase in number of colonies of desired bacterium
EMB – E.coli

11. • DIFFERENTIAL MEDIA
• Substances incorporated in it enabling it to distinguish between bacteria.
• Eg: Mac conkey’s medium
Lactose fermenters – Pink colonies
Non lactose fermenters – colourless colonies

12. SUGAR MEDIA
• Media containing any fermentable substance.
• Eg: Glucose, Arabinose, Lactose, Starch etc.
• Media consists: 1% of the sugar in peptone water + indicator
• Contain a Durham's tube for the detection of gas by the bacteria

13. • TRANSPORT MEDIA
For transporting the samples.
• Eg: Stuart’s medium

14. CULTURE METHODS
PURPOSE
• To isolate bacteria in pure cultures.
• To demonstrate their properties.
• To determine sensitivity to antibiotics
• To estimate viable counts.
• Maintain stock cultures.

16. • STREAK CULTURE
• Used for the isolation of bacteria in pure culture from clinical
specimens.
• Platinum wire or Nichrome wire is used.

17. • LAWN CULTURE
• Provides a uniform surface growth of the bacterium.
• Uses – for bacteriophage typing.
– Antibiotic sensitivity testing.
– In the preparation of bacterial antigens and vaccines.
• Lawn cultures are prepared by flooding the surface of the plate with a
liquid suspension of the bacterium

19. • STAB CULTURE
Prepared by puncturing a suitable medium – gelatin or glucose agar with a
long, straight, charged wire.
• Uses
– Demonstration of gelatin liquefaction.
– Oxygen requirements of the bacterium under study.
– Maintenance of stock cultures.

20. • STROKE CULTURE
• Made in tubes containing agar slopes (slant).
• Employed for providing a pure growth of the bacterium for slide
agglutination and other diagnostic tests.

21. POUR - PLATE METHOD
• In this, successive dilutions of inoculum
is added to molten agar in respective petriplates.
• Individual colonies are picked for sub culturing.
SPREAD – PLATE METHOD
 In this, dilute sample is placed onto solidified agar
and is spread uniformly with sterile ,bent glass rod.

23. SPREAD PLATE POUR PLATE

24. • LIQUID CULTURE
• For blood culture & for sterility tests.
• Preferred when large yields are desired.

25. •
Anaerobic
culture
methods
Production
of vacuum
Displacement
of oxygen
with other
gases (H, N2
He & CO2)
Chemical
method
McIntosh-
Filde's
anaerobic
jar
Gas Pak
Biological
method

26. MCINTOSH – FILDES’ANAEROBIC JAR

27. • GAS PAK JAR
• Palladium aluminium coated pellets
- Catalyst
- Chemically reduces O2
- Reacts with residual O2 in
the presence of H2 to form H2O

29. • SIMPLE STAINING DIFFERENTIAL STAINING
For visualization of
morphological
Shape &
arrangement Identification Visualization of
structure
Gra
m
stain
Acid fast
Stain Spore
stain
Capsule
stain

30.  Simple staining
 Methylene blue , Carbol fuchsin or Crystal violet
 Determination the size ,shape and arrangement
of bacterial cell.

32. GRAM’S STAINING
Gram stain is fundamental to the phenotypic
characterization of bacteria.
1. Primary stain (Crystal violet)
2. Mordant (Gram’s iodine),
3. Decolourisation with ethanol or acetone
4. Counter staining with safranin

33. Gram +ve cocci
Staphylococcus aureus
Gram –ve bacilli E.coli

34. •ACID-FAST STAINING-
ZIEHL NEELSEN METHOD
• Diagnosis of tuberculosis and leprosy.
• E.G., Mycobacterium tuberculosis – causes tuberculosis
• E.G., Mycobacterium leprae – causes leprosy
• Primary stain- carbol fuchsin
• Mordant heating
• Decolorizing agent-acid alcohol solution
• Counter stain- methylene blue
Mycobacterium tuberculosis

35. •SPORE STAINING
Used to visualize bacterial endospores.
Endospores are formed by a few genera of bacteria, such as bacillus
• Primary stain- Malachite green
• Mordant -heating
• Decolorizing agent - H2O
• Counter stain- safranin
Bacillus cereus

36. CAPSULE STAINING
• Detects presence of bacterial capsule
• Place a small drop of a India ink, Congo red, Nigrosin on the
slide.
• Drain the dye by the slide.
Klebsiella pneumoniae

37. NEGATIVE STAINING
Used for determining cell size and arrangement
Requires an acidic dye such as
India ink or Nigrosin.

38. MOTILITY TEST
 Bacteria are suspended in a drop
of liquid they can be seen by
light microscopy
 Motile bacteria swim in straight line
non motile bacteria vibrate
a bit Brownian motion

39. METHYLENE BLUE DYE REDUCTION TEST
• Quick method
• To assess microbiological quality of raw and pasteurized milk.
• Based on the blue colour of the dye solution added to the milk
get decolourized when the oxygen present in the milk get exhausted due to
microbial activity.
• The sooner the decolourization, more inferior is the bacteriological quality
of milk

40. 5 hrs and above Very good
3 to 4 hrs Good
1 to 2 hrs Fair
Less than ½ hrs Poor
10 ml milk in a
sterile test tube
1ml MBRT dye
(0.005%)
Placed in
water bath
(37+/-1ºC)

41. RESAZURIN DYE REDUCTION TEST
Also used for testing
microbiological quality of milk
10 milk in a sterile test tube
1ml resazurin dye
Placed in water bath(37±1ºC)
Compare the colour of test tube
with standard disc in comparator

42. Bio chemical tests
Single
enzyme
tests
Catalase
test
Coagulase
test
Oxidase
test
Urease
test

43. • CATALASE TEST
• To differentiate members of the family Microcococcaceae (including
Staphylococcus) and Streptococcus species which are catalase negative
catalase
2 H202 -------------- 2 H20 + O2
bubbles or effervescence
A. Positive – Staphylococcus aureus.
B. Negative – Streptococcus pyogenes

44. • COAGULASE
To determine the ability of the organism to produce coagulase
which clots plasma
Fibrinogen Fibrin
coagulase
A. Negative – Staphylococcus epidermidis
B. Positive – Staphylococcus aureus
A. Positive – Staphylococcus aureus
B. Negative – Staphylococcus epidermidis

45. • OXIDASE TEST
Identify bacteria that produce cytochrome c oxidase
Tetramethyl-p-phenylene
diamine hydrochloride Purple colourCytochrome
oxidase+atm.o2
oxidation
A. Positive – Staphylococcus aureus
B. Negative – Escherichia coli

46. • UREASE TEST
• To determine the ability of an organism to produce the enzyme, urease,
which hydrolyzes urea.
Urea Ammonia
Positive - Proteus spp.
Negative - Escherichia coli

49. • OXIDATION FERMENTATION TEST
To study oxidative & Fermentative
breakdown of carbohydrate

50. Fermenter – Escherichia coli Oxidizer – Pseudomonas aeruginosa
Non utilizer- Alcaligenes faecalis

51. • TRIPLE SUGAR IRON AGAR TEST
Glucose/lactose/sucrose ACID/GAS/H2S

52. NITRATE NITRITE
NITRATE REDUCTION TEST
Ability of a bacteria to reduce nitrate to nitrite
Nitrate reductase
Positive- E.coli

54. 1. Ananthanarayan and paniker, r. Ananthanarayan, c. K. Jayaram paniker
2005.,ananthanarayan and paniker's textbook of microbiology 9th edition
Pg no 5-9, 10-15, 37-45
1. joanne m. Willey, linda sherwood, christopher j. Woolverton – 2011
Prescott's microbiology pg no 45-50, 49-55, 333-335
1. ‘https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
1. www.biologydiscussion.com/food-microbiology/microbiological-examination-of-foods