MICROBIOLOGICAL TESTS (CONVENTIONAL METHODS )
- 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
- 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
- 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