MICROBIOLOGICAL TESTS (CONVENTIONAL METHODS )

RAHI KRISHNA
COLLEGE OF VETERINARY & ANIMAL SCIENCES MANNUTHY

1. Microbiological examination of foods ( CONVENTIONAL METHOD) RAHI KRISHNA 16-MSVM-014

2. Microbiological examination

3. Culture media Sugar media

4. Simple media / basal media : Most common in routine diagnostic laboratories E g: nutrient broth, nutrient agar

5. COMPLEX MEDIA • Media other than basal media. • A dded complex ingredients (Yeast extract) Provide special nutrients SYNTHETIC OR DEFINED MEDIA * Prepared from pure chemical substances * Used for special studies, E g . Metabolic requirements

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

7. 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

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

9. 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

10. 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

11. TRANSPORT MEDIA F or transporting the samples . • Eg : Stuart’s medium

12. 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.

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

14. 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

15. 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.

16. 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.

17. 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.

18. SPREAD PLATE POUR PLATE

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

20. MCINTOSH – FILDES’ ANAEROBIC JAR

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

22. Simple staining Differential staining For visualization of morphological Shape & arrangement Identification Visualization of structure Gram stain Acid fast Stain Spore stain Capsule stain

23. Simple staining Methylene blue , C arbol fuchsin or Crystal violet Determination the size ,shape and arrangement of bacterial cell.

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

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

26. 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

27. 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

28. Capsule staining D etects presence of bacterial capsule Place a small drop of a  I ndia ink, Congo red, Nigrosin on the slide.  Drain the dye by the slide . Klebsiella pneumoniae

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

30. 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

31. METHYLENE BLUE DYE REDUCTION TEST Q uick 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

32. 5 hrs and above Very good 3 to 4 hrs Good 1 to 2 hrs Fair Less than ½ hrs Poor

33. Resazurin dye reduction test Also used for testing microbiological quality of milk

34. Bio chemical tests

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

36. 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

37. Oxidase test Identify bacteria that produce cytochrome c  oxidase Tetramethyl-p- phenylene diamine hydrochloride Purple colour Cytochrome oxidase+atm.o 2 oxidation A . Positive – Staphylococcus aureus B. Negative – Escherichia coli

38. Urease test To determine the ability of an organism to produce the enzyme, urease, which hydrolyzes urea . Urea Urease Ammonia P ositive - Proteus spp. Negative - Escherichia coli

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

40. Fermenter – E scherichia coli Oxidizer – Pseudomonas aeruginosa Non utilizer- Alcaligenes faecali s

41. TRIPLE SUGAR IRON AGAR TEST Glucose/lactose/sucrose Acid/gas/H 2 S

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

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