Lecture 5
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This document discusses various methods for enumerating and testing for microorganisms in food. It describes total plate counts, coliform tests, and tests for mesophilic bacteria, staphylococci, Salmonella, Shigella, and other pathogenic bacteria. Specific procedures are outlined, including enrichment, plating, screening, and confirmation steps. A variety of media are used, such as violet red bile agar, Baird-Parker agar, triple sugar iron agar, and lysine iron agar. Colonies are examined for characteristics like color, zone formation, and biochemical reactions to identify microorganisms.
Lecture 5
- 1. Methods of enumerating microorganisms food MIC 204
- 2. Ways to enumerate microorganisms enumeration Total plate count Coliform test Testing for mesophilic bacteria Testing for staphylococci Testing for pathogenic bacteria
- 3. Procedures involving in plate counts Streak dilution plate Spread plate Pour plate
- 5. Spread plate Method of quantifying the number of viable cells (or colony forming units) in a sample after appropriate dilution
- 7. Pour plate A known volume of cell suspension is mixed with this molten agar distributing the cells throughout the medium Disadvantages: 1. Typical colony morphology seen in surface cultures will not be observed for those colonies that develop within the agar medium 2. Some of the suspension will left behind in the screw capped bottle
- 9. Count the accurate result which contain 30 - 300 colonies ↓ Count the mean ↓ Calculate the colony count per ml of that particular dilution by dividing by the volume (in ml) of liquid transferred to each plate (v) Example: Count per ml (unit in CFU) = C/V x M C= Mean of colony eg: 5.5 colonies V= Volume of transferred to plate eg: 0.05ml M= Multiplication factor eg: for a dilution of 10'7, the multiplication factor would be 107 or in other words: mean colony count of 5.5 colonies per plate for volume of 0.05ml at a dilution of 10'7, the count would be: 5.5 / 0.05 x 10^ 1.1 xlO^7UmI-1
- 10. Coliform test Coliform bacteria – a gram negative nonspore forming bacilli usually found in the human and animal intestine. They ferment lactose to acid and gas Coliform test been carried out to test the presence of enteropathogenic bacteria such as Salmonella, Shigella and others. The test normally use coliform bacteria as indicator As a result, testing for coliform bacteria can be a reasonable indication of whether other pathogenic bacteria are present
- 11. Why? Because: They are able to survive for extensive periods of time in the environment Relatively easy to cultivate in the laboratory and numerous
- 12. The ideal indicator organism should: a) Be present whenever the pathogens concerned are present b) Be present only when there is a real danger of pathogen being present c) Occur in greater numbers than the pathogen to provide a safety margin d) Survive in the environment as long as the potential pathogens e) Be easy to detect with a high degree of realibility
- 13. Stages of coliforms test a) Presumptive tests b) Confirmation tests c) Completed tests
- 14. a) Presumptive test Example in solid sample like food Use plating method Food is added to Petri dishes (in duplicated) Then molten violet red bile agar is added, mix, then allowed to harden The plate then incubates at 35°C for 18-24 hours. Positive result: dark red colonies with a surrounding zone of precipitated bile at least 0.5mm in diameter Violet red bile agar
- 15. Most Probable Number (MPN) method In this method, samples were inoculated in 10ml (101), 1ml (100) and 0.1ml (10-1) into lactose broth tubes. The tubes are incubated-and coliform organisms are identified by their production of gas from lactose. Referring to an MPN table, a statistical range of the number of coliform bacteria is determined by observing how many broth tubes showed gas. However presumptive test for coliforms does not constitute absolute identification of these organisms. All positive result should be confirmed by further testing which is confirmation test. Example for drinking water:
- 16. b) Confirmation test This test being carried out because gas formation in lactose broth at 37°C is characteristic not only of fecal Salmonella, Shigella and E.coli strains but also of non fecal coliform like Enterobacter aerogenes and some Klebsiella species. As a result, in this second stage test (confirmation), the presence of enteric bacteria is confirmed by reculture the positive result: Positive result from plating method: Those colonies from violet red bile agar are transfer to a separate tube of brilliant green lactose bile broth and then incubate for 48 hours under 35° C before examined for the gas presence.
- 17. c) Completed test ◦ Positive tube from Brilliant Green Lactose bile broth cultures are streaked and stabbed on slant of nutrient agar ◦ After incubation for 18-24 hours at 35 °C, the slant is examined for the growth on the surface and in the stabbed portion of the slant ◦ Gram stain smear was made from agar slant: ◦ Positive result showed: Gram negative, non-sporing rods Brilliant green lactose bile broth
- 18. Test for mesophilic bacteria Mesophilic bacteria – bacteria that can grow at the opt T (btw 20°C- 50°C) However many mesophiles have an optimal temperature of about 37° C, corresponds to human body temperature. Many of the normal resident microorganisms of the human body, such as Escherichia coli are mesophiles.
- 19. Testing method for mesophilic bacteria Pour plate aliquots serially diluted sample using non selective media such as Plate Count Agar (PCA), tryptic soy agar, NA and others However, PCA is recommended for colony forming units (CFU) determination The temperature and time of plate incubation required for the colonies to develop differ with the microbial groups being enumerated. For mesophilic bacteria, normally incubation temperature is 37ºC for 24-48 hours.
- 20. Testing for staphylococci Why Staphylococci? Because certain strains of Staphylococci like S.aeureus can grow on food and produce heat-stable enteroxins, which cause food poisoning. Characteristics of Staphylococci 1) Gram positive, cocci arranged in clusters. 2) Biochemical test: Oxidase (negative) Catalase(positive) 3) Some strains {S.aereus) are coagulase test (positive) but some are coagulase (negative)
- 21. Testing methods for staphylococci • Several methods involving direct plating and or / enrichment techniques have been proposed for the detection of coagulase-positive Staphylococci in foods. Medium used for Staphylococci is Baird- Parker agar medium. • In this medium, it contains all the nutrients that Staphylococci required Baird- Parker agar
- 22. Presumptive test (more 100 Staphylococci) • When greater than 100 Staphylococci aureus / gram are expected, direct plating of a diluted sample on Baird- Parker medium followed by incubation for 30 hour at 37° C is recommended. • Result: Staphylococci grow on this medium as a black colony surrounded by a clear zone
- 23. Presumptive test (low Staphvlococci aureus) When count of less than 100 / Staphylococci aureus are expected, the most probable number (MPN) enrichment technique is recommended. How to carry out the test? Using 0.1g, 1g and 10g samples of the food to be analyzed. Growth in anaerobic tellurite glycine mannitol pyruvate broth and incubated at 37°C is determined. The positives tubes are plated on Baird-Parker agar based medium and typical colonies are examined for the producing coagulase as described.
- 24. Confirmatory test These tests include: 1)The utilization of glucose anaerobically, which separates the Staphylococci aureus from other species of Staphylococci.' 2) The ability to produce coagulase, which separates Staphylococci aureus from other species of Staphylococci How to carry out the coagulase test? • The tests involve inoculation samples into blood serum and incubation for certain period under certain temperature to determine coagulation of the serum. 3) Also, S. aureus is separated from other Staphylococci based on its property of utilizing mannitol anaerobically.
- 25. Testing for pathogenic bacteria Pathogenic bacteria – bacteria which have ability to cause infection to the organisms. Example: Salmonella, Shigella and others. These are common pathogenic bacteria that cause food poisoning
- 26. Detection of Salmonella in food The detection method of Salmonella involves several stages: a) Enrichment of samples b) Plating of enrichment cultures c) Screening d) Confirmation test
- 27. Enrichment of sample • The purpose to do enrichment of samples is to supply all the nutrients that required by Salmonella to grow. How to carry out the enrichment method? • The food sample are weighed and put into screw-capped jars. • Nutrients broth was added into jars. •Then the lids of the jars are now tightened and the jars are shaken vigorously after which they are incubated for 24 hours at 35°C.
- 28. Plating of enrichment cultures After incubation, a loop full of enrichment culture is streaked onto a plate of: 1) Salmonella-Shigella agar 2) Brilliant green agar, The plates are then incubated for 22-24 hours at 35°C Salmonella-Shigella agar: Salmonella colonies are usually colorless, transparent and usually larger than 5 mm in diameter and may have black centers. Brilliant green agar: Salmonella colonies are pink to deep red in color.
- 30. Screening • From Salmonella-Shigella and Brilliant green agar, at least 5 typical colonies are selected and transferred in each case to a triple sugar iron agar slant. • The slant being streaked and the butt stabbed. • The slant are incubated overnight at 35° C and then examined. • Result: For Salmonella positive, cultures the slant should be alkaline (purplish), the butt showed show acid (yellow).
- 31. Confirmation test • Triple sugar iron agar positive cultures are now examined for their reaction to polyvalent antiserum. • Result: Positive result showed a sign of agglutination with antiserum.
- 32. Testing for Shigella The testing methods for Shigella involved a) Enrichment of sample b) Plating the enrichment culture c) Screening test d) Confirmation test Enrichment test The enrichment procedure for Shigella and the same are those for Salmonella
- 33. Plating the enrichment culture After enrichment, the cultures were streaked on: 1) Salmonella-Shigella agar 2) Mc Conkey agar
- 34. 1. Salmonella-Shigella agar A. Klebsiella pneumoniae B. Escherichia coli Klebsiella pneumoniae & Escherichia coli are positive for acid production from fermentation of the carbohydrate(s) present. C. Salmonella sp. D. Proteus mirabilis Both Salmonella sp. & Proteus mirabilis product hydrogen sulfide. E. Pseudomona aeruginosa The Pseudomonas colonies are nearly colorless.
- 35. 2. Mc Conkey agar The plate is incubated for 24hours at 35° C. Result in agar: Typical colonies are colorless and transparent
- 36. Screening Typical colonies are transferred into 1) Triple sugar iron slants, incubated at 35°C, at 24 to 48 hours. Result: On this medium the slant is alkaline (purple) and the butt acid (yellow) 2) Lysine iron agar in which the slant is streaked and the butt stabbed Result: The slant is alkaline (purple) and file butt of the lysine iron agar is acid (yellow)
- 37. Triple sugar iron slants Lysine iron agar A. Salmonella sp. B. Escherichia coli C. Unknown D. Proteus mirabilis