MICR20010 Lecture 9 Theory behind Practical 2 PDF

MICR20010 Lecture 9 Theory behind Practical 2 Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science To be carried out in Laboratory: 1. Streaking bacteria onto selective Media (Baird Parker and McConkey Agar) using Strea...

MICR20010 Lecture 9 Theory behind Practical 2 Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science To be carried out in Laboratory: 1. Streaking bacteria onto selective Media (Baird Parker and McConkey Agar) using Streak Plate technique 2. Enumeration of bacteria by serial Dilution Method followed by the Spread Plate technique 3. Personal Hygiene experiment. Extra material to be viewed online with demonstrations in lab: Most Probable Number test for Water Sampling Membrane Filtration The Dye Reduction test Streaking bacteria onto selective Media (Baird Parker and McConkey Agar) using Streak Plate technique SELECTIVE AND DIFFERENTIAL MEDIA Many media have been developed in order to select for certain organisms and to differentiate among them. These media are called selective or differential media. A selective medium is one which favours the growth of certain organisms and represses the growth of others. The choice of a selective medium for a given experiment depends on two factors: o Growth characteristics of the desired organism. o Nature of the material from which it is being isolated. A differential medium is one in which certain bacterial species produce characteristic colony morphology which may be easily recognised. The colonies may be characteristic because of their unusual size, colour or their effect on the immediate environment. Many of the selective media are also differential. In lab 2 we examine 2 commonly used selective and differential media: MacConkey agar and Baird-Parker agar. McConkey Agar MacConkey broth/agar is routinely used for the isolation and differentiation of coliforms and other intestinal bacteria in water, dairy products and biological specimens. The basic ingredients are lactose as sole carbon source, peptone as nitrogen source and bile salts as selective agent. Colonies of organisms capable of fermenting lactose produce a localized drop in pH and the inclusion of the pH indicator, neutral red, causes these organisms to appear as dark red colonies. MacConkey agar allows the isolation and differentiation of lactose fermenting, bile salt tolerant bacteria. McConkey Agar Ingredient g/L Peptone 17 g Polypeptone 3g Lactose 10 g Bile salts 1.5 g Sodium chloride 5g (Agar) (13.5 g) Neutral red 0.03 g Crystal violet 0.001 g Distilled water 1L Final pH 7.1 BAIRD-PARKER AGAR Baird-Parker agar is used principally for the detection and enumeration of staphylococci, particularly coagulase positive organisms such as S. aureus. It is widely used in food safety applications. Lithium chloride and tellurite are included to inhibit many organisms and the presence of glycine and pyruvate enhance staphylococcal growth. Reduction of tellurite yields a grey to black colour and coagulase positive organisms form a clear halo around the colonies. BAIRD-PARKER AGAR Ingredient g/L Casein Peptone 10 g Meat Extract 5g Yeast Extract 1g Lithium Chloride 5g Glycine 12 g Sodium pyruvate 10 g Agar 15 g Final pH (at 25 °C) 6.8±0.2 Cool to 50°C and aseptically add 50ml of Egg Yolk Emulsion and 3ml of Potassium Tellurite 3.5% STREAK BACTERIA ONTO SELECTIVE AND DIFFERENTIAL MEDIA 1. Using the streak plate method carried out in Practical 1 streak one plate of MacConkey agar, with the two organisms provided making sure not to cross-contaminate. 2. Streak one plate of Baird Parker agar, with the two organisms provided making sure not to cross-contaminate. 3. Incubate at 37oC. 4. Photos of plates on brightspace after 48 hours. Note and discuss for practical write up.  Streak each of 2 organisms provided on to 2 different types of media Staphylococcus aureus and Escherichia coli MacConkey agar (1x) and Baird-Parker agar (1x) Plates 1 1 2 2 Half plates for each organism 4 4 3 3 Note: Make sure not to cross-contamination E. coli S. aureus  Selective MacConkey Agar Selects enteric bacteria Bile salts inhibit many bacteria Differential Lactose + pH indicator (Neutral red) Lactose fermenters - Pink Enumeration of Bacteria Serial dilution- agar plate method 0.1 Plate counts ml (CFU/ml) Add sample (milk or contaminated water) 1 to diluent (water) ml Mix Add diluted sample to fresh diluent (fresh tip) Repeat n times Samp Samp H2O H2O H2O H2O Spread dilutions on agar plates le le 9ml 9ml 9ml 9ml Incubate 10- 10-2 10- 10- Count colonies 1 3 4 Dilutions Calculate original concentration  Spread plate technique Isolate colonies of interest Counting Enumerate bacteria in a sample of milk Create 10-fold dilution series Use spread Nutrient Agar Plate to detect bacteria in 10-2, 10-3, 10-4 dilutions Calculate number per ml in original sample 10 x ( 288.33 x 1000) (28 X 10000) Personal Hygiene- transfer of microorganisms The potential to transfer the microorganisms through tissue paper Use of hand washing 2  Agar 2 day incubation Agar plate plate 3 day incubation results results Plate 1 Malt Extract Agar Touch plate with Touch Touch unwashed unwashed gloved Glovedfingers fingers glovedFingers fingers to to plate plate Plate 2 # # 1 2 Wrap gloved fingers in tissue Wrap Remove tissue Ppapergloved fingers and touch in tissue plate paper Paper and touch plate aper and touch plate # Plate 3 3 Wrap gloved fingers in tissue Touch Wrap Ppapergloved fingers and touch in tissue plate, remove fingers to plate tissue and then wash with soap provided. Touch  Display for students Dye reduction test Membrane filtration Methylene blue: redox indicator Cellulose acetate or cellulose nitrate: Pore size Strepotcoccus lactis: change from blue to of 0.45 – 0.2 μm colourless upon reduction Bacteria become trapped on the filter, which can be removed and placed on appropriate medium. Advantages: Quick (24 h), field testing, greater reproducibility, etc. Poor milk – decolourised inside 2 h; High quality milk – not decolourised after 8 h. Methylene blue Leuco-Methylene blue  Most Probable Number (MPN) Coliform bacteria are indicators of faecal contamination No. of coliforms in a given water sample can be determined by MPN method double-strength MacConkey broth 2 1 1