Water and Milk Bacteriology

Cards (24)

  • Water Bacteriology
    Used to determine the presence of Escherichia coli and other coliforms (Citrobacter, Enterobacter, Klebsiella) if there fecal contamination present in water
  • Water-borne Diseases
    • Cholera
    • Typhoid fever
    • Shigellosis
    • Salmonellosis
    • Gastroenteritis
  • Water Collection Procedure
    1. Wipe the mouth of the drinking fountain with alcohol
    2. Heat the mouth of the fountain
    3. Heat the mouth of the tube & collect water
  • Methods of Water Bacteriology
    • Most Probable Number (MPN) Test
    • Presence-Absence (P-A) Coliform Test
    • Membrane Filter Technique for Fecal Coliforms and Fecal Streptococci
    • KONFIRM Test for Fecal Coliforms
  • Most Probable Number (MPN) Test

    A statistical estimation of the number of coliform bacteria per 100 ml of water sample
  • Parts of MPN Test
    1. Presumptive Test
    2. Confirmed Test
    3. Completed Test
  • Brilliant Green Lactose Bile Broth (BGLB) with Durham's Fermentation Tube
    A selective and differential medium for coliforms. It has 2% bile to inhibit Gram positive, Brilliant Green to inhibit Gram negative except the coliforms, Lactose as carbohydrate source. Turbidity + gas in DFT is indicative of a positive result
  • Turbidity with gas is confirmatory for the presence of coliform bacteria
  • Presence-Absence (P-A) Coliform Test
    A modification of the MPN Procedure in which a large water sample (100 ml) is incubated in 50 ml P-A broth which contains Lactose, Sodium lauryl sulfate, Bromcresol purple. Production of acid from lactose fermentation results in change of color from purple to yellow
  • Presence-Absence (P-A) Coliform Test

    1. Presumptive Test: Incubate at 35oC for 24-48 hours
    2. Confirmed Test: Inoculate BGLB with DFT from (+) P-A broth, Incubate at 35oC for 24-48 hrs
  • Milk Bacteriology is used to detect the sanitary quality of milk and to prevent outbreaks
  • Diseases transmitted from contaminated milk samples (Bovine origin)
    • Q Fever (Coxiella burnetii)
    • Undulant fever (Brucella abortus)
    • Anthrax (Bacillus anthracis)
    • Cowpox
    • Foot and mouth disease
    • Bovine Tuberculosis (M. bovis)
  • Diseases transmitted from contaminated milk samples (Human origin)
    • Salmonellosis/Typhoid fever (Salmonella typhi and Salmonella sp.)
    • Scarlet Fever (Group A Strep)
    • Cholera – Vibrio sp.
    • Diphtheria (C. diphtheriae)
    • Septic sore throat (Group A Strep)
    • Human tuberculosis (M. tuberculosis)
    • Bacillary dysentery (Shigella sp., Salmonella, Campylobacter)
    • Listeriosis
  • Normal Flora of Milk
    • Lactobacillus acidophilus
    • Bifidobacterium bifidum
    • Lactobacillus casei
    • Lactobacillus bulgaricus – yoghurt
    • Lactococcus (Streptococcus) lactis – cheese production; resp. for the normal souring of milk
    • Alcaligenes viscolactis or Alcaligenes viscosus – slimy or ropy milk
    • Bacillus subtilis – exerts proteolytic action on coagulated milk
    • Clostridium butyricum and Clostridium perfringens – "stormy fermentation" or strong-smelling acids
  • Chromogenic/Pigment Producing bacteria in Milk
    • Pseudomonas syncyanea (blue milk)
    • Serratia marcescens (red milk)
    • Pseudomonas aeruginosa (blue-green milk)
    • Flavobacterium synxanthum (yellow milk)
  • Pasteurization
    A heat process that is gentle enough to preserve the physical and nutrient properties of milk but sufficient to destroy pathogenic organisms. Batch Method: 62.9oC for 30 mins, Flash Method/Continuous Flow: 72oC for 15 seconds
  • A Grade "A" Pasteurized milk – bacterial content is below 75,000 CFU/ml when raw and should not exceed 15,000 CFU/ml when pasteurized
  • Certified Milk – produced under close medical supervision. It contain < 10,000 CFU/ml
  • Colony Count
    Pour plate method using: Undiluted milk, 1:10 dilution of milk in NSS, 1:100 dilution of milk in NSS. Violet Red Bile Agar (VRBA) is used - Crystal violet and bile salts inhibit Gram positive, Lactose is the carbohydrate source, Neutral red is the indicator
  • VRBA Agar
    Lactose fermenters: pink to red colonies with halo, Non-lactose fermenters: colorless
  • Methylene Blue Reductase Test
    An indirect method for the detection of bacteria in milk. When methylene blue is added to milk, the dye is decolorized (reduced) by the oxygen-consuming bacteria present in the sample. The speed of this reduction is directly proportional to the number of bacteria present in the sample indicating the bacterial content of milk
  • Procedure for Methylene Blue Reductase Test
    10 ml of milk + 1 ml methylene blue 1/25,000, mix, incubate 37oC water bath for 5 mins. Observe the tube at 30 minute intervals for 8 hours. Endpoint Reduction: at least 4/5 of the tube is white
  • Quality of Milk Samples based on Methylene Blue Reductase Test

    • Class I (Good): 6 to > 8 hours
    • Class II (Fair): 2-6 hours
    • Class III (Poor): 30 mins -2 hours
    • Class IV (Very poor): w/in 30 mins
  • Bacterial Identification
    1. A. Pathogenic StreptococciGram stain a milk smear for the presence of long-chain streptococci together with neutrophils and do culture on BA to observe for hemolytic properties
    2. B. Mycobacterium bovis – tuberculin test of cows demonstrates the infection; Guinea pig inoculation
    3. C. Brucella abortus – subcutaneous guinea pig inoculation and perform autopsy 3-5 wks later; Cultures of milk for Brucella are frequently unsuccessful; Agglutination test (using animal serum) or skin test (brucellergin) is more reliable
    4. D. Corynebacterium diphtheriae – Culture in Loeffler's Serum Agar Slants and Tinsdale medium
    5. E. Coliform Group – presumptive evidence for the presence of coliform is gas formation within 24 hrs in Lactose broth with Durham's fermentation tubes
    6. F. Typhoid/Parathyphoid (Salmonella Group) – use enrichment media first (tetrathionate broth) then inoculate in SSA and BSA and other selective media