Water and Milk Bacteriology

• 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 Streptococci – Gram 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