Microbiology: Testing for Bacteria Linda Wolf Glencoe High School SWRP Teacher for 12 years

Pathogens Pathogens are organisms capable of causing disease The following are some of the “bad guys”: Protozoa: Giardia, Cryptosporidium Bacteria: Salmonella typhi, Legionella, Shigella, Vibrio cholerae, Vibrio vulnificus Virus: Hepatitis, Polio

Sizes Bacteria are µm Viruses are µm For reference: 10 6 microns (or micrometer, µm) = 1 meter 1000 µm = 1 mm

Testing for Pathogens Direct testing for pathogens is impractical Pathogens are usually found in low numbers Can’t survive for very long outside the warm confines of a human or animal body Too many methods are too sophisticated and expensive

Indicator Bacteria Some bacteria can be good indicators of human pollution – the source for most pathogens Bacteria present in sewage pollution Survive longer than pathogens Easily detectable

Common Indicator Bacteria Total Coliforms Fecal Coliforms E. coli Enterococci E. coli Total Coliforms Fecal Coliforms

Total coliforms Rod-shaped, gram negative bacteria Ferment lactose at 35°C Found in intestinal tracts of cold and warm- blooded animals Group members: Escherichia, Klebsiella, Enterobacter, Serratia, Citrobactera, Edwardsiella

Fecal coliforms Subset of Total coliform group Present in sewage and indicate possibility of human pathogens Distinguished from Total coliform by ability to ferment lactose at 44.5°C Group members: E. coli and Klebsiella (not always fecal often associated with paper, textile & pulp waste)

Fecal coliforms Common in the intestines of both warm and cold-blooded animals If fecal coliforms are present it is presumed that human or animal excrement is present Diseases such as typhoid fever, hepatitis, gastroenteritis, dysentery and ear infections can be contracted in water with high Fecal coliform levels

E. coli Escherichia coli is a specific species within the Fecal coliform group Specific to intestines of mammals and other warm blooded animals Only specific strains (i.e. O157:H7) are pathogenic According to EPA, is the best indicator of health risks from water contact recreation

Enterococci Survives in salt water More human specific Found primarily in the intestinal tract of warm-blooded animals Used in some states as indicator organism in estuarine and marine waters

Bacterial Measurement Membrane Filtration Methods Quantify bacteria numbers by filtering water, growing bacteria, and counting Most Probable Number Methods Estimate bacterial numbers based upon a color change or amount of gas produced through a specific bacterial metabolic process

Membrane Filtration Known volume of water is filtered through a filter (0.45 µm) that is capable of trapping all bacteria Filter transferred to Petri dish containing growth media Individual bacterial cells will grow on the filter into visible colonies in 24 hours

Membrane Filtration m-ColiBlue24 broth Due to the metabolism of the bacteria on the media: Blue colonies indicate E. coli Red colonies indicate other Total coliform bacteria E. coli turn blue from the action of β- glucuronidase enzyme on 5-bromo-4-chloro- 3-indolyl-Beta-B-glucuronide

Procedure Collect water in a sterile container Filter water within 6 hours* Place sample in cooler if taking to lab *6 hours is standard holding time, but samples should definitely be filtered within 24 hours

Prepare plates Determine amount of water to filter 5 plates for each site For each site label one plate 0 mL for a “blank” # platesLabelVolume 10 mL10 mL sterile 210 mL10 mL sample 230 mL30 mL sample

Prepare Plates Label bottom of plate with: Date and time Sampling site Volume to be filtered Use sterile forceps to place sterile absorbent pad in each plate, if plates don’t already have them Place about 2 mL of broth on each pad, using either a sterile pipette or by shaking and pouring ampule

Filter Samples Sterilize forceps, place membrane filter on filter holder Use sterile water for small samples of water (1 mL) to wet the filter Pump until most of water is through filter Release pressure Sterilize forceps and place filter grid-side- up on the absorbent pad

Plates Put cover on plate Leave upright until all plates are filtered Incubate upside down for 24 hours in an incubator at 35° C

Calculating Results Count the blue and red colonies on each plate Blue colonies are E. coli Red + Blue = Total Coliforms If there are greater than 200 colonies report that plate as TNTC (Too numerous to count)

Most accurate plates The best are when the colony counts are in the range of: 20 – 80 colonies per plate for E. coli, and 50 – 200 for Total coliforms

Calculating Results Standard Units = CFU/100 mL (Colony Forming Units) Average colony counts x 100 = CFU/100 ml Volume Filtered (mL) If fewer than 20, estimate CFU/ 100 ml using all plates. Add total number of colonies and total volume Total colony counts x 100 = CFU/100 ml Total mL filtered

Other problems If over 200, but colonies are clearly countable, use the same general formula. Conflicting colony counts: go with the smaller sample size

0 mL filtered1 mL filtered 10 mL filtered30 mL filtered 24 colonies counted 6 colonies counted 0 colonies counted 67 colonies counted The 10 mL plate would be used for calculating CFU/100 mL: 24 / 10 x 100 = 240 CFU/100 mL

0 mL filtered 30 mL filtered 1 mL filtered 23 colonies counted 10 mL filtered 18 colonies counted 0 colonies counted 52 colonies counted 1 mL plate has more colonies that 10 mL plate. Possible problem(s): mislabeled plate contaminated apparatus The 1 mL plate would be used for calculating CFU/100 mL: 23 / 1 x 100 = 2300 CFU/100 mL

0 mL filtered 1 mL filtered30 mL filtered 23 colonies counted 6 colonies counted 7 colonies counted Estimate or report as Too Numerous to Count (TNTC) Possible problem: finger on filter or contaminated forceps Possible problem: “sterile” water not sterilized Possible problem: filter not wetted with sterilized water before filtering low volume sample – sample concentrated in one area of filter. Example of plate with more than 200 colonies. Colonies could be counted or estimated, and results flagged as “estimate”. The sterile water “blank” or 0 mL plate is a quality control measure – bacterial growth on the blank makes the other plate counts suspect.

Water Quality Standards In Oregon, based upon contact recreation 126 CFU/100 mL for 5 samples within a 30 day period 406 CFU/100 mL for a single sample