Analytical Methods for Bacteria & Fungi

Bacteria

Analytical Method

Heterotrophs (Enumeration)

Idexx SIM (Spread) Plate; SM9215 C (Spread Plate); SM9215 B (Pour Plate)

Hydrocarbon Degrading Bacteria

ASTM D 6974-04 modified

Iron & Sulfur Utilizers

SM9240 BCD (microscope / culture)

Pathogenic Bacteria

 

Enteric

 

Coliform, Total & E Coli  (+/-)

SM9223 B (EST)

Coliform, Total & E Coli (Enumeration)

SM9223 B (EST – Idexx Colilert); SM9221 B (MTF/MPN); SM9222 B (MF)

Enterococci (Enumeration)

SM9230 B (MTF/MPN); Idexx Enterolert (EST)

Fecal Coliform

SM9221 E (MTF/MPN); SM9222 D (MF)

Fecal Streptococci

SM9230 B (MTF/MPN)

Klebsiella

SM9222 F (MF)

Salmonella (+/-)

SM9260 B (media culture, presence or absence with serological confirmation)

Salmonella (Enumeration)

SM9260 D (media culture with serological confirmation)

Shigella (+/-)

SM9260 E (MF/Centrifugation)

Staphylococcus aureus

FDA BAM (Direct Spread Plate Count with serological confirmation)

Other

 

Aeromonas hyrophila

SM9260 L (SP, MF, MTF)

Legeonella

SM9260 J (MF)

Pseudomonas aeruginosa (Enumeration)

SM9213 E (MF or Direct Spread Plate Count)

Fungi

 

Yeast and Mold Count, Total

FDA BAM (Direct Spread Plate Count)

Algae

 

Algae Identification

SM10900 CD (microscope)

 


Definitions:

Enzyme Substrate Method:    Method using hydrolysable substrates that form either a distinct color or fluorescence in the presence of certain bacteria.  Results are reported in MPN.

 

Membrane Filtration (MF):   Method in which sample is filtered through a sterile, 0.45mm-pore, gridded membrane.  All of the bacteria in the sample are “trapped” on the surface of the membrane, which is then placed on appropriate media.  This allows each bacterium to form a colony on the surface of the membrane.  Results are reported in cfu.

 

MPN (Most Probable Number) – Indirect rather than direct enumeration or colony count such as that given by membrane filtration. MPN means that the reported number of coliform bacteria has a higher probability than any other number of being correct, and of being reproducible by duplicate testing.

 

Multiple-Tube Fermentation (MTF):  Method in which replicate tubes of several dilutions are inoculated with sample.  Each tube has an inverted vial that is used to detect gas formation resulting from the fermentation of an appropriate carbon-source.  Results are reported in MPN.

 

Pour Plate Method:    Method in which sample is mixed with agar prior to solidification.  The sample and agar are poured into sterile petri dish that is then allowed to solidify.  Each individual bacterium forms a single colony within the agar.  Results are reported in cfu.

 

Presence / Absence (+/-) – Determination of the presence or absence of bacteria without enumeration / MPN. All Total Coliform positives MUST be tested for the presence of E Coli or fecal coliforms.

 

Spread Plate Method: Method in which sample is spread evenly on an agar plate.  Each individual bacterium forms a single colony on the surface of the agar plate.  Results are reported in colony-forming units (cfu).

 

Bacteria, Fungi & Algae Tested By MAI:

Algae: Testing is available for the isolation and identification of algae in various environmental samples.

 

Aeromonas hyrophila: Testing is available for Aeromonas hydrophila. This group of bacteria is a natural inhabitant of aquatic environments worldwide. They are not enteric. They have been isolated from groundwater, treated drinking water, surface water, wastewater, sludge, sediments and foods. They are known to cause gastrointestinal illness to human & can infect through wounds or ingestion. Enumeration of Aeromonas spp. is done by spread plate, membrane filtration or multiple tube fermentation. Positive results from the enumeration techniques are confirmed by screening tests for Aeromonads. Results are expressed in CFU or MPN.

 

Coliform, Total, & E Coli Bacteria: Testing available for water, soil and food & reported as +/- , CFU or MPN. Coliform are a group of bacteria present in the gut and feces of warm-blooded animals as well as in other environments. Non-fecal coliforms are expected to be further distributed from the source than fecal coliforms, so that it is possible to have a positive total coliform but negative fecal / E Coli. Total coliform positive (without E Coli confirmation), E Coli positive, and Fecal coliform positive results found in drinking, bathing & recreational waters MUST be reported to the responsible party within 24 hours. Escherichia coli & Enterobacter aerogenes are positive culture controls; Staphylococcus aureus, Proteus vulgaris & Pseudomonas aeruginosa are negative culture controls.

E Coli are a sub-group of coliform bacteria whose presence is deemed by regulation to be diagnostic of the presence of fecal coliform, i.e. positive E Coli confirms the presence of fecal coliform bacteria. Escherichia coli (MUG positive strain) is the positive culture control; Enterobacter aerogenes & Klebsiella pneumoniae (thermotolerant MUG(-) strain) are the negative culture controls (ELAP).

 

Enterococci: Testing available for water & soil & reported as +/- or MPN. Enteroccoci are a sub-group of the Fecal Streptococci group and that group is designated in Standard Methods for monitoring of fecal contamination in recreational waters. Enterococcus Faecalis & Enterococcus faecium are the positive control cultures for this group; Staphylococcus aureus (nalidixic acid sensitive), Escherichia coli (sodium azide sensitive) & Serratia marcescens (fluorogenic compound not hydrolyzed) are negative culture controls (ELAP). Enterococci are can be associated with both human and animal fecal origins.  These bacteria are considered to be more specific as indicator organisms of the sanitary quality of recreational waters than the fecal streptococci or the fecal coliform bacteria.  Some important clinical infections caused by Enterococcus include bacterial endocarditis, urinary tract infections, bacteremia, and meningitis to name a few.

 

Fecal Coliform Bacteria: Testing available for water, soil and some food & reported as MPN or CFU. These bacteria are a sub-group of coliform bacteria defined by regulation as those bacteria capable of producing gas from lactose in a suitable culture medium at 44.5 ±0.2°C.  These bacteria are present in the fecal excrement of humans, livestock and wildlife.  A positive result for fecal coliform bacteria in a sample indicates fecal contamination, which can result from several sources including the direct discharge of waste from mammals and birds, from both agricultural and storm runoff, and from untreated human sewage.  Fecal coliform bacteria are used as indicator organisms, and their presence can indicate a higher risk of pathogens being present in the contaminated sample.  These pathogens can cause many diseases including, but not limited to, dysentery, typhoid fever, viral or bacteria gastroenteritis, and Hepatitis A.  Escherichia coli & Klebsiella pneumoniae (thermotolerant strain) are positive culture controls; Enterobacter aerogenes is the negative culture control (ELAP).

 

Fecal Streptococci: Testing available for water & soil & reported as MPN. Enterococcus faecalis is a positive control culture for this group; Staphylococcus aureus & Escherichia coli are negative culture controls (SM Table 9002:V). Enterococci positives are diagnostic of the presence of Fecal Streptococci but Enterococci negatives are not definitive of the absence of Fecal Streptococci. For example, equine or bovine fecal material may contain or be characterized by other Streptococci bacteria.

 

Heterotrophic Plate Count (HPC): Testing available for water, soil & food & reported as CFU or MPN Provides an approximate enumeration of total numbers of viable bacteria yielding useful information about water quality and water treatment efficiency.

 

Hydrocarbon Degrading Bacteria: Testing is available for the enumeration of petroleum degrading bacteria in soil and water matrices. Results are expressed as Total Recoverable Heterotrophs (TRH) in CFU.  A qualitative testing for industrial fluids such as metalworking coolants, cutting oils, engine-lubricating oils, emulsions, slurry pools and cooling tower process water is also available. This test gives an early warning of microbial growth in process fluids.

 

Iron and Sulfur Bacteria: Testing is available for iron and sulfur bacteria. The method applied is microscopic examination and culture. Various culture media are used to isolate most if not all the iron and sulfur bacteria in a sample. Results are expressed either +/- or CFU.

 

Klebsiella: MF testing is available for Klebsiella to differentiate this bacterium from other fecal coliforms. Results are expressed in CFU.

 

Legionella: Testing is available for the enumeration of Legionella in water and other environmental samples. Various Legionella selective agar and different antibiotic supplements are used to successfully isolate the bacterium. Results are expressed in CFU.

 

Pseudomonas aeruginosa: Testing available for water & soil. These bacteria are commonly found in of soil and water, in addition to the surfaces of plants.  Pseudomonas aeruginosa is a pathogen of plants as well as humans.  Pseudomonas aeruginosa is an opportunistic pathogen that can cause urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections in immuno-compromised and immuno-suppressed patients.  People who are seriously affected by Pseudomonas aeruginosa include AIDS patients, cancer patients, burn victims, and people with cystic fibrosis. Results are reported in CFU.

 

Salmonella: Enumerative testing available for water only, but +/- testing of Salmonella in both soil and water samples. Salmonellae live in the intestinal tracts of both warm and cold-blooded animals.  These organisms cause enteric fever and gastroenteritis in humans.  Environmental sources of the organism include water, soil, animal waste, raw meat, raw poultry, and raw seafood.

 

Shigella: Testing available for the detection (+/-) of Shigella in soil and water samples. Shigella is the enteric bacteria that causes Shigellosis in humans.  People affected by Shigellosis have diarrhea (often bloody) and severe stomach cramps.  The diarrhea can affect the young and the elderly so severely that they need to be hospitalized due to dehydration.  The organism is commonly found in water that has been polluted with human fecal material.

 

Staphylococcus aureus: Testing available for enumeration of S. aureus in food, soil, and water samples. S. aureus is found most commonly in human nasal passages.  This bacteria is considered a pathogen of humans, as it causes skin infections and infections associated with wounds, pneumonia, mastitis, and toxic shock syndrome.  S. aureus also causes food poisoning by releasing enterotoxins in food and is one of the most common causes of hospital-acquired infections in patients.  Foods that are frequently sources of staphylococcal food poisoning are meat and meat products, poultry, eggs,  mayonnaise-based salads including egg, tuna, chicken, potato, and macaroni, and various bakery products.

 

Yeast and Mold Count, Total: Aerobic plate count of yeast and molds in food, soil, and water samples.