9218 AEROBIC ENDOSPORES*
9218 A. Introduction
1. Description Analysis for aerobic endospores can be used to evaluate a
variety of water treatment processes, including physical removal
Endospores are dormant, environmentally resistant structures processes (e.g., coagulation, clarification) and disinfection.1–3
formed by certain genera of bacteria. The majority of aerobic- Most surface water sources (except some lakes and reservoirs)
endospore-forming bacteria are harmless, saprophytic organisms contain sufficient levels of endospores to determine order-of-
found in soil and water. Vegetative cells of aerobic-endospore- magnitude removal efficiencies. Because endospores are highly
forming bacteria sporulate in response to adverse environmental resistant structures, they are also used for determining the effi-
conditions. The bacteria can persist in the endospore form for an cacy of such processes as halogen, ozone, and ultraviolet inac-
extended period. Under favorable conditions, such as a readily tivation. The procedure also has been used to evaluate the
available nutrient supply, endospores convert back to vegetative efficacy of riverbank infiltration. In addition, the presence of
bacterial cells in a process known as germination. Unlike veg- endospores may be used to determine the physical integrity of
etative cells, endospores resist environmental pressures, such as drinking water distribution systems that may have been compro-
heat, desiccation, disinfection, and irradiation. Endospores are mised via pipeline breaks or maintenance procedures.
generally even-sized and refractile.
3. References
2. Detection and Applications
1. Rice, E.W., K.R. Fox, R.J. Miltner, D.A. Lytle & C.H. Johnson.
Aerobic endospores are ubiquitous in the environment. Indig- 1996. Evaluating plant performance with endospores. J. Amer. Water
enously occurring aerobic endospores in water are detected by Works Assoc. 88:122.
exposing the sample to a heat treatment to inactivate any vege- 2. BARBEAU, B., L. BOULOS, R. DESJARDINS, J. COALLIER & M. PREVOST.
1999. Examining the use of aerobic spore-forming bacteria to assess
tative cells; heat treatment does not inactivate the endospores.
the efficiency of chlorination. Wat. Res. 33:2941.
The sample is then plated onto a nonselective nutrient medium 3. NIEMINSKI, E.C., W.D. BELLAMY & L.R. MOSS. 2000. Using surrogates
and incubated aerobically at 35°C. The endospores germinate to improve plant performance. J. Amer. Water Works Assoc. 92:67.
and form bacterial colonies. Most of these organisms will be
species of Bacillus. 4. Bibliography
* Approved by Standard Methods Committee, 2007.
SCHUBERT, R.H.W. 1975. The detection of spores of the Bacillus species
Joint Task Group: 21st Edition—Clifford H. Johnson (chair), Kathleen E. Atter- within the scope of the hygienic control of water pollution. Zbl.
bury. Bakt. Hyg., I. Abt. Orig. B 160:155.
9218 B. Membrane Filter Method
1. Apparatus Agar...................................................................................... 15.0 g
Reagent-grade water ............................................................ 1.0 L
a. Bottles or Erlenmeyer flasks with closures, suitable for Heat to boiling to dissolve agar. Sterilize by autoclaving for
sample size chosen and capable of withstanding hot and cold 15 min at 121°C. Final pH should be 6.8 ⫾ 0.2. After autoclav-
temperatures. ing, cool to 50°C and dispense aseptically into 50- ⫻ 9-mm
b. Culture dishes: See Section 9222B.1e. plastic culture plates with loose-fitting lids. The pH should be
c. Filtration units: See Section 9222B.1f. 6.8 ⫾ 0.2. Refrigerated medium can be held for 20 d at 4 to 8°C.
d. Water bath, preferably shaking style, capable of heating
samples to 80°C. 3. Procedure
e. Thermometer.
a. Sample size: See Section 9222A.2.
2. Materials and Culture Media b. Heat treatment of sample: Distribute samples into bottles or
Erlenmeyer flasks (9218B.1a). Loosen caps. Prepare a pilot
Nutrient agar plus trypan blue: control flask that contains the same volume as that of the water
Peptone................................................................................. 5.0 g sample in the test flasks. The control flask must have a closure
Beef extract.......................................................................... 3.0 g that includes a thermometer. Place control flask into the water
Trypan blue.......................................................................... 0.015 g bath along with the samples. Ensure that the level of water in the
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AEROBIC ENDOSPORES (9218)/Membrane Filter Method
bath is higher than that of the samples. When using a water bath endospores. Count colonies with a low-power (10 to 15⫻) bin-
with shaker, agitate samples at 60 to 80 rpm. If a shaking bath is ocular wide-field dissecting microscope or other optical device.
not available, manually swirl samples periodically. Monitor tem- Trypan blue is added to impart a colored background on the
perature in pilot control flask. When pilot flask reaches desired membrane. Consider any bacterial colonies growing on the
temperature, begin timing and hold sample at that temperature membrane as aerobic spore-forming bacteria.
for the specified time. At a temperature of 75°C, heat samples for d. Interpretation: For colony counting procedure, see Section
15 min; at a temperature of 80°C, heat samples for 10 min.1,2 9215A.8b. Colonies should catalase positive.
Then, cool samples immediately in a bath containing a slurry of
wet ice. Let samples cool to approximately room temperature. 4. References
As a quality control procedure, spike samples and then examine
for the presence of spores via staining or phase contrast micros- 1. RICE, E.W., K.R. FOX, R.J. MILTNER, D.A. LYTLE & C.H. JOHNSON.
copy. 1996. Evaluating plant performance with endospores. J. Amer. Water
c. Filtration of samples and counting: Filter samples as de- Works Assoc. 88:122.
scribed in 9222B.4c. Place membrane filter on agar surface and 2. BARBEAU, B., L. BOULOS, R. DESJARDINS, J. COALLIER, M. PREVOST &
incubate for at least 24 ⫾ 2 h at 35 ⫾ 0.5°C. Some colonies D. DUCHESNE. 1997. A modified method for the enumeration of
require incubation for 5 to 7 d for intracellular formation of aerobic spore-forming bacteria. Can. J. Microbiol. 43:976.
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