Pathogenic Free-Living Amoebae and Recreational Waters
87
they are filtered through 10μ membranes. Each membrane is placed on the wall of a
sterile beaker and flushed repeatedly with a small amount of dilution water with the aid
of a capillary pipette. The washings are surveyed for types and concentration of amoebae
by examining under low power one or more drops placed on a microslide. The number of
amoebae gal"
1 is obtained by multiplying the average number of amoebae drop"
1 by the
number of drop in ml and number of ml in a gallon.
Fiberglass prefllters can be used to replace the 10μ membranes. If the strained water is
too turbid, two or more membranes of fiberglass filters may be used for each sample. The
washings are pooled before they are examined for amoebae. If the volume of the pooled
washings is too large, it can be reduced by centrifuging and resuspending the sediment in
a desired amount of dilution water.
6. Examination of Concentrated Samples for Amoebae by Plaquing on Plates
This technique is quite similar to the plaque counting of bacteriophage and has been
described elsewhere (Chang, 1971). In essence, a concentrated sample is diluted with a
suspension of B. bronchoseptica such that each drop from a capillary pipette contains not
more than 3 amoebae. The entire sample is plaqued by placing 10 drops on each poured
BST agar plate and smearing the mixed suspension evenly over the whole plate with a
sterile golf-club-shaped glass rod. After the liquid is absorbed, the plates are turned over
and incubated at 25°-28°C. Examination of plates for plaques starts on the 3rd day and
covers a period of 2 weeks.
Wild strains of N. gruben and other schizopyrenid amoebae grow fast on bacteria and
generally show plaques in 3 to 4 days of incubation; Hartmannella and Acanthamoeba
spp. grow slower and take 7-10 days to show plaques. The pathogenic strains of Naegleria
grow so poorly in the presence of bacteria growth that their plaques are not visible until
after 12-15 days. These differences have already been observed with several strains of wild
and pathogenic Naegleria and other species of free-living amoebae (Chang, 1971).
Microscopic examination of the plaques for trophozoites and cysts enables one to
place the amoebae in general groups mentioned above. To establish cultures of single
strains of amoebae the center of a plaque is touched with an inoculation needle or loop
and the amoebae so picked up are placed in the center of a BST agar plate preseeded with
B. bronchoseptica or on an inorganic agar plate preseeded with A. aerogenes. The
amoebae will move away from the site of inoculation, and transfer of the migrated
amoebae with the bacteria-associate onto a new plate results in the formation of a pure
amoeba culture with a known bacteria-associate.
7. Preliminary Test for Pathogenicity
This is done by demonstrating the CPE (cytopathic effect) of the amoebae on PMK
cells. The amoebae of an established plate culture were suspended in sterile dilution or
distilled water, and were repeatedly centrifuged and washed to remove the associated
bacteria. The sediment was finally suspended in water containing 200 Mgm each of
tetracycline and streptomycin. After 2-4 hours of storage in a refrigerator the suspension
was centrifuged again and the supernate discarded. The sediment was transferred to a
tube of PMK cell cultures. Generally, one trial was enough to establish a positive culture.
Occasionally, 1 or 2 trials were needed before a positive culture was prepared. Similar
procedure was used for preparing amoebic cultures in Hep-2 or Vera cell tubes.
As soon as the cultures were established, they were observed for the CPE on the
mammalian cells. In the preceding report (Chang, 1971) it has been shown that 4 pathogenic
87
they are filtered through 10μ membranes. Each membrane is placed on the wall of a
sterile beaker and flushed repeatedly with a small amount of dilution water with the aid
of a capillary pipette. The washings are surveyed for types and concentration of amoebae
by examining under low power one or more drops placed on a microslide. The number of
amoebae gal"
1 is obtained by multiplying the average number of amoebae drop"
1 by the
number of drop in ml and number of ml in a gallon.
Fiberglass prefllters can be used to replace the 10μ membranes. If the strained water is
too turbid, two or more membranes of fiberglass filters may be used for each sample. The
washings are pooled before they are examined for amoebae. If the volume of the pooled
washings is too large, it can be reduced by centrifuging and resuspending the sediment in
a desired amount of dilution water.
6. Examination of Concentrated Samples for Amoebae by Plaquing on Plates
This technique is quite similar to the plaque counting of bacteriophage and has been
described elsewhere (Chang, 1971). In essence, a concentrated sample is diluted with a
suspension of B. bronchoseptica such that each drop from a capillary pipette contains not
more than 3 amoebae. The entire sample is plaqued by placing 10 drops on each poured
BST agar plate and smearing the mixed suspension evenly over the whole plate with a
sterile golf-club-shaped glass rod. After the liquid is absorbed, the plates are turned over
and incubated at 25°-28°C. Examination of plates for plaques starts on the 3rd day and
covers a period of 2 weeks.
Wild strains of N. gruben and other schizopyrenid amoebae grow fast on bacteria and
generally show plaques in 3 to 4 days of incubation; Hartmannella and Acanthamoeba
spp. grow slower and take 7-10 days to show plaques. The pathogenic strains of Naegleria
grow so poorly in the presence of bacteria growth that their plaques are not visible until
after 12-15 days. These differences have already been observed with several strains of wild
and pathogenic Naegleria and other species of free-living amoebae (Chang, 1971).
Microscopic examination of the plaques for trophozoites and cysts enables one to
place the amoebae in general groups mentioned above. To establish cultures of single
strains of amoebae the center of a plaque is touched with an inoculation needle or loop
and the amoebae so picked up are placed in the center of a BST agar plate preseeded with
B. bronchoseptica or on an inorganic agar plate preseeded with A. aerogenes. The
amoebae will move away from the site of inoculation, and transfer of the migrated
amoebae with the bacteria-associate onto a new plate results in the formation of a pure
amoeba culture with a known bacteria-associate.
7. Preliminary Test for Pathogenicity
This is done by demonstrating the CPE (cytopathic effect) of the amoebae on PMK
cells. The amoebae of an established plate culture were suspended in sterile dilution or
distilled water, and were repeatedly centrifuged and washed to remove the associated
bacteria. The sediment was finally suspended in water containing 200 Mgm each of
tetracycline and streptomycin. After 2-4 hours of storage in a refrigerator the suspension
was centrifuged again and the supernate discarded. The sediment was transferred to a
tube of PMK cell cultures. Generally, one trial was enough to establish a positive culture.
Occasionally, 1 or 2 trials were needed before a positive culture was prepared. Similar
procedure was used for preparing amoebic cultures in Hep-2 or Vera cell tubes.
As soon as the cultures were established, they were observed for the CPE on the
mammalian cells. In the preceding report (Chang, 1971) it has been shown that 4 pathogenic