86
S.L. Chang
animals or birds serve as carriers of the pathogenic Naegleria! (7) Why should an indoor
swimming pool be involved in the occurrence of the disease?
It is intended in this report to present data that have been obtained both in the
laboratory and the field to answer some of these questions and to discuss the direction of
future research in order that preventive measures can be formulated.
MATERIALS AND METHODS
1. Naegleria Amoebae
Nine pathogenic strains of Naegleria, all isolated from autopsy or cerebrospinal fluid,
were carried in PMK (primary monkey kidney) cell cultures. Four of these strains were
also carried on the BST (buffered sucrose tryptose) agar plates in association with a strain
of Bordetella bronchoseptica. Ten wild strains of N gruben, isolated from swimming
pools, river or lake waters, and sewage effluents, were carried on the BST agar plates in
association with a strain of Aerobacter aerogenes. All cell cultures were kept at 35°C and
plate cultures at room temperature (25°-27°C).
2. Bacteria-Associates
The B. bronchoseptica, Gram-negative and rod-shaped, were isolated from a culture on
inorganic agar of one of the pathogenic strains of Naegleria. It was apparently a
contaminant but found to support the growth of both wild and pathogenic strains of
Naegleria. Stock cultures were carried on heart infusion agar slants. The A. aerogenes was
isolated from a sewage effluent. It supported the growth of wild strains of Naegleria on
the BST agar plates and that of the pathogenic strains on inorganic agar. Stock cultures
were carried on nutrient agar slants.
3. Mammalian Cell Cultures
Primary MK cells were purchased from a commercial source. Preparation of tube and
bottle cultures has been described elsewhere (Chang et aL, 1958; Berg et al., 1963). Hep-2
cells (oesophageal cancer cell line) was obtained from Dr. Robert Sullivan of the Milk
Sanitation Research Section, FDA, Public Health Service, and Vera cells (African green
monkey kidney cell line) from Dr. Gerald Berg of the Advanced Waste Treatment
Research, Cincinnati Water Research Laboratory. Both cell lines were originally
purchased from the American Type Culture Collections. Tube and bottle cultures of each
cell line were prepared in the same manner as the PMK cells except that the BME
(medium with Earl's base) was used for growth and maintenance of the Hep-2 cells.
4. BST Agar
The BST agar was developed primarily for cultivation of free-living amoebae in
laboratory (Chang, 1958). It is a phosphate-buffered (pH 7.4-7.5) agar containing 1%
(w/v) sucrose and 0.2% try ptose. The low concentration of try ptose is necessary because
of its inhibitive effect on amoebae at concentrations commonly employed in
bacteriological media. Agar plates were poured with 25 ml of the medium per petri dish.
5. Concentration of Amoebae from Water or Wastewater
The method for concentrating amoebae in water has been previously described (Chang,
1971). In brief, gallon-size samples are allowed to pass through 25μ microstrainers before
S.L. Chang
animals or birds serve as carriers of the pathogenic Naegleria! (7) Why should an indoor
swimming pool be involved in the occurrence of the disease?
It is intended in this report to present data that have been obtained both in the
laboratory and the field to answer some of these questions and to discuss the direction of
future research in order that preventive measures can be formulated.
MATERIALS AND METHODS
1. Naegleria Amoebae
Nine pathogenic strains of Naegleria, all isolated from autopsy or cerebrospinal fluid,
were carried in PMK (primary monkey kidney) cell cultures. Four of these strains were
also carried on the BST (buffered sucrose tryptose) agar plates in association with a strain
of Bordetella bronchoseptica. Ten wild strains of N gruben, isolated from swimming
pools, river or lake waters, and sewage effluents, were carried on the BST agar plates in
association with a strain of Aerobacter aerogenes. All cell cultures were kept at 35°C and
plate cultures at room temperature (25°-27°C).
2. Bacteria-Associates
The B. bronchoseptica, Gram-negative and rod-shaped, were isolated from a culture on
inorganic agar of one of the pathogenic strains of Naegleria. It was apparently a
contaminant but found to support the growth of both wild and pathogenic strains of
Naegleria. Stock cultures were carried on heart infusion agar slants. The A. aerogenes was
isolated from a sewage effluent. It supported the growth of wild strains of Naegleria on
the BST agar plates and that of the pathogenic strains on inorganic agar. Stock cultures
were carried on nutrient agar slants.
3. Mammalian Cell Cultures
Primary MK cells were purchased from a commercial source. Preparation of tube and
bottle cultures has been described elsewhere (Chang et aL, 1958; Berg et al., 1963). Hep-2
cells (oesophageal cancer cell line) was obtained from Dr. Robert Sullivan of the Milk
Sanitation Research Section, FDA, Public Health Service, and Vera cells (African green
monkey kidney cell line) from Dr. Gerald Berg of the Advanced Waste Treatment
Research, Cincinnati Water Research Laboratory. Both cell lines were originally
purchased from the American Type Culture Collections. Tube and bottle cultures of each
cell line were prepared in the same manner as the PMK cells except that the BME
(medium with Earl's base) was used for growth and maintenance of the Hep-2 cells.
4. BST Agar
The BST agar was developed primarily for cultivation of free-living amoebae in
laboratory (Chang, 1958). It is a phosphate-buffered (pH 7.4-7.5) agar containing 1%
(w/v) sucrose and 0.2% try ptose. The low concentration of try ptose is necessary because
of its inhibitive effect on amoebae at concentrations commonly employed in
bacteriological media. Agar plates were poured with 25 ml of the medium per petri dish.
5. Concentration of Amoebae from Water or Wastewater
The method for concentrating amoebae in water has been previously described (Chang,
1971). In brief, gallon-size samples are allowed to pass through 25μ microstrainers before