LIST OF ABBREVIATIONS
CHAPTER 3: MATERIALS AND METHODS
3.4 Physico-chemical and microbiological parameters
3.4.2 Microbiological parameters
3.4.2.1 Enumeration of indicator bacteria
Faecal streptococci, E. coli, total coliform and faecal coliforms were enumerated via the membrane filtration method. Triplicate samples (100 mL) were filtered through 0.45 µm pore size
agar (Merck, Germany), MLG agar (Oxoid, UK), and KF-Streptococcus agar with an added supplement of 1 mL of 2,3,5-Triphenyltetrazolium chloride (TTC) per 100 mL of media (Sigma and Aldrich, Germany). TTC was added to KF-Streptococcus agar media at a temperature of 60°C.
The filters placed on the m-FC agar plates were incubated at 44.5°C for 18 – 24 hours. The filters on the MLG agar plates were incubated at 37°C for 18 – 24 hours, and the KF-Streptococcus agar media were incubated at 37°C for 48 hours. After incubation, blue and yellow colony growth on m-FC agar plates was counted. Yellow colonies represented total coliforms and blue colonies faecal coliforms. The green colonies on MLG agar plates (E. coli) were counted, and light pink or flat dark red colonies on the KF-Streptococcus agar were presumptively represented as enterococci. Colony numbers were recorded, and colony-forming units (CFU) per 100 mL calculated.
3.4.2.2 Incubation and isolation of Clostridium
Clostridium is an anaerobic micro-organism and had to be cultured in capped test tubes (16 mm x 125 mm; Pyrex) filled with 7 mL of double-strength Clostridium perfringens agar base. After autoclaving, the test tubes were cooled to approximately 50°C, and 1 mL of water sample and 32 µL of TSC supplement with D-cycloserine (Oxoid; UK) were mixed with the agar in the test tube. An autoclaved glass inserter tube with a diameter of 8 mm was inserted into the test tube with the liquid content and sealed with the screw-cap to ensure anaerobic conditions. This was done in triplicate for each water sample taken, a total of 24 test tubes for each sample period.
The sealed test tubes were incubated at 44°C for six hours (longer incubation will result in overgrowth). After incubation, black colonies were counted and documented as CFU/mL.
Numbers exceeding 300 colonies were recorded as too numerous to count (TNTC) and given a value of 300 for statistical reasons. (White et al., 2010).
To isolate Clostridium spp. the contents of the Fung double tubes were emptied into a sterile petri dish, and black colonial growth was pierced with a sterile wooden pick. The pierced colonies served as the culture to prepare streak plates of the culture, on TSC agar plates. To ensure that the streak plates could grow in anaerobic conditions, the plates were placed in an AnaeroJar (AG0025; Oxoid), with an AnaeroGen sachet (AN0025; Thermo Scientific) and an anaerobic indicator (BR0055B; Oxoid). The AnaeroJar and the plates were incubated for 24 hours at 44°C.
These streak plates were sub-cultured three times on Reinforced Clostridia agar to ensure that the bacterial cultures were pure enough for further analysis.
3.4.2.3 Enumerating heterotrophic plate count bacteria
To enumerate the Heterotrophic Plate Count (HPC) bacteria, a dilution series (to 10-10) was made of each of the water samples. Each of the dilutions was aseptically spread onto R2A agar plates (Becton, Dickinson & Company, France) and incubated at room temperature (approximately 23°C) for seven days. After incubation, the total colony growth of each dilution was counted and documented. Colony numbers were converted to CFU/mL. For further antibiotic and molecular studies, various morphologically different colonies, be they different in colour, shape or form, were aseptically streaked onto R2A agar plates. To ensure purity, the selected bacterial cultures were successively streaked (at least three times) on R2A agar plates.
3.4.2.4 Primary characterisation and biochemical screening of faecal enterococci, Clostridium sp. and HPC bacteria
3.4.2.4.1 Gram staining
The first step in any microbial characterisation is to distinguish between Gram-positive (G+) and Gram-negative (G-) bacteria and to ensure the purity of the enterococci isolates. A Gram stain was performed by preparing a bacterial smear on a glass slide and heat-fixing it before staining it with crystal violet for one minute. The crystal violet was rinsed off with distilled water. Secondly, Gram iodine was dropped onto the smear and left for one minute before rinsing it off with distilled water. The smear was de-stained with ethanol (96%) for 10 seconds and again rinsed with distilled water. Finally, the counterstain safranin was applied to the smear for one minute and rinsed off with distilled water once more. Schleifer and Kilpper-Balz (1984) described faecal streptococci as Gram-positive, elongated, ovoid-shaped cells in pairs or short chains. The presumptive result for faecal enterococci is deep purple-stained pair and short-chain cocci.
The same procedure for Gram staining was followed for the characterisation of HPC bacteria and Clostridium spp. However, Clostridium was also tested for endospore formation by using the Schaeffer and Fulton’s method for endospore staining. Clostridium is a rod-shaped, Gram- positive endospore producing bacteria (Willey et al., 2011a).
3.4.2.4.2 Catalase activity
Obligate aerobes and facultative anaerobes, such as enterococci, can produce enzymes such as catalase and peroxidase (Rolfe et al., 1978). These enzymes enable chemical reactions like hydrolyse, where hydrogen peroxide (H2O2) is converted into water and free oxygen as end products. To test for catalase activity in Enterococci, a cleaned microscope slide was used.
Bacterial isolates that produced gas after exposure to 3% H2O2 were catalase positive. It is known that faecal streptococci (Enterococci) are catalase-negative.
3.4.2.4.3 Triple sugar iron test
The Triple Sugar Iron (TSI) agar (Merck, Germany) test was used to confirm the presence of E.
coli. Purified presumptive E. coli isolates were inoculated with a stab and streak (continuous S- shaped streak) technique, in a TSI agar slant, using an inoculation needle. Slants were incubated at 37°C for 18 – 24 hours. A colour change in the agar slant from red to yellow was documented as well as gas production inside the test tube. When a black precipitate was present, this was indicative of Hydrogen sulphide (H2S) production.