Occupational Health - Public Health Poster Session
Materials and Methods
Breast-milk samples were collected from lactating mothers at various postpartum (PP) periods, by manual expression or a breast pump. The samples were placed in sterile plastic tubes and transported to the laboratory on ice and processed immediately or stored in small aliquots at -70° or -80°C until use. One set of milk samples was collected in 20mM EDTA solution, while the other set of samples from the same mother was collected in an equivalent volume of PBS buffer only. The samples were classified according to postpartum period of lactation as follows (11); colostrum (1-4 days PP, 5 samples), transitional milk (5-30 days PP, 4 samples) and mature milk (>30 days PP, 4 samples).
De-fatted milk samples were obtained by dividing whole milk samples into smaller portions and centrifuging them consecutively at rates of 500g and 3,000g at 4°C for 15 min, aspirating the aqueous phase of the milk each time. During centrifugation, the milk separates into three layers, i.e. a cell pellet, a middle aqueous layer and an upper fat layer. The aqueous layers were collected each time and processed immediately or stored as small aliquots at -70 or -80°C. Certain portions of the de-fatted breast-milk were also further clarified by centrifugation twice at 20,000g for 15 minutes each at 4°C. The clarified phases were collected and stored in 1 ml aliquots at -70°C until required.
Portions of the breast-milk were stored at 4°C and -20°C for different times after which they were collected and either immediately processed or stored at -70 or -80°C in small aliquots until processed.
Readings of pH values were taken using a glass electrode, with combined sensing and reference elements, inserted into the samples, connected to a research pH meter. The Radiometer (PHM84), (Radiometer, Copenhagen, Denmark), was capable of measuring temperature-dependent pH values, to an accuracy of three-decimal places. The Radiometer was calibrated daily, before and after sample measurements, by measuring its response to buffers of known pH.
Samples of de-fatted and whole breast-milk, as well as serum or plasma, were heated in a water bath at 56°C for 30 min to inactivate the complement activity.
A complement-sensitive strain of Escherichia coli NCTC 8007, serotype 0111 K58(B4) H2 was used (25). Bacteria were cultured overnight in blood agar and suspended in sterile normal saline adjusted to 3 x 108 colony-forming units (CFU) per ml using the McFarland method (26). 20µl of the adjusted bacteria was added to round-bottom microtiter wells (Nunc A/S, Roskilde, Denmark) with 80µl of the milk or serum sample to be tested. The trays were thoroughly mixed on an orbital mixer before and after incubation at 37°C for 2 hrs. Samples of 20µl were taken from each well before and after incubation for viable counts using poured plate method (27). Whole-milk and de-fatted (or clarified) milk samples, stored at -70°C or -80°C, were tested heat-treated or not, with or without addition of Mg2+ EGTA (to support the alternative pathway of C activation), and compared with was tested unde-fatted and de-fatted, heat-treated (56°C, 30 min) and non heat-treated, with and without added. normal human serum and heat-inactivated serum. The contribution of the complement system was confirmed from the difference between the bactericidal effect of fresh milk and the milk heated at 56°C for 30 minutes and fresh milk sample collected in 20mM EDTA.
Bacteria (same as above) were cultured overnight in blood agar and suspended in sterile normal saline adjusted to 3 x 108 colony-forming units (CFU) per ml using the McFarland method (26). 20µl of the adjusted bacteria was added to round-bottom microtiter wells (Nunc A/S, Roskilde, Denmark) with 80µl of the milk sample to be tested. The trays were thoroughly mixed on an orbital mixer. Samples of 20µl were then taken from each well for bacteria counts to assess the level of sequestration from suspension by milk fat globule membranes (MFGM), using poured agar plate method (27). The level of bacteria recovered from de-fatted milk and saline were taken as baseline, to determine the level of bacteria sequestered by MFGM in whole-milk samples.
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|Ogundele, M.O.; (1998). EFFECTS OF STORAGE ON THE PHYSICOCHEMICAL AND ANTI-BACTERIAL PROPERTIES OF HUMAN BREAST-MILK. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/occupational/ogundele0300/index.html|
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