MATERIALS AND METHODS
This study followed the bacterial growth rate of human milk that was
expressed, stored in a 4-6° C refrigerator for 12-36 hours, warmed to 37°
C, partially fed to an infant and then stored in the refrigerator for 48
hours. It was important to emulate realistic storage conditions, therefore
clean, but not aseptic techniques were followed during expression, initial
storage and feeding. However, standard aseptic techniques were followed
during plate preparation and bacterial analysis.
Collection: Six women ages 17-26 with breastfed babies age 1-9 months
participated in the study. One week prior to the study, women were given a
packet which included detailed instructions (Appendix A), an Informed
Consent Form (Appendix B), a questionnaire (Appendix C), two 8-ounce clean
bottles with lids, a small cooler with ice, and a thermometer.
Women were instructed to express 6-8 ounces of breastmilk 12-36 hours
before coming into the laboratory to feed to their infant. Milk was
collected by electric breast pumps. Before pumping, women were instructed
to wash their hands in hot water and soap for one minute. The bottles
provided to the women in their kits were cleaned by a dishwasher, because
this is the method many women use to clean bottles. The breasts were not
sanitized prior to collection because it did not fit “real life” criteria.
There are conflicting reports as to whether discarding the first 5 ml of
expressed milk will decrease the total amount of bacteria present in human
milk (Asquith 1979 and West 1979). I chose to use the entire sample of milk
because most women use it all for future feedings. The milk was stored
12-36 hours in a 4-6° C refrigerator. The participants used a small cooler
with ice pack for transport to the laboratory the next day.
Feeding: After arrival at the laboratory, the expressed breastmilk was
poured into two clean, 4-ounce bottles and warmed in a 37° C hot water bath
for 10-20 minutes. Each bottle was labeled E for experimental or C for
control. Individual participants were assigned a letter, A - F. A clean
nipple was placed on all bottles. The experimental bottle was fed to the
infant for one minute, or until one ounce was gone. The control bottle was
not fed to the infant. Plastic nipple covers were placed on the bottle
before transport down the hallway to the lab for storage and culturing.
Plate Preparation, and Bacterial Analysis: One day before the study all
agar plates were prepared in accordance with the Difco Manual using 100 mm
disposable plastic petri dishes. All plates were labeled with the
participant’s code letter (A - F), the milk sample used [control (C) or
experimental (E)], time of collection (0, 12, 24, 36 or 48), type of media
[plate count (Pc), 5% sheep blood (Bl), mannitol salt (Mn) or MacConkey
(Mc)], and amount of milk plated (10 µl or 100 µl). Plates were stored in a
4° C refrigerator until use.
Storage: All bottles (control and experimental) were stored at 4° C for a
total of 48 hours. Cultures from all bottles were analyzed at 0, 12, 24, 36
and 48 hours post-feeding. Bottles were removed one at a time for analysis.
Each bottle was inverted 25 times, or until the milk was homogeneous. Care
was taken to return each bottle to the refrigerator as quickly as possible
( Colony Counts: Tryptone glucose extract agar (TGEA) was used to perform
colony counts. This media is used by the dairy industry to do standard
plate counts (Richardson, 1985). 1:10 and 1:100 serial dilutions of all
milk were plated onto correspondingly labeled plates. Plates were incubated
under aerobic conditions at 35° C for 48 hours (Difco, 1969).
Mannitol Salt Agar Plates with 7.5% NaCl and Phenol Red indicator: 100 µl
of milk was pipetted and distributed onto correspondingly labeled plates.
100 µl was used, because it should identify bacteria that are present in
concentrations of 10 CFU/ml or higher. Plates were incubated under aerobic
conditions at 37° C for 36 hours.
MacConkey Agar Plates: 100 µl of milk was pipetted and distributed onto
correspondingly labeled plates. Plates were incubated under aerobic
conditions at 37° C for 16-18 hours.
5% Sheep Blood Agar Plates: 100 µl of milk was pipetted and distributed
onto correspondingly labeled plates. Plates were incubated under aerobic
conditions at 37° C for 36 hours.
Previous microbiological studies with human milk incubated plates in 10%
CO2 conditions (Jocson 1997), 5% CO2 conditions (El-Mohnades 1993), and
aerobic conditions (West 1979 and Pardou 1994). Because of the expense of
equipment needed and scope of the study, I chose to incubate all plates in
aerobic conditions.
To aid in the removal of plates from the incubator, a table was created and
posted on the incubator (Appendix D).
RESULTS
Results of the Questionnaire are recorded in Table 1.
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Table 1 - Questionnaire Results (n = 6)
Average Range
Age of Mother (years) 22.8 17-26
Child's Age (months) 5.9 2-10
Amount of Water Mother Drinks Per Day (8 ounce Glasses) 3.5 0-8
Amount of Sleep Mother Gets Per 24-hour Day (hours) 6.0 4-8
Amount of Times Per Day Child is Breastfed 5.5 2-10
Time Milk was Refrigerated Prior to Feeding (Hours) 22.5 12-36
Total Amount of Milk Collected (Ounce) 6.2 6-8
Temperature Milk was Stored at Prior to Feeding (°C) 5.3 4-6
Mothers who Take Prenatal Vitamins 50 %
Mothers who smoke Cigarettes 16.6%
Mothers on Medications 16.6% (antibiotics)
16.6 % (thyroid
medication)
Mothers with Current Breast Infections 16.6%
Mothers who Exercise 16.6 %
Infant’s Crawling 33.3%
Infant’s Walking 16.6%
Infants Supplemented with expressed breastmilk 50 % yes
Infants Supplemented with formula 50 % yes
Infants Supplemented with Other foods 66.6 %
Infants with Recent Illness 50 % (flu, stuffy nose)
Education Completed 33 % High School 33 % Some College 33% College
Income Bracket 16.6 %
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