Oops! Part 3 was 368 words. I'll send a Part 3 and Part 4.
Pat
__________________________________________________________________-
-
Results of the microbiological analysis are as follows.
Plate Count Agar: Colony counts on the TGEA plates were performed using a
Quebec colony counter. The 10 µl plates (1:100 dilution) were counted
first. Each colony on the 10 µl plates represents 100 live bacterium per
one ml of milk, commonly referred to as CFU/ml. If the 10 µl plate had less
than 25 colonies, then the corresponding 100 µl plate was counted. Each
colony on the 100 µl plates represents 10 individual bacterium in one
milliliter of milk. Average CFU/ml in the control and experimental milk
were compared for each time period. T-tests were performed to establish if
there was a significant difference in the amount of bacteria found before
the child drank the milk and after. The average CFU/ml and results of the
t-tests can be found in Table 2. The numbers in Table 2 were calculated by
two methods. First, all of the participants were averaged together, and
CFU/ml were calculated for each time period. Second, individual
participants CFU/ml were calculated using all time periods. More
detailed counts and t-test calculations can be found in Appendix E.
------------------------------------------------------------------------
Table 2 - Average Total Bacteria Counts Before and After Infant Feeding
Analysis by Individual Storage Times
Time Milk Before Feeding Milk After Feeding t-value Critical
t-value Significantly
(post-feeding) (mean CFU/mL) (mean
CFU/mL) alpha=.10 Different?
0
hours 11,900 12,137 0.15 2.13
no
12
hours 10,665 13,355 1.43 2.13
no
24
hours 12,032 13,627 1.78 2.13
no
36
hours 12,053 12,472 0.42 2.13
no
48
hours 12,347 12,062 -0.16 2.13
no
Analysis by Individual Participants
Time Milk Before Feeding Milk After Feeding t-value Critical
t-value Significantly
(post-feeding) (mean CFU/mL) (mean
CFU/mL) alpha=.10 Different?
Participant
A 810 1,000 1.28 2.02
no
Participant
B 1,028 980 -.037 2.02 no
Participant
C 618 762 1.96 2.02 no
Participant
D 11,920 9,320 -0.12 2.02 no
Participant
E 26,180 25,880 -0.12 2.02 no
Participant
F 30,240 36,040 3.62 2.02 yes
------------------------------------------------------------------------
Mannitol Salt Agar Plates: Any colony with a yellow ring around it was
considered a positive mannitol fermenter (Difco, 1969). 11 out of the 30
mannitol salt plates inoculated with milk the infant had partially eaten
were positive for mannitol fermentation. Similarly, 11 out of the 30
mannitol salt plates inoculated with control milk were also positive for
mannitol fermentation. Colonies within the yellow zones were gram stained
and tested for coagulation and catalase activity. All colonies tested were
coagulase-negative, catalase-positive, gram-positive cocci.
MacConkey Agar Plates: Any pink or red colony grown on MacConkey agar was
considered a positive lactose fermenter. 6.6% of the control plates, and
10% of the experimental plates contained positive lactose fermenters.
Organisms were identified as Klebsiella pneumoniae, Acinatobacter sp., and
Escherichia coli.
5% Sheep Blood Plates: All of the blood agar plates had various colonies
growing on them, however no hemolytic zones were formed around any of the
colonies. A b-hemolytic bacteria (Staphylococcus aureus) from the
laboratory culture stock was plated onto one of the blood agar plates. It
showed clear zones of b-hemolysis.
DISCUSSION
Plate Count Agar: To determine if the total bacteria levels I found in the
study were safe, I need to first define “safe”. Currently, “no agreed-upon
guidelines exist regarding the acceptable microbiological quality of
collected human milk” (El-Mohandes, 1993). The human milk banking industry
does have standards, but they are very strict, because most of the milk
from human milk banks is fed to pre-term infants with compromised immune
systems (Hamosh, 1996).
I found varying requirements for “safe” milk. Three examples are: 103
CFU/ml with no enteropathogens (Sauve 1984), 105 CFU/ml excluding
Staphylococcus aureus, group ß Streptococci, Pneumonocci or coliforms
(Tyson 1982), and 2.5 X 104 CFU/ml with no enterobacteria, (except
non-lactose-fermenting enterobacteria), with Staphylococcus aureus levels
below 1.0 X 103 CFU/ml (Williamson 1978).
With exception of participant F there was no change in total bacteria found
in expressed human milk that has been partially fed to an infant. Where I
did see variance was between the individual participants. For example both
the control and experimental milk from participants A-C ranged from
600-1,000 CFU/ml, while participants D-F ranged from 12,000 to 36,000
CFU/ml. It is interesting to note that due to personal scheduling problems,
participants A and B’s experiment was performed two days prior to C, D, E
and F. It is also interesting to note that C, D, E and F’s code letters
were assigned randomly and did not reflect the order in which any tests
were performed.
Since participants E-F were showing a significantly higher amount of growth
than participants A-C I examined the questionnaire to see if I could find
any common factors among each group. Due to small sample size it was not
possible to statistically analyze the information, but I could not find
anything outstanding that participants A-C did differently from
participants E-F. For example both groups had children that were crawling
and eating solids. The general health and average sleep amongst mothers was
consistent throughout. Although I clearly specified that freshly expressed
refrigerated milk was to be provided for the study, participant F’s milk
was frozen. In future studies I would suggest the use of a more detailed
questionnaire to help identify the sources of variance.
According to Margit Hamosh, an accomplished human milk researcher, the
“great individual variations among lactating women” found in my study was
similar to other studies (Hamosh, 1998). A 1987 study by Jan Barger
illustrates this point. The study showed expressed, refrigerated milk to
have an average of 2000 CFU/ml with a range of 0-113,000 CFU/ml. This
variation can be explained with a variety of reasons.
There may be several sources of contamination, including mothers’ hands,
nasopharyngeal secretions, breast skin flora, and distal milk ductules as
well as collection and storage equipment (El-Mohanas, 1993). In attempt to
reduce contamination from the hands and nose I asked the mothers to wash
their hands prior to collection. Since I did not have the women rinse their
breasts with water maybe milk from a previous feeding remained on the
breast, or bra, which contaminated the results. A probable source of
contamination could be due to the collection technique. Perhaps the breast
pumping equipment was not clean. Due to the difficulty of cleaning the
hollow, bent collection devise, it is possible that the apparatus was not
thoroughly sanitized between uses. When I questioned how the participants
cleaned their pumping equipment, I found that participants A-C cleaned by
hand with bottle brushes, and D used a steaming disinfectant made for
bottles, and E-F used the dishwasher. Could it be possible
that cleaning the equipment by hand may be a more effective method for
cleaning the pump?
Mannitol Salt Agar: The mannitol salt agar was used to identify the
possible presence of Staphylococcus aureus. The high salt content inhibits
gram-negative organisms, and many gram-positive organisms other than staff.
Many Staphylococci ferment mannitol, therefore the second coagulase test
was done to confirm the presence of S. aureus, which also forms a
b-hemolytic zone on 5% sheep blood agar. Since all samples taken from the
yellow areas of the mannitol salt agar plates were coagulase-negative, and
because none of the blood agar plates showed b-hemolytic zones, I will
conclude that S. aureus was not present in any of the samples. Since this
test’s intent was to rule out the presence of S. aureus, and not to
identify every organism in the milk, I did no further testing with these
plates.
5% Sheep Blood Agar: Since the 5% sheep blood agar did not show any
hemolytic zones, I will also conclude that there were no b-hemolytic
Streptococci in any of the samples.
MacConkey Agar: MacConkey agar is used for detection and isolation of
coliforms and enteric pathogens. It inhibits gram-positive organisms.
Participants A-C had zero growth at all time periods for both the control
and experimental milk. Participants D-F had a lactose fermenting
enterobacteriaceae, commonly known as coliforms.
To sum up my results I found total average bacterial counts of 1.2 X 104
CFU/ml, with no Staphylococcus aureus, or group ß Streptococci. Two of the
participants had the presence of coliforms. Although high bacterial levels,
and high coliform levels were found in the milk, it is important that they
were found in the control sample as well as the experimental sample. This
study showed high variability among participants, but no significant
difference between the quality or quantity of bacteria found in breastmilk
that has been partially consumed.
It would be interesting to see how bacterial counts would be affected if
the nipple were stored off of the bottle, the bottle were stored at room
temperature, the milk were previously frozen before fed to child, and the
milk were warmed again before plating. It would also be interesting to see
how bacterial counts in stored, used human milk compared to stored, used
infant formula. The major flaw in this experiment was the small sample
size. I cannot be confident that the control and experimental samples were
actually statistically similar, or just appeared that way because of the
low number of participants.
The most important lesson we can learn from this data is that in spite of
high bacterial levels found both control and partially consumed milk, none
of the babies became ill. This provides some evidence that different
standards need to be made for healthy full-term infants. “The rationale for
less stringent recommendations for storage of a mother’s own milk that is
fed to her (own) healthy, full-term infant ... is that the microorganisms
are probably less hazardous than the organisms from an unrelated donor,
because a mother secretes antibodies in her milk that reflect her own
immunologic experience” (Hamosh 1996). It is believed that protection is
provided by secretory IgA that mothers produce in their milk against
potential pathogens in their gastrointestinal tracts (Narayanan, 1981).
On a personal note this researcher, and mother, never throws breastmilk
down the drain. If there is any left over, I feel confident refrigerating
the milk for future feedings.
------------------------------------------------------------------------
Mailto:[log in to unmask]
***********************************************
To temporarily stop your subscription: set lactnet nomail
To start it again: set lactnet mail (or digest)
To unsubscribe: unsubscribe lactnet
All commands go to [log in to unmask]
The LACTNET mailing list is powered by L-Soft's renowned
LISTSERV(R) list management software together with L-Soft's LSMTP(R)
mailer for lightning fast mail delivery. For more information, go to:
http://www.lsoft.com/LISTSERV-powered.html
|
