INTRODUCTION
Powdered milk or dried milk is a manufactured dairy product made by evaporating milk to
dryness. One purpose of drying milk is to preserve it; milk powder has a far longer shelf life than liquid
milk and does not need to be refrigerated, due to its low moisture content. Another purpose is to reduce
its bulk for economy of transportation. Once the milk powder is blended with water (preferably hot
water), it must be consumed immediately as it promotes growth of microbes (Keynes, 2015).
In order to isolate bacteria in milk, streaking and plating can produce pure colonies of bacteria.
Through Gram-staining, we can identify whether the bacteria is Gram-positive or Gram-negative. Gram
staining or Gram stain, also called Gram's method, is a method of staining used to distinguish and
classify bacterial species into two large groups: gram-positive and gram-negative. This method was
developed by the Danish bacteriologist named Hans Christian Gram. The said Gram stain provides
preliminary results on whether bacteria are present and the general type, such as the shape (Gould,
2014). It is used mainly to make a preliminary morphologic identification or to establish if there are
significant numbers of bacteria in a given sample. It is also usually performed when the result of a
culture is positive, on a sample of the bacteria grown in the culture (in this case, milk).
In this lab activity, this experiment will be used to study community succession in milk. We
will be using different types of milk which should lead to different organisms growing at different times.
Community succession is caused when different organisms change over a period of time. The growth
of one community changes the environment which leads to that community’s death and the emergence
of a new community which continues to occur in succession (Jiao & Chen, 2016). Both the environment
and the resources available will influence succession. We serially diluted aged milk—refrigerated and
not refrigerated for 8 days, 4 days, and 1 day and plated and streaked them in agar plates. Then, we
identified whether it is Gram-positive or –negative through Gram staining.
METHODOLOGY
Lab 1. Serial dilutions and plating:
1. Solutions in four flasks were used for the cultivation of milk bacteria. Aliquots of 1 mL were
distributed to each test tube to serially dilute the lactic acid bacteria.
2. Aliquot 1 mL of the first test tube into the second test tube and so on until the fourth test tube
to serially dilute the milk bacteria.
3. The same was done for the remaining three flasks and petri plates.
4. Plate 10-8 of the dilution by depositing 1 mL to prepoured TGY plates and spreading with an
alcohol sterilized wire loop.
5. Plates were incubated at 30 ⁰C for 24-48 hrs.
6. After incubation period, new TGY plates were streaked with the bacteria from each incubated
petri plates.
Lab 2. Streaking:
1. Flame the wire loop and a plate.
2. Streak a small part of the incubated petri plates using the wire loop near the flame. Bacteria is
transferred to the wire loop.
3. Streak the new TGY plates with the wire loop containing the bacteria following a pattern near
the flame.
4. Same is done for the rest of the plates.
Lab 3. Gram staining:
1. Drop bacterial isolate on a slide with an inoculating loop. Add a drop of water to the slide.
2. Flame heat to dry the smear.
3. Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for thinly prepared
slides, it is usually acceptable to pour the stain on and off immediately. Pour off the stain and
gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. Note
that the objective of this step is to wash off the stain, not the fixed culture.
4. Add the iodine solution on the smear, enough to cover the fixed culture. Let stand for 10 to 60
seconds. Pour off the iodine solution and rinse the slide with running water. Shake off the excess
water from the surface.
5. Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with water
after 5 seconds. The exact time to stop is when the solvent is no longer colored as it flows over
the slide. Further delay will cause excess decolorization in the gram-positive cells, and the
purpose of staining will be defeated.
6. Counterstain with basic fuchsin solution for 40 to 60 seconds. Wash off the solution with water.
Blot with bibulous paper to remove the excess water. Alternatively, the slide may be shaken to
remove most of the water and air-dried.
RESULTS AND DISCUSSION
Characteristics of Colony Microscopic
(as visible by unaided eye or w/ dissecting microscope) Characteristics
(100X oil immersion
objective)
Age Edge Sheen Silhouette Color Transparency Solidity
Gram-
8 days Smooth Shiny Flat Light Transparent Solid Bacteria negative
Ref Green
Dirty
8 days Smooth Dull Flat White Transparent Solid Bacteria Gram-
RT with Light positive
Green
Gram-
4 days Smooth Shiny Flat Dirty Transparent Solid Bacteria positive
RT White and
negative
Gram-
24 hours Smooth Semi- Flat Dirty Transparent Solid Bacteria positive
RT Shiny White and
negative
Table 1. Physical properties of milk bacterial colonies
Age/ Organisms Present on TGY
Type of Environmental Characteristics of Milk Culture Plates
Milk
pH Consistency Odor Color Gram +/ - Shape
Light
8 days Ref 6 Consistent; Sweet brown on Gram- Cocci
Homogenous top negative
Dark
brown on
boom
Visible layer Yellowish
of solidified in the
substance; Rotten middle
middle layer Dull layer; Gram-
8 days RT 5 with Unpleasant light positive Cocci
yellowish brown on
liquid; bottom top; dark
layer with brown at
precipitate the
bottom
Gram-
Visible layer Mildly Light positive and
4 days RT 5 on top only Rotten brown negative Bacilli
with little Unpleasant
clumps of
milk
Consistent; no Normal Fresh Gram-
24 hours 6 visible layers; chocolate light positive and Bacilli and
RT no clumps milk smell brown, negative Cocci
same
color in
its first
day
Table 2. Physical feature of bacterial communities of aged chocolate milk
Hypothesis: Succession results in an increase in microbial populations. The chocolate milk that
has not been treated and is kept at room temperature for 8 days will have the most bacterial
growth while the one in 24 hours will generate the least bacteria over time. The chocolate milk
that kept for 8 days in refrigerator will have a lesser change and bacterial content comparing to
the 8 days one kept in room temperature.
1. Describe the changing sequence of organisms and corresponding environmental changes during
succession in plain milk. Do the results of your investigation match your hypothesis?
Over time, the closed environment eventually becomes anaerobic because aerobic organisms
consume and deplete oxygen in the air. Yeasts (cocci) consume glucose or specifically lactose in milk
as a source of energy in order to survive. The rods (bacilli) were abundant at 8 days room temperature
milk because of the bacteria that is produced when the milk begins to ferment. The chemicals released
by this bacteria cause a strong and unpleasant (to most people) odor that is one of the methods by which
we determine whether or not milk has gone bad. Also, the lactobacillus are abundant in8 days room
temperature milk because they are facultative bacteria which they dominate as the oxygen depleted over
time and the aerobic bacteria seems to decrease. The refrigerated milk seems to have a lesser amount
of bacteria and spoilage than the room temperature one. This is due to the niche environment of bacteria.
The ideal temperature for bacteria to grow is around 37 to 40 degrees, less than that will lessen the
bacterial growth.
2. Describe the changing sequence of organisms and corresponding environmental changes during
succession in chocolate milk. Do the results of your investigation match your hypothesis?
The aerobic organisms inside the milk uses the oxygen in order to survive. Since the milk is a closed
environment, the oxygen supply is limited. Over time, the oxygen will deplete and the facultative remain
and anaerobic organisms will start to dominate, as the aerobic organism’s growth rate will slow down
and some will die. Based on the table, the 8 day one have a strong spoiled milk, 4th day and lastly, no
odor at all is the 24 hour one. The odor is caused by metabolic end-products of the anaerobic organisms,
which are mostly organic acids. This is an evidence that shows that 8 days chocolate milk kept in room
temperature have the most bacteria that uses anaerobic respiration. Not only that but also the comparison
of two chocolate milk stored for 8 days in refrigerator and room temperature have different outcome.
The one stored in refrigerator have a lesser changes than that of room temperature. As refrigeration
inhibits or slows down the growth rate of bacteria because the bacteria tends to grow the most in the
warmer temperature but above 120 Fahrenheit because it will kill them except of those thermophilic
one. Overall, the results matched our hypothesis made.
3. Compare succession in plain and chocolate milk. Propose reasons for differences.
A plain milk tend to have a lesser sweet like taste than the chocolate one. This because chocolate
milk tend to have a higher content of sugar like glucose and galactose. This promotes the growth of
lactose-eating or glucose-eating bacteria. That is why there is an abundant amount of gram-positive
bacteria that we suspect that are lactobacillus related bacteria than the plain one. Not only that but
also there are many nutrient component in the chocolate milk which boosts the bacterial growth
rate. Lastly, changes in chocolate milk over time is more evident than the plain one because can see
the yellow acid, curd cheese like and settlement of chocolate component in the chocolate milk while
the milk has lesser amount of separation of curd cheese like and yellow water.
4. Propose an experiment to test the environmental factors and/or organisms changing in your
proposed scenario for milk succession.
I would like to perform an experiment comparing the succession changes in different types of
milk like buttermilk, evaporated milk, sterilized milk, full milk, chocolate milk, fresh milk, skim
milk etc. Because each of them have different nutrient content which may affect the bacterial
growth. Just for example, the chocolate milk tend to have higher number of bacteria than that plain
one because it have a higher content of glucose content. I would like to compare which one will
results a faster changes and what bacteria species would grow in each of them. I would like to know
the difference of the changes in each bottle like it might be the buttermilk have higher or lower pH
after 8 days than the plain milk.
CONCLUSION
Succession results in an increase in microbial populations. Aging milk promotes the rapid
growth of bacteria such as lactic acid bacteria (LAB). LAB such as Lactobacillus were found upon
microscopic observation in 100X oil immersion objective. However, there is a change of organisms that
survives overtime due to the changes of the environment. Since the experiment bottles is a closed
environment, the evidence implies that overtime, the oxygen will deplete and the aerobic organisms’
growth rate will decrease giving a rise to organisms that use anaerobic respiration. This is the reason
why the 8 day milk stink so much or give the most evident foul smell due to the presence of the end-
products of anaerobic bacteria. This is evident of the presence of rod shaped gram positive in the 8 days
room tempered shown in table 3 which we suspect as lactobacillus related species. And overtime, the
pH of the bottle tend to be more acidic due to the end product that lactic acid bacteria produce called
lactic acid. Not only that but also refrigeration inhibits bacterial grow. The changes is very evident in
8 days room tempered bottle than the refrigerated one. Comparing to the plain milk, there is a faster
change and separation of yellow water and curd like cheese formed in chocolate milk. This is due to the
glucose and lactose content is higher than the plain one. That is why chocolate milk tend to taste sweeter
than the plain one. Due to that, the bacteria have more energy source in chocolate milk which promotes
their growth rate faster. Because of that, chocolate milk had the faster succession changes. Based on the
bacterial counting table, we are unable to calculate specifically due to too much bacterial colonies
developed. We suspect that it might due to contamination or the culture was overly stored for a long
time. In the gram staining, we saw a large amount of cocci shaped and positive gram bacteria. We also
saw an evidence of gram positive and bacillus shape bacteria which might be a lactobacillus related
species. Overall, the results gathered shows the succession and changes of organisms in the closed
system due to the different needs of each organisms. The knowledge gathered is useful for awareness
in proper storage of food and the potential content or organisms that build up depending on the
environment.
PHOTOS/ DOCUMENTATION
8 days Ref
8 days RT
4 days RT
24 hours
Table 1. Physical changes of chocolate milk
8 days Ref
8 days RT
4 days RT
24 hours
Table 2. Bacterial isolates
8 days Ref
8 days RT
4 days RT
24 hours
Table 3. Gram-stained bacterial isolates
REFERENCES
Gould, S. E. (2014). How does gram staining works? Retrieved from Scientific American:
[Link]
Jiao, S., & Chen, W. (2016). Microbial succession in response to pollutants in batch-
enrichment culture. Scietific Reports, 2-3.
Keynes, R. (2015). How Long Can Milk Really Keep After Being Opened? Retrieved from
Men's Health: [Link]
after-being-opened
