Bacteria in Milk

							                                             STAGE 1 HUMAN BIOLOGY




                             Bacteria in Milk
                                     A series of Practicals
                                                     Bec Hooper
                                                      1/1/2012




This series of Practicals is designed to increase student understanding on microbial growth as part of the Public
Health and Diseases unit of work. ‘Bacteria in Milk’ consists of two Formative Practicals which will provide the
laboratory skills and background knowledge necessary to complete the final Summative Design Practical.
Overview

Background
Fresh food is important for our health and wellbeing. In order to ensure a constant supply of fresh foods, it must be
prevented from spoiling. Spoilage is the result of the action of bacteria and other microorganisms. Bacteria can be
found in varying degrees in all fresh foods.

Milk can have bacteria in it, either from the cow itself (if it is ill) or from its handling and storage. In Australia all milk
sold for consumption must be ‘Pasteurised’. Pasteurisation is the process of heating then rapidly cooling the milk. In
order to kill as many bacteria as possible, while preserving the taste of the milk, pasteurised milk is heated to 63-
65oC for 30 minutes or 71oC for 15 seconds followed by rapid cooling and refrigeration. This does not prevent the
milk from spoiling, it simply extends its shelf life, the degree of which depends on the temperature at which it is
stored (Practical 1).

This process kills almost all the bacteria, but some still remain. If these bacteria are given an environment that
promotes growth, they will rapidly multiply. When milk spoils it changes in consistency, appearance, and smell.
Spoiled milk also undergoes a chemical change. As the milk spoils, the bacteria produce acid. It is the acid that causes
the milk to clot. You can compare the acidity of the milk samples by using indicator strips.

There is a variety of bacteria that remain in milk after pasteurisation. The different bacteria that remain will grow
and multiply optimally at different temperatures. Some grow better in cold temperatures, other prefer warmer
temperatures. Observing the physical changes of the milk is not sufficient to determine how many different kinds of
bacteria remain in milk after pasteurisation. One way to observe the variety of bacteria is to spread the material on
a special material known as a Nutrient Agar. Here bacterial growth becomes dependant on the nutrients provided
(type of agar) and the temperature at which it is stored (Practical 2).

Following pasteurisation the milk will still spoil. Factors that affect the rate of spoilage include: further treatment
measures (cooling, heating, salting and dehydrating, adding preservatives), storage (container, temperature) and the
type of milk used (fresh, UHT, Organic, Cow, Goat, etc). The ability to maintain the flavour of the milk, while
prolonging its shelf like is an important consideration in maintaining our populations fresh food supply (Practical 3).

Curriculum Links
This series of practicals is part of the Public Health and Diseases unit of work. During this unit, students will study:

       Microorganisms, including reproduction
       Infectious Diseases
       Public Health, including food handling
Practical 1 – Formative Practical – The effect of temperature on Bacterial Growth
in Milk
Background
Fresh food is important for our health and wellbeing. In order to ensure a constant supply of fresh foods, it must be
prevented from spoiling. Spoilage is the result of the action of bacteria and other microorganisms. Bacteria can be
found in varying degrees in all fresh foods.

Milk can have bacteria in it, either from the cow itself (if it is ill) or from its handling and storage. In Australia all milk
sold for consumption must be ‘Pasteurised’. Pasteurisation is the process of heating then rapidly cooling the milk. In
order to kill as many bacteria as possible, while preserving the taste of the milk, pasteurised milk is heated to 63-
65oC for 30 seconds or 71oC for 15 seconds followed by rapid cooling and refrigeration. This does not prevent the
milk from spoiling, it simply extends its shelf life, the degree of which depends on the temperature at which it is
stored.

Materials and Methods
    1. Work in Groups of two or three, your group will be assigned a number, Groups 1-3 will use fresh milk, groups
        4-6 will use expired milk (milk which has passed its expiry date).
    2. Using the marking pen, label each 10ml test tube with your group number and a let A-C.
    3. Using the 250ml beaker, carefully measure 60ml of Full Cream Milk
    4. Using the 25ml measuring cylinder, measure 10ml of Full Cream Milk into each of the three test tubes and
        return test tubes to the test tube holder
    5. Dispose of any remaining milk and thoroughly rinse beaker in water
    6. Dip a small piece of indicator paper in each test tube. Record results.
    7. Cover all test tubes with plastic wrap
    8. Place test tube A in the test tube holder marked ‘Fridge’
    9. Place test tube B in the test tube holder marked ‘Room Temp’
    10. Place test tube C in the test tube holder marked ‘Incubator’
    11. Pack up work area and clean bench
    12. Write predictions for each sample.

Results
Your results need to be detailed and recorded in a table. You will need to observe the changes in colour, odour,
consistency and pH for each sample. Observe the agar plates on Tuesday and Wednesday/Thursday.

Discussion
The results for each group will be shared with the class. Assess which milk (fresh or expired) and which temperature
produced the bacterial growth across the observation period. State possible sources of errors and ways that these
could be avoided next time.

Conclusion
State the main results.
Practical 2 – Formative Practical – Growing milk bacteria on Agar Plates
Background
Pasteurising milk kills almost all the bacteria, but some still remain. If these bacteria are given an environment that
promotes growth, they will rapidly multiply. When milk spoils it changes in consistency, appearance, and smell.
Spoiled milk also undergoes a chemical change. As the milk spoils, the bacteria produce acid. It is the acid that causes
the milk to clot. You can compare the acidity of the milk samples by using indicator strips.

There is a variety of bacteria that remain in milk after pasteurisation. The different bacteria that remain will grow
and multiply optimally at different temperatures. Some grow better in cold temperatures, other prefer warmer
temperatures. Observing the physical changes of the milk is not sufficient to determine how many different kinds of
bacteria remain in milk after pasteurisation. One way to observe the variety of bacteria is to spread the material on
a special material known as a Nutrient Agar. There are a number of different agar plates available, each providing
specific nutrients. Bacterial growth becomes dependant on the nutrients provided (type of agar) and the
temperature at which it is stored.

The aim of this practical is to investigate the effect of different nutrients agar plates on growth rates of bacteria in
milk.

Materials and Methods

1.    Collect a small sample of spoilt milk in a petri dish.
2.    Sterilise the bench area by wiping the area with ethanol (use paper toweling and ethanol). Remove the
      ethanol bottle from near the Bunsen burner.
3.    Light the Bunsen burner so the blue flame can sterilise the air in the work area.
4.    Dip the glass “hockey stick” spreader in ethanol and then flame for 3 seconds to sterilise.
5.    Allow the hockey stick to cool by holding it in the air.
6.    Dip the cooled hockey stick into the spoilt milk sample.
7.    Evenly spread a thin layer over a pre-prepared agar plate and replace the lid.
8.    Seal agar plates with parafilm and label with a permanent marker or sticky label.
9.    Repeat steps 3 to 7 with remaining plates.
10.   Tip all labelled agar plates upside down.
11.   Agar plates will be incubated at either in the fridge, at room temperature or in the incubator at 40OC.

Results
Your results need to be detailed and recorded in a table, including a sketch of the plates. You will need to count the
number of different types of bacteria and mould that appear to have grown. Each bacterial colony grows and
spreads from only one bacteria when conditions are suitable for growth. Bacterial colonies can show different
colours, textures, edges and manner of covering a space. The sooner and more often the agar plates can be viewed
after inoculation, the easier it is to identify and count separate colonies. You will probably see both bacterial colonies
and moulds growing. Describe each one and count how many other colonies of that type appear to be present.
Observe the agar plates on Monday and Thursday/Friday.

Discussion
The results for each group will be shared with the class. Assess which nutrient agar and which temperature for
growth is the best to use across the observation period. State possible sources of errors and ways that these could
be avoided next time.

Conclusion
State the main results.
Stage 1: Human Biology 2012
Mrs Reimers and Ms Hooper

Design Due: Tuesday 24 April 2012
Draft Due: Thursday 10 May 2012                                                  Final Due: Monday: 14 May 2012
Practical 3 – Summative Design Practical – Bacterial Growth Rates in Milk
Background:
Pasteurisation kills most, but not all bacteria in milk. A variety of bacteria remain and will increase rapidly in the
right conditions. Factors that affect the rate of spoilage include: further treatment measures (cooling, heating,
salting and dehydrating, adding preservatives), storage (container, temperature) and the type of milk used (fresh,
UHT, Organic, Cow, Goat, etc). The ability to maintain the palatability (flavour) of the milk and prolonging its shelf
life while removing harmful bacteria are important considerations in maintaining our population’s fresh food supply.

Pasteurisation involves heating the milk to a specified temperature for a specified amount of time in order to kill as
many bacteria as possible without affecting the palatability (flavour) of the milk. Commercially pasteurised milk is
heated to 63-65oC for 30 seconds or 71oC for 15 seconds followed by rapid cooling and refrigeration; this delays the
spoiling by a few weeks.

For this summative assessment, you must design a practical to investigate the effect of heat on the amount of
bacteria in milk.

Option 1: Investigate the effect of time at a specific heat (65OC). By heating the milk to 65OC and keeping it here for
4 different lengths of time between 10-60 seconds, then rapidly cooling it in an ice bath.

Option 2: Investigate the effect of heat for a specific time (15 seconds). By heating the milk to 4 different
temperatures between 30-100OC for 15 seconds each, then rapidly cooling it in an ice bath.

Aim:




Independent Variable:

Dependant Variable:

Controlled Variables:

Hypothesis:

Materials and Method
Include safety. List materials and write a detailed method clearly showing how the samples of milk were treated.
One sample should be untreated; it will act as your control. You sample will be stored at room temperature and
should be checked daily for changes.

You need to decide how you will determine the effect of heat on the milk; this must include the measurement of pH.

Safety:
Materials:




Method - Treating Milk Samples:




Method – Measuring changes including pH:




RESULTS:
Record the pH and description of the test tube samples in a suitable table. Draw a graph of the changes in pH results
on attached graph paper.

Discussion
Attach extra sheets as required to complete your discussion questions.
1. Description of pattern of results (description of samples, pH) including analysis of the graph.
2. Evaluation of support of the hypothesis, use relevant theory to explain results.
3. Describe the pattern shown in the graph and hence state the effect of the independent variable on the
   dependent variable.
4. Discuss 2 sources of random error that could have affected the results and caused variations in measured values
   even though the same procedure was followed each time. Clearly explain how the error affects the results.
5. Discuss 1 source of systematic error that could have affected every result in the same way.
6. Describe 1 improvement to the method that would reduce each type of error in this experiment (2 in total).
7. Discuss the importance of sterile techniques in food preparations, hospitals or other community domains

Conclusion:
State the main results and whether or not they supported your hypothesis

Collaborative Paragraph:
Clearly identify any issues encountered and how they were resolved. Discuss how the individual members of your
team worked together. For design prac how were the independent, dependant and controlled variables decided. It
may be relevant to mention other communication/collaboration/distribution of tasks that enabled suitable
collection of data.