FSHN2100 Lab 5 1 LABORATORY SESSION 5 TITLE: EXAMINATION OF MILK FOR BACTERIA PREREQUISITES: Required readings: • Text: “Prescott, Harley, & Klein’s Microbiology” (9th, 10th Ed.). • Laboratory notes...

FSHN2100 Lab 5
1
LABORATORY
SESSION 5
TITLE: EXAMINATION OF MILK FOR BACTERIA
PREREQUISITES:
Required readings:
• Text: “Prescott, Harley, & Klein’s Microbiology” (9th, 10th Ed.).
• Laboratory notes
OBJECTIVES: At the conclusion of this unit the student will be expected to be able to:
1. Explain the purpose of pasteurisation
2. Determine the sanitary quality of milk by performing a coliform analysis and
methylene blue reductase test.
LABORATORY SEQUENCE:
1. Pre-lab discussion on the program for the session.
2. Set up coliform analysis of pasteurised vs. unpasteruized milk samples.
3. Set up and perform methylene blue reductase assay.
EVALUATION/FOLLOW UP:
Demonstrate the understanding and ability to perform assays to assess the quality of
pasteurised vs. unpasteruized milk samples.
Prepare a laboratory report. THIS LAB REPORT IS WORTH 15%
FSHN2100 Lab 5
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Examination of Milk for Bacteria
Safety Considerations
Be careful with the Bunsen burner flame and the boiling water bath.
No mouth pipetting.
Refer to risk assessment at the end of this laboratory.
Materials per Group of Students
1. Pasteurised milk sample
2. Unpasteurized milk may be obtained from any dairy farm. If a sample is not
available, pasteurised milk incubated at 35ºC may be used or the instructor
may wish to contaminate the sample with coliforms such as Escherichia coli,
Klebsiella pneumoniae, or Enterobacter aerogenes.
3. 32ºC incubator
4. 6 x 15ml and 6 x 5ml violet red bile agar tubes
5. 2 x 9ml and 2 x 9.9ml saline blanks
6. 6 petri plates
7. wax pencil
8. boiling water bath
9. methylene blue solutions (1/25,000)
10. 2 screw cap test tubes
11. sterile 10ml and 1ml pipettes with pipettor
12. 37ºC water bath
13. Bunsen burner
14. Test tube rack
15. Colony counter
16. 48º to 50ºC water bath for cooling tubes
Suggested Reading in Textbook
1. The Use of Physical Methods in Control, Chapter 7
2. Dairy Products, Chapter 43
3. Diseases Transmitted by Foods, Chapter 43
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Pronunciation Guide
Enterobacter aerogens (en-ter-oh-BAK-ter-a-RAH-jen-eez)
Klebsiella pneumoniae (kleb-se-EL-lah nu-MO-ne-ah)
Principles
Pasteurization is a means of processing raw milk, before it is distributed, to assure
that it is relatively free of bacteria and safe for human consumption. It is a heat
process gentle enough to preserve the physical and nutrient properties of milk, but
sufficient to destroy pathogenic microorganisms. The two methods most commonly
used for pasteurisation of milk are (1) heating at 62.9ºC (145ºF) for 30 minutes, or (2)
heating to 71.6ºC (161ºF) for a minimum of 15 seconds.
The presence of coliforms in milk (and milk products) is a major indicator of the
sanitary quality of milk. Their presence can be determined by a coliform plate count.
A high count means that there is the possibility of a presence of disease-causing
bacteria. A low count decreases this possibility but does not completely rule out the
absence of disease-causing bacteria. A low count decreases this possibility but does
not completely rule out the absence of disease-causing bacteria. In the first part of
this exercise, each group of students will do a plate count on a pasteruized and
unpasteurized milk sample.
Milk that contains a large number of growing bacteria will have a lower concentration
of O2 (a lower oxidation-reduction potential) compared to milk with few bacteria.
This is because growing aerobic and facultatively anaerobic bacteria use oxygen as a
final electron acceptor in cellular respiration. The dye, methylene blue, is a redox
indicator. It loses its blue colour in an anaerobic environment and is reduced to
leuco-methylene blue (Figure 3.3 below). As a result, the methylene blue reductase
test can be used to rapidly screen the quality of milk for the load of coliforms and
Lactococcus (Streptococcus) lactis, strong reducers of methylene blue and indicators
of contamination. The larger the bacterial load, the more quickly the milk will spoil.
FSHN2100 Lab 5
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The speed at which the reduction occurs and the blue colour disappears indicates the
quality of milk as follows:
a) Reduction within 30 minutes – very poor milk quality (class 4 milk)
b) Reduction between 30 minutes and 2 hours – poor milk quality (class 3
milk)
THEREFORE: REDUCTION UNDER 2 HOURS = VERY POOR/POOR
QUALITY
c) Reduction between 2 to 6 hours – fair quality (class 2 milk)
d) Reduction between 6 to 8 hours – good quality (class 1 milk) – CHECK
DEMONSTRATION TUBE SET UP FOR YOUR REFERENCE
THEREFORE: REDUCTION OVER 2 HOURS = FAIR/GOOD QUALITY
In the second part of this exercise, each group of students will perform a
methylene blue reductase test on a pasteurised milk sample.
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Figure 3.3: Biochemistry of the methylene blue reductase assay.
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Procedure:
FIRST (From last week): Analyse you Oxidation Fermentation (OF) test tubes to
assess whether your unknown organism can respire or ferment glucose (Lab 4, last
week). This is necessary to complete your investigations and determine the identity of
your unknown.
Coliform Analysis
1. Shake the milk sample 25 times. Make dilutions (100
, 10-1
, 10-2
) of the
pasteurised and unpasteruized milk samples as indicated in figure 3.4
(below).
2. Use the wax pencil or marker pen to label the petri plates with your name,
date the respective dilution (100
, 10-1
, 10-2
), and either pasteurised or
unpasteurized milk.
3. Pipette 1ml milk aliquots of each dilution into the appropriate plates.
4. Melt and cool the violet red bile agar (VRBA) tubes and add 15ml of it to
each of the plates (Pour plate technique - Lab 3). Swirl gently on a flat
surface and allow the agar to solidify. Afterward, add 5 ml of VRBA to
each plate, swirl gently, and allow to solidify.
5. Incubate all plates at 32ºC for 24 hours.
Follow-up Examination
1. For accuracy, select the plate that has between 25 to 250 colonies, which
are located below the surface, are lens-shaped, deep red, and surrounded
by a pink halo. Record these as the coliform count per ml of milk.
FSHN2100 Lab 5
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Figure 3.4: Coliform Analysis and Methylene Blue Reductase Set-up
METHYLENE BLUE REDUCTASE TEST
1. Label the two screw cap tubes with your name, as well as pasteurised or
unpasteurized, respectively.
100µL
9.9mL
Blank
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2. Using the 10 ml pipette, transfer 10ml of unpasteurized milk to one screw
cap tube and with another pipette, 10ml of pasteurised milk to the other
tube (Figure 3.4 above)
3. Add 1 drop of methylene blue to each tube.
4. Cap tightly and invert the tubes several times.
5. Place the tubes in a test tube rack and place the rack in the 37ºC water
bath. After a 5-minute incubation, remove the tubes from the water bath
and invert several times to mix again and return to waterbath.
6. Observe the tubes at 30-minute intervals for 6-8 hours. Reduction is
demonstrated by a change in colour of the milk sample from blue to white.
When at least 80% of the tube has turned white, the end point of reduction
has been reached, and the time should be recorded.
7. Record your results and the class of milk in your Laboratory book.