TANSIAN UNIVERSITY, UMUNYA

(OBA CAMPUS)
FACULTY OF NATURAL AND APPLIED SCIENCES
DEPARTMENT OF MICROBIOLOGY

EXERCISE 1

STANDARD QUALITATIVE ANALYSIS OF WATER

The three basic tests to detect coliform bacteria in water are presumptive,
confirmed and completed. They detect the presence of coliform bacteria (indicators
of fecal contamination). The Gram-negative, nonspore forming bacilli hat ferment
lactose with the production of acid and gas that is detectable following a 24 hours
incubation period at 37oC.

Presumptive Test: Determination of the most probable number of coliform

bacteria.

You are provided with water samples from sewage plant, pond and tap. 12 double
strength lactose fermentation broths (LB2x) and 30 single-strength lactose
fermentation broths (LB1x).

1. Examine all tubes after 24 and 48 hours of incubation. Record your results in
the chart as follows:
a. Positive: 10% or more of gas appears in a tube in 24 hours.
b. Doubtful: Gas develops in a tube after 48 hours.
c. Negative: There is no gas in a tube after 48 hours.
2. Determine and record the MPN using the table provided.
 GAS
LB 2x -10 LB 1x -1 LB 1 x – 0.1 Reading MPN Range 95%
Probability
Tube Tube Tube
Water 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
sample
Sewag
e
Pond
Tap

EXERCISE 11

AN ENZUMATIC TEST TO DETERMINE THE QUALITY OF A MILK

SAMPLE

A milk sample that contains a large population of activity metabolizing

microorganisms will contain a markedly decreasing concentration of dissolved
oxygen because of the vigorous growth of the organisms. In other words, the
oxidation reduction potential of the sample is greatly lowered. The dye methylene
blue (MB), a redox indicator, uses its colour in an anaerobic environment and is
then said to be reduced.

The methylene blue reductase test is designed to screen the quality of raw milk
which may contain large populations of enteric organism s and Lactocossu lactis,
which are potent reducer of the dye. The speed at which reduction occurs
following addition of MB to a sample of milk indicates the milks quality.

This determination is made as follows:

1. Reduction within 30 minutes is indicative of very poor quality.

2. Reduction occurring between 30 minutes and 2 hours is indicative of poor
quality.
 3. Reduction occurring between 2 and 6 hours is indicative of fair quality.
4. Reduction occurring between 6 and 8 hours is indicative of good quality.

You are provided with one raw milk sample and one pasteurized milk sample
stored at room temperature for 48 hours, sterile screw-cap test tubes, water bath.

1. Label the test tubes as raw milk and pasteurized milk.

2. Using a different 10-ml pipette each time, transfer 10ml of each type of milk
into its appropriately labeled test tube.
3. Add 1ml of methylene blue dye to each test tube.
4. Insert stoppers, invert the test tubes gently about four times, and place in
water bath. Record the time of incubation, that is, record the amount of time
elapsed for the colour to turn white.
5. Allow the tubes to stabilize for 5 minutes, remove them from the water-bath,
invent them gently once and replace them in the water-bath.
a. Observe the milk sample for methylene blue reduction every 30 minutes
for 3 to 6 hours. Reduction is demonstrated by change in the colour of the
sample to white.
b. Record in the chart the time required for reduction in both milk samples.
c. Based on your observation, determine and record in the chart the quality
of each sample as very poor, poor, fair, or good.

RAW MILK PASTEURIZED MILK

Reduction Time
Quality of Milk Sample