MICROBIOLOGICAL

METHODS FOR LIQUID

SUGAR
MESOPHILIC BACTERIAL
The majority of laboratories found
nutrient agar with a pH of 6.7-7 to be the
best substrate and an optimum incubation
temperature of 28-37°C.
Wort agar incubated in 4 days at 25°C
was the most suitable medium for mold
fungi; the recommended concentration was
2.5 mL of a 20% sugar solution on four
plates.
MOLECULAR MEMBRANE
FILTERS
- adopted by ICUMSA for
enumeration of mesophilic bacteria,
yeasts, molds and thermophilic spore
forming bacteria.
General Procedure:
1. Dissolve 10- 20 g crystallize sugar in 100
ml cold sterile water.

2. A portion of the solution containing 10 g

sugar (liquid sugar, 10 g solids) is passed
through a membrane filter (0.2 – 0.6µ).

3. Microorganism are retained on the

membrane, which is then transferred to petri
dish with a suitable nutrient medium.
Enumeration of Mesophilic
Bacteria, Yeasts, and Molds

For mesophilic bacteria, the dishes

are incubated for 48 h at 30°C.
For yeasts and molds, the dishes
are incubated at 30°C for 72 h.
 Enumeration of Thermophilic
Spore-Forming Bacteria

1. Vegetative cells are first killed by heat

treatment. Then, follow the procedure being
described.
2. Add an indicator to distinguished the acid
and non-acid producing organisms. Using the
specific indicator, flat sour shows a yellow halo
around the colony.
 In estimating anaerobic spore- forming
bacteria, the membranes must be rolled and put
into a tube or a deep sterile cup and covered with
a thick layer of nutrient medium.

In estimating the total number of

thermophilic spores and flat source spores, the
membrane filter is incubated for 48 h at 55°C ;
for anaerobes, the incubation time is increased to
72 h.
 If more than 100 colonies grow on one
membrane, the test is repeated with a smaller
quantity of sugar .

To avoid false results due to airborne

organisms, permanent sterile conditions are
essential; all metals and glass must be flamed
frequently.
 Glass materials are autoclaved at 121 C for 15
min or sterilized by dry heat at 160 C for 2 h.
Membrane filters are sterilized according to the
manufacturer’s instructions.
The membrane filtration equipment (porous
plate) is sterilized by swabbing with alcohol
followed by flaming.
The nutrient media and the water used to
dissolve the sugar are autoclaved at 121 C for 15
min.
 TEST FOR SALMONELLA
- test commonly employed for lower
purity liquid products.

Salmonella – pathogenic bacteria that are

inhabitants of the intestinal tract of man and
animals.
DIRECT MICROSCOPIC
EXAMINATION

- the total number counted represents

both viable and non-viable organisms
I. Testing Equipment

1. Microscope with eyepiece and

objective (430 magnifications)
2. Glass Microscope slides
3. Cover Glasses
4. Type AA Millipore filters w/
adsorbent
5. Millipore filter assembly
II. Solutions

1. Methylene Blue
Dissolve 0.2 g of methylene blue in 60
mL of 95% ethanol, add 2 mL N/ 10 KOH and
make up to 200 mL with distilled water.

2. Tannic Acid
Dissolve 50 g of commercial grade
tannic acid and 0,3 g of sodium benzoate in 200 ml
water.
 3. Carbon Fuschin

Dissolve 0.2 g of carbon- fuschin in 6o ml

of 95% ethanol and make up to 200 ml distilled
water.
III. Method
1. Filter 250 ml of sugar by vacuum through a
type AA aerosol white molecular membrane.

2. The disk is then washed with 15 mL water.

3. The disk is placed for 3-5 min on absorbent

pad wet with methylene blue solution and
theen washed with 15 mL water.
4. Wet a second pad with a boiling tannic acid
solution and place the disk on this pad for 5 min.

5. Wash as before and place on a pad wet with

the carbon-fuschin solution , leaving it there for 3
min, then washing with 10 mL of liquid sugar.

6. After final washing, cut out section of the filter

and mount on a clean microscope slide.
7. A drop of liquid sugar is placed on both sides
of the section and covered with a cover glass.

8. The section is then examined with

approximately 430 magnifications.

9. Calculate the number of organisms per

milliliter sample by dividing the total number
counted by a factor K.
 𝐴𝑓 (𝑁𝑓)(𝑉𝑠)
K=
𝐴𝑡

Where: Af – area of one field

Nf – number of field
Vs – volume of sample(mL)
At – total area of filter
membrane