REUTILIZATION OF USED AGAR MEDIA, I
By E. V. SESHACHARYULU and ZAL R. KOTHAVALLA, Imperial Dairy Research Institute, Bangalore
(Received for publication on 22 January 1943)
MILK agar suggested by the Ministry of Agricul-
ture [1934] is commonly employed for determining
the number of bacteria in milk by the plate method.
Due to the present war conditions the prices of the
constituents of this medium, namely peptone,
lemco and agar, have enormously increased and
there is already a great shortage of agar in this
country.
The reutilization of used agar has been the sub-
ject for serious investigation in the present war
time. The foremost workers in this field are
Huang, Shen and Tang [1941] who have recom-
mended the addition of used agar, that was pre-
viously washed for 24-28 hours in running water,
to fresh agar in the ratio of 1 : 3. Macmorine
[1942] reclaimed agar from the used stuff by che-
mical methods. He claims that this reclaimed
agar has no bactericidal effect and can be safely
used. These workers have, however, ignored the
other constituents of the used media, namely lemco
and peptone, as the continuous washing of used
agar in running water adopted by the former work-
ers removes the soluble constituents, peptone and
lemco and in the method used by Macmorine to
reclaim agar, he autoclaved the agar solution with
potassium oxalate and potassium carbonate which
completely decompose peptone and lemco. Por-
tions of peptone and lemco undecomposed by
bacteria may still remain in the used media.
Even the break-down products of peptone and
lemco may also serve as energy materials for bac-
terial growth, provided that no toxic substances are
formed. With a view, therefore, to ascertaining
whether once used agar media as a whole can be
reutilized for estimating the bacterial counts of
milk, the present investigation was undertaken.
It is hoped that the information obtained in this
work will be found useful by laboratories engaged
in the routine bacteriological examination of milk.
Milk agar (Ministry of Agriculture) and nutrient
agar recommended by the Ministry of Health
[1929] were tried out in this investigation. The
counts obtained by the use of once used media of
these types as reclaimed by the present authors
were compared with their respective fresh media.
The samples of milk examined for total bacterial
counts by the plate method were from (a) individual
cow's and buffalo's, (b) cow herd and buffalo herd
milk, (c) mixed milk (cows' and buffaloes'), (e) milk
handled by town dairies, (e) village produced milk
and (f) pasteurized milk. The experiments were
first carried out with milk agar and once used milk
agar and then with nutrient agar and once used
nutrient agar. On the whole 324 samples were
examined in the former case and 151 samples with
the latter media.
Experimental
One hundred and eighty ml. of tap water were
added to used agar medium*, collected from 40
petri dishes, in a beaker. The solid medium was
melted and boiled for one minute. Next it was
passed through a pad of sterile cotton wool, wetted
with boiling water, and then kept in an air oven at
a temperature of 95°-100°C. for 1½ to 2
hours. After this, the requisite amount of tap
water was added to make up for the loss caused by
the evaporation, boiled for one minute and then
filtered through a double layer of clean muslin
cloth wetted with boiling water. The pH of this
medium was then adjusted to 6.8. After this it
was filled in 8 to 10 ml. portions in sterile test tubes
and sterilized at 15 lb. pressure for 25 minutes.
The dilution technique of the Ministry of Agri-
culture [1934] was used. The platings were done
in duplicate and the averages of the duplicates
were taken for the counts in each case. The
arithmetic means, standard deviations and co-
efficients of variation of the counts obtained for
each set of samples by the use of milk agar and
once used milk agar and nutrient agar and once
used nutrient agar are given in Tables I and II.
The differences between the arithmetic means of
the counts and their standard errors in each case
are also given in the same tables.
* Ten ml. of fresh medium were originally used in
each petri dish.
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