160
A . SARAGEA E l ' AL.
C . diphtheriae and C. ulcerans produce black colonies with characteristic
Atypical corynebacteria develop with black-greyish dry colonies,
Diphtheroids: smooth brownish to shiny black colonies.
morphology for each type: gravis, mitis, intermedius or C. ulcerans.
deeply irregular in margins (atypical daisy head).
5. Loeffler medium (slants)
I n 100 ml peptone broth, containing 1% glucose, adjusted to p H = 7.6,
add 300 ml sterile normal bovine serum. After thorough mixing, the
medium is distributed either in 5-6 ml amounts in 16 x 160 tubes, or in
3 ml aliquots in 12 x 120 ml tubes; then the tubes are inspissated for
30 min at 90°C in a slanting position.
6. Hiss medium for carbohydrate fermentation
(a) Preparation of basic medium. Dissolve 7 g peptone and 1.4 g disodium
phosphate (NaZHP04) in 1400 ml distilled water. Boil and filter through
filter paper, autoclave for 20 min at 120°C. Cool and add 250 normal
bovine serum. Then steam at 100°C for 20 min; add 11 ml of sterile
solution of Andrade indicator.
The basic medium distributed in bottles keeps indefinitely at + 4°C.
Different carbohydrate solutions may be added ; 1% for all carbohydrates
except starch which is used at 0.4%.
(b) Preparation of Andrade indicator. T o 100 ml distilled water add:
0.5 acid fuchsin
10 m l 1 N NaOH
After autoclaving for 30 min at 115°C this keeps indefinitely at +4"C.
(c) The carbohydrate solutions used are : glucose, sucrose, maltose, levulose,
dextrine and glycogen 10% solutions, and starch 4%.
The carbohydrate solutions are sterilized at 105°C for 20 min.
For sucrose, filtration is recommended, due to the tendency to hydrolyse
during heating.
The final medium is prepared by adding sterile carbohydrates to each
90 ml basic Hiss medium, 10 ml of each carbohydrate solution. The p H
should be 8.1-8.2. The final medium is distributed in 2-3 ml quantities in
12 x 120 mm tubes.
For practical purposes, glycogen is recommended instead of starch.
At acid pH, the medium turns pink.
7. Pisu's medium (modified by Maximescu and Nicoara', 1964) for cystinase test
T o 80 ml nutrient agar, with 0.4% agar (Difco) containing 2% peptone,
pH = 74-7.6 melted and cooled at 45"C, add:
A . SARAGEA E l ' AL.
C . diphtheriae and C. ulcerans produce black colonies with characteristic
Atypical corynebacteria develop with black-greyish dry colonies,
Diphtheroids: smooth brownish to shiny black colonies.
morphology for each type: gravis, mitis, intermedius or C. ulcerans.
deeply irregular in margins (atypical daisy head).
5. Loeffler medium (slants)
I n 100 ml peptone broth, containing 1% glucose, adjusted to p H = 7.6,
add 300 ml sterile normal bovine serum. After thorough mixing, the
medium is distributed either in 5-6 ml amounts in 16 x 160 tubes, or in
3 ml aliquots in 12 x 120 ml tubes; then the tubes are inspissated for
30 min at 90°C in a slanting position.
6. Hiss medium for carbohydrate fermentation
(a) Preparation of basic medium. Dissolve 7 g peptone and 1.4 g disodium
phosphate (NaZHP04) in 1400 ml distilled water. Boil and filter through
filter paper, autoclave for 20 min at 120°C. Cool and add 250 normal
bovine serum. Then steam at 100°C for 20 min; add 11 ml of sterile
solution of Andrade indicator.
The basic medium distributed in bottles keeps indefinitely at + 4°C.
Different carbohydrate solutions may be added ; 1% for all carbohydrates
except starch which is used at 0.4%.
(b) Preparation of Andrade indicator. T o 100 ml distilled water add:
0.5 acid fuchsin
10 m l 1 N NaOH
After autoclaving for 30 min at 115°C this keeps indefinitely at +4"C.
(c) The carbohydrate solutions used are : glucose, sucrose, maltose, levulose,
dextrine and glycogen 10% solutions, and starch 4%.
The carbohydrate solutions are sterilized at 105°C for 20 min.
For sucrose, filtration is recommended, due to the tendency to hydrolyse
during heating.
The final medium is prepared by adding sterile carbohydrates to each
90 ml basic Hiss medium, 10 ml of each carbohydrate solution. The p H
should be 8.1-8.2. The final medium is distributed in 2-3 ml quantities in
12 x 120 mm tubes.
For practical purposes, glycogen is recommended instead of starch.
At acid pH, the medium turns pink.
7. Pisu's medium (modified by Maximescu and Nicoara', 1964) for cystinase test
T o 80 ml nutrient agar, with 0.4% agar (Difco) containing 2% peptone,
pH = 74-7.6 melted and cooled at 45"C, add: