- Email: [email protected]
Occurrence of Clostridia in White Pickled Domiati and Gouda Cheeses
Zbl. Bakt. Abt. II, Bd. 128, S. 8/,-87 (1973)
[N ational Research Centre, Dokki, Cairo, and Dept. of Bacteriology, Faculty of Veterinary Medicine, Cairo University, Giza, Cairo, Egypt]
Occurrence of Clostridia in White Pickled Domiati and Gouda Cheeses Khayria Naguib and T. Shauman Summary Commercial white pickled" Domiati" and "Gouda" cheeses were examined for the presence of cIosuidia. These organisms were detected in 72% of the Domiati and 80% of the Gouda samples. Their identification revealed that Clostridium tyrobutyricum, followed by Cl. per{ringens, predominated in the Domiati cheese, while Cl. tyrobutyricum and Cl. sporogenes were the most prevalent in the Gouda cheese.
Introduction A very often observed defect in the quality of hard and white pickled cheeses is blowing. It is usually connected with flavour deterioration and texture defects. In hard and white pickled cheeses blowing is caused by species of bacteria, e. g., Escherichia, Aerobacter (DOLEZALEK and DOCEKALOV.,;\. 1962 and REINEKE 1965), and Clostridium (DOLEZALEK and DOCEKALOVA 1962, STOIAN and UNGUREANA 1962, IKONOMOV and TODOROV 1963, PULA Y 1966, KUTZNER 1966, BERGERE et al. 1969, and .YIATTEAZZI and ANNIBALDI 1970). The presence of clostridia in cheese was shown by many investigators to be favoured by the suitable oxidation-reduction potential, created by the growth of both E. coli and lactic acid bacteria (GALESLOOT and Kooy 1960) and/or the metabolic stimulatory substance, produced by some starter organisms used in cheese-making (SWARTLING and LINDGERD 1960, and HUNTER and FRAZIER 1961). High numbers of clostridia were found by MABBIT et al. (1959) and SIMONART et al. (1966) in hard cheese, produced from pasteurized milk in aseptic vat or under normal conditions. On the other hand, CHOMAKOV (1966a) reported that soft cheeses were less susceptible to "late gassy fermentation" on account of their high content of lactic acid and low pH (below 5). In white pickled cheese, IKONOMOV and TODOROV (1963) found that late gas formation occurred only when milk was very heavily contaminated with Cl. butyricum. CHOMAKOV (1966b) found Cl. tyrobutyricum in the same cheese type. However, Ct. tyrobutyricum was the main species reported to be able to grow in hard cheeses because of its tolerance of low pH (LIND 1953, GALESLOOT 1961, KUTZNER 1963, GOUDKOV and SHARPE 1965) and of comparatively high salt content (SJOSTROM 1943). On the other hand, BURBIANKA (1965) found that in hard Polish cheese, including Gouda, Ct. sporogenes, followed by Cl. perfringens, predominated. It is the aim of the present work to assess the incidence and types of clostridia, occurring in white pickled Domiati and Gouda cheeses under the conditions of cheese production in Egypt. Materials and Methods Samples of pickled white Domiati (25) and Gouda (25) cheeses (:1-1, weeks old) were taken at random from a factory in Cairo, Egypt. Cheeses were prepared from pasteurized lnilk, but no starter was used in the case of Domiati cheese. The samples were examined for connts and species of clostridia, pH, and salt content.
85
Occurrence of Clostridia in White Pickled Domiati Counting, isolation, and identification of clostridia
Ten gramme portions of cheese were taken aseptically, ground in a sterile mortar, and diluted serially up to 10- 5 with 2% sodium citrate. Inocula from the sel'ial dihltions were transferred to sterile tubes of freshly prepared melted sulphite-iron agar, as recommended by MOSSEL et a1. (1956). The tubes were subjected to heating at 80°C for 15 minutes, then cooled, scaled immediately with agar containing 0.05% cysteine hydrochloride or vaspar (mixture of vaseline and para rein wax), and incubated at 37°C for 3 days. Colonies surrounded by distinct black zones wel'C rounted. Representative colonies wel'e picked, transferred to reinforced clostridial medium HCM (HIRSCH and GRINSTEAD 1954), and the tubes incubated for 48 hI'S. at :37°C. A loopful from each tube was strcaked on liver-sheep blood agar (SHOUMAN 1966), fortified with 0.05% cysteine l,ydrochloride. Plates were anaerobically incubated in a McIntosh Jar at 37 of: for 3-4 days. Isolated single colonies were identified according to BERGEY'S scheme (1957) und the methods of NARAYAN (1967), modified by NAGUIB and SHOUMAN (197'2). Chelllical Analysis The salt con tent of the two types of cheese was estima ted by the method of DAVIES (1932). For pH measurements, a Bekman zeromatic pH meter was used.
Hesults and Discussion
Percentage incidence and counts of clostridia as well as pH and salt content of samples of Domiati and Gouda cheeses are summarized in Table 1. Clostridia were present in 76% of Domiati cheese samples in counts ranging from 150 to 240 and averaging 210 per g. As for the Gouda cheese, clostridia were detected in 80% of the Ta ble '/. Incidelll:c and Counts of Clostl'idia, pH, and Salt Content of \Vhite Pickled Domiati and Gouda C1wcses
Type of cheese
-
.\Tumbel' of samples examined
Samples revealing Clostridia No. %
.. - - - -
pH
Clostl'idial counts Min.
--_."-,".
Max.
Avr.
Min.
Max.
Salt content Avr.
)J.in.
Max. Avr.
'--
Ilomiati 25
18
72
150
240
210
5.30
5.55
5.40
5.50
6.15
5.70
(;oud[\
20
80
225
:370
.'300
5.-'l5
6.60
5.50
2.80
3.20
3.00
25
Table 2 Types of Clostridia, Isulated from ''''hite Pickled Domiati and Gouda Cheeses Domiati Types of Clostridia No.
%
Cl. tyrobutyricum Cl. perfringens Cl. sporogenes n. bifermentans
61.80 25.45 7.27 5.47
-----
-~-,-,.
Total
__._--,
.....
34 14 4 3 ..
"-._-_.
55
Gouda Types of Clostridia - --
Cl. Cl. Cl. C1. C1. _._..-
tyrobutyricum sporogenes butyricum per(ringens beijerinkii ....... -
No.
%
30 20
40.54 27.02 13.51 12.16 6.75
10 9 5 74
86
Khayria Naguib and T. Shauman
samples examined, the counts ranging from 225 to 370 with an average of 300 per g. The presence of clostridia in hard and white pickled cheeses was reported by IKoNoMOV and TODOROV (1963), STOIAN and UNGUREANA (1962), BURBIANKA (1965), GOUDKOV and SHARPE (1965), CHOMAKOV (1966b), SIMONART et aI. (1966), SIAPANTES (1967), MATTEAZZI and ANNIBALDI (1970). Their incidence in both the Domiati and Gouda cheeses examined may be partly due to heavy contamination of the raw milk with clostridia. Being in the highly resistant sporeform, they withstand heat during pasteurization. However, contamination during processing could also be involved. The types of clostridia isolated from the two types of cheese examined are presented in Table 2. From Domiati cheese, Cl. tyrobutyricum (34 cultures) and Cl. perfringens (14 cultures) were the most commonly isolated, with Cl. sporogenes (4 cultures) and Cl. bi{ermentans (3 cultures) following in order of frequency. As for the Gouda cheese, Cl. tyrobutyricum (30 cultures), then Cl. sporogenes (20 cultures) predominated, followed by Cl. butyricum (10 cultures), Cl. perfringens (9 cultures), and Cl. beijerinkii (5 cultures). The presence of Cl. tyrobutyricum in white pickled cheese was previously shown by CHOMAKOV (1966b), and in hard cheeses by LIND (1953), STOIAN and UNGUREANA (1962), GOUDOKOV and SHARP (1965), and MATTEAZZI and ANNIBALDI (1970). The predominance of Cl. tyrobutyricum in both types of cheeese examincd might be attributed in part to its tolerance of low pH (LIND 1953, GALESLOOT 1961, KUTZNER 1963, GOUDKOV and SHARPE 1965) and to comparatively high NaCI concentrations (SJOSTROM 1943). The pH of the Domiati cheese was somewhat lower, and its salt content was higher than that of the Gouda cheese (Table :1). However, the incidence of Cl. tyrobutyricum in the latter cheese was slightly lower than that in the former. These results indicate heavy contamination of the raw milk with this type of clostridia and/or contamination during processing. The considerably lower incidcnce of Cl. sporogenes in the Domiati cheese, compared with Gouda cheese, mig'ht be due to the comparatively lower pH and higher salt content of the former cheese which are not favourable for the growth of Cl. sporogenes (GALESLOOT 1961, GOUDKOV and SHARP 1965). The relatively lower incidence of Cl. perfringens in Gouda cheese than that in Domiati cheese could be explained on the basis that Cl. tyrobutyricum and Cl. sporogenes possibly had overgrown Cl. perfringens under the prevailing conditions. Formerly, LIND (1953) had reported that the general opinion of several research workers was that hlowing defects in chceses arise only in thc presence of Ct. tyrobutyricum which is capablc of fermenting laetatcs cven at low pH (KUTZNER 1963). However, the butyric acid bacteria, containing proteolytic enzymes which do not ferment lactates, e. g. Cl. perfringens and Cl. sporogenes, and cause proteolysis in cheese (GOUDKOV and SHARPE 1965) might justify the assumption that they are dangerous micro-organisms with respect to cheese production. To avoid the presence of these organisms in cheese, the logic start must be the application of hygienic measures in the production of milk to be used in manufacture, and cleanliness of cheese rooms as well as of all utensils concerned in processing. Zusammenfassung .
'WeiBel' gepokelter, handelsiiblicher "Domiati"- und "Gouda"-Kase wurde auf Anwesenheit von Clostridien gepriift. Diese Organismen konnten in 72% del' "Domiati"- tllld in 80% del' "Gouda"-Kiiseprohen nachgewiesen werden. Die Identifizierung diesel' Organismen zeigte
Occurrence of Clostl,idia in '''hite Piekle,[ Domiati
87
die vorherrsehende Anwesenheit yon Clostridium tyrobutyriculII, gefolgt VOll Cl. perfringens illl Domiati-Kase, walu'end Cl. tyrobutyricuTn und Cl. sporogenes die Illeist yorkomlllenden im C; ou,la- Kiise waren.
Literature BERGERE, J. L., BARS, D. Le, a lid' VASSAL, L.: Industrie Lait 267 (1969),463. - Bergey's .\Iallual of Determinative Bacteriology, 7th Ed. Edited by R. S. BREED, F. G. D. MURRAY, and 1'\. H. SMITH, The \Villiams and Co., Baltimore 1957. - BURBIANKA, MARIA: Rocznpanst. Zakl. I-lig. 16 (196;")), 309. - CHOMAKOV, Ch.: Milchwiss. 21 (I06Ga), 215. - Ibid.: Nauehni Trud. yissh. selskostop. lnst. GeOl'gi Dimitrov. 16 (1966b), 21. - DAVIES, W. L.: Analyst 57 (IHJ2), 7!J. - DOLEzALEK . .T., and DOCEKALOVA, II.: Int. Dairy Congr. B (1962), 617. GALESLOOT, TH. E.: Neth. Milk Dairy J. 15 (1961), 263. - GALESLOOT, TH. E., and Kooy, J. S.: .\'eth. Milk ])ail'~' J. 14 (JHGO), I. - GOUDKOV, A. V., and SHARPE, ELISABETH: J. appl. Bae!. 28 ( 1965),63. - HIRSCH, .\., alld GRINSTEAD, ELISABETH: J. Dairy Hes. 21 (1954), 101. HUNTER, J. E., and FRAZIER, "-. C: .J. Dairy Sci. 44 (1961), 217(j. - IKoNOMov, L., ami TODOROV, B. W.: Zhivolill. Produkti 3 (1963), 119. - KUTZNER, H . .T.: ZbL Bakt. I Orig. 191 (l!lGJ), 441. - I bid.: Int. Dairy Cong". XVII D (1.9GG) , 647. - LIND, c.: Int. Dail'y Congr. XIII (W.-,;l), (j2!J. - ~IABBITT, L. .\., CHAPMAN, H. R., and SHARPE, lL E.: J. Dairy Res. 26 (1959), 10."). - .\IATTEAZZI. D., and .\NNIBALDI, S.: Int. Dairy Congr. XVIII IE (1970),143. - MOSSEL, I) .•\ .•\., DE BRUIN, .\. S., VAN IhEPEN, H. SI. J., VEND RIG , C.lI. A., and ZOUTEWELLE, G.: .J. appL Bact. 19 (1.9;")(j), H2. - NAGUIB, KHAYRIA, and SHOUMAN, T.: J. AppL Bact. (1972, in p,·ess). - XARAYAN,K. C.: ZbL Bah. I Orig. 202 (1967), 212. - PULAY, G.: Int. Dairy Cong!'. XVII D (19(j6), 41. - HEINEKE, Il.: Inaug.-Diss. 1965, 71. - SHOUMAN, T.: lnaug.Diss. Vet. Med. Fakultiit, .T usius Liebig- U ni vel'sitat GieLlen 1$)66. - SJOSTROM, G.: Milchw. Forsell. 21 (1943), 272. - STOIAN, C, and UNGUREANA, C.: Lait 42 (1962), 415. - SIAPANTAS, L. C.: Diss. Abstr., Sect. 13 28 (Hl67), -leOR. - SIMONART, P., POFFE, R.,WEcKx, M., and HEUSSEL, A. VAN: lut. Dairy Congr. XVII (H)(j(j), 21. - SWARTLING, P., and LINDGREN, B.: ltep..\Jilk Dairy Hes. Alnarp, Sweden G2 (lU60) .
.\ lItllOl'S' addresses: .\ssoc. Prof. Dr. Khayria Saguill, X"tiullal R"search Centre, Dokki, Cail'O, and Dr.}\!. T. Shouman, Departllleut of Bacteriology, Faeulty of Veterinary lledieine, Cairo University, Giza, Cairo (Egypt).
[N ational Research Centre, Dokki, Cairo, and Dept. of Bacteriology, Faculty of Veterinary Medicine, Cairo University, Giza, Cairo, Egypt]
Occurrence of Clostridia in White Pickled Domiati and Gouda Cheeses Khayria Naguib and T. Shauman Summary Commercial white pickled" Domiati" and "Gouda" cheeses were examined for the presence of cIosuidia. These organisms were detected in 72% of the Domiati and 80% of the Gouda samples. Their identification revealed that Clostridium tyrobutyricum, followed by Cl. per{ringens, predominated in the Domiati cheese, while Cl. tyrobutyricum and Cl. sporogenes were the most prevalent in the Gouda cheese.
Introduction A very often observed defect in the quality of hard and white pickled cheeses is blowing. It is usually connected with flavour deterioration and texture defects. In hard and white pickled cheeses blowing is caused by species of bacteria, e. g., Escherichia, Aerobacter (DOLEZALEK and DOCEKALOV.,;\. 1962 and REINEKE 1965), and Clostridium (DOLEZALEK and DOCEKALOVA 1962, STOIAN and UNGUREANA 1962, IKONOMOV and TODOROV 1963, PULA Y 1966, KUTZNER 1966, BERGERE et al. 1969, and .YIATTEAZZI and ANNIBALDI 1970). The presence of clostridia in cheese was shown by many investigators to be favoured by the suitable oxidation-reduction potential, created by the growth of both E. coli and lactic acid bacteria (GALESLOOT and Kooy 1960) and/or the metabolic stimulatory substance, produced by some starter organisms used in cheese-making (SWARTLING and LINDGERD 1960, and HUNTER and FRAZIER 1961). High numbers of clostridia were found by MABBIT et al. (1959) and SIMONART et al. (1966) in hard cheese, produced from pasteurized milk in aseptic vat or under normal conditions. On the other hand, CHOMAKOV (1966a) reported that soft cheeses were less susceptible to "late gassy fermentation" on account of their high content of lactic acid and low pH (below 5). In white pickled cheese, IKONOMOV and TODOROV (1963) found that late gas formation occurred only when milk was very heavily contaminated with Cl. butyricum. CHOMAKOV (1966b) found Cl. tyrobutyricum in the same cheese type. However, Ct. tyrobutyricum was the main species reported to be able to grow in hard cheeses because of its tolerance of low pH (LIND 1953, GALESLOOT 1961, KUTZNER 1963, GOUDKOV and SHARPE 1965) and of comparatively high salt content (SJOSTROM 1943). On the other hand, BURBIANKA (1965) found that in hard Polish cheese, including Gouda, Ct. sporogenes, followed by Cl. perfringens, predominated. It is the aim of the present work to assess the incidence and types of clostridia, occurring in white pickled Domiati and Gouda cheeses under the conditions of cheese production in Egypt. Materials and Methods Samples of pickled white Domiati (25) and Gouda (25) cheeses (:1-1, weeks old) were taken at random from a factory in Cairo, Egypt. Cheeses were prepared from pasteurized lnilk, but no starter was used in the case of Domiati cheese. The samples were examined for connts and species of clostridia, pH, and salt content.
85
Occurrence of Clostridia in White Pickled Domiati Counting, isolation, and identification of clostridia
Ten gramme portions of cheese were taken aseptically, ground in a sterile mortar, and diluted serially up to 10- 5 with 2% sodium citrate. Inocula from the sel'ial dihltions were transferred to sterile tubes of freshly prepared melted sulphite-iron agar, as recommended by MOSSEL et a1. (1956). The tubes were subjected to heating at 80°C for 15 minutes, then cooled, scaled immediately with agar containing 0.05% cysteine hydrochloride or vaspar (mixture of vaseline and para rein wax), and incubated at 37°C for 3 days. Colonies surrounded by distinct black zones wel'C rounted. Representative colonies wel'e picked, transferred to reinforced clostridial medium HCM (HIRSCH and GRINSTEAD 1954), and the tubes incubated for 48 hI'S. at :37°C. A loopful from each tube was strcaked on liver-sheep blood agar (SHOUMAN 1966), fortified with 0.05% cysteine l,ydrochloride. Plates were anaerobically incubated in a McIntosh Jar at 37 of: for 3-4 days. Isolated single colonies were identified according to BERGEY'S scheme (1957) und the methods of NARAYAN (1967), modified by NAGUIB and SHOUMAN (197'2). Chelllical Analysis The salt con tent of the two types of cheese was estima ted by the method of DAVIES (1932). For pH measurements, a Bekman zeromatic pH meter was used.
Hesults and Discussion
Percentage incidence and counts of clostridia as well as pH and salt content of samples of Domiati and Gouda cheeses are summarized in Table 1. Clostridia were present in 76% of Domiati cheese samples in counts ranging from 150 to 240 and averaging 210 per g. As for the Gouda cheese, clostridia were detected in 80% of the Ta ble '/. Incidelll:c and Counts of Clostl'idia, pH, and Salt Content of \Vhite Pickled Domiati and Gouda C1wcses
Type of cheese
-
.\Tumbel' of samples examined
Samples revealing Clostridia No. %
.. - - - -
pH
Clostl'idial counts Min.
--_."-,".
Max.
Avr.
Min.
Max.
Salt content Avr.
)J.in.
Max. Avr.
'--
Ilomiati 25
18
72
150
240
210
5.30
5.55
5.40
5.50
6.15
5.70
(;oud[\
20
80
225
:370
.'300
5.-'l5
6.60
5.50
2.80
3.20
3.00
25
Table 2 Types of Clostridia, Isulated from ''''hite Pickled Domiati and Gouda Cheeses Domiati Types of Clostridia No.
%
Cl. tyrobutyricum Cl. perfringens Cl. sporogenes n. bifermentans
61.80 25.45 7.27 5.47
-----
-~-,-,.
Total
__._--,
.....
34 14 4 3 ..
"-._-_.
55
Gouda Types of Clostridia - --
Cl. Cl. Cl. C1. C1. _._..-
tyrobutyricum sporogenes butyricum per(ringens beijerinkii ....... -
No.
%
30 20
40.54 27.02 13.51 12.16 6.75
10 9 5 74
86
Khayria Naguib and T. Shauman
samples examined, the counts ranging from 225 to 370 with an average of 300 per g. The presence of clostridia in hard and white pickled cheeses was reported by IKoNoMOV and TODOROV (1963), STOIAN and UNGUREANA (1962), BURBIANKA (1965), GOUDKOV and SHARPE (1965), CHOMAKOV (1966b), SIMONART et aI. (1966), SIAPANTES (1967), MATTEAZZI and ANNIBALDI (1970). Their incidence in both the Domiati and Gouda cheeses examined may be partly due to heavy contamination of the raw milk with clostridia. Being in the highly resistant sporeform, they withstand heat during pasteurization. However, contamination during processing could also be involved. The types of clostridia isolated from the two types of cheese examined are presented in Table 2. From Domiati cheese, Cl. tyrobutyricum (34 cultures) and Cl. perfringens (14 cultures) were the most commonly isolated, with Cl. sporogenes (4 cultures) and Cl. bi{ermentans (3 cultures) following in order of frequency. As for the Gouda cheese, Cl. tyrobutyricum (30 cultures), then Cl. sporogenes (20 cultures) predominated, followed by Cl. butyricum (10 cultures), Cl. perfringens (9 cultures), and Cl. beijerinkii (5 cultures). The presence of Cl. tyrobutyricum in white pickled cheese was previously shown by CHOMAKOV (1966b), and in hard cheeses by LIND (1953), STOIAN and UNGUREANA (1962), GOUDOKOV and SHARP (1965), and MATTEAZZI and ANNIBALDI (1970). The predominance of Cl. tyrobutyricum in both types of cheeese examincd might be attributed in part to its tolerance of low pH (LIND 1953, GALESLOOT 1961, KUTZNER 1963, GOUDKOV and SHARPE 1965) and to comparatively high NaCI concentrations (SJOSTROM 1943). The pH of the Domiati cheese was somewhat lower, and its salt content was higher than that of the Gouda cheese (Table :1). However, the incidence of Cl. tyrobutyricum in the latter cheese was slightly lower than that in the former. These results indicate heavy contamination of the raw milk with this type of clostridia and/or contamination during processing. The considerably lower incidcnce of Cl. sporogenes in the Domiati cheese, compared with Gouda cheese, mig'ht be due to the comparatively lower pH and higher salt content of the former cheese which are not favourable for the growth of Cl. sporogenes (GALESLOOT 1961, GOUDKOV and SHARP 1965). The relatively lower incidence of Cl. perfringens in Gouda cheese than that in Domiati cheese could be explained on the basis that Cl. tyrobutyricum and Cl. sporogenes possibly had overgrown Cl. perfringens under the prevailing conditions. Formerly, LIND (1953) had reported that the general opinion of several research workers was that hlowing defects in chceses arise only in thc presence of Ct. tyrobutyricum which is capablc of fermenting laetatcs cven at low pH (KUTZNER 1963). However, the butyric acid bacteria, containing proteolytic enzymes which do not ferment lactates, e. g. Cl. perfringens and Cl. sporogenes, and cause proteolysis in cheese (GOUDKOV and SHARPE 1965) might justify the assumption that they are dangerous micro-organisms with respect to cheese production. To avoid the presence of these organisms in cheese, the logic start must be the application of hygienic measures in the production of milk to be used in manufacture, and cleanliness of cheese rooms as well as of all utensils concerned in processing. Zusammenfassung .
'WeiBel' gepokelter, handelsiiblicher "Domiati"- und "Gouda"-Kase wurde auf Anwesenheit von Clostridien gepriift. Diese Organismen konnten in 72% del' "Domiati"- tllld in 80% del' "Gouda"-Kiiseprohen nachgewiesen werden. Die Identifizierung diesel' Organismen zeigte
Occurrence of Clostl,idia in '''hite Piekle,[ Domiati
87
die vorherrsehende Anwesenheit yon Clostridium tyrobutyriculII, gefolgt VOll Cl. perfringens illl Domiati-Kase, walu'end Cl. tyrobutyricuTn und Cl. sporogenes die Illeist yorkomlllenden im C; ou,la- Kiise waren.
Literature BERGERE, J. L., BARS, D. Le, a lid' VASSAL, L.: Industrie Lait 267 (1969),463. - Bergey's .\Iallual of Determinative Bacteriology, 7th Ed. Edited by R. S. BREED, F. G. D. MURRAY, and 1'\. H. SMITH, The \Villiams and Co., Baltimore 1957. - BURBIANKA, MARIA: Rocznpanst. Zakl. I-lig. 16 (196;")), 309. - CHOMAKOV, Ch.: Milchwiss. 21 (I06Ga), 215. - Ibid.: Nauehni Trud. yissh. selskostop. lnst. GeOl'gi Dimitrov. 16 (1966b), 21. - DAVIES, W. L.: Analyst 57 (IHJ2), 7!J. - DOLEzALEK . .T., and DOCEKALOVA, II.: Int. Dairy Congr. B (1962), 617. GALESLOOT, TH. E.: Neth. Milk Dairy J. 15 (1961), 263. - GALESLOOT, TH. E., and Kooy, J. S.: .\'eth. Milk ])ail'~' J. 14 (JHGO), I. - GOUDKOV, A. V., and SHARPE, ELISABETH: J. appl. Bae!. 28 ( 1965),63. - HIRSCH, .\., alld GRINSTEAD, ELISABETH: J. Dairy Hes. 21 (1954), 101. HUNTER, J. E., and FRAZIER, "-. C: .J. Dairy Sci. 44 (1961), 217(j. - IKoNOMov, L., ami TODOROV, B. W.: Zhivolill. Produkti 3 (1963), 119. - KUTZNER, H . .T.: ZbL Bakt. I Orig. 191 (l!lGJ), 441. - I bid.: Int. Dairy Cong". XVII D (1.9GG) , 647. - LIND, c.: Int. Dail'y Congr. XIII (W.-,;l), (j2!J. - ~IABBITT, L. .\., CHAPMAN, H. R., and SHARPE, lL E.: J. Dairy Res. 26 (1959), 10."). - .\IATTEAZZI. D., and .\NNIBALDI, S.: Int. Dairy Congr. XVIII IE (1970),143. - MOSSEL, I) .•\ .•\., DE BRUIN, .\. S., VAN IhEPEN, H. SI. J., VEND RIG , C.lI. A., and ZOUTEWELLE, G.: .J. appL Bact. 19 (1.9;")(j), H2. - NAGUIB, KHAYRIA, and SHOUMAN, T.: J. AppL Bact. (1972, in p,·ess). - XARAYAN,K. C.: ZbL Bah. I Orig. 202 (1967), 212. - PULAY, G.: Int. Dairy Cong!'. XVII D (19(j6), 41. - HEINEKE, Il.: Inaug.-Diss. 1965, 71. - SHOUMAN, T.: lnaug.Diss. Vet. Med. Fakultiit, .T usius Liebig- U ni vel'sitat GieLlen 1$)66. - SJOSTROM, G.: Milchw. Forsell. 21 (1943), 272. - STOIAN, C, and UNGUREANA, C.: Lait 42 (1962), 415. - SIAPANTAS, L. C.: Diss. Abstr., Sect. 13 28 (Hl67), -leOR. - SIMONART, P., POFFE, R.,WEcKx, M., and HEUSSEL, A. VAN: lut. Dairy Congr. XVII (H)(j(j), 21. - SWARTLING, P., and LINDGREN, B.: ltep..\Jilk Dairy Hes. Alnarp, Sweden G2 (lU60) .
.\ lItllOl'S' addresses: .\ssoc. Prof. Dr. Khayria Saguill, X"tiullal R"search Centre, Dokki, Cail'O, and Dr.}\!. T. Shouman, Departllleut of Bacteriology, Faeulty of Veterinary lledieine, Cairo University, Giza, Cairo (Egypt).