Article
Vol. 8, No. 7
597
Eur. J. Clin. Microbiol. Infect. Dis., July 1989, p. 597-602 0934-9723/89/07 0597-06 ~ 3.00/0
Influence of Media Supplements on Growth and Survival of
Campylobacterpylori S. L. H a z e l l * , D. C. M a r k e s i c h , D. J. E v a n s , D . G . E v a n s , D. Y . G r a h a m
Experiments were designed to determine the role of heme and the importance of other factors in the growth of Campylobacter pylori. Campylobacter pylori strains were tested for their ability to synthesize porphyrin, for their ability to grow and be maintained on basal m e d i u m and basal medium supplemented with b l o o d o r b l o o d products, and for the influence o f bovine serum albumin and eatalase on viability. Results indicated that Campylobacter pylori does not require heme as a souree o f porphyrin. Growth o f Campylobacter pylori could not be sustained on media containing starch or hemoglobin, b u t was sustained on media containing erythrocytes, serum, bovine serum albumin or catalase. The ability to grow on media containing bovine serum albumin and catalase suggests that protection from toxic fatty acids and the prevention o f toxic p r o d u c t formation m a y be i m p o r t a n t factors in the growth and survival o f Campylobacter pylori in vitro. Both bovine serum albumin and catalase combined provide t h e m i n i m u m requirements which allow the spectrum of Campylobacter pylori present in a single culture to grow on blood-free media.
Although there have been reports on the enzyme profile o f the gastric bacterium Campylobacter pylori with a heavy emphasis on urease, there is only limited information on the factors that are important for growth o f Campylobacter pylori in vitro ( i - 4 ) . A clear understanding o f the nutritional requirements o f a bacterium can provide important insights into microbe-environment interactions. In their early studies Marshall and associates (5) noted that whole blood enhanced the growth o f Campylobacter pylori. Subsequently it was reported that heine stimulated the growth o f Campylobacter pylori on basal medium (6), however the claim that this microorganism needs heme as a growth factor is not universally accepted (7). Heme may play a variety o f roles in bacterial growth as a source o f porphyrins (e.g. in the case ofHaemophilus influenzae) for incorporation into hemoproteins, as a source o f iron in situations where free iron is restricted, or as an alternate growth factor (8, 9). In addition, heine can function as a protective agent by catalyzing the degradation o f hydrogen peroxide (10, 11). Starch has also been reported to stimulate the growth o f Campylobacter pylori (7), suggesting that adsorptive detoxification o f substances such as fatty acids present in the microbial environment may be important.
Digestive Diseases Section 111D, Department of Medicine, Veterans Affairs Medical Center and Baylor College of Medicine, 2002 Holcombe Blvd., Houston, Texas, 77030, USA.
We report the results o f experiments designed to determine the role o f heme and the importance o f other nutritional factors in the growth o f Campylo-
bacter pylori.
Materials and Methods
Bacteria. Campylobacter pylori strains were isolated from biopsy material from patients undergoing endoscopy at the Veterans Affairs Medical Center. Campytobacter ]e]uni strains were isolated from the faeces of diarrhea patients and were provided from the microbiology department culture collection by Dr. J. Clarridge. Campylobacterpylori strains were characterized as previously described (6). Six strains of Campylobacter pylori were used, being given the designatians 8826 and 8831 (laboratory adapted isolates), and 8801, 8802, 8803 and 8804 (fresh isolates). Unless otherwise specified, cultures were grown on 7 % (v/v) horse blood agar and incubated at 37 °C in an environment of 10 % CO2 in air and 99 % relative humidity.
Chemicalsand Reagents. Unless otherwise specified chemicals and reagents were obtained from Sigma Chemical, USA.
Porphyrin Test. The porphyrin test was performed according to tile method of Kilian (8). Briefly, strains (2 Campylobacter ]e/uni and 6 Campylobacter pylori) were removed from the surface of a blood agar plate after incubation for 2 or 4 days and inoculated into sterile glass vials containing 300 ul of a test solution (33 mg of delta-aminolevulinic acid and 20 mg of MgSO4. 7H20 dissolved in 100 ml of 0.1M sodium phosphate buffered saline, pH 7.2). The vials and appropriate controls were incubated lbr 6 - 8 h at 37 °C. Whole cells were removed by centrifugation for 2 min in a Beckman Microfuge 12, and 250 ~ulof the supernatant mixed into 2.75 ml of PBS. Porphyrin was detected by fluorescence
598
Eur. J. Clin. Microbiol. I n f e c t . Dis.
Table 1: Supplements added to Iso-Sensitest agar as basal medium. Supplement
Abbreviation
Concentration
Citrated human blood Human serum Human erythrocytes a Bovine liver catalase Human apotransferrin
BA SERA RBC CATALASE T
5% 5% 5% 0.05 % 0.002 %
T plus ferric ammonium citrate
TFe
0.001%
Ferric ammonium citrate Human hemoglobin Hb plus apotransferrin Oleic acid albumin complex b OAC plus T OAC plus TFe OAC plus Fe OAC plus Hb OAC plus HbT
Fe Hb HbT OAC OACT OACTFe OACFe OACHb OACHbT
0.001% 0.01% 0.002 % 10 % (v/v)
5 % (w/v) neutral bovine serum albumin c
BSA
10 % (v/v)
5 % (w/v) fatty acid free bovine serum albumin d
FAF-BSA
10 % (v/v)
BSA plus bovine liver catalase
BSA/Catalase
0.1%
FAF-BSA plus bovine liver catalase
FAF-BSA/Catalase
0.1%
aCells washed in Alsever's solution. bAs in reference 13. cSigma No. A-2153. dSigma No. A-6003.
spectrophotometery (excitation 396 nm, emission 615 nm; Perkin-Elmer model LS-5). Porphobilinogen was detected by adding 0.5 ml of modified Ehrlich's reagent (12) directly to the inoculated vials. Cultures of Campylobacter ]ejuni incubated for two days were used as positive controls.
Test Media. Test media were prepared using Iso-Sensitest Agar (ISA) (Oxoid, USA) as the base (Table 1). Blood or blood products were obtained fresh from a healthy Campylobacter pylori negative volunteer. All heat sensitive reagents were filter sterilized through a 0.2 tzm membrane filter. Growth on Basal Medium Supplemented with Blood or Blood Products. Strains of Campylobacter pylori were grown on basal medium which was either unsupplemented or supplemented with blood or blood tractions. Following incubation for 24, 48, 72 and 96 h or at 72 h only the relative yield of cells for each of the media was determined. Inocula were prepared by suspending the growth of an approximately 3-day-old culture in sterile PBS (~ 1 ml per plate). Plates were inoculated with 25 t~l of the suspension (~ 107 CFU) streaked in a uniform manner. Yields were determined by transferring all surface growth with a glass ("hockey stick") rod into PBS. Where necessary, cells from several plates were pooled. The yield per plate was expressed as the dilution volume of cell suspension required to obtain an absorbance of 1.0-+ 0.01 at a wavelength of 600 nm against a PBS blank.
Effect of Supplements on Viability. Cultures were repeatedly passaged on media of the same composition (up to seven passages). Growth was recorded, and where no colonies were evident, viability was determined by subculture on horse blood agar. bTfluence of Bovine Serum Albumin and Bovine Serum Albumin/Catalase on the Viable Count. The capacity of
bovine serum albumin, fatty acid-free bovine serum albumin and fatty acid-free bovine serum albumin plus catalase to support the entire population of viable bacteria grown and maintained on horse blood agar was determined by means of culture dilution experiments. Campylobacter pylori were taken from horse blood agar and the viable count determined on blood agar, basal medium (Iso-Sensitest agar), basal medium containing bovine serum albumin, fatty-acid-free bovine serum albumin and fatty-acid-free bovine serum albumin plus catalase.
Viable Counts. Counts were performed after the cultures had been incubated for a minimum of seven days. Bacteria were vortexed (30 s) and diluted tenfold in 1% (w/v) peptone (Difco Laboratories, USA). Viable counts were determined by the method of Miles and Misra (14).
Results Porphyrin
Test. Cultures i n c u b a t e d for t w o days b e f o r e testing were positive for b o t h p o r p h o b i l i n o g e n ( m o d i f i e d E h r l i c h ' s r e a g e n t ) a n d p o r p h y r i n (fluoresc e n c e ) . Cultures o f Campylobacter pylori i n c u b a t e d for f o u r d a y s gave negative results for the p o r p h o bilinogen t e s t and negative or equivocal results w h e n s c a n n e d in the f l u o r i m e t e r f o r p o r p h y r i n .
Growth on Basal Medium Supplemented with Blood or Blood Products. A f t e r o n e passage, t h e g r o w t h o f t h e l a b o r a t o r y a d a p t e d Campylobacter pylori strains 8 8 2 6 a n d 8831
o n the basal m e d i u m c o n -
Vol. 8, 1989
599
taining serum was found to be equivalent to that on basal medium supplemented with whole blood, better than that on basal medium containing washed h u m a n erythrocytes and markedly better than that on the unsupplemented Iso-Sensitest agar. Both strains generated almost identical growth patterns, the peak yields on blood agar and serum agar occurring after 48 h of incubation (data for 8831 shown in Figure 1). When the yield from two fresh isolates of Campylobacter pylori (8801 and 8802) incubated for 72 h were compared (Figure 2), it was found that the total yield on blood agar was in the order of 10 % o f that of strains 8826 and 8831 (Figure 1). Further, the yield obtained on serum agar was less than that obtained on blood agar, being up to 50 % less for strain 8802.
50~ • El ~.
40
~ m.z
30
T,
~.~ ~ 15
ISA/SE|IA
• BLOOD AGAR • ISA/RBC ' o ISA
//
2o
where blood, blood products or catalase were used as supplements, only the basal Iso-Sensitest agar failed to sustain growth of the bacteria (Table 2), although growth on the medium containing catalase was inferior compared to blood agar. When h u m a n apotransferrin, h u m a n hemoglobin, iron or combinations of these supplements were tested for their ability to maintain the growth of Campylobacter pylori, only strains 8801 and 8831 were sustained by any additives for two passages more than the basal medium (Table 3). This series of experiments was repeated using all six strains of Campylobacter pylori with the addition of oleic acid albumin complex to the supplemented media (including a control consisting of the base supplemented with oleic acid albumin complex alone). Using these combinations it was found that oleic acid albumin complex alone could maintain the cultures for a m i n i m u m of seven passages, the growth appearing more confluent after each passage (data for 4 strains and 6 passages on Iso-Sensitest agar/ oleic acid albumin complex is given in Table 3 for comparison).
Influence of Bovine Serum Albumin and Bovine Serum Albumin/Catalase on Viable Count. When
10
2 4'
Effect of Supplements on Viability. In the series
4 8~
7 2=
96 ~
Incubation Period (h) Figure 1: Culture yields up to 96 h for Campylobacterpylori
media supplemented with bovine serum albumin, fatty acid-free bovine serum albumin and fatty acid-free bovine serum albumin plus catalase were tested to determine if they could support the entire population of viable bacteria maintained on horse blood agar, it was found that in all cases (Figure 3) the medium containing fatty acid free bovine serum
8831 grown on the basal medium (ISA) and the basal medium containing human serum (SERA), blood (BLOOD AGAR) or erythrocytes (RBC). Table 2: Growth and viability of Campylobacter pylori on various media over multiple passages. • [] []
u
BLOOD AGAR
ISA/SERA ISA/RBC
[] ISA
"0
Passage b Mediuma
. . . . . . . . . . . . . . . . .
8802
8801
Strain
Figure 2: Culture yields at 72 h for Campylobacter pylori 8801 and 8802 grown on the basal medium (ISA) and the basal medium containing human serum (SERA), blood (BLOOD AGAR) or erythrocytes (RBC).
1
2
3
4
5
6
7
+ +
+ +
+ +
+ + + +
BA
8826 8831
+ +
+ +
ISA
8826 8831
+ +
+ + -/+
-
ISA/SERA
8826 8831 8826 8831
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
8826 8831
+ +
+ +
+ +
+ +
+ +
+ + + +
ISA/RBC 0
Strain
ISA/CATALASE
+ + + +
aRefer to Table 1. b+ visible viable colonies; - no visible colonies, no viable cells; -/+ no visible colonies, viable cells isolated on blood agar.
600
Eur. J. Clin. Microbiol. Infect. Dis.
Table 3: Growth and viability of four strains of Campylobacter pylori grown on basal media and media supplemented with iron and/or blood factors.
A
Passage b Medium a tSA
ISA/Fe
ISA/TFe
ISA/T
ISA/Hb
ISA/HbT
ISA/OAC
O HBA BII ISNFAF-BSAJCatalase ~KN ISA/BSA ISA/FAF-BSA r ~ t fSA tSA/eSAJCatalase
Strain 1
2
8801 8802 8826 8831
+ -/+ + +
-/+ +
8801 8802 8826 8831
+ + + +
-/+ -/+ -
8801 8802 8826 8831
+ + + +
-/+ +
8801 8802 8826 8831
+ + + +
-/+ -
8801 8802 8826 8831
+ + + +
-/+ -/+
8801 8802 8826 8831 8801 8802 8826 8831
+ + + + + + + +
3
4
5
6
U) +1
Plis|ge NO.27
PIIssageNO 3
PlllSlgl NO.27 I
B
133 O u3
==
2 +
"b
"t-
-t_o
O
P~sa~No 3
C3
I
nigeNo.2? I P u ~ i No, 27 p~| b
C -/+
-/+
-
-
+ -/+ -/+ -/+ + + + +
+
"t-
+
"1-
--/+ PllsIlglS NO 23
-1+
-/+
-/+ -t+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
aRefer to Table 1. b+ visible viable colonies, - no visible colonies, no viable cells; - / + no visible colonies, viable cells isolated on blood agax.
albumin plus catalase s u p p o r t e d m o r e bacteria than all o t h e r s e x c e p t horse b l o o d agar. F o r high passage cultures ( > 20) the m e d i u m containing fatty-acid-free bovine serum albumin plus catalase s u p p o r t e d as m a n y bacteria as horse b l o o d agar. It was n o t e d , particularly in the l o w passage cultures on the basal m e d i u m plus fatty-acid-free bovine serum albumin and catalase, that t w o c o l o n y types were present; a large c o l o n y f o r m and a small c o l o n y form. The small c o l o n y f o r m p r e d o m i n a t e d at the higher dilutions o n the fatty-acid-free bovine serum albumin plus catalase m e d i u m . On the m e d i u m containing bovine serum albumin alone t h e large c o l o n y f o r m appeared to p r e d o m i n a t e . F u r t h e r g r o w t h on the basal m e d i u m (Iso-Sensitest agar) and the basal m e d i u m plus bovine serum a l b u m i n o r fatty-acid-free bovine serum albumin t e r m i n a t e d abruptly b e t w e e n dilutions, as t h o u g h a m i n i m u m "critical mass" o f cells was required to sustain the cultures.
PISllhgl NO 47
Pi*sag~ No. r0
Figure 3: Viable counts for three strains of Campylobacter pylori (A: 8801, B: 8802 and C: 8826). After various growth cycles on horse blood agar, cultures were harvested, diluted and counted on horse blood agar (HBA), basal medium (ISA), or basal medium containing bovine serum albumin (ISA/BSA), fatty acid free bovine serum albumin (ISA/FAFBSA), fatty acid free bovine serum albumin plus bovine liver catatase (ISA/FAF-BSA/Catalase) or bovine serum albumin plus bovine liver catalase (ISA/BSA/Catalase). In two cases, at passage number 27, strains were passed through three cycles on either a) the basal medium containing bovine serum albumin plus bovine liver catalase or b) the basal medium containing bovine serum albumin alone. Cultures maintained on horse blood agar were transferred every 3 - 4 days (= one passage). *** indicates no detectable colonies.
When the effect o f three passages on m e d i u m containing bovine serum albumin and catalase was c o m p a r e d to the effect o f three passages on m e d i u m containing bovine s e r u m a l b u m i n alone, it was n o t e d t h a t there was a significant increase in the comparative viable c o u n t for the strain g r o w n on the latter m e d i u m containing bovine serum a l b u m i n but n o t for the strain grown on the f o r m e r m e d i u m (Figure 3 A and B).
Heat Inactivation o f CataIase. When catalase was added bovine it was strains
to the basal m e d i u m before autoclaving, serum albumin being added u p o n cooling, f o u n d that the viable c o u n t s for the three tested were significantly greater than for
Vol. 8,1989
medium containing bovine serum albumin alone. Further, the counts approached or equatled those obtained using filtered catalase and bovine serum albumin, although the colony size was markedly reduced compared to the size on this latter medium.
Discussion it has previously been reported that the growth of Campylobacter pylori on basal media (Blood Agar Base No. 2, Oxoid) could be stimulated by the use of X-factor (heine) impregnated discs (6). Buck and Smith (7) showed that Campylobacter pylori would grow on basal media supplemented with starch, charcoal, serum or hemin, but considered that hemin was not an absolute requirement for growth. We investigated which of the potential roles of heine in the growth of Campylobacter pylori might be important, i.e. as a source of tetrapyrrole groups, a source of iron, an alternative growth factor or a detoxifier. When tested for their ability to synthesize porphyrins from delta-aminolevulinic acid, all strains of Campylobacter pylori gave a positive result early in the growth cycle, older cultures giving a false negative response. [Interestingly, similar effects of culture ageing have been noted for the urease activity of Campylobacterpylori (4)]. From this finding it is apparent that Campylobacterpylori does not require heine as a source of tetrapyrrole groups. Iso-Sensitest agar is a semi-defined medium which contains starch and as such was considered a good base to test the effect of supplements on the growth of Campylobacter pylori. When, on a single passage, laboratory-adapted strains were examined for their ability to grow on iso-Sensitest agar or Iso-Sensitest agar supplemented with blood products, serum was found to be an equivalent supplement to fresh whole blood. Although Campylobacter pylori grew on Iso-Sensitest agar, the yield from this medium was less than on any of the supplemented media. Similar observations were made with fresh isolates (8801 and 8802), except that these strains did not grow as well on media supplemented with serum, suggesting that fresh isolates are more fastidious than laboratory adapted strains. As nutrient carryover may support growth through several generations (15), the laboratory strains were passed through up to seven cycles on the basal medium (Iso-Sensitest agar) and Iso-Sensitest agar containing blood products or catalase. The growth of Campytobacter pylori could not be maintained on lso-Sensitest agar alone. Thus, although starch, or other factors, in this medium permit initial growth of the bacterium, the medium is not sufficient for long-term maintenance of Campylobacter pylori.
601
Media containing erythrocytes, serum and bovine catalase maintained the growth of the test strains, suggesting that common or alternate factors found in these media were sufficient for culture maintenance. To test whether the common factor was iron, four strains of Campylobacterpylori were passaged on media containing apotransferrin, hemoglobin, iron or combinations of these factors. The results for each strain were different, some responding to a few supplements where others did not. Hence these supplements appeared insufficient to maintain the growth of Campylobacter pylori. This may be influenced by the supplement's concentration or mode of presentation, or by the degree of bacterial adaptation to growth in an artificial environment. Dubos and Middlebrook (13) found that a neutral complex of oleic acid and bovine serum albumin could be substituted for serum for the growth of some mycobacteria. When the oleic acid albumin complex was incorporated in Iso-Sensitest agar, either alone or in combination with a range of other supplements, all media were found to sustain the growth of Campylobacterpylori. This data suggested that the oleic acid albumin complex was sufficient for the successful culture of Campylobacter pylori in a blood-free system. However, when tested for the ability to isolate Campylobacter pylori from biopsy material, the number of colonies formed on lso-Sensitest agar containing oleic acid albumin complex, relative to horse blood agar, were few (unpublished data). Thus, although oleic acid albumin complex-containing media could maintain the growth of Campylobacter pylori over extended passages it was possible that the oleic acid albumin complex was supporting a sub-population of bacteria with less fastidious growth requirements. Quantitative studies supported this conclusion for it was demonstrated that only basal media containing both bovine serum albumin and catalase could support a population of bacteria equivalent to that of blood agar. It was found that at least two populations of cells appear to be present in the cultures of Campylobacter pylori, a small colony of the "catalase dependant" type, and a large colony of the "catalase independent" type. Further, it was possible to enrich cultures for the "catalase independent" large colony form by multiple passage on the basal medium supplemented with bovine serum albumin. Dubos and Middlebrook (13) proposed that the bovine serum albumin detoxified fatty acids thus making them available for cell metabolism. The deletion of oleic acid from the media used in later studies with Campylobacter pylori did not appear to adversely affect the growth of this bacterium. Neither did the use of fatty-acid-free bovine serum albumin, although some of the high passage cultures appeared to grow better on bovine serum albumin
602
than fatty-acid-free bovine serum albumin. Thus, the bovine serum albumin was n o t necessarily a source o f fatty acids per se. However, Campylobacter pylori has been reported as having lipase activity (1) and thus m a y be able to obtain fatty acids from the media or from cell membranes in vivo. Knapp and Melly (16) have noted that polyunsaturated fatty acids are bactericidal, this effect being dependant u p o n t i m e , concentration and degree of unsaturation (the long chain saturated fatty acids having little toxic effect). They found some protective effects when high concentrations o f bovine serum albumin were added to media, but near complete protection occurred with the addition o f catalase. They concluded that the bactericidal effects o f polyunsaturated fatty acids were mediated by peroxidation of the fatty acids involving H2 02 and bacterial iron. We contend that bovine serum albumin and catalase act in concert to protect Campylobacter pylori from fatty acid toxicity by both adsorption and prevention o f the conversion o f the unsaturated fatty acids into toxic species. When catalase was added to the base medium prior to autoclaving there remained a beneficial effect. This m a y represent a secondary effect o f catalase or the removal o f H20~ present in the medium before autoclaving. This study establishes that Campytobacter pylori does not require heine as a source o f porphyrins. The ability o f heme to stimulate growth to a limited extent may relate to its weak capacity to break down H 2 0 2 . Any capacity for heine to act as an alternate growth factor is over shadowed by the ability o f bovine serum albumin plus catalase to provide conditions suitable for the growth o f Campylobacter pylori. Whereas we do not necessarily recommend that media supplemented with bovine serum albumin and catalase be used in the routine primary isolation o f Campylobacter pylori, these supplements may prove valuable where cultivation o f Campylobacter pylori in a blood-free environment is desired.
Acknowledgements Stuart Hazell is a C.J. Martin Fellow supported by the National Health and Medical Research Council of Australia. This work was supported in part by Veterans Affairs and in part by grants DK-35369 and DK-39919 from the National Institute of Digestive and Kidney Diseases.
Eur. J. Clin. Microbiol. Infect. Dis.
References 1. Megraud, F., Bonnet, F., Gamier, M., Lamouliatte, H.: Characterization of Campylobacterpyloridis by culture, enzymatic profile, and protein content. Journal of Clinical Microbiology 1985, 22: 1007-1010. 2. McNulty, C. A. M., Dent, J. C.: Rapid identification of Campylobacter pylori (Campylobacter pyloridis) by preformed enzymes. Journal of Clinical Microbiology 1987, 25: 1683-1686. 3. Mobley, H. L.T., Cortesia, M.J., Rosenthal, L. E., Jones, B. D.: Characterization of urease from Campylobacter pylori. Journal of Clinical Microbiology 1988, 26: 831-836. 4. Ferrero, R. L., Hazell, S. L., Lee, A.: The urease enzymes of Campylobaeter pylori and a related bacterium. Journal of Medical Microbiology 1988, 27: 33-40. 5. Marshall, B.J., Royce, H., Annear, D. I., Goodwin, C. S., Pearman, J.W., Warren, J.R., Armstrong, J. A.: Original isolation of Campylobacter pytori from human gastric mucosa. Microbios Letters 1984, 25: 83-88. 6. Hazell, S. L., Lee,A., Brady, L., Hennessy,W.: Campylobacter pyloridis and gastritis: association with intercellular spaces and adaption to an environment of mucus as important factors in colonization of the gastric epithelium. Journal of Infectious Disease 1986, 153: 658-663. 7. Buck, G. E., Smith, J. S.: Medium supplementation for growth of Campylobacter pyloridis. Journal of Clinical Microbiology 1987, 25: 597-599. 8. Kilian, M.: A rapid method for the differentiation of Haemophilus strains. Acta Pathologica, Microbiologica Scandinavica 1974, 82: 835-842. 9. Burnham, B. F., Neiland, J.B.: Studies on the metabolic function of the ferrichrome compounds. Journal of Biological Chemistry 1961, 236: 554-559. 10. Lascelles, J.: Synthesis of tetrapyrroles by microorganisms. Physiological Reviews 1961, 41: 417-441. 11. Myers, W. F., Osterman, J. V., Wisseman, C. L.: Nutritional studies of Rickettsia quintana: nature of the hematin requirement. Journal of Bacteriology 1972, 109: 89-95. 12. Mauzerall, D., Granick, S.: The occurrence and determination of d-aminolevulinic acid and porphobilinogen in urine. Journal of Biological Chemistry 1956, 219: 435-446. 13. Dubos, R.l., Middlebrook, G.: Media for tubercle bacilli. American Review of Tuberculosis 1947, 56: 334-345. 14. Miles, A.A., Misra, S.S.: The estimation of the bactericidal power of the blood. Journal of Hygiene 1938, 38: 732-748. 15. Mellencamp, M.W., McCabe, M.A., Kochan, I.: The growth-promoting effect of bacterial iron for serum exposed bacteria. Immunology 1981, 43: 483-491. 16. Knapp, H.R., Melly, M.A.: Bactericidal effects of polyunsaturated fatty acids. Journal of Infectious Disease 1986, 154: 84-94.