NUTRITIONAL REQUIREMENTS FOR THE GROWTH OF LEPTOSPIRES
The nutritional requirement of leptospires are unique although simple. Vit B1 and B 12 and long-chain fatty acids are the only organic compounds that are known to be required. Fatty acids are their source of energy and carbon, and are required as a source of cellular lipids since leptospira cannot synthesize fatty acids de novo. Owing to the inherent toxicity of free fatty acids, these must be supplied to the leptospires either bound to albumin. Carbohydrates are not a suitable source of energy or carbon. Even though amino acids are utilized to a limited extent, they cannot satisfy the nitrogen requirements of these organisms.
The nonessential nutrient, pyruvate, enhances the initiation of growth of the fastidious leptospires.
Leptospires incorporate purine bases but not pyrimidine bases into their nucleic acids. Because of this, they are resistant to the antibacterial activity of the pyrimidine analogue, 5-fluoro-uracil. This compound is used in selective media for the isolation of leptospires from contaminated sources.
A wide variety of media have been described for the cultivation of leptospires. Basically these can be divided into four groups:
While a number of these media are adequate for the recovery of the less fastidious leptospires, they are totally inadequate for the isolation of the more fastidious leptospires like hardjo. The two media which were found most useful for the growth of more fastidious leptospires are EMJH + 1% fresh rabbit serum and a Tween 80/40 medium containing sodium-pyruvate/lactalbumin hydrolysate/superoxide dismutase and bovine serum albumin.
Three forms of medium:
Essential for isolation of leptospires and growing cultures to be used as antigen in agglutination tests. Pieces of agar present in the semi-solid media interfere with the interpretation of these reactions.
Growth of leptospires: turbidity, granular appearance or deposit forms.
SEMI-SOLID Contains 0.1-0.5% agar, (w/v)
Preferred for isolating strains and medium-term maintenance. (up to several years). Growth is readily initiated in these media and usually is easily visualized as one or more rings of dense growth, several mm to cm below the surface of the medium. The lack of rings does not necessarily mean the absence of leptospires. Semi-solid media in screw capped tubes are generally used for the maintenance of stock-cultures. Cultures are stored at RT and transferred at 6 month intervals.
SOLID Contains 0.8-1.3% agar (w/v)
Dispense in tubes or plates. The lower the concentration of agar the more likely is the growth tendency of leptospires to swarm across the plate and through the medium; the higher the concentration the smaller the colonies.
Growth occurs below the surface. Plates have to be sealed to prevent dehydration, i.e. moist chamber.
This method is used for isolating strains from contaminated natural materials, contaminated cultures or cloning of mixed leptospira infections. The colonies in 1% agar are subsurface and become visible within 7-14 days in most serovars. The shape of the colony of motile strains changes with time and, for a given strain, the colony size is directly related to the agar concentration. Subsurface colonial morphology has not proved to be a useful characteristic for differentiating the strains of leptospira in routine laboratories.
|GROWTH REQUIREMENTS OF LEPTOSPIRES|
|Asparagine||Can replace peptone|
|Beef extract||Protein source|
|Fatty acids||Leptospira growth depends on long chain fatty acids for energy and carbon source|
|Sodium-pyruvate||Addition of 50-400 ug/ml sodium pyruvate is optimal for reducing the lag periods seen with lower inoculum|
|Glycerin||Addition of 50-400 ug/ml of glycerol decreases the generation
times and increases growth rates;
Remark: Acetate permits full utilization of fatty acids of Tween, i.e. 200 ug/ml medium of each sodium pyruvate, sodium acetate and glycerol
|Salts||Higher salt concentrations might be essential for membrane stabilization and growth of cells|
|Na/K||Stimulatory effects of higher levels of Na and K. The requirements for higher K could be replaced by equimolar amounts of Na although the use of K permitted shorter generation times|
|Ca/Mg||Virulent cells resond to higher levels of Mg. Increased levels of Ca did not replace the requirements for higher levels of Mg|
|Fe 2+||Iron is known to be active in oxidation-reduction functions and may contribute to the reduction of peroxides which results from the accumulation of organic peroxides resulting from auto-oxidation of long-chain, unsaturated fatty acids. Fe sulfate solution should be freshly prepared. Iron is not only an adequate substitute for hemoglobin but also retains its effectiveness longer|
|Ammonium||Ammonium salts are an effective source of cellular nitrogen|
|Rabbit serum||Schuffner noted that growth was often improved when rabbit serum was slightly haemolysed. Rabbit sera used in media: Increasing percentages of serum up to and including 12% results in a progressively steeper growth curve. This stimulation is seen in the first four days and is significant in attainment of high early growth levels. 6-8% of rabbit serum-enrichment results in total growth response is linear up to 6%|
|5 Fluoro-uracil||Uracil is a pyrimidine, pyrimidine is lethal to various micro-organisms, but not to leptospires. Add a smaller amount of 5 FU when continued subcultures are made. Concentration of 200-500 ug/ml|
It is generally assumed that animal serum is one of the most important growth factors for culture of leptospires. Most of the media described require rabbit serum as the protein source. In cultivating leptospires one of the most striking features is the irregularity in growth pattern according to the kinds of media used.
An amount of serum albumin will bind and detoxify only a certain amount of Tween 80. Investigators have divided rabbit serum into 3 fractions: albumin, globulin and whole serum ultra filtrate.
Albumin alone supported fair growth but all three fractions were required for good growth. The major function of the globulin fractions seemed to be the provision of some lipid material while albumin was required to bind and detoxify the lipid.
Pathogens could grow in lower Tween 80 concentrations (100 ug/ml). They could also be grown from small inoculate to high yields if the Tween 80 was added periodically in exceedingly small increment during the growth curve. Unsaturated fatty acids are at least 10 fold more toxic than their saturated counterparts.
3-7 days (30°C) + 3-7 days (37°C)
Until one month at RT before use. Check microscopically. If necessary use blood agar or other solid media.
Treatment of distilled water to be used in preparation of stock solutions, additives and basal media:
METHOD OF PREPARATION
PREPARATION OF FINAL MEDIUM
|EMJH (ELLINGHAUSEN AND MCCULLOUGH, MODIFIED BY JOHNSON AND HARRIS)|
The stock solution quantities are given for 20 liters of EMJH medium.
ALBUMIN FATTY ACID SUPPLEMENT STOCK SOLUTIONS
METHOD OF PREPARATION
ALBUMIN FATTY ACID SUPPLEMENT
PREPARATION OF FINAL MEDIUM
Basal medium 9 parts = 18 liters
Albumin fatty acid supplement 1 part = 2 liters ____________
Total = 20 liters
Filtration is performed through a Millipore or Seitz filter (0.22 um). We use a Millipore filter holder type 316, 142 mm, in combination with a 20 liter dispensing pressure vessel. On the support screen put a 0.22 um type GSWP 14200 Millipore (glossy side of the filter up) followed by a gauze type AP 3212400 and a 0.45 um type HAWP 14200 filter (Millipore). The two prefilters type AP 2512450 are put on it whereafter all filters are wetted with distilled water. The choice of filters evidently depends on the medium and quantity to filter.
|Enriched media (for isolation)|
|Basal medium (Method for 1 L):
Na2HPO4 1 g KH2PO4 0.3 g NaCl 1 g Distilled water 998 ml
Add following stock solutions:
Glycerol (10% vol/vol) 1 ml NH4Cl (25%) 1 ml
Semi-solid medium: Agar 1.5 g Solid medium: Agar 15 g
Stock solutions (per 100 ml of distilled water)
ZnSO4 x 7H2O 0.4 g CaCl2 x 2H2O 1 g MgCl2 x 6H2O 1 g Vit B1 0.5 g Vit B12 0.02 g MnSO4 x 4 H2O 0.3 g
Following stock solutions are made up on same day as preparation of supplement:
FeSO4 x 7H2O 0.4 g Tween 80 20 ml + 180 ml distilled water Tween 40 20 ml + 180 ml distilled water
Method for 1 L:
Bovine serum albumin 100 g Vit B1 stock solution 10 ml CaCl2 stock solution 10 ml MgCl2 stock solution 10 ml ZnSO4 stock solution 10 ml MnSO4 stock solution 1 ml FeSO4 stock solution 100 ml Vit B12 stock solution 10 ml Tween 80 stock solution 90 ml Tween 40 stock solution 35 ml Lactalbumin hydrolisate 10 g SOD (Superoxide dismutase)0.01 g Sodium pyruvate 0.4 g Rabbit serum 40 ml
Preparation of final medium (for 1 L)
- 100 ml of supplement is added to 900 ml of basal medium.