NUTRITIONAL REQURIMENTS FOR THE GROWTH OF LEPTOSPIRES

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.

LEPTOSPIRA MEDIA
A wide variety of media have been described for the cultivation of leptospires. Basically these can be divided into four groups:

1. The traditional media containing approximately 8-10 per cent rabbit serum: Stuart, Korthof, Fletcher and Vervoort. Why rabbit serum? Rabbit serum contains the highest concentration of bound Vitamin B 12 essential for multiplication of leptospires. Proportions of rabbit sera which contain leptospira agglutinins is usually low compared with other animals (pool sera/dilute/check for antibodies). Schuffner and Korthof media have the disadvantage of phosphate precipitation which is undesirable for the microscopic agglutination test.
2. The Tween 80 bovine serum albumin of Ellinghausen and McCullough (1965) and the modification of it by Johnson and Harris (1967). The BSA component of the leptospira medium is the most expensive ingredient.
3. Low protein media or protein free media, often used for the preparation of vaccines.
4. Enriched media
5. With the addition of calf serum (FCS), rabbit serum, with or without agar, with addition of 5 Fluoro-uracil (and/or antibiotics) with or without agar.

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:

LIQUID
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
Peptone | Protein source
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.

MEDIUM CHECK

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.

LITERATURE

1. Ellis, W.A. and T.W.A. Little (Ed.) The present state of leptospirosis diagnosis and control, Martinus Nyhoff Publishers, 1986.
2. Turner, L. H. Maintenance, isolation and demonstration of leptospires. Transactions Roy. Soc. Trop. Med. Hy., Vol. 64, No. 4, 623-646, 1980.
3. Staneck, J.L. Henneberry, R.C. Cox, C.D. Growth requirements of pathogenic leptospires. Infection and Immunity, Vol. 7, No. 6, 886-897, 1973.
4. Lawrence, J.J. The growth of leptospirae in semi-solid media. Austr. J. Exp. Biol. Med. Science, Vol. XXIX, 195-199, 1951.
5. Ellinghausen, H.C. Some observations on cultural and biochemical characteristics of Leptospira pomona. J. Inf. Dis., Vol. 106, 237-244, 1960.
6. Vaneseltine, W.P. Staples, S.A. Nutritional requirements of leptospirae. I. Studies on oleic acid as a growth factor for a strain of Leptospira pomona. J. Inf. Dis., Vol. 108, 262-269, 1961.
7. Wolff, J. The Laboratory Diagnosis of Leptospirosis. 1954.
8. Thiermann, A.B. Use of solid medium for isolation of leptospires of the Hebdomadis serogroup from bovine milk and urine. Am.J.Vet.Res., Vol. 42, No.12, 2143-2145, 1981.
9. Wood, J. Johnson, R.C. Palin, K. Surface colonies of Leptospira interrogans. J. Clin. Micobiol., Vol. 13, No. 1, 102-105, 1981.

MEDIA PRESCRIPTIONS

Necessery equipment

- Balance (analytical, 2 decimal places, 300 g)
- Pipettes (different volumes)
- Magnetic stiereer
- pH meter
- Filtration equipment (it is better to use pressure filtration instead of vaccum)
- Liquid handling equipment for media transfer

Treatment of distilled water to be used in preparation of stock solutions, additives and basal media:

- Filter through 0.22 um filter
- Sterilize by autoclaving at 121 C for 20 minutes.

Fletcher media
REQUIREMENTS Na2HPO4 KH2PO4 NaCl Peptone Beef extract Agar noble STOCK SOLUTIONS Sorensen buffer A: Disolve 11.876 g Na2HPO4 in 1 liter distilled water Sorensen buffer B: Disolve 9.078 g KH2PO4 in 1 liter distilled water Autoclave above and store at 4 degrees C METHOD OF PREPARATION Disolve the following reagents in 820 ml distilled water: 0.3 g Peptone, 0.5 g NaCl, 0.2 g beef extract and 1.5 g agar noble; Add 80.8 ml Sorensen buffer A and 19.2 g Sorensen buffer B; Mix thoroughly; Adjust to pH 7.6 - 8.0; Heat until all ingredients are dissolved; Dispense the medium in bottles, 184 ml per bottle; Place the bottles in pressure cooker; Heat until boiling; Shake the bottles with medium vigorously until an even suspension is obtained; Place the lid on the pressure cooker and autoclave during 30 minutes at 121 degrees C; After autoclaving and before the medium has cooled shake the bottles again vigorously. The shaking is needed to prevent the agar to settle down at bottom; Store the bottles at 4 degrees C. PREPARATION OF FINAL MEDIUM Collect rabbit serum with the little bit of red blood cells; Inactivate the serum at 56 C, 30 minutes in water bath; Add 16 ml of this serum to 184 ml medium; Dispense the Fletcher's medium in 4/5 ml volumes in tubes; Check for sterility by incubating the tubes for 48 hours at 37 C and 48 hours at 30 C Store the medium at 4 C until use.

EMJH (ELLINGHAUSEN AND MCCULLOUGH, MODIFIED BY JOHNSON AND HARRIS)
REQUIREMENTS

STERILE GLASSWARE

- 5 flasks of 5 liters volume each
- 1 flask of 3 litres volume
- 1 cilinder of 2 liters volume each
- 1 cilinder of 1 liter volume
- Pasteur pipettes

CHEMICAL REAGENTS

- Na2HPO4 Merck 1.06586.0500
- KH2PO4 Merck 1.04873.1000
- NaCl Merck 1.06404.1000
- NH4Cl Merck 1.01145.0500
- Thiamine Merck 1.08181.0025
- Glycerol Merck 1.04093.1000
- Bovine Serum Albumin Fraction V Sigma A 8022
- CaCl2.2H2O Merck 1.02382.0500
- MgCl2.6H2O Merck 1.05833.0250
- FeSO4.7H2O Merck 1.03965.0100
- CuSO4.5H2O Merck 1.02790.0250
- ZnSO4.7H2O Merck 1.08883.0100
- Vitamin B 12 (cyanocobalamin) Merck 5.24950.0010
- Tween 80 Merck 8.22187.0500
- Na-pyruvate Merck 1.06619.0050

STOCK SOLUTIONS

The stock solution quantities are given for 20 liters of EMJH medium.

ALBUMIN FATTY ACID SUPPLEMENT STOCK SOLUTIONS

Reagents | Contents flask | Grams per 100 ml H2O | Storage
CaCl2 x 2H2O + MgCl2 x 6H2O | 30 ml | 1 (each) | -20 C
ZnSO4 x 7H2O | 20 ml | 0.4 | -20 C
CuSO4 x 5H2O | 2 ml | 0.3 | +4 C
Vitamin B12 | 20 ml | 0.02 | -20 C
Tween 80 | 250 ml | 10.0 | -20 C
NH4Cl | 20 ml | 25.0 | -20 C
Thiamine (Vitamin B1) | 20 ml | 0.5 | -20 C
Glycerol | 20 ml | 10.0 | -20 C

METHOD OF PREPARATION

ALBUMIN FATTY ACID SUPPLEMENT

1. Dissolve 200 grams of bovine serum albumin (BSA) in 1200 ml sterile glass distilled water by gently stirring on a magnetic stirrer (avoid foaming). It can take several hours depending on the batch of BSA.
2. Take all necessary stock solutions out of the frezzer
3. Add 30 ml calcimchloride and magnesiumchloride stock solution.
4. Add 20 ml zinc sulphate stock solution.
5. Add 2 ml copper sulphate stock solutin.
6. Add 1 gram ferrous sulphate and 0.8 gram of sodiumpyruvate.
7. Add 20 ml Vitamin B 12 stock solution.
8. Add 250 ml Tween 80 stock solution.
9. Add sterile distilled water up to 2 liters.
10. . Adjust pH to 7.4 - 7.6 with 1N NaOH using pH paper or pH meter.

BASAL MEDIUM

1. Dissolve 20 grams of Na2HPO4 together with 6 grams of KH2PO4 and 20 grams of NaCl in 2 liters glass distilled water.
2. Add 20 ml stock solution ammonium chloride (NH4Cl)
3. Add 20 ml stock solution Thiamin B 1
4. Add 20 ml stock solution glycerol
5. Add glass distilled water to a total volume of 4 liters
6. Divide these four liters equally over 4 flasks of 5 liter each, i.e. each flask contains 1 liter of solution.
7. Add another 4 liters glass distiled water to each of the four flasks.
8. Adjust pH to 7.4.
9. Autoclave durign 30 minutes at 121°C
10. 18 liters of basal medium will be used for 20 liters EMJH medium

PREPARATION OF FINAL MEDIUM

    Basal medium 9 parts =                18 liters

    Albumin fatty acid supplement 1 part = 2 liters
                                         ____________

    Total =                               20 liters

FILTRATION

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
Stir ingredients (magnetic stirrer) Adjust pH to 7.4 using 10% NaOH Discard 100 ml leaving 900 ml basal medium Add purified agar (if needed) Autoclave at 121 C for 20 minutes If final media contains agar it should be placed in waterbath at 56 C until supplement is to be added Cool to approximatelly 40 C before adding supplement Note:
    Semi-solid medium:
    Agar                       1.5 g
    Solid medium:
    Agar                        15 g
Supplement: 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
Add 500 ml sterile distilled water Add ingredients in order as above Adjust pH to 7.4 using 10% NaOH Make up the final volume (1 L) Sterilize by filtration - negative pressure filtration (prefiltration 0.47 um) - (vacuum should not exceed 5 psi) - possitive pressure filtration - (prefilter 0.47 um screen filter 0.22) Dispense asseptically in 100 ml amounts in 250 ml tissue culture flasks Store at -20 C Before use defrost it completely. Preparation of final medium (for 1 L) - 100 ml of supplement is added to 900 ml of basal medium.