Carlos Gusils, Adriana Pérez Chaia, Guillermo Oliver, and Silvia González
Certain gut species are pathogens, but a number of other resident bacteria may be of some benefit to host health. Examples include enterococci, lactobacilli, propionibacteria, and bifidobacteria, which are present in the colon in significant numbers. Identification and classification are not identical. A group can be identified only after it has been classified, based on a pattern of properties shown by all the members of the group that other groups do not possess. The properties used in identification are often different from those used in classification. Biochemical, nutritional, and physiological characterization tests (usually carried out in bottles and tubes of solid and liquid media and on plates) have been developed and modified since the earliest days of bacteriology. Generally, the characteristics chosen for an identification plan should be easily deter-minable, whereas those used for classification (such as DNA homology) may be quite difficult to determine. Genera and species identification might not be based on only a few tests, but rather on the pattern given by a whole battery of tests. The members of the family Lactobacillaceae represent one example of this (1). Some probiotic strains can be selected for their benefical properties as active antimicrobial agents against pathogenic microorganisms, hydrophobic ability, presence of substances with a capacity for adherence to epithelium, and so on. After isolation, identification is an important step before selecting probiotic strains.
These identifications can be facilitated through microtechniques. To alleviate the need to inoculate large numbers of tubes with media (conventional test), some rapid multitest systems have been devised and are commercially available (such as the API or Biolog systems). Although they are expensive for large studies and not always sufficiently versatile, these kits do offer the advantages of convenience, miniaturization, rapidity, and, above all, strict standardization. Other methods (2) such as ribotyping or randomly amplified polymorphic DNA do not take into account the phenotypic char-
acteristics (biotyping). In this chapter, we describe a simple, rapid, and economical microplates technique to identify lactic acid bacteria and enterobacteria based on general metabolic characteristics, adapted for routine study of a large number of strains.
1. LAPTg broth (3): This medium is used for culture of lactic acid bacteria. It contains 15 g/L peptone, 10 g/L yeast extract, 10 g/L glucose, 10 g/L triptone, and 1% Tween-80; pH 6.4 ± 0.2. Sterilize in an autoclave at 121°C for 15 min (see Note 1).
2.2. Study of Sugar Fermentations
1. Physiological solution (PS): 8.5 g/L sodium chloride. Sterilize in an autoclave at 121°C for 15 min (see Note 2).
2. Sugar solutions (20%, w/v): D-amygdaline, D-arabinose, D-cellobiose, D-erythritol, D-fruc-tose, D-galactose, D-glucose, glycerol, inositol, D-lactose, maltose, D-mannitol, D-man-nose, D-melibiose, D-melezitose, D-salicine, D-raffinose rhamnose, D-ribose, sorbitol, D-sucrose, D-trehalose, and D-xylose, sterilized by filtration (see Note 3).
a. For lactobacilli, use MRS (4) broth: 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetato, 2 g/L ammonium citrate, 2 g/L K2HPO4, 0.2 g/L MgSO4-7H2O, 0.05 g/L MnSO4-4H2O, and 1.08 g/L Tween-80; with 0.2 g/L of aggregated of clorophenol red, pH 6.5. Sterilize in an autoclave at 121°C for 15 min (see Note 4).
b. For cocci, use base medium (5): 1 g/L meat extract, 10 g/L protease peptone, 0.5 g/L sodium chloride, and 0.2 g/L phenol red, pH 7.6. Sterilize in an autoclave at 121°C for 15 min (see Note 4).
4. Sterile microplates (see Note 5).
5. Sterile Vaseline. Sterilize in autoclave at 121°C for 15 min.
2.3. Biochemical Assays (see Note 6)
a. Lactobacilli: 3 g/L arginine, 4 g/L triptone, 4 g/L yeast extract, 1 g/L glucose, 6 g/L peptone, and 1 g/L Tween-80, pH 6.5.
b. Cocci: 3 g/L arginine, 5 g/L triptone, 2.5 g/L yeast extract, 0.5 g/L glucose, 6 g/L peptone, and 2 g/L K2HPO4, pH 7.0.
c. Sterilize in an autoclave at 121°C for 15 min.
d. Test reagent: Nessler's reagent: A) 7 g potasium iodide, B) 19 g mercuric iodide, C) 10 g Potassium hydroxide, D) distilled water (to 100 mL).
i. Dissolve A and B in 40 mL of distilled water and C in 50 mL of distilled water.
ii. Mix the two solutions and add distilled water to 100 mL.
iii. Allow the precipitate to settle, decant the clear supernatant liquid into a reagent bottle, and discard the precipitate.
a. 1 g/L Esculin, 4 g/L triptone, 4 g/L yeast extract, 1 g/L glucose, 6 g/L peptone, and 1 g/L Tween-80, pH 6.5. Sterilize in autoclave at 121°C for 15 min.
3. Milk with methylene blue: 100 g/L skim milk, and 0.1 and 0.3% methylene blue. Sterilize in autoclave at 121°C for 15 min.
a. 10 g/L Triptone. Sterilize in autoclave at 121°C for 15 min.
b. Test reagent: Gracian I: formaldehyde, H2SO4conc. (1:1); Gracian II: 0.5 % K2Cr2O4.
a. 10 g/L Peptone, 5 g/L sodium chloride, 1 g/L KNO3, and inverted Durham tube. Sterilize in autoclave at 121°C for 15 min.
b. Test reagent: Reagent 1: 1 g sulphanilic acid, 100 mL 5 N acetic acid; Reagent 2: 1 g a-naphtol, 100 mL ethanol.
6. Gluconate: 1.5 g/L triptone, 1 g/L yeast extract, 1 g/L K2HPO4, 40 g/L gluconate. Sterilize in autoclave at 121°C for 15 min.
7. Gibson's medium: 80 g/L skim milk, 2.5 g/L yeast extract, 50 g/L glucose, 10 mL 4% MnSO4, 20 mL nutritive agar (see Note 7), pH 6.5. Sterilize in an autoclave at 121°C for 15 min.
a. 10 g/L Triptone, 3 g/L yeast extract, 2 g/L K2HPO4, 0.5 g/L glucose, and 20 g/L sodium hippurate. Sterilize in an autoclave at 121°C for 15 min.
b. Test reagent: H2SO4 conc.
9. 2,3,5-Triphenyltetrazolium chloride (TTC): 10 mL skim milk and 0.01% 1mL 2,3,5-triphenyltetrazolium chloride. Sterilize in an autoclave at 121°C for 15 min.
10. LAPTg broth with different conditions: final pH of 9.2, 9.4, and 9.6; 0.02% of sodium azide; 1 and 4% bile salts; 4 and 6.5% sodium chloride.
3.1. Sugar Fermentation Assays
1. Grow the strains of lactic acid bacteria in LAPTg broth at 37°C for 16 h.
2. Harvest the cells by centrifugation at 4000g for 5 min.
3. Wash the pellet obtained with PS twice (see Note 8).
4. Resuspend the cells with the PS by vortexing to optical density (OD)540 nm = 0.5 (see Note 9).
5. Inoculate (1%) fermentation media (cocci or lactobacilli) with the bacterial suspension.
6. Dispense 10 ^L of each sugar solution in wells of microplates under sterile conditions (see Note 10, and Fig. 1).
7. Add 200 ^L of the specific inoculated medium and 50 ^L of sterile Vaseline (see Note 11, and Fig. 1).
8. Incubate the microplates for 48 h at 37°C.
9. After 24 and 48 h of incubation, evaluate fermentation capacity (see Note 12).
3.2. Biochemicals Assays
1. Prepare the culture inoculum as described in steps 1—4 from Subheading 3.1.
2. Dispense 200 ^L of each sterile media in wells of microplates under sterile conditions (see Note 13).
3. Inoculate (1%) each well containing specific media with the bacterial suspension.
4. Incubate the microplates for 48 h at 37°C.
5. Inoculate (1%) another three microplates containing LAPTg broth with the bacterial suspension. In wells with gluconate, add 50 ^L of Vaseline-paraffin (1:1).
6. Incubate these microplates for 48 h at different temperatures (15, 45, and 50°C).
7. After 1, 2, 5, and 7 d of incubation, evaluate growth capacity, gas production, substance reduction, and other parameters in the media assays (see Table 1).
a. Arginine: Add 0.1 mL bacterial culture and 0.1 mL Nessler's reagent.
b. Esculin: Add 0.1 mL reagent and 0.1 mL bacterial culture.
c. Indol: Add 0.1 mL Gracian I reagent and 0.1 mL bacterial culture, and then add 0.1 mL Gracian II reagent.
d. Nitrate: Add 0.1 mL bacterial culture and 0.1 mL reagent 1, and then add 0.1 mL reagent 2. Negative reaction: Add 0.1 mL bacterial culture and add a small quantity of zinc.
e. Hippurate: Add 0.1 mL bacterial culture and 0.1 mL reagent.
1. de Man-Ragosa-Sharpe (MRS) broth (4), instead of LAPTg broth, can be use to aid lactic acid bacteria growth. MRS broth: 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetato, 2 g/L ammonium citrate, 2 g/L K2HPO4, 0.2 g/L MgSO4-7H2O, 0.05 g/L MnSO4-4H2O, and 1.08 g/L Tween-80. Sterilize in an autoclave at 121°C for 15 min.
2. Phosphate buffer, instead of physiological solution, can be use to wash cells
3. D-arabinose, D-erythritol, D-fructose, D-galactose, inositol, D-lactose, maltose, D-mannose, rhamnose, D-ribose, sorbitol, D-sucrose and D-xylose should be sterilized by filtration and another sugars by autoclaving at 121°C for 15 min.
Tests, Test reagents and Results for Identification of Bacteria
Hydrolysis of arginine
Hydrolysis of esculine Methylene blue milk Production of indole
Reduction of nitrate Nitrite
Residual nitrate Gluconate Gibson Hippurate
LAPTg broth pH
Sodium azide Bile salts Sodium chloride
Nessler's reagent 1% FeCl3 in 2% HCl
Development of an orange to brown color Black precipitated Color reduction and growth Gracian I and Gracian II Development of a deep red color
Reagent 1 and reagent 2 Small quantity of zinc
Development of a red color Development of a red color Presence of gas Disruption of the medium White precipitated (5-10 min) Reduction of the colorless compound yields a red-colored compound Bacterial growth
4. For determining the sugar fermentation capacity, it is important to use both pH indicators at 10-fold concentration, to obtain a better visualization of the change of color of the indicator.
5. Microplates can be sterilized by UV light for 1 h.
6. Studies of the biochemical properties of lactobacilli included hydrolysis of arginine, esculin growth in the presence of 0.1 and 0.3% methylene blue added to milk, indol, reduction of nitrate, gluconate, and Gibson's medium to study formation of carbon dioxide from glucose, and growth at different temperatures (15, 37, 45, and 50°C). Studies of the biochemical properties of cocci included arginine, esculin, hippurate, hydrolysis, 2,3,5-triphenyltetrazolium chloride, potassium tellurite, and nitrate reductions, growth in LAPTG at initial pH of 9.2, 9.4, and 9.6, growth at 4 and 6.5% sodium chloride, growth in the presence of 0.1 and 0.3% methylene blue added to milk, growth in 0.02% sodium azide, growth in the presence of 1 and 4% bile salts, and growth at different temperatures (15, 37, 45, and 50°C).
7. Nutritive agar: 1 g/L meat extract, 2 g/L yeast extract, 5 g/L peptone, 5 g/L sodium chloride, 3 g/L agar, pH 7.4.
8. Cell washes are important to minimize carryover of nutrient from culture.
9. These suspensions were used for inoculating biochemical studies or diluted 1:10 in adequate media for sugar fermentation.
10. The microplates with sugars can be stored for 60 d, but recent cell suspensions must be used.
11. Addition of the sterile Vaseline over fermentation media is very important to avoid contamination and to obtain an anaerobic atmosphere.
12. Fermentation is considered positive when the pH indicator in the medium becomes yellow after incubation; any shade of red is considered negative.
13. Studies of the biochemical properties of lactobacilli included arginine, esculin, methylene blue milk, indol, esculine, nitrate, gluconate, Gibson's medium, and growth at different temperatures (15, 37, 45, and 50°C). For cocci, studies included arginine, esculin, hippurate, tetrazolium chloride, potassium tellurite, nitrate, growth in LAPTg broth at initial pH of 9.2, 9.4, and 9.6; 4 and 6.5% sodium chloride; 0.1 and 0.3% methylene blue milk; 0.02% sodium azide; 1 and 4% bile salts; and at different temperatures (15, 37, 45, and 50°C).
1 Fagnant, J. E., Sanders, C. C., and Sanders, E. (1982) Development and evaluation of a biochemical scheme for identification of endocervical lactobacilli. J. Clin. Microbiol. 16, 926-934.
2 Du Plessis, E. M. and Dicks, L. M. T. (1995) Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, L. crispatus, L. amyglovorus, L. gallinarum, L. grasseri and L. johnsonii. Curr. Microbiol. 31, 114-118.
3. Raibaud, P., Caulet, M., Galpin, J. V., and Mocquot, G. (1961) Studies on the bacterial flora of the alimentary tract of pigs II. Streptococci: selective enumeration and differentiation of the dominant group. J. Appl. Bacteriol. 24, 285-291.
4. De Man, J. C., Rogosa, M., and Sharpe, E. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-155.
5. Larpent, J. P. and Larpent-Gourgaud, M. (1975) Mémento Technique de Microbiologie Technique et Documentation, Paris, pp. 1-200.
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